Polypeptide tags, compositions and methods for site-specific coupling of proteins

Abstract

The present invention provides polypeptide tags and tag modified proteins that can react with a chemical to specifically bind the chemical to the tag, thereby producing protein-chemical conjugates, including antibody-drug conjugates. The invention also provides a method for preparing the tag, the tag modified protein or the protein-chemical conjugate. In addition, the present invention provides compositions and methods for treating diseases and cancer. The present invention achieves significant advantageous effects in binding a chemical substance to a desired position of a protein with high specificity and effectiveness. The antibody-drug conjugate according to the present invention can target a target cell significantly and more efficiently compared to an antibody alone or a drug alone.

Description

[0001] Cross - Reference to Related Applications

[0002] This application claims priority to PCT International Application PCT / CN 2022 / 130722, filed on November 8, 2022, the entire content of which is incorporated herein by reference in its entirety. Field of the Invention

[0003] The present invention relates to polypeptide tags, compositions and methods for protein site-specific conjugation. Background of the Invention

[0005] Protein site-specific conjugation has little impact on protein function and is highly desirable in practical applications. Current protein site-specific conjugation is mainly achieved through unnatural amino acid insertion or enzymatic transformation. Site-specific chemical conjugation through natural amino acids is more attractive due to its simplicity. However, as of the filing date of this application, only a few cysteine-based site-specific chemical conjugation methods have been reported, and conjugation methods that do not rely on cysteine are extremely rare, although they are widely used in protein labeling and the preparation of therapeutic conjugates. The development of such conjugation methods is one of the most challenging topics in the field of chemistry.

[0006] Transition metals can catalyze a variety of chemoselective reactions on natural amino acids. However, since it is difficult to distinguish the same amino acids at different sites, except for a few terminal-selective reactions, transition metal-catalyzed reactions generally lack site-specificity. In addition, it is unclear whether site-specific conjugation catalyzed by transition metals can be achieved under biocompatible conditions.

[0007] We recently reported that Ni(II) can act as a protease to achieve protein site-specific cleavage by binding to a specific polypeptide motif (SNAC tag). In this process, the SNAC tag activates the cleavage reaction as a metal ion ligand and simultaneously determines the cleavage site.

[0008] We envisioned that protein site-specific conjugation could be achieved through a similar mechanism: a polypeptide sequence could specifically bind a metal ion, enabling the metal ion to catalyze a conjugation reaction with the polypeptide itself. Therefore, we selected several biocompatible transition metal-catalyzed reactions, including the Chan-Lam reaction, to test this hypothesis. Summary of the Invention

[0009] On the one hand, the present invention provides a polypeptide tag that can be inserted into the C-terminus or N-terminus of a protein, or inside a protein, to generate a tagged protein. The tagged protein can react with a chemical small molecule in the presence of a metal ion, thereby specifically conjugating a chemical functional group to the polypeptide tag, thus generating a protein conjugate, and the protein chemical conjugate can be an antibody-drug conjugate. On the other hand, the present invention provides a DNA encoding the above-mentioned tag or tagged protein of the present invention. On the other hand, the present invention provides a composition comprising the tag, tagged protein or protein chemical modification product of the present invention. On the other hand, the present invention provides a method for preparing the tag, tagged protein or protein chemical modification product. On the other hand, the present invention provides a composition and method for treating diseases and cancers.

[0010] The present invention has significant advantageous effects in conjugating a chemical substance to a protein target site with high specificity and high efficiency. Compared with using an antibody or a drug alone, the antibody-drug of the present invention has a significantly higher specific targeting effect on target cells.

[0011] The first aspect of the present invention provides a polypeptide tag comprising the following sequence: [Xaa1] a -[Xaa2] b -[Xaa3] c -[Xaa4] d -Xaa5-Xaa6-His-[Xaa7] e -[Xaa8] f ;

[0012] where a = 0 or 1, b = 0 or 1, c = 0 or 1, d = 0 or 1, e = 0 or 1, and / or f = 0 or 1; and

[0013] Xaa1 = phenylalanine, histidine, arginine, asparagine, proline, glutamine, tryptophan, aspartic acid, glutamic acid, lysine, isoleucine, methionine, serine, threonine or tyrosine;

[0014] Xaa2 = phenylalanine, leucine, histidine, methionine, asparagine, serine, threonine, proline, tyrosine, alanine, aspartic acid, lysine, glutamine, arginine, tryptophan or isoleucine;

[0015] Xaa3 = lysine, alanine, aspartic acid, glutamic acid, phenylalanine, histidine, isoleucine, asparagine, threonine, serine, proline, arginine, glycine, glutamine, leucine, tryptophan or methionine;

[0016] Xaa4 = aspartic acid, lysine, glutamine, glycine, histidine, asparagine, threonine, serine, methionine, lysine, glutamic acid, leucine, tryptophan, proline, alanine, phenylalanine, isoleucine, arginine or tyrosine;

[0017] Xaa5 = aspartic acid, threonine, serine or asparagine;

[0018] Xaa6 = aspartic acid, histidine, serine, threonine, glutamic acid, alanine or asparagine;

[0019] Xaa7 = alanine, tryptophan, isoleucine, leucine, methionine, tyrosine, aspartic acid, glutamine, serine, threonine, aspartic acid, lysine, glutamic acid, histidine, tryptophan, arginine, phenylalanine, proline or glycine; and

[0020] Xaa8 = alanine, glycine, isoleucine, leucine, aspartic acid, serine, threonine, aspartic acid, arginine, lysine, valine, phenylalanine, tryptophan, tyrosine, glutamine, glutamic acid or histidine.

[0021] The second aspect of the present invention provides a labeled protein, which comprises the polypeptide tag of claim 1 linked to a protein.

[0022] The third aspect of the present invention provides a protein - chemical molecule conjugate, which comprises a chemical substance and the labeled protein of the present invention, wherein the chemical substance is conjugated to the polypeptide tag through a copper(II)-catalyzed Chan - Lam reaction.

[0023] In certain embodiments, the chemical molecule is conjugated to Xaa6 of the polypeptide tag.

[0024] In certain embodiments, the chemical molecule is conjugated to the polypeptide tag through a linker.

[0025] In certain embodiments, the copper(II)-catalyzed Chan - Lam reaction is a reaction between a boronic acid group or a boronic acid - derived group and Xaa6 of the polypeptide tag.

[0026] In certain embodiments, the boronic acid can be a substituted or unsubstituted alkyl boronic acid, cycloalkyl boronic acid, alkenyl boronic acid, cycloalkenyl boronic acid, alkynyl boronic acid, cycloalkynyl boronic acid, aryl boronic acid, heteroaryl boronic acid or cycloalkyl - alkenyl boronic acid, and the boronic acid - derived group is a boronic acid pinacol ester or a trifluoroborate.

[0027] In certain embodiments, the boronic acid can be a substituted or unsubstituted vinyl boronic acid, cyclohexenyl boronic acid or styryl boronic acid.

[0028] In certain embodiments, the chemical substance includes a drug, a label, a linker, a reactive group, an antigen, a hapten, a ligand of a protein, or any combination thereof.

[0029] In certain embodiments, the drug is a cytotoxic agent.

[0030] In certain embodiments, the protein is an antibody.

[0031] A fourth aspect of the present invention provides a protein-protein complex composed of any one of the above protein-chemical conjugates and a second protein; wherein the second protein binds to the chemical substance in the protein-chemical conjugate.

[0032] In certain embodiments, the second protein is avidin and the chemical moiety is biotin, or the second protein is an antibody and the chemical moiety is an antigen specifically recognized by the antibody.

[0033] A fifth aspect of the present invention provides the above polypeptide tag, the above tagged protein, any one of the above protein-chemical conjugate products or any protein-protein complex, wherein the tag sequence is selected from the group consisting of SEQ ID NOs: 4-281 and DDH.

[0034] A sixth aspect of the present invention provides DNA encoding the above polypeptide tag or the above tagged protein.

[0035] A seventh aspect of the present invention provides a method for preparing a tagged protein, comprising adding or inserting the above polypeptide tag into a protein to obtain the tagged protein.

[0036] An eighth aspect of the present invention provides a method for preparing a protein-chemical conjugate, comprising conjugating a chemical moiety to the above tagged protein by a copper(II)-catalyzed Chan-Lam reaction.

[0037] In certain embodiments, the method comprises: reacting a chemical moiety containing a boronic acid group or a boronic acid derivative group (or a chemical moiety conjugated to the group) with the tagged protein under conditions suitable for a copper(II)-catalyzed Chan-Lam reaction to generate a protein-chemical conjugate.

[0038] In certain embodiments, the chemical moiety is conjugated to the boronic acid group or the boronic acid derivative group through a linker.

[0039] In some embodiments, the method comprises:

[0040] (i) reacting a first linker containing a boronic acid group or a boronic acid derivative group with the labeled protein under conditions suitable for a copper(II)-catalyzed Chan-Lam reaction to generate a protein-linker conjugate;

[0041] (ii) Couple the chemical with the first linker in the protein-linker conjugate.

[0042] In some embodiments, the chemical is pre-coupled with a second linker, and step (ii) comprises coupling the conjugate of the chemical with the second linker to the first linker.

[0043] In some embodiments, the boronic acid is a substituted or unsubstituted alkyl boronic acid, cycloalkyl boronic acid, alkenyl boronic acid, cycloalkenyl boronic acid, alkynyl boronic acid, cycloalkynyl boronic acid, aryl boronic acid, heteroaryl boronic acid, or cycloalkyl-alkenyl boronic acid, and the boronic acid derivative group is a pinacol boronic ester or a trifluoroborate.

[0044] In certain embodiments, the boronic acid can be a substituted or unsubstituted vinyl boronic acid, cyclohexenyl boronic acid, or styryl boronic acid.

[0045] In certain embodiments, the chemical comprises a drug, a label, a linker, a reactive group, an antigen, a hapten, a ligand of a protein, or any combination thereof.

[0046] In some embodiments, the chemical is a cytotoxic agent.

[0047] In some embodiments, the chemical is linked to the linker by a click reaction or a bioorthogonal reaction.

[0048] In certain embodiments, the protein is an antibody.

[0049] The ninth aspect of the present invention provides a method for preparing a protein-protein complex, comprising:

[0050] (i) Prepare a protein-chemical conjugate according to any of the above methods, wherein the chemical in the protein-chemical conjugate is capable of specifically binding to a second protein; and

[0051] (ii) React the protein-chemical conjugate with the second protein to form a protein-protein complex.

[0052] In certain embodiments, the second protein is avidin and the chemical moiety is biotin, or the second protein is an antibody and the chemical moiety is an antigen specifically recognized by the antibody.

[0053] In some embodiments, the sequence of the label is selected from the group consisting of SEQ ID NOs: 4-281 and DDH. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] Figure 1: a) LC-MS analysis of the reaction mixture of peptide YFLHQSHHWG (SEQ ID NO: 308) and boric acid 1. The starting material is abbreviated as SM, and the modified product is abbreviated as Mod. b) Q-Exactive MS / MS spectrum of (E)-styreneboronic acid-modified peptide YFLHQSHHWG. The b-ions and y-ions fragments observed in the MS / MS spectrum have been correspondingly labeled.

[0055] Figure 2 : LC-MS analysis of the coupling reactions on a) peptide mixture RWYFLHQX1HHWG (SEQ ID NO: 309), b) peptide mixture RWYFLHQSHHX2G (SEQ ID NO: 310), c) peptide mixture RWYFLHQSX3HWG (SEQ ID NO: 311), d) peptide mixture RWYFLHX4SHHWG (SEQ ID NO: 312), and e) peptide mixture RWYFLX5QSHHWG (SEQ ID NO: 313).

[0056] Figure 3 : LC-MS analysis of the coupling reactions of a) peptide RWYFLKQSDHWG (SEQ ID NO: 314) at 37 °C for 3 hours, b) peptide RWYFLKQDDHWG (SEQ ID NO: 315) at 25 °C for 2 hours, c) peptide RWYFLKGDDHAG (SEQ ID NO: 316) at 37 °C for 1 hour, and d) peptide RWYFFKKDDHAA (SEQ ID NO: 317) at 25 °C for 15 minutes.

[0057] Figure 4 : Development and characterization of CAST binding. a) Sequence optimization process. b) 214 nm absorption chromatogram of the reaction of RWYFFKKDDHAA (SEQ ID NO: 317) with boric acid 1 obtained from LC-MS analysis. c) Reaction kinetics. d) Partial NOESY spectrum of the modified peptide. e) LC-MS / MS analysis of the modified peptide. f) Copper-binding ATCUN (copper and nickel recognition amino terminus) motif.

[0058] Figure 5 : a) Tested boric acid reagents and their corresponding reaction yields. b) LC-MS analysis of the binding reactions between different boric acid reagents and peptide RWYFFKKDDHAA (SEQ ID NO: 317).

[0059] Figure 6 : a) LC-MS analysis of the coupling reactions between boric acid 1 and peptide RWYFFKKDDHAA (SEQ ID NO: 317) in the presence of different metal ions. b) LC-MS analysis of the coupling reactions between boric acid 1 and peptide RWYFFKKDDHAA (SEQ ID NO: 317) in the presence of different molar equivalents of Cu(II).

[0060] Figure 7 : LC-MS analysis of the coupling reaction between styrene boronic acid molecules with different modifications and the polypeptide RWYFFKKDDHAA (Sequence No.: 317).

[0061] Figure 8 : a) LC-MS analysis of the coupling reaction between boronic acid 1 and the polypeptide with the tag FFKKDDHAA (Sequence No.: 192) at different positions. b) LC-MS analysis of the coupling reaction between boronic acid 1 and cysteine-protected RWCFFKKDDHAA (Sequence No.: 318).

[0062] Figure 9 : LC-MS analysis of the coupling reaction between boronic acid 1 and polypeptides with different shortened versions of the CAST tag.

[0063] Figure 10 : Stability comparison of boronic acid 1-modified RWYFFKKDDHAA (Sequence No.: 317) and maleimide-modified DLAAEIAKHCG (Sequence No.: 324) under different conditions.

[0064] Figure 11 : ESI ion-trap / deconvolution mass spectrometry: Reaction of small ubiquitin-like modifier proteins with boronic acids 1, 1A, 1B, and 1C. a) Small ubiquitin-like modifier proteins. b) Reaction of small ubiquitin-like modifier proteins with boronic acid 1 (no modification observed). c) Small ubiquitin-like modifier protein-CAST. d) Reaction of small ubiquitin-like modifier protein-CAST with boronic acid 1. e) Reaction of small ubiquitin-like modifier protein-CAST with 1A. f) Reaction of small ubiquitin-like modifier protein-CAST with 1B. g) Reaction of small ubiquitin-like modifier protein-CAST with 1C.

[0065] Figure 12 : ESI ion-trap / deconvolution mass spectrometry: Reaction of nanobodies with boronic acids 1, 1A, 1B, and 1C. a) Nanobodies. b) Reaction of nanobodies with boronic acid 1 (no modification observed). c) Nanobody-CAST. d) Reaction of nanobody-CAST with boronic acid 1. e) Reaction of nanobody-CAST with 1A. f) Reaction of nanobody-CAST with 1B. g) Reaction of nanobody-CAST with 1C.

[0066] Figure 13: ESI ion tandem / deconvolution mass spectrometry: Reactions of maltose-binding protein with boronic acids 1, 1A, 1B, and 1C. a) Maltose-binding protein. b) Reaction of maltose-binding protein with boronic acid 1 (no modification observed). c) Maltose-binding protein-CAST. d) Reaction of maltose-binding protein-CAST with boronic acid 1. e) Reaction of maltose-binding protein-CAST with 1A. f) Reaction of maltose-binding protein-CAST with 1B. g) Reaction of maltose-binding protein-CAST with 1C.

[0067] Figure 14 : ESI ion tandem / deconvolution mass spectrometry: Reactions of trigger factor with boronic acids 1, 1A, 1B, and 1C. a) Trigger factor. b) Reaction of trigger factor with boronic acid 1 (no modification observed). c) Trigger factor-CAST. d) Reaction of trigger factor-CAST with boronic acid 1. e) Reaction of trigger factor-CAST with 1A. f) Reaction of trigger factor-CAST with 1B. g) Reaction of trigger factor-CAST with 1C.

[0068] Figure 15 : ESI ion tandem / deconvolution mass spectrometry: Reactions of maleimide-modified sortase with boronic acids 1, 1A, 1B, and 1C. a) Maleimide-modified sortase. b) Reaction of maleimide-modified sortase with boronic acid 1 (no modification observed). c) Maleimide-modified sortase-CAST. d) Reaction of maleimide-modified sortase-CAST with boronic acid 1. e) Reaction of maleimide-modified sortase-CAST with 1A. f) Reaction of maleimide-modified sortase-CAST with 1B. g) Reaction of maleimide-modified sortase-CAST with 1C.

[0069] Figure 16 : The CAST fusion proteins are quantitatively modified by various styrylboronic acid derivatives. Deconvoluted mass spectra of protein peaks show a) small ubiquitin-like modifier protein, b) nanobody, c) maltose-binding protein, d) trigger factor, and e) sortase in their starting states and after modification with 1, 1A, 1B, or 1C.

[0070] Figure 17 : ESI ion tandem / deconvolution mass spectrometry. a) Trastuzumab. b) Reaction of trastuzumab with boronic acid 1 (no modification observed in either the light or heavy chain). c) Trastuzumab, treated with EndoS prior to ESI-MS analysis. d) Reaction of trastuzumab with boronic acid 1, treated with EndoS prior to ESI-MS analysis (no modification observed in either the light or heavy chain).

[0071] e) Tra-CAST. f) Tra-CAST, treated with Endos before ESI-MS analysis. g) Reaction of Tra-CAST with boronic acid 1. h) The product modified with 1 in g), treated with EndoS before ESI-MS analysis.

[0072] i) Reaction of Tra-CAST with 1A. j) The product modified with 1A in i), treated with EndoS before LC-MS analysis. k) Reaction of Tra-CAST with 1B. l) The product modified with 1B in k), treated with EndoS before ESI-MS analysis.

[0073] m) Reaction of Tra-CAST with 1C. n) The product modified with 1C in m), treated with EndoS before ESI-MS analysis. o) Reaction of Tra-CAST with SBA-MMAE.

[0074] p) Tra-CASTi. q) Reaction of 1B with Tra-CASTi. r) Reaction of Tra-CASTi modified with 1B with DBCO-MMAE.

[0075] Figure 18 : Antibody site-specific conjugation mediated by CAST. a) Deconvolution mass spectra of the starting sample and Tra-CAST light chain modified with 1A, 1B, 1C, or SBA-MMAE and deglycosylated heavy chain peaks. b) Binding affinity characteristics of Tra-CAST (K D = 0.43 nM) and Tra-CAST-MMAE (K D = 0.49 nM) with HER2 measured by biolayer interferometry.

[0076] Figure 19 : In vitro antitumor activity of Tra-CAST-MMAE.

[0077] Figure 20 : In vitro plasma stability assessment. IAPDDHAA has higher plasma stability in vitro than FFKKDDHAA (SEQ ID NO: 192). a) Control: Phenyl-PEG2-FFKKDDHAA. b) Phenyl-PEG2-IAPDDHAA. c) IAPDDHAA modified with boronic acid 1 under standard conditions.

[0078] Figure 21: Functions and characteristics of Tra-CASTi-MMAE. a) In vitro anti-tumor activity of Tra-CASTi-MMAE. b) Detection of the stability of Tra-CASTi-MMAE in mouse plasma using anti-human IgG or anti-MMAE. c) SKOV-3 xenograft tumor model. Left panel: Tumor volume measurement; Right panel: Survival rate (female Balb / c nude mice, n = 5 for the 12 mg / kg Fc isotype control group, n = 5 for the 12 mg / kg Tra-CASTi group, n = 4 for the 6 mg / kg Tra-CASTi-MMAE group, n = 6 for the 12 mg / kg Tra-CASTi-MMAE group). Black arrows indicate the dosing time, and error bars represent standard error. d) Changes in body weight after administration of Tra-CASTi-MMAE (female Balb / c nude mice, n = 5 for the 12 mg / kg Fc isotype control group, n = 5 for the 12 mg / kg Tra-CASTi group, n = 4 for the 6 mg / kg Tra-CASTi-MMAE group, n = 6 for the 12 mg / kg Tra-CASTi-MMAE group).

[0079] Figure 22 : Electrospray ionization ion series / deconvolution mass spectrometry. a) Reaction of maltose-binding protein-CAST with 1E. b) ESI-MS analysis of the reaction product of Tra-CAST with 1E after treatment with EndoS.

[0080] Figure 23 : LC-MS analysis of the coupling reaction of boric acid 1 with further shortened polypeptide segments.

[0081] Figure 24 : LC-MS analysis of the coupling reactions of boric acid reagents with WLGFFKKDDHAA (a) and FFKKDDHAA (b). Detailed Description of the Invention

[0083] Unless otherwise defined, all technical and scientific terms used herein have the meanings commonly understood by those of ordinary skill in the art to which this invention pertains. The entire text of this application incorporates by reference publications, patent applications, patents, and other references. In case of conflict, the present specification and its definitions shall prevail. In addition, the materials, methods, and examples described herein are illustrative only and not restrictive.

[0084] When the terms "about" and "approximately" are used in conjunction with numerical variables, they generally represent the value of the variable and all values within the range of measurement or experimental error (e.g., the 95% confidence interval of the mean), or a specified deviation value within a broader range (e.g., ±10%).

[0085] Unless otherwise specified in the text, the terms "a" and "the" as used herein may also include plural contents.

[0086] The term "comprising" and its variants such as "consisting of" and "containing", as well as "include", "contain", "have", "possess", "comprise" and "include", mean including the stated steps or elements and not excluding others. "Comprising" means not excluding any steps or elements not stated. "Essentially comprising" means not excluding steps or elements that have no substantial influence on the basic and novel features of the claimed invention. The term "comprising" and its variants also cover the cases of "containing" and "essentially comprising".

[0087] Where a numerical range is provided, the reader should understand that the upper and lower limits, and each smaller range between the upper limit (or lower limit) and any intermediate value, or between any two intermediate values within the range, are considered specifically disclosed. Any intermediate range and all individual numerical values can be excluded from the numerical range.

[0088] The term "and / or" means any one, several or all of the elements connected by this term.

[0089] Unless otherwise specified, nucleic acid sequences are written from left to right in the 5' to 3' direction; amino acid sequences are written from left to right in the N-terminal to C-terminal direction, respectively.

[0090] The terms "coupled", "connected", "bonded" or similar expressions mean that one element (such as a compound) is directly bound to another element (such as another compound) in a covalent or non-covalent manner, or indirectly bound to another element (such as another compound) through one or more intermediate groups.

[0091] The terms "first" and "second" are only used to distinguish different elements or steps and do not imply a specific order of precedence or positional relationship. It should be understood that when referring to the "second" element or step, it only represents a literal distinction from other elements or steps and does not necessarily imply the existence of a corresponding "first" element or step.

[0092] On the one hand, the present invention provides a polypeptide tag which comprises the sequence [Xaa1] a -[Xaa2] b -[Xaa3] c -[Xaa4] d -Xaa5-Xaa6-His-[Xaa7] e -[Xaa8] f, where a, b, c, d, e, and f are each independently 0 or 1; where Xaa1 is one of the following amino acids: histidine, arginine, asparagine, proline, glutamine, tryptophan, aspartic acid, glutamic acid, lysine, isoleucine, methionine, serine, threonine, and tyrosine; Xaa2 is one of the following amino acids: phenylalanine, leucine, histidine, methionine, asparagine, serine, threonine, proline, tyrosine, alanine, aspartic acid, lysine, glutamine, arginine, tryptophan, and isoleucine; Xaa3 is one of the following amino acids: lysine, alanine, aspartic acid, glutamic acid, phenylalanine, histidine, isoleucine, asparagine, threonine, serine, proline, arginine, glycine, glutamine, leucine, tryptophan, and methionine; Xaa4 is one of the following amino acids: aspartic acid, lysine, glutamine, glycine, histidine, asparagine, threonine, serine, methionine, lysine, glutamic acid, leucine, tryptophan, proline, alanine, phenylalanine, isoleucine, arginine, and tyrosine; Xaa5 is one of the following amino acids: aspartic acid, threonine, serine, and asparagine; Xaa6 is one of the following amino acids: aspartic acid, histidine, serine, threonine, glutamic acid, alanine, and asparagine; Xaa7 is one of the following amino acids: alanine, tryptophan, isoleucine, leucine, methionine, tyrosine, asparagine, glutamine, serine, threonine, aspartic acid, lysine, glutamic acid, histidine, tryptophan, arginine, phenylalanine, proline, and glycine; Xaa8 is one of the following amino acids: alanine, glycine, isoleucine, leucine, asparagine, serine, threonine, aspartic acid, arginine, lysine, valine, phenylalanine, tryptophan, tyrosine, glutamine, glutamic acid, histidine

[0093] In certain embodiments, the polypeptide tag comprises the sequence Xaa1-Xaa2-Xaa3-Xaa4-Xaa5-Xaa6-His-Xaa7-Xaa8, where Xaa1, Xaa2, Xaa3, Xaa4, Xaa5, Xaa6, Xaa7, and Xaa8 are as defined above. In some embodiments, the polypeptide tag comprises the Xaa1-Xaa2-Xaa3-Xaa4-Xaa5-Xaa6-His sequence, where Xaa1, Xaa2, Xaa3, Xaa4, Xaa5, and Xaa6 are as defined above. In some embodiments, the polypeptide tag comprises the Xaa2-Xaa3-Xaa4-Xaa5-Xaa6-His-Xaa7-Xaa8 sequence, where Xaa2, Xaa3, Xaa4, Xaa5, Xaa6, Xaa7, and Xaa8 are as defined above. In some embodiments, the polypeptide tag comprises the Xaa2-Xaa3-Xaa4-Xaa5-Xaa6-His sequence, where Xaa2, Xaa3, Xaa4, Xaa5, and Xaa6 are as defined above. In some embodiments, the polypeptide tag comprises the Xaa4-Xaa5-Xaa6-His-Xaa7-Xaa8 sequence, where Xaa4, Xaa5, Xaa6, Xaa7, and Xaa8 are as defined above. In some embodiments, the polypeptide tag comprises the Xaa3-Xaa4-Xaa5-Xaa6-His sequence, where Xaa3, Xaa4, Xaa5, and Xaa6 are as defined above. In some embodiments, the polypeptide tag comprises the Xaa4-Xaa5-Xaa6-His sequence, where Xaa4, Xaa5, and Xaa6 are as defined above. In some embodiments, the polypeptide tag comprises the Xaa5-Xaa6-His-Xaa7-Xaa8 sequence, where Xaa5, Xaa6, Xaa7, and Xaa8 are as defined above. In some embodiments, the polypeptide tag comprises the Xaa5-Xaa6-His-Xaa7 sequence, where Xaa5, Xaa6, and Xaa7 are as defined above. In some embodiments, the polypeptide tag comprises the Xaa5-Xaa6-His sequence, where Xaa5 and Xaa6 are as defined above. The polypeptide tag of the present technology patent can be covalently linked to a chemical substance through a copper(II)-catalyzed Chan-Lam reaction.

[0094] In certain embodiments, the polypeptide tags of the present invention do not contain the following sequences: FLGGSHHTD (SEQ ID NO: 325), FLPGSRHWG (SEQ ID NO: 326), FLPGSHHWG (SEQ ID NO: 327), GSHHTDLP (SEQ ID NO: 328), GSRHW (SEQ ID NO: 329), GSHHW (SEQ ID NO: 330), PGSHHW (SEQ ID NO: 331), HNSHHW (SEQ ID NO: 332), GSHHTDLP (SEQ ID NO: 333), GSHHSSPN (SEQ ID NO: 334), PGSKHNCG (SEQ ID NO: 335), SGSHHNYS (SEQ ID NO: 336), LGSQHQAQ (SEQ ID NO: 337), NGSSHFRT (SEQ ID NO: 338), NGSHHFMN (SEQ ID NO: 339), SGSKHDIS (SEQ ID NO: 340), KGSLHHAF (SEQ ID NO: 341), AGSVHATS (SEQ ID NO: 342), RGSSHGDR (SEQ ID NO: 343), TGSQHTMS (SEQ ID NO: 344), or VGSSHDGS (SEQ ID NO: 345).

[0095] In some embodiments, the polypeptide tag comprises a sequence selected from SEQ ID NOs: 4 - 281 and DDH. In some embodiments, Xaa5 - Xaa6 - His - [Xaa7] e - [Xaa8] f is DDHAA.

[0096] The N - terminus of the polypeptide tag can be Xaa1, Xaa2, Xaa3, or Xaa4, and / or the C - terminus of the polypeptide tag can be His, Xaa7, or Xaa8, depending on the values of a, b, c, d, e, and f.

[0097] In some embodiments, the tag sequence is selected from SEQ ID NOs: 4 - 281 and DDH. In another aspect, the present invention provides a tagged protein (i.e., a protein modified by the tag), comprising the polypeptide tag of the present invention linked to a protein. The tag and the protein can form a fusion protein through covalent linkage, and the tag can be linked to the N - terminus or C - terminus of the protein, or inserted into the protein sequence. It should be understood that when the tag is inserted into the protein sequence, its position should not affect the protein activity. As used herein, proteins include monomers composed of a single polypeptide chain or multimers composed of two or more polypeptide chains. When the protein is a multimer, the tag can be linked to any one, several, or all of the two or more polypeptide chains. Each polypeptide chain can be linked to one, two, or more tags. In some embodiments, the tag is heterologous to the protein. The tagged protein can be an antibody, an enzyme, a receptor, a ligand, etc. including but not limited to small ubiquitin - like modifier protein (SMT3), maltose - binding protein (MBP), nanobody, trigger factor, and sortase. The molecular weight of the protein of the present invention can range from 1 kilodalton to about 150 kilodaltons, for example, about 5 kilodaltons to about 70 kilodaltons or about 10 kilodaltons to about 50 kilodaltons.

[0098] It should be understood that in the context of a tagged protein, the terms "linked to" and "inserted" only mean that the sequence of the tagged protein is the same as the sequence obtained by linking or inserting the tag, and do not imply that the linking or insertion operation must be performed when preparing the tagged protein. The tagged protein can be prepared by any method known in the art, such as expressing the modified protein in a host cell by DNA recombinant technology and then isolating and purifying it.

[0099] In some embodiments, the protein modified by the tag is an antibody, particularly a monoclonal antibody, scFv, or nanobody. In some embodiments, the antibody is trastuzumab. When the antibody contains multiple polypeptide chains (such as heavy and light chains), the tag can be linked to the heavy chain, the light chain, or both.

[0100] In another aspect, the present invention provides a protein - chemical conjugate comprising the tagged protein of the present invention and a chemical substance, wherein the chemical substance is linked to the tag of the tagged protein. In other words, the conjugate is formed by conjugating the chemical substance to the tag of the tagged protein of the present invention. In some embodiments, the chemical substance is conjugated to Xaa6 of the tag. In some embodiments, the chemical substance can be conjugated to the tag via a copper(II)-catalyzed Chan - Lam reaction, for example, by the reaction of boric acid or a boric acid derivative with -NH, -OH, -SH in Xaa6 of the tag. Before the copper(II)-catalyzed Chan - Lam reaction occurs, the chemical substance can comprise or be linked to a boric acid group or a boric acid derivative group. It should be understood that after the copper(II)-catalyzed Chan - Lam reaction with the polypeptide tag, the boric acid group or the boric acid derivative group no longer exists, and the protein - chemical conjugate comprises the Chan - Lam reaction product, i.e., the product formed by the reaction of boric acid or a boric acid derivative with Xaa6 (specifically -NH, -OH, -SH of Xaa6) of the tag.

[0101] As used herein, the term boric acid generally refers to an organic boric acid having the general formula R - B(OH)2, wherein R is an organic substituent containing at least one carbon atom, and the boron atom is directly bonded to the carbon atom of the substituent. R can be an organic substituent including, but not limited to, a substituted or unsubstituted alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, heteroaryl, or cycloalkyl - alkenyl. In certain embodiments, R can be a substituted or unsubstituted vinyl or cyclohexyl - vinyl, styryl.

[0102] In certain embodiments, the boric acid can be a substituted or unsubstituted alkyl boric acid, cycloalkyl boric acid, alkenyl boric acid, cycloalkenyl boric acid, alkynyl boric acid, cycloalkynyl boric acid, aryl boric acid, heteroaryl boric acid, or cycloalkyl - alkenyl boric acid. In certain embodiments, the alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, heteroaryl, or cycloalkyl - alkenyl can have 2 - 10 carbon atoms. In certain embodiments, the alkenyl can be vinyl. In certain embodiments, the boric acid can be vinyl boric acid, cyclohexyl - vinyl boric acid, or styryl boric acid. In certain embodiments, the cycloalkenyl can be cyclohexenyl, cyclopentenyl, cyclobutenyl, or cyclopropenyl. In certain embodiments, the boric acid can be cyclohexenyl boric acid, cyclopentenyl boric acid, cyclobutenyl boric acid, or cyclopropenyl boric acid. In certain embodiments, the cycloalkyl - alkenyl can be cyclohexyl - vinyl, cyclopentyl - vinyl, cyclobutyl - vinyl, or cyclopropyl - vinyl. In certain embodiments, the boric acid can be cyclohexyl - vinyl boric acid, cyclopentyl - vinyl boric acid, cyclobutyl - vinyl boric acid, or cyclopropyl - vinyl boric acid.

[0103] As used herein, the term "boronic acid derivative" includes, but is not limited to, boronic esters (such as pinacol boronate, trimethylene glycol borate) and trifluoroborates (such as R-BF3K). In certain embodiments, the boronic acid derivative can be vinylboronic acid pinacol ester, potassium vinyltrifluoroborate, cyclohexyl-vinylboronic acid pinacol ester, cyclohexyl-potassium vinyltrifluoroborate, styrylboronic acid pinacol ester or styryl-potassium trifluoroborate. In certain embodiments, the boronic acid derivative group can be pinacolboronyl or trifluoroborate.

[0104] The chemical substance can be a small molecule, including, but not limited to, drugs, markers, linkers, reactive groups, antigens, haptens, and protein ligands. The chemical substance of the present invention can contain more than one functional moiety. The functional moiety can be selected from, but is not limited to, drugs, markers, reactive groups, antigens, haptens, and protein ligands. The chemical substance can contain one or more drugs, markers, reactive groups, antigens, haptens, or protein ligands, or any combination thereof. In certain embodiments, the drug can be a cytotoxic agent. In certain embodiments, the marker can be a detectable marker or an affinity tag (or purification tag), such as a fluorescent marker. In certain embodiments, the protein ligand can be biotin. In certain embodiments, the reactive group can be a bioorthogonal reaction pair or a click reaction pair.

[0105] In certain embodiments, the chemical substance is conjugated to a polypeptide tag through a linker, wherein the chemical substance is linked to the linker, the linker is linked to the polypeptide tag of the tagged protein, and the linker is located between the chemical substance and the polypeptide tag. That is to say, the protein-chemical conjugate is formed by conjugating the chemical substance and the linker to the tag of the tagged protein of the present invention. In certain embodiments, the linker is conjugated to the Xaa6 site of the tag. In certain embodiments, the linker can be conjugated to the tag through a copper(II)-catalyzed Chan-Lam reaction (such as the reaction between a boronic acid or a boronic acid derivative and -NH, -OH, -SH at the Xaa6 site of the tag). Before the copper(II)-catalyzed Chan-Lam reaction occurs, the linker can contain or be conjugated to an additional boronic acid group or a boronic acid derivative group.

[0106] In certain embodiments, the linker can be a cleavable linker (such as an enzyme-cleavable polypeptide linker, an acid-sensitive hydrazone linker, a glutathione polypeptide-sensitive disulfide linker, a cathepsin B-sensitive linker, etc.) or a non-cleavable linker (such as SMCC). The linker can be a linker commonly used in antibody-drug conjugates (ADCs). The linker can be a linker containing a PEG moiety. In certain embodiments, the linker contains Val-Cit-PAB. In certain embodiments, the linker can contain Val-Cit-PAB, Glu-Val-Cit-PAB, DBCO-(PEG)3-Val-Cit-PAB, or DBCO-Glu-Val-Cit-PAB.

[0107] In certain embodiments, the linker can contain a reactive group capable of reacting with a chemical substance. In certain embodiments, the chemical substance can be linked to the linker by any chemical reaction. In certain embodiments, the chemical substance can be linked to the linker by a click reaction or a bioorthogonal reaction.

[0108] In certain embodiments, the linker for linking a chemical substance to a tagged protein can contain more than two linkers, which can be referred to as the first linker, the second linker, etc. In certain embodiments, the first linker is used to conjugate with a polypeptide tag, and the second linker is used to conjugate with a chemical substance. The first linker and the second linker can be connected to each other by any chemical reaction (such as a click reaction or a bioorthogonal reaction).

[0109] The chemical substance can be linked to the linker before or after the linker is conjugated with the tag sequence. In certain embodiments, the conjugate of the tagged protein and the first linker is linked to the conjugate of the chemical substance and the second linker to form a protein-chemical substance conjugate.

[0110] In certain embodiments, the protein-chemical substance conjugate contains a protein-drug or a protein-linker-drug structure. In certain embodiments, the protein-chemical substance conjugate contains a protein-label or a protein-linker-label structure.

[0111] In certain embodiments, the protein-chemical substance conjugate contains any of the following structures:

[0112]

[0113]

[0114] wherein the definitions of Xaa1, Xaa2, Xaa3, Xaa4, Xaa5, Xaa6, Xaa7, Xaa8, and a, b, c, d, e, f are as described above.

[0115] The N-terminus of the structure (i.e., Xaa1, Xaa2, Xaa3 or Xaa4) or the C-terminus of the structure (i.e., His before Xaa7, Xaa7 or Xaa8) is located at the end of the protein-chemical conjugate; or both the N-terminus and C-terminus of the structure are linked to amino acids in the protein-chemical conjugate, i.e., the structure is inserted into the interior of the protein-chemical conjugate;

[0116] wherein n is an integer from 0 to 12 (inclusive).

[0117] In another aspect, the present invention provides a protein-protein complex comprising a protein-chemical conjugate as described above and a second protein, wherein the second protein specifically binds to the chemical substance. The chemical substance and the second protein can bind through any interaction, including covalent binding or non-covalent binding. Examples of non-covalent binding include electrostatic force, hydrogen bond, hydrophobic effect, van der Waals force, etc., such as the interaction between a protein and its ligand or between an antigen and an antibody. In some embodiments, the chemical substance in the protein-protein complex is an antigen or a protein ligand. In some embodiments, the second protein can be avidin and the chemical substance can be biotin. In some embodiments, the second protein can be an antibody and the chemical substance can be an antigen specifically recognized by the antibody.

[0118] In another aspect, the present invention provides a DNA encoding the tag or tag-modified protein of the present invention.

[0119] In another aspect, the present invention provides a composition comprising the tag, tag protein, protein-chemical conjugate, protein-protein complex or DNA of the present invention. In some embodiments, the composition may further comprise a pharmaceutically acceptable excipient. In some embodiments, the composition comprising the protein-chemical conjugate can be used for treating or preventing diseases, tumors or cancers.

[0120] In another aspect, the present invention provides a method for preparing the tag protein of the present invention, comprising adding or inserting the polypeptide tag of the present invention into a protein to obtain the tag protein. The sequence of the tag can be linked to the N-terminus or C-terminus of the protein, or inserted at any position in the protein sequence.

[0121] In another aspect, the present invention provides a method for preparing the protein-chemical conjugate of the present invention, comprising conjugating a chemical substance with the tag protein of the present invention through a copper(II)-catalyzed Chan-Lam reaction to obtain the protein-chemical conjugate.

[0122] The method can be achieved through one or more steps, as long as the protein-chemical conjugate of the present application can be obtained. The reaction conditions of the copper(II)-catalyzed Chan-Lam reaction are well-known to those skilled in the art.

[0123] For example, in certain embodiments, the method may include contacting a chemical substance containing or bound to a boronic acid group or a boronic acid derivative group with the labeled protein of the present invention under conditions suitable for the copper(II)-catalyzed Chan-Lam reaction, thereby generating a protein-chemical conjugate.

[0124] The chemical substance and / or labeled protein containing or bound to a boronic acid group or a boronic acid derivative group in the present invention can be prepared by known technical methods, such as chemical synthesis, DNA recombination technology, etc.

[0125] In certain embodiments, the chemical substance is bound to a boronic acid group or a boronic acid derivative group through a linker. The method may include contacting the conjugate of the chemical substance and the linker with the labeled protein of the present invention under conditions suitable for the copper(II)-catalyzed Chan-Lam reaction, thereby generating a protein-chemical conjugate; wherein the chemical substance is connected to the linker, and the linker contains or is bound to a boronic acid group or a boronic acid derivative group.

[0126] The chemical substance can be connected to the linker through any chemical reaction, for example, through a click reaction or a bioorthogonal reaction.

[0127] In certain embodiments, the method may include:

[0128] (i) contacting a linker containing or bound to a boronic acid group or a boronic acid derivative group with the labeled protein under conditions suitable for the copper(II)-catalyzed Chan-Lam reaction, thereby generating a protein-linker conjugate; and

[0129] (ii) binding the chemical substance to the linker of the protein-linker conjugate.

[0130] The chemical substance can be connected to the linker through any chemical reaction, for example, through a click reaction or a bioorthogonal reaction.

[0131] In certain embodiments, the method may include:

[0132] (i) contacting a first linker containing a boronic acid group or a boronic acid derivative group with the labeled protein under conditions suitable for the copper(II)-catalyzed Chan-Lam reaction, thereby generating a protein-linker conjugate;

[0133] (ii) binding the conjugate of the chemical substance to the first linker of the protein-linker conjugate.

[0134] The second linker is connected to the first linker and is located between the chemical substance and the linker of the protein-linker conjugate. The second linker can be connected to the first linker by any chemical reaction, such as by click reaction or bioorthogonal reaction.

[0135] In another aspect, the present invention provides a method for preparing a protein-protein complex, comprising: (i) preparing a protein-chemical conjugate according to the preparation method of the present invention; (ii) reacting the protein-chemical conjugate with a second protein to form a protein-protein complex.

[0136] In another aspect, the present invention provides a molecule prepared by the method of the present invention, including a labeled protein, a protein-chemical conjugate, and a protein-protein complex.

[0137] In another aspect, the present invention provides a method for treating a disease, tumor or cancer, which includes administering a therapeutically effective dose of the protein-chemical conjugate of the present invention to a subject.

[0138] Definitions:

[0139] Polypeptide tag: The terms "polypeptide tag" and "tag" can be used interchangeably and refer to a short amino acid sequence that can be used to conjugate a chemical substance to a polypeptide or protein. The term "tag" as used in the present invention refers to a polypeptide sequence that can be attached to another polypeptide or protein to provide a certain function. The minimum length of the tag of the present invention is at least about 3 amino acids, for example, at least about 4 amino acids, at least about 5 amino acids, at least about 6 amino acids, at least about 7 amino acids, at least about 8 amino acids or at least about 9 amino acids. In certain embodiments, the length of the tag of the present invention ranges from about 3 to about 100 amino acids, for example, from about 3 to about 50 amino acids, from about 3 to about 20 amino acids, or from about 3 to about 10, 11, 12, 13, 14, 15, 16, 17, 18 or 19 amino acids. In certain embodiments, the length of the tag of the present invention is about 3 to about 9 amino acids, about 4 to about 9 amino acids, about 5 to about 9 amino acids, about 6 to about 9 amino acids or about 7 or 8 amino acids.

[0140] Protein: The term "protein" refers to a polymer of amino acids of any length, which may include coded and non-coded amino acids, chemically or biochemically modified or derived amino acids, and proteins with modified polypeptide backbones. The proteins of the present invention may be monomers consisting of a plurality of polypeptide chains, or a multimeric protein. Monomeric proteins may also be used interchangeably with the term "polypeptide". The term "multimeric protein" refers to a protein that may exist in a multimeric form consisting of two or more polypeptide chains. In a multimer, two or more polypeptide chains may be linked by covalent bonds (such as disulfide bonds), or by non-covalent bonds (such as hydrogen bonds and hydrophobic interactions), or by a combination of both. The multimer preferably contains one or more intermolecular disulfide bonds. The multimer may be a homomultimer consisting of a single type of polypeptide chain, or may be a heteromultimer consisting of two or more types of polypeptide chains.

[0141] Heterologous: The term "heterologous" means that a first entity and a second entity are combined in a manner that does not normally exist in nature. The "heterologous" tag means that the protein sequence linked to the tag does not originally contain the sequence of the tag.

[0142] Antibody: The term "antibody" refers to any antigen-binding molecule or molecular complex that contains at least one complementarity-determining region (CDR) that specifically binds to or interacts with a particular antigen. The term "antibody" includes various forms of antibodies, including but not limited to monoclonal antibodies, polyclonal antibodies, antigen-binding fragments, and engineered antibodies such as multispecific antibodies (e.g., bispecific or trispecific antibodies). The antibodies of the present invention can also be chimeric antibodies, humanized antibodies, or human antibodies. The terms "monoclonal antibody", "intact antibody", and "immunoglobulin" are used interchangeably and refer to a glycoprotein that contains at least two heavy (H) chains and two light (L) chains linked by disulfide bonds. Each heavy chain consists of a heavy-chain variable region (abbreviated herein as VH) and a heavy-chain constant region. The heavy-chain constant region consists of three domains, CHI, CH2, and CH3. Each light chain consists of a light-chain variable region (abbreviated herein as VL) and a light-chain constant region. The light-chain constant region consists of one domain, CL. The VH and VL regions can be further subdivided into hypervariable regions called complementarity-determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs, arranged in the order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain the binding domains that interact with the antigen. The constant region of the antibody can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system (Clq). The antibodies of the present invention can be of any isotype (e.g., IgA, IgD, IgE, IgG, or IgM, i.e., α, δ, ε, γ, or μ heavy chains). In the IgG isotype, the antibody can be an IgG1, IgG2, IgG3, or IgG4 subclass. The antibodies of the present invention can have a κ or λ light chain. The term "monoclonal antibody" as used herein refers to an antibody derived from a single clone, including any eukaryotic, prokaryotic, or phage clone, obtained by any means available or known in the prior art and is not limited to antibodies produced by hybridoma technology. Monoclonal antibodies relevant to the present disclosure can be prepared using a variety of techniques known in the prior art, including the use of hybridomas, recombinant DNA, and phage display techniques, or combinations thereof. The term "antigen-binding fragment" refers to one or more fragments of an intact antibody, and the fragment(s) referred to should retain the ability to specifically bind to a given antigen. Antigen-binding fragments can be obtained by proteolytic digestion of intact antibodies, recombinant DNA, or phage display techniques.Examples of the term "antigen-binding fragment" include (i) Fab fragment, a monovalent fragment consisting of VL, VH, CL, and CH1 domains; (ii) Fab' fragment, essentially Fab with a portion of the hinge region; (iii) F(ab')2 fragment, a divalent fragment consisting of two Fab fragments linked by a disulfide bridge in the hinge region; (iv) Fd fragment, consisting of VH and CH1 domains; (v) Fv fragment, consisting of VL and VH domains of a single arm of an antibody; (vi) single-chain Fv fragment (scFv), a single protein chain in which the VL and VH regions pair to form a monovalent molecule; (vii) disulfide-stabilized Fv fragment (dsFv), an Fv with engineered intermolecular disulfide bonds to stabilize the VH-VL pair; (viii) single-domain antibody (sdAb), also known as nanobody, consisting of a single monomeric variable antibody domain (e.g., a single heavy-chain variable region VHH), and so on.

[0143] Conjugate: The term "conjugate" refers to any compound formed by the covalent attachment of two or more individual compounds, where one individual compound can be directly covalently linked to another individual compound or indirectly covalently linked to another individual compound through one or more intervening moieties.

[0144] Copper(II)-catalyzed Chan-Lam reaction: The Chan-Lam reaction is a cross-coupling reaction between an organoboric acid and a (thio)alcohol or amine, including an amide NH bond. Traditionally, in an ambient air environment at atmospheric pressure, copper(II) (such as Cu(OAc)2) is used as a catalyst and acts together with an amine base in a chlorinated solvent to carry out the Chan-Lam coupling reaction at room temperature.

[0145] Boric acid: The terms "boric acid" and "organoboric acid" are used interchangeably herein, and its general formula is: R-B(OH)2, where R is a substituent containing at least one carbon atom and the boron atom is directly bonded to the carbon atom of the substituent. R can be an organic substituent, including but not limited to substituted or unsubstituted alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, heteroaryl, or cycloalkyl-alkenyl. In certain embodiments, R can be substituted or unsubstituted vinyl or cyclohexyl-vinyl, styryl.

[0146] Boric acid derivative: The term "boric acid derivative" refers to a compound obtained by replacing one or both of the two hydroxyl groups of boric acid with one or two other functional groups. Boric acid derivatives include boronic esters (such as pinacol borate and triglycol borate) and trifluoroborates (such as R-BF3K).

[0147] Chemical Substance: The term "chemical substance" refers to any molecule having a specific chemical structure, including but not limited to drugs, small molecule markers, and reactive handles. The chemical substance may contain a boronic acid or boronic acid derivative group capable of reacting with a polypeptide tag.

[0148] Linker: The term "linker" generally refers to a group that connects two other functional moieties. The linker of the present invention may contain at least one reactive group. The linker of the present invention comprises a bifunctional linker.

[0149] Functional Moiety: The term "functional component" refers to any part that exhibits one or more functions, including but not limited to therapeutic functions, detectable functions, acting as a linker, having reactivity, having enzymatic activity, acting as an affinity tag, antigen, or hapten.

[0150] Bifunctional Linker: The term "bifunctional linker" refers to a linker containing two reactive groups, wherein each reactive group can react with a corresponding reactive group on the functional moiety to which the linker is attached.

[0151] Reactive Group: The term "reactive group" as used herein refers to a group capable of reacting with another chemical group to form a covalent bond, i.e., having covalent reactivity under suitable reaction conditions, and generally represents a point of attachment to another substance. Reactive groups include but are not limited to click reaction pairs and bioorthogonal reaction pairs.

[0152] Protein Ligand: The term "protein ligand" as used herein refers to a small molecule capable of interacting with a protein through non-covalent binding (such as electrostatic force, hydrogen bond, hydrophobic effect, van der Waals force, etc.). Examples of protein ligands include but are not limited to biotin.

[0153] Antigen: The term "antigen" refers to a substance capable of inducing an immune response in an organism (referring to an animal, such as a human). The antigen of the present invention includes small molecule antigens (such as haptens), including but not limited to polysaccharides, lipids, nucleic acids, as well as small molecule compounds and drugs.

[0154] Hapten: The term "hapten" refers to a small molecule that can elicit an immune response only when attached to a large carrier such as a protein.

[0155] Drug: A drug refers to any chemical substance capable of causing physiological or psychological changes in an organism and producing a therapeutic or preventive effect when ingested or administered.

[0156] Cytotoxic Agent: A cytotoxic agent refers to a compound that is toxic to cells. Cytotoxic agents can be used as chemotherapeutic drugs for treating tumors or cancers.

[0157] Small molecule: The term "small molecule" refers to a compound having a molecular weight less than or equal to about 4000 Daltons, preferably less than or equal to about 1000 Daltons, about 900 Daltons, 800 Daltons, 700 Daltons, 600 Daltons or 500 Daltons.

[0158] Label: A label refers to any small molecule that, when attached to other chemical substances, aids in their identification or purification. Labels include fluorescent labels, affinity tags for protein purification, etc.

[0159] Click reaction: A click reaction refers to any reaction that allows a chemical substance to be attached to a biomolecule (such as a protein, glycan, lipid, and metabolite). Click reactions include copper(II)-catalyzed azide-alkyne cycloaddition, strain-promoted azide-alkyne cycloaddition, and strain-promoted alkyne-nitrone cycloaddition (SPANC).

[0160] Click reaction pair: A click reaction pair refers to one of the two chemical groups that interact in a click reaction and is responsible for linking a chemical substance to a biomolecule.

[0161] Bioorthogonal reaction: A bioorthogonal reaction refers to any chemical reaction that can occur within a living system without interfering with natural biochemical processes. Bioorthogonal reactions include nitrone dipole cycloaddition, norbornene cycloaddition, oxanorbornadiene cycloaddition, tetrazine ligation, [4+1] cycloaddition, tetracyclane ligation, and the Staudinger reaction.

[0162] Bioorthogonal reaction pair: A bioorthogonal reaction pair refers to one of the two chemical groups that interact in a bioorthogonal reaction and is responsible for linking two molecules together.

[0163] Protein-linker: A protein-linker refers to a substance formed after a labeled protein containing a polypeptide tag reacts with a linker, where the protein and the linker are covalently linked.

[0164] Protein-chemical substance: A protein-chemical substance refers to a substance formed after a protein reacts with a chemical substance, where the protein and the chemical substance are covalently linked.

[0165] Antibody-drug conjugate (ADC): An ADC refers to any substance in which an antibody is linked to a drug.

[0166] Therapeutically effective dose: The term "therapeutically effective dose" refers to a dose sufficient to provide the desired therapeutic benefit. The effective dose range of an active agent is typically from 0.1 mg of the active agent / kg body weight to about 50 mg of the active agent / kg body weight. However, the dose level needs to consider multiple factors, including the type of injury, the age, weight, gender, health status, severity of the condition, route of administration, and the specific active agent used. Therefore, the dosing regimen may vary widely, but a surgeon can routinely determine it by standard methods.

[0167] Pharmaceutically acceptable excipients: The term "pharmaceutically acceptable excipients" refers to materials such as carriers, diluents, stabilizers, dispersants, suspending agents, thickening agents, etc. that enable the active pharmaceutical ingredient (API) to be processed into a suitable dosage form. Such excipients should not significantly impair the expected biological activity or properties of the compound (i.e., the API), and should be relatively non-toxic, that is, the material should not cause adverse biological effects when administered to an individual, nor should it have harmful interactions with any component in the formulation. Examples

[0168] Example 1: Optimization of the tag sequence

[0169] a. Preliminary screening

[0170] We initiated the study by screening several metal ion-binding polypeptides. The sequences of the different polypeptides used are shown in Table 1. To facilitate HPLC and mass spectrometry detection, amino acid Y was added to the N-terminus of the sequences shown in Table 1. The boric acid reagents used were as follows:

[0171]

[0172] Standard reaction conditions for Examples 1a and 1b: To the polypeptide solution (8.6 μL of 5 mM aqueous solution, final concentration 0.43 mM) in N-methylmorpholine buffer (82.4 μL of 5 mM stock solution, pH 7.4), the boric acid reagent (2 μL of 50 mM DMSO solution, final concentration 1.0 mM) and CuCl2·2H2O (7 μL of 5 mM aqueous solution, final concentration 0.35 mM) were added successively. After vortex mixing, the reaction was shaken at 37 °C for the specified time. The reaction was quenched with tetrasodium ethylenediaminetetraacetate (Na4-EDTA·2H2O) (2 μL of 500 mM aqueous solution, final concentration 20 mM), and the supernatant was taken for analysis after centrifugation to remove the precipitate.

[0173] The different metal ion-binding polypeptides were reacted with different boric acid reagents at 37 °C for 12 hours under the standard reaction conditions. The yield was determined by LC-MS analysis of the reaction mixture, analyzed using HPLC, and calculated by dividing the peak area of the reaction product by the sum of the peak areas of the starting material and the product. The results are shown in Table 1.

[0174] The LC-MS results of the reaction mixture of boric acid 1 with YFLHQSHHWG (sequence number: 308) are shown in Figure 1 a (the starting material is abbreviated as "SM" and the modified product is abbreviated as "Mod").

[0175] The preliminary results of the Chan-Lam reaction are encouraging: there are significant differences in the reaction yields of similar polypeptide sequences. This indicates that the Cu(II)-mediated Chan-Lam coupling reaction is indeed sequence-specific. However, the reaction rate is slow, and the best sequence (FLHQSHHWG at 0.43 mM) only produces about 55% of the product after 12 hours (Table 1 and Figure 1 a).

[0176] (E)-The Q-Exactive MS / MS spectrum of styreneboronic acid-modified YFLH1QSH2H3WG (where H1, H2, and H3 all represent the amino acid H, and the subscripts 1, 2, or 3 are used to distinguish H at different positions) is as shown in Figure 1 b. This mass spectrometry analysis indicates that the reaction site is located at residue H2, which, based on previous research results, means that residue H3 is the anchoring residue ( Figure 1 b).

[0177] Table 1. Reaction yields of different boronic acid reagents binding to polypeptides with different metal ions

[0178] Sequence 1 2 3 4 FLGGSHHWG (Sequence No.: 1) Trace 0％ Trace 0％ FLGHSRHWG (Sequence No.: 2) 0％ 0％ 0％ 0％ FLHGSRHWG (Sequence No.: 3) Trace 0％ Trace 0％ FLGHSHHWG (Sequence No.: 4) 21％ 0％ 11％ 0％ FLPHSHHWG (Sequence No.: 5) 50％ 0％ 12.5％ 0％ FLHQSHHWG (Sequence No.: 6) 55％ 0％ 9％ 0％

[0179] b. Screening of mixed polypeptide segments

[0180] Subsequently, we randomized each residue around the H3 site in FLH1QSH2H3WG respectively to identify the best residues that can improve the reaction rate. For efficient liquid chromatography (HPLC) and mass spectrometry detection, the amino acids RWY were added to the N-terminus of each sequence shown in Tables 2a, 3, 4, and 5.

[0181] First, different amino acids at the X1 site in FLHQX1HHWG (sequence number: 319) were screened. The test sequences and numbers are listed in Table 2a.

[0182] Table 2a.

[0183]

[0184] Table 2b.

[0185]

[0186] The mixtures of all polypeptides listed in Table 2a were prepared using an automated parallel polypeptide synthesizer (Syro II, Biotage). During the cycle of synthesizing X1 in RWYFLHQX1HHWG (sequence number: 309), a mixture of amino acids A, D, E, H, L, K, M, P, S, N, Q, Y, W, I, and R in specific proportions (as shown in Table 2b) was used to ensure the uniform distribution of the synthesized polypeptides.

[0187] The polypeptide mixture was reacted with boric acid 1 at 37 °C for 4 hours under standard reaction conditions, and the reaction mixture was analyzed by LC-MS.

[0188] As Figure 2 shown by the LC-MS results of

[0189] a, amino acids Asp, His, and Ser were preferentially selected at the X1 position.

[0190] Table 3.

[0191]

[0192]

[0193] Mixtures of all the polypeptides listed in Table 3 were prepared by the above method. The polypeptide mixture was reacted with boric acid 1 at 37 °C under standard reaction conditions for 2 hours, and the reaction mixture was analyzed by LC-MS.

[0194] As Figure 2 shown by the LC-MS results of

[0195] b, amino acids Asp, Glu, His, Leu, Ser, Tyr, Trp, and Arg were preferentially selected at the X2 site.

[0196] Table 4.

[0197]

[0198] Mixtures of all the polypeptides listed in Table 4 were prepared by the above method. The polypeptide mixture was reacted with boric acid 1 at 37 °C under standard reaction conditions for 2 hours, and the reaction mixture was analyzed by LC-MS.

[0199] As Figure 2 shown by the LC-MS results of

[0200] c, amino acids Asp, Asn, and Ser were preferentially selected at the X3 position.

[0201] Table 5.

[0202]

[0203]

[0204] The mixture of all the polypeptides listed in Table 5 was prepared by the above method. The polypeptide mixture was reacted with boric acid 1 under standard reaction conditions at 37 °C for 2 hours, and the reaction mixture was analyzed by LC-MS.

[0205] As Figure 2 shown by the LC-MS results of d, the amino acids Gln, Asp, and Ser were preferentially selected at the X4 site.

[0206] Fifth, different amino acids at the X5 position in FLX5QSHHWG (SEQ ID NO: 323) were screened. The sequences used are listed in Table 6.

[0207] Table 6.

[0208]

[0209] The mixture of all the polypeptide segments listed in Table 6 was prepared by the above method. The polypeptide segment mixture was reacted with boric acid 1 under standard reaction conditions at 37 °C for 2 hours, and then the reaction mixture was analyzed by LC-MS.

[0210] As Figure 2 shown by the LC-MS results of e, the amino acids Asp, Lys, and His were preferentially selected at the X5 site.

[0211] c. Combinations of preferred amino acids and further optimization

[0212] The results showed that Asp, Asn, and Ser were often preferentially selected at multiple sites. Therefore, we combined the preferred amino acids at different sites in order to obtain a synergistic effect. For the convenience of HPLC and mass spectrometry detection, the amino acids RWY were added to the N-terminus of each sequence shown in Table 7 and Tables 9 - 13.

[0213] Standard reaction conditions for Example 1c: To a mixed system of an aqueous polypeptide solution (1 μL of a 5 mM stock solution, final concentration 0.05 mM) and an N-methylmorpholine buffer solution (97 μL of a 5 mM stock solution, pH 7.4), boric acid 1 (1 μL of a 50 mM DMSO stock solution, final concentration 0.5 mM) and CuCl2·2H2O (1 μL of a 5 mM aqueous solution, final concentration 0.05 mM) were added successively. After vortexing, the reaction was carried out at the specified temperature and time according to the experiment. The reaction was quenched by adding tetrasodium ethylenediaminetetraacetate (Na4-EDTA·2H2O) (2 μL of a 500 mM aqueous solution, final concentration 20 mM). After centrifuging to remove particulate matter, the supernatant was taken for LC-MS analysis to determine the reaction yield.

[0214] First, test the sequences shown in Table 7. Each polypeptide segment reacts with boric acid 1 under standard conditions at 37 °C, and the reaction yields are measured at 3, 6, and 18 hours respectively.

[0215] Table 7.

[0216]

[0217] Further experiments were carried out on the sequences that gave the best reaction yields, as shown in Table 7.

[0218] The polypeptide RWYFLKQSDHWG (Sequence No.: 314) reacts with boric acid 1 under standard reaction conditions at 4 °C, room temperature, and 37 °C. The reaction yields at reaction times of 0.5, 1, 2, 3, and 18 hours are shown in Table 8. The LC-MS data of the reaction mixture at 37 °C for 3 hours are as Figure 3 shown in a. This polypeptide achieved a yield of 72% within 3 hours at a concentration of 50 μM.

[0219] Table 8.

[0220]

[0221]

[0222] We then performed further sequence optimization based on FLKQSDHWG (Sequence No.: 85) by site-directed mutagenesis. The tested sequences and reaction yields are shown in Table 9.

[0223] Each polypeptide reacts with boric acid 1 for 2 hours under standard reaction conditions at 25 °C. Then the reaction yield is determined. The LC-MS data of the polypeptide RWYFLKQDDHWG (Sequence No.: 315) are shown in Figure 3 b.

[0224] Table 9.

[0225]

[0226]

[0227] Subsequently, we performed another round of sequence optimization by replacing Q in FLKQDDHWG (Sequence No.: 111) with other amino acids. The tested sequences and reaction yields are shown in Table 10.

[0228] To the mixed system of the polypeptide solution (2 μL of 5 mM aqueous stock solution, final concentration 0.1 mM) and N-methylmorpholine buffer (94 μL of 5 mM stock solution, pH 7.4), boric acid 1 (2 μL of 50 mM DMSO stock solution, final concentration 1 mM) and CuCl2·2H2O (2 μL of 5 mM aqueous stock solution, final concentration 0.1 mM) were added successively. The mixture was vortexed and reacted at 25 °C for 1 hour, and then the reaction yield was measured.

[0229] Table 10.

[0230]

[0231]

[0232] Subsequently, we performed another round of sequence optimization by replacing the amino acids in FLKGDDHWG with alanine (A). The tested sequences and reaction yields are shown in Table 11.

[0233] Each polypeptide was reacted with boric acid 1 at 37 °C for 1 hour under standard reaction conditions. Then the reaction yield was measured. The LC-MS data of polypeptide RWYFLKGDDHAG (sequence number: 316) are as Figure 3 shown in

[0234] Table 11.

[0235]

[0236]

[0237] Then, we performed another round of sequence optimization by replacing A or the terminal G in FLKGDDHAG (sequence number: 143) with other amino acids. The tested sequences and reaction yields are shown in Table 12.

[0238] Each polypeptide was reacted with boric acid 1 at 37 °C for 1 hour under standard reaction conditions. Then the reaction yield was determined.

[0239] Table 12.

[0240]

[0241]

[0242] Subsequently, we performed another round of sequence optimization based on FLKKDDHAA by point mutation. The tested sequences and reaction yields are shown in Table 13.

[0243] Each polypeptide was reacted with boric acid 1 at 25 °C for 30 minutes under standard reaction conditions, and then the reaction yield was measured.

[0244] Table 13.

[0245]

[0246]

[0247]

[0248]

[0249] In addition, to a mixed system of an aqueous solution of the polypeptide RWYFFKKDDHAA (sequence number: 317) (1 μL of a 5 mM stock solution, final concentration 0.05 mM) and N-methylmorpholine buffer (97 μL of a 5 mM stock solution, pH 7.4), boric acid 1 (1 μL of a 50 mM DMSO stock solution, final concentration 0.5 mM) and CuCl2·2H2O (1 μL of a 15 mM aqueous solution, final concentration 0.15 mM) were successively added. The mixture was vortexed and reacted at 25 °C for 15 minutes, and then LC-MS analysis was performed. The results are as Figure 3 shown in

[0250] d. The superior reaction kinetics of this tag

[0251] The above research finally obtained the polypeptide segment FFKKDDHAA (sequence number: 192) with the highest reactivity with styrenylboronic acid ( Figure 4 a-b). This polypeptide (sequence number: 192) was named CAST (copper-assisted sequence-specific coupling tag). Trace (<2%) secondary boronic acid coupling was observed on CAST during the reaction ( Figure 4 b and Figure 3 d), and MS / MS analysis showed that the secondary coupling reaction site was located on the anchored histidine residue.

[0252] We further studied the reaction kinetics of CAST. To a mixed system of the polypeptide RWYFFKKDDHAA (sequence number: 317) (1 μL of a 5 mM aqueous solution, final concentration 0.05 mM) and N-methylmorpholine buffer (97 μL of a 50 mM stock solution, pH 7.4), boric acid 1 (1 μL of a 50 mM DMSO stock solution, final concentration 0.5 mM) and CuCl2·2H2O (1 μL of a 15 mM aqueous solution, final concentration 0.15 mM) were successively added. After the mixture was vortexed and mixed evenly, it was reacted with shaking at 37 °C, and samples were taken for LC-MS analysis at 0 minute, 2 minutes, 5 minutes, 8 minutes, 10 minutes, and 15 minutes respectively.

[0253] The kinetic study showed that when the concentration of the CAST polypeptide was 50 μM, the rate constant was 8.1 M -1 s -1 , and the half-life t 1 / 2 was less than 2 minutes (Figure 4 c).

[0254] MS / MS and two-dimensional NMR experiments confirmed that the reaction site is located on the Asp amide nitrogen atom before the anchored histidine in the CAST polypeptide segment ( Figure 4 d - e). As previously hypothesized, Cu(II) may form the "ACTUN" motif by binding to the histidine-containing CAST polypeptide segment, thereby activating the backbone N-H bond of the residue at the i-1 position (the residue before histidine) ( Figure 4 f).

[0255] Based on the sequence optimization data, we additionally designed 15 sequences similar to the CAST polypeptide. These polypeptide segments all exhibited reaction kinetics comparable to those of the CAST polypeptide (Table 14). The details of each sequence and its reaction yield are shown in Table 14. For the convenience of HPLC and mass spectrometry detection, a phenyl-PEG2-SPG group was added to the N-terminus of all sequences.

[0256] To a mixture of a polypeptide segment (1 μL of 5 mM aqueous solution stock, final concentration 0.05 mM) and N-methylmorpholine buffer (97 μL of 50 mM stock solution, pH 7.4), boric acid 1 (1 μL of 50 mM DMSO stock solution, final concentration 0.5 mM) and CuCl2·2H2O (1 μL of 15 mM aqueous solution stock, final concentration 0.15 mM) were added successively. After vortexing the mixture, it was reacted with shaking at 37 °C for 5 minutes, and the reaction yield was calculated by LC-MS.

[0257] Table 14.

[0258]

[0259]

[0260] e. Further improve the coupling yield by changing the coupling conditions.

[0261] For the same tag sequence, we compared the yields under two different coupling conditions. To make it better identifiable in high-performance liquid chromatography (HPLC) and mass spectrometry analysis, the amino acids RWY were added to the N-terminus of each sequence shown in Table 15.

[0262] Condition a: To N-methylmorpholine buffer (97 μL of 5 mM stock solution, pH 7.4) containing a polypeptide segment (1 μL of 5 mM aqueous solution stock, final concentration 0.05 mM), boric acid reagent (1 μL of 50 mM DMSO stock solution, final concentration 0.5 mM) and CuCl2·2H2O (1 μL of 5 mM aqueous solution stock, final concentration 0.05 mM) were added successively. After vortexing and shaking the mixture, it was reacted at 25 °C for 30 minutes, and the reaction yield was determined by LC-MS analysis.

[0263] Condition b: To the N-methylmorpholine buffer solution (92.7 μL of 5 mM mother liquor, pH 7.4) containing the polypeptide segment (4 μL of 5 mM aqueous mother liquor, final concentration 0.2 mM), boric acid reagent (2 μL of 100 mM DMSO mother liquor, final concentration 2 mM) and CuCl2·2H2O (1.3 μL of 75 mM aqueous mother liquor, final concentration 1 mM) were successively added. After vortexing the mixture, the reaction was carried out at 37 °C for 2 hours, and the reaction yield was determined by LC-MS analysis.

[0264] The results are shown in Table 15.

[0265] Table 15.

[0266]

[0267]

[0268] The results show that the reaction yield can be further improved by prolonging the reaction time, increasing the reagent concentration and / or raising the temperature.

[0269] Coupling reaction of boric acid reagent with f.FFKKDDHAA (SEQ ID NO: 192) and WLGFFKKDDHAA (SEQ ID NO: 346).

[0270] To the N-methylmorpholine buffer solution (97 μL of 5 mM mother liquor, pH 7.4) of the polypeptide WLGFFKKDDHAA (1 μL of 5 mM aqueous mother liquor, final concentration 0.05 mM), styrenylboronic acid (1 μL of 50 mM DMSO mother liquor, final concentration 0.5 mM) and CuCl2·2H2O (1 μL of 25 mM aqueous mother liquor, final concentration 0.25 mM) were successively added. The mixture was vortexed and reacted at 37 °C for 15 minutes. Figure 24 a shows the liquid chromatography-mass spectrometry analysis results of the coupling reaction of the polypeptide WLGFFKKDDHAA with the boric acid reagent.

[0271] To the N-methylmorpholine buffer solution (97 μL of 5 mM mother liquor, pH 7.4) containing the polypeptide segment FFKKDDHAA (1 μL of 5 mM aqueous mother liquor, final concentration 0.05 mM), styreneboronic acid (1 μL of 50 mM DMSO mother liquor, final concentration 0.5 mM) and CuCl2·2H2O (1 μL of 25 mM aqueous mother liquor, final concentration 0.25 mM) were successively added. After vortexing the mixture, the reaction was carried out at 37 °C for 15 minutes. The LC-MS analysis results of the coupling reaction of the polypeptide segment FFKKDDHAA with the boric acid reagent are as Figure 24 shown in a.

[0272] Example 2: Reagent screening

[0273] a. Screening of boric acid reagents

[0274] Screening was carried out through the reaction of the polypeptide segment RWYFFKKDDHAA (SEQ ID NO: 317) with different boric acid reagents.

[0275] To the N-methylmorpholine buffer solution (97 μL of 50 mM mother liquor, pH 7.4) containing the polypeptide segment (1 μL of 5 mM aqueous mother liquor, final concentration 0.05 mM), the boric acid reagent shown in Figure 5 a (1 μL of 50 mM DMSO mother liquor, final concentration 0.5 mM) and CuCl2·2H2O (1 μL of 15 mM aqueous mother liquor, final concentration 0.15 mM) were successively added. After the mixture was vortexed and reacted at 37 °C for 15 minutes, Na4-EDTA (2 μL of 500 mM aqueous mother liquor, final concentration 20 mM) was added to quench the reaction. After the crude product was centrifuged to remove the precipitate, the supernatant was analyzed by LC-MS to determine the reaction yield.

[0276] The corresponding yields of each boric acid reagent are shown in Figure 5 a marked, and some LC-MS results are shown in Figure 5 b.

[0277] Vinylboronic acid and its derivatives showed relatively high activities, among which styreneboronic acid had the best effect.

[0278] b. Metal salt screening

[0279] Screening was carried out through the reaction of the polypeptide segment RWYFFKKDDHAA (SEQ ID NO: 317) with boric acid 1 under different metal salt conditions.

[0280] To the N-methylmorpholine buffer solution (97 μL of 50 mM mother liquor, pH 7.4) containing the polypeptide segment (1 μL of 5 mM aqueous mother liquor, final concentration 0.05 mM), boric acid 1 (1 μL of 50 mM DMSO mother liquor, final concentration 0.5 mM) and different metal ions (CuSO4·5H2O, Cu(OAc)2·H2O, CuCl2·2H2O, NiCl2·6H2O, FeCl3·6H2O, FeCl2·4H2O or CoCl4·6H2O) (1 μL of 15 mM aqueous mother liquor, final concentration 0.15 mM) were successively added. After the mixture was vortexed and reacted at 37 °C for 15 minutes, Na4-EDTA (2 μL of 500 mM aqueous mother liquor, final concentration 20 mM) was added to quench the reaction. After the crude product was centrifuged to remove the precipitate, the supernatant was analyzed by LC-MS to determine the reaction yield. The results are shown in Figure 6 a.

[0281] We studied the binding of different molar equivalents of Cu(II) to the polypeptide segment RWYFFKKDDHAA (SEQ ID NO: 317).

[0282] To a mixed system of a polypeptide solution (1 μL of a 5 mM aqueous stock solution, final concentration 0.05 mM) and an N-methylmorpholine buffer solution (97 μL of a 50 mM stock solution, pH 7.4), boric acid 1 (1 μL of a 50 mM DMSO stock solution, final concentration 0.5 mM) and copper(II) chloride dihydrate (final concentrations of 0.05 mM, 0.15 mM, 0.25 mM or 0.5 mM) were added successively. After vortexing and mixing, the reaction was shaken at 37 °C for 15 minutes. Na4-EDTA (2 μL of a 500 mM aqueous stock solution, final concentration 20 mM) was added to quench the reaction, and the precipitate was removed by centrifugation of the crude product. The supernatant was subjected to LC-MS analysis. The results are as Figure 6 shown in

[0283] The results showed that increasing the molar equivalent of Cu(II) could increase the reaction rate. When using 3 equivalents of Cu(II), the reaction reached equilibrium within 10 minutes. If the reaction time was extended, the same yield could still be obtained with less Cu(II) usage.

[0284] Example 3: Reaction of modified styreneboronic acid

[0285] We then synthesized alkynyl-, azide- or biotin-modified styreneboronic acid molecules.

[0286] To a mixed system of the polypeptide RWYFFKKDDHAA (SEQ ID NO: 317) (1 μL of a 5 mM aqueous stock solution, final concentration 0.05 mM) and an N-methylmorpholine buffer solution (96 μL of a 50 mM stock solution, pH 7.4), modified styreneboronic acid (2 μL of a 25 mM DMSO stock solution, final concentration 0.5 mM) and copper(II) chloride dihydrate (1 μL of a 15 mM aqueous stock solution, final concentration 0.15 mM) were added successively. After vortexing and mixing, the reaction was shaken at 37 °C for 15 minutes. Na4-EDTA (2 μL of a 500 mM aqueous stock solution, final concentration 20 mM) was added to quench the reaction, and the precipitate was removed by centrifugation of the crude product. The supernatant was subjected to LC-MS analysis. The results are as Figure 7 shown.

[0287] The results showed that styreneboronic acids conjugated with different functional groups did not change the coupling reaction rate.

[0288] Example 4: Testing of different positions of the tag

[0289] The above polypeptide tag can be placed at any position, C-terminus or N-terminus of another polypeptide chain and react with a boric acid-containing molecule.

[0290] For example, the polypeptide FFKKDDHAA (SEQ ID NO: 192) was inserted at the N-terminus, C-terminus or in the middle of another polypeptide chain to form a longer polypeptide chain. The long polypeptide (1 μL of a 5 mM aqueous stock solution, final concentration 0.05 mM) was mixed with N-methylmorpholine buffer (97 μL of a 50 mM stock solution, pH 7.4), and boric acid 1 (1 μL of a 50 mM DMSO stock solution, final concentration 0.5 mM) and CuCl2·2H2O (1 μL of a 15 mM aqueous stock solution, final concentration 0.15 mM) were added successively. After vortexing and mixing, the reaction was shaken at 37 °C for 15 minutes. Na4-EDTA (2 μL of a 500 mM aqueous stock solution, final concentration 20 mM) was added to quench the reaction, and the precipitate was removed by centrifugation of the crude product. The supernatant was analyzed by LC-MS. The results are as Figure 8 shown in

[0291] The results showed that regardless of the position of the tag in the polypeptide chain, it exhibited the same high reactivity and yield towards the boric acid reagent.

[0292] In addition, the compatibility of this coupling reaction with disulfide bonds was also investigated.

[0293] To Figure 8 a N-methylmorpholine buffer (97 μL of a 50 mM stock solution, pH 7.4) containing the Cys-protected polypeptide shown in Figure 8 b (1 μL of a 5 mM aqueous stock solution, final concentration 0.05 mM), boric acid 1 (1 μL of a 50 mM DMSO stock solution, final concentration 0.5 mM) and CuCl2·2H2O (1 μL of a 25 mM aqueous stock solution, final concentration 0.25 mM) were added successively. After vortexing and shaking the mixture, the reaction was carried out at 37 °C for 15 minutes. Na4-EDTA (2 μL of a 500 mM aqueous stock solution, final concentration 20 mM) was added to quench the reaction. After centrifuging the crude product to remove the precipitate, the supernatant was analyzed by LC-MS. The results are as shown in

[0294] b. The results showed that the disulfide bond was compatible with the coupling reaction between the tag and boric acid. Therefore, if necessary, solvent-exposed Cys could be temporarily protected with a disulfide bond during coupling.

[0295] Example 5: Shortening of the tag sequence

[0296] This experiment aimed to verify whether the polypeptide segment FFKKDDHAA (SEQ ID NO: 192) could be further shortened while maintaining its specific reactivity with the boric acid molecule.

[0297] Different truncated versions of FFKKDDHAA (SEQ ID NO: 192) were specifically tested. To facilitate the HPLC analysis of the reaction yield, the WLG amino acid sequence was added to the N-terminus of all polypeptide segments. To Figure 9The N-methylmorpholine buffer (97 μL of 50 mM stock solution, pH 7.4) of the indicated polypeptide segment (1 μL of 5 mM aqueous stock solution, final concentration 0.05 mM) was successively added with boric acid 1 (1 μL of 50 mM DMSO stock solution, final concentration 0.5 mM) and CuCl2·2H2O (1 μL of 15 mM aqueous stock solution, final concentration 0.15 mM). After vortexing the mixture, it was reacted at 37 °C for 15 minutes, and Na4-EDTA (2 μL of 500 mM aqueous stock solution, final concentration 20 mM) was added to quench the reaction. After centrifuging the crude product to remove the precipitate, the supernatant was subjected to LC-MS analysis. The results are as Figure 9 shown.

[0298] The results showed that the tag could be shortened to six amino acids FKKDDH (SEQ ID NO: 276) without affecting the reactivity.

[0299] We tested various further shortened versions of this polypeptide tag. To facilitate the HPLC analysis of the reaction yield, a naphthalene group was linked to the N-terminus of each polypeptide segment. To the N-methylmorpholine buffer (97 μL of 50 mM stock solution, pH 7.4) containing the polypeptide segment (1 μL of 5 mM aqueous stock solution, final concentration 0.05 mM), boric acid 1 (1 μL of 50 mM N,N-dimethylformamide stock solution, final concentration 0.5 mM) and CuCl2·2H2O (1 μL of 25 mM aqueous stock solution, final concentration 0.25 mM) were successively added. After vortexing the mixture, it was reacted at 37 °C for 3 hours, and Na4-EDTA (2 μL of 500 mM aqueous stock solution, final concentration 20 mM) was added to quench the reaction. After centrifuging the crude product to remove the precipitate, the supernatant was subjected to LC-MS analysis. The results are as Figure 23 shown. The results showed that the tag could be shortened to 6, 5, 4, 3 amino acids: KDDHAA (SEQ ID NO: 277), KKDDH (SEQ ID NO: 278), KDDH (SEQ ID NO: 279), DDHA (SEQ ID NO: 280), DDHA (SEQ ID NO: 281), DDH, and all of them did not affect the reactivity.

[0300] Example 6: Stability of the conjugate

[0301] This experiment aimed to study the stability of the conjugate obtained from the reaction of the tagged polypeptide with the boric acid reagent.

[0302] The specific operations are as follows: To RWYFFKKDDHAA (SEQ ID NO: 317) (1 μL of 5 mM aqueous stock solution, final concentration 0.05 mM) in N-methylmorpholine buffer (97 μL of 50 mM stock solution, pH 7.4), boric acid 1 (1 μL of 50 mM DMSO stock solution, final concentration 0.5 mM) and CuCl2·2H2O (1 μL of 15 mM aqueous stock solution, final concentration 0.15 mM) are added successively. The mixture is vortexed and reacted at 37 °C for 15 minutes. Subsequently, the modified RWYFFKKDDHAA (SEQ ID NO: 317) is incubated at 95 °C for 1 hour, or incubated at 95 °C in 10 mM cysteine solution for 1 hour, and finally analyzed by LC-MS. The results are as Figure 10 shown in

[0303] As a control, to PBS buffer (89 μL of 10 mM stock solution, pH 7.4) containing polypeptide DLAAEIAKHCG (1 μL of 5 mM aqueous stock solution, final concentration 0.05 mM), maleimide (10 μL of 50 mM DMSO stock solution, final concentration 5 mM) is added successively. The mixture is vortexed and reacted at room temperature for 30 minutes. After removing the excess maleimide, the product is incubated at 95 °C for 1 hour or incubated at 95 °C in 10 mM cysteine solution for 1 hour, and finally analyzed by LC-MS. The results are as Figure 10 shown in

[0304] The results show that the coupling products generated by this tag remain stable under both high temperature and excess thiol nucleophile (cysteine) conditions. While the products of the prior art tags undergo hydrolysis in high temperature and / or excess thiol nucleophile environments.

[0305] Example 7: Preparation of boric acid derivatives

[0306] a. Synthesis of boric acid derivatives

[0307]

[0308] tert-Butyl (E)-(4-(2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)vinyl)benzyl)carbamate (S1)

[0309] Refer to the literature method 3Synthetic starting material tert-butyl (E)-(4-(2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)vinyl)benzyl)carbamate. To a nitrogen-protected 25 mL sealed reaction flask, tert-butyl (4-ethynylbenzyl)carbamate (4.00 mmol), pinacolborane (4.40 mmol, 0.640 mL), and H3B·THF (0.40 mmol, 1 M THF solution, 0.40 mL) were added successively, and the reaction was stirred at 60 °C for 4 hours. After the reaction was completed, it was concentrated under reduced pressure, and the crude product was purified by basic flash column chromatography (SiO2: n-hexane / ethyl acetate = 15:1 - 10:1, yellow oil, yield 70%).

[0310] 1 H NMR (600 MHz, chloroform-d) δ 7.37 (d, J = 8.0 Hz, 2H), 7.30 (d, J = 18.4 Hz, 1H), 7.17 (d, J = 7.9 Hz, 2H), 6.07 (d, J = 18.4 Hz, 1H), 4.23 (d, J = 6.0 Hz, 2H), 1.38 (s, 9H), 1.24 (s, 12H). 13 C NMR (150 MHz, CDCl3) δ 156.01, 149.14, 139.84, 136.75, 127.78, 127.42, 83.48, 79.65, 44.53, 28.53, 24.93. HRMS calculated for C 20 H 31 BNO4 [M+H] + 360.2341, found 361.2568.

[0311]

[0312] (E)-(4-(((tert-butoxycarbonyl)amino)methyl)styryl)boronic acid (S2)

[0313] tert-Butyl (E)-(4-(2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)vinyl)benzyl)carbamate (4.00 mmol), NaIO4 (12 mmol), and NH4OAc (12 mmol) were added to the reaction flask, followed by acetone (32 mL) and water (16 mL), and the mixture was stirred at room temperature for 12 hours. The reaction solution was quenched with saturated brine and extracted with ethyl acetate (3 × 40 mL). The combined organic phases were dried over sodium sulfate and concentrated under reduced pressure, and the product was directly used for the next step (white solid, yield 90%).

[0314] 11H NMR (500 MHz, DMSO-d6) δ 7.75 (s, 2H), 7.42 (d, J = 8.1 Hz, 2H), 7.22 (m, 3H), 6.08 (d, J = 18.4 Hz, 1H), 4.11 (d, J = 6.1 Hz, 2H), 3.34 (m, 1H), 1.39 (s, 9H). 13 13C NMR (125 MHz, DMSO-d6) δ 155.69, 145.48, 140.43, 136.06, 127.16, 126.42, 122.58, 77.69, 43.04, 28.14. HRMS calculated for C 14 H 20 BNO4 [M+Na] + 300.1383, found 300.1377.

[0315]

[0316] (E)-(4-(Aminomethyl)styryl)boronic acid hydrochloride (S3)

[0317] (E)-(4-(((tert-Butoxycarbonyl)amino)methyl)styryl)boronic acid was added to a reaction flask and the boronic acid substrate was dissolved in (1,4-dioxane solution of 4.0 M hydrochloric acid), and stirred at room temperature for 1 - 2 hours. After completion of the reaction, it was concentrated under reduced pressure to quantitatively obtain (E)-(4-(aminomethyl)styryl)boronic acid hydrochloride (white solid).

[0318] 1 1H NMR (500 MHz, DMSO-d6) δ 8.51 (s, 4H), 7.81 (s, 1H), 7.50 (m, 4H), 7.26 (d, J = 18.4 Hz, 1H), 6.16 (d, J = 18.4 Hz, 1H), 4.00 (m, 2H). 13 13C NMR (125 MHz, DMSO-d6) δ 144.99, 137.63, 134.03, 129.22, 126.56, 123.94, 41.74. HRMS calculated for C9H 10 BO2 [M-NH2] + 161.0768, found 161.0757.

[0319]

[0320] (E)-(4-(Hex-5-ynamidomethyl)styryl)boronic acid (1A)

[0321] 5-Hexynoic acid (20.2 mg, 0.18 mmol) and HATU (60.8 mg, 0.16 mmol) were dissolved in N,N-dimethylformamide (1.0 mL). N,N-Diisopropylethylamine (31.3 μL, 0.18 mmol) was added at 0 °C, and the mixture was stirred at room temperature for 10 minutes. After the reaction solution was cooled with an ice bath, S3 (21.3 mg, 0.1 mmol) was added. The mixture was stirred at room temperature overnight, and all volatiles were removed by a vacuum pump. The crude product was purified by reverse-phase HPLC (5.0–65% aqueous acetonitrile solution, 40-minute gradient). The fractions were collected and lyophilized to obtain the product.

[0322] 1 H NMR (500 MHz, dimethyl sulfoxide-d6) δ 8.35 (t, J = 5.9 Hz, 1H), 7.75 (s, 2H), 7.42 (d, J = 8.1 Hz, 2H), 7.21 - 7.24 (m, 3H), 6.08 (d, J = 18.4 Hz, 1H), 4.25 (d, J = 5.9 Hz, 2H), 2.79 (t, J = 2.6 Hz, 1H), 2.21 - 2.24 (m, 2H), 2.15 - 2.17 (m, 2H), 1.73 - 1.67 (m, 2H). 13 C NMR (125 MHz, DMSO-d6) δ 171.36, 145.44, 139.90, 136.12, 127.42, 126.44, 122.65, 83.96, 71.42, 41.67, 33.97, 24.15, 17.29 HRMS calculated for C 15 H 18 BNO3 [M + H] + 272.1413, found 272.1474.

[0323]

[0324] (E)-(4-((2-Azidoacetamido)methyl)styryl)boronic acid (1B)

[0325] 2-Azidoacetic acid (18.2 mg, 0.18 mmol) and HATU (60.8 mg, 0.16 mmol) were dissolved in N,N-dimethylformamide (1.0 mL). N,N-Diisopropylethylamine (31.3 μL, 0.18 mmol) was added at 0 °C, and the mixture was stirred at room temperature for 10 minutes. After the reaction solution was cooled with an ice bath, S3 (21.3 mg, 0.1 mmol) was added. The mixture was stirred at room temperature overnight, and all volatiles were removed by a vacuum pump. The crude product was purified by reverse-phase HPLC (5.0–65% aqueous acetonitrile solution, 40-minute gradient). The fractions were collected and lyophilized to obtain the product.

[0326] 1 1H NMR (500 MHz, dimethyl sulfoxide-d6) δ 8.62 (t, J = 5.9 Hz, 1H), 7.76 (s, 2H), 7.43 (d, J = 8.1 Hz, 2H), 7.21 - 7.27 (m, 3H), 6.09 (d, J = 18.4 Hz, 1H), 4.30 (d, J = 5.9 Hz, 2H), 3.89 (s, 2H). 13 13C NMR (125 MHz, DMSO-d6) δ 167.24, 145.37, 139.11, 136.34, 127.62, 126.49, 122.82, 50.68, 41.88. HRMS calculated for C 11 H 13 BN4O3 [M+H] + 261.1114, found 261.1170.

[0327]

[0328] ((E)-4-((5-((4S)-2-Oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamido)methyl)styryl)boronic acid (1C)

[0329] Dissolve biotin (44.0 mg, 0.18 mmol) and HATU (60.8 mg, 0.16 mmol) in N,N-dimethylformamide. N,N-Diisopropylethylamine (31.3 μL, 0.18 mmol) was added at 0 °C and the mixture was stirred at room temperature for 10 minutes. The reaction solution was cooled in an ice bath and S3 (21.3 mg, 0.1 mmol) was added. The mixture was stirred at room temperature overnight and all volatiles were removed by a vacuum pump. The crude product was purified by reverse-phase HPLC (5.0–65% aqueous acetonitrile, 40-minute gradient). The fractions were collected and lyophilized to give the product.

[0330] 11H NMR (500 MHz, dimethyl sulfoxide-d6) δ 8.30 (t, J = 5.8 Hz, 1H), 7.75 (s, 2H), 7.42 (d, J = 8.0 Hz, 2H), 7.23 (m, 3H), 6.43 (s, 1H), 6.36 (s, 1H), 6.08 (d, J = 18.4 Hz, 1H), 4.29 - 4.32 (m, 1H), 4.25 (d, J = 5.8 Hz, 2H), 4.11 - 4.13 (m, 1H), 3.07 - 3.11 (m, 1H), 2.81 - 2.84 (m, 1H), 2.57 - 2.60 (m, 1H), 2.14 (t, J = 7.3 Hz, 2H), 1.47 - 1.60 (m, 4H), 1.30 - 1.36 (m, 2H). 13 13C NMR (125 MHz, dimethyl sulfoxide-d6)) δ 171.94, 162.59, 145.45, 139.99, 136.09, 127.41, 126.43, 122.62, 60.93, 59.07, 55.33, 41.64, 39.74, 35.04, 28.13, 27.93, 25.20. HRMS calculated for C 19 H 26 BN3O4S [M + H] + 404.1771, found 404.1836.

[0331]

[0332] Reagents and conditions: (i) Pd(pinacolato)vinylboronate, triethylamine, toluene, 90 °C, 16 h; (ii) sodium periodate, hydrochloric acid, tetrahydrofuran / water, room temperature, 2 h; (iii) piperidine-4-carboxylic acid, sodium triacetoxyborohydride, dichloroethane, 30 °C, overnight.

[0333] (E)-4-(2-(4,4,5,5-Tetramethyl-1,3,2-dioxaborolan-2-yl)vinyl)benzaldehyde (a)

[0334] To a mixture of 4-bromobenzaldehyde (2 g, 10.9 mmol) and 4,4,5,5-tetramethyl-2-vinyl-1,3,2-dioxaborolane (2.2 mL, 13 mmol, 1.2 equiv), toluene (20 mL) and triethylamine (3 mL, 21.8 mmol, 2 equiv) were added, and then tetrakis(tri-tert-butylphosphine)palladium(0) (0.28 g, 1.1 mmol, 10 mol%) was added. The resulting mixture was purged with nitrogen and then heated at 90 °C for 18 h. After cooling to room temperature, the reaction was quenched with saturated NaHCO3 (20 mL) and extracted with ethyl acetate (30 mL × 3). After evaporation of the solvent, the residue was purified by a Biotage column system (ethyl acetate / petroleum ether: 0 - 10%) to give a white solid. Yield: 60%. 1 1H NMR (500 MHz, DMSO-d6) δ 10.01 (s, 1H), 7.90 (d, J = 8.2 Hz, 2H), 7.81 (d, J = 8.2 Hz, 2H), 7.38 (d, J = 18.5 Hz, 1H), 6.36 (d, J = 18.5 Hz, 1H), 1.26 (s, 12H). 13 13C NMR (125 MHz, DMSO-d6) δ 192.52, 147.68, 142.31, 136.11, 129.78, 127.57, 120.47, 83.19, 24.53. HRMS calculated for C 15 H 19 BO3 [M + H] + 259.1461, found 259.1489.

[0335] (E)-(4-Formylstyryl)boronic acid (b)

[0336] (E)-4-(2-(4,4,5,5-Tetramethyl-1,3,2-dioxaborolan-2-yl)vinyl)benzaldehyde (500 mg, 1.9 mmol) was dissolved in 20 mL of a THF:water (4:1) mixture. To this solution, NaIO4 (1.2 g, 3.0 equiv) was added and stirred for 5 min. Then aqueous HCl (2.0 M, 2.0 mL) was added and stirred for 1 h until TLC monitoring showed complete consumption of the boronate. The reaction mixture was extracted with ethyl acetate (20 mL × 3), and the combined organic layers were washed with H2O. After evaporation of the solvent, the residue was purified by a Biotage column system (ethyl acetate / petroleum ether: 0 - 20%) to give a pale yellow solid. Yield: 88%. 1HNMR (500 MHz, dimethyl sulfoxide-d6) δ 10.00 (s, 1H), 7.93 (s, 2H), 7.90 (d, J = 8.2 Hz, 2H), 7.69 (d, J = 8.2 Hz, 2H), 7.32 (d, J = 18.4 Hz, 1H), 6.32 (d, J = 18.4 Hz, 1H). 13 C NMR (125 MHz, dimethyl sulfoxide-d6) δ 192.48, 144.31, 143.18, 135.65, 129.91, 127.38, 127.09. HRMS calculated for C9H9BO3 [M+H] + 177.0678, found 177.0703.

[0337] (E)-1-(4-(2-Boronoethenyl)benzyl)piperidine-4-carboxylic acid (c)

[0338] Piperidine-4-carboxylic acid (100 mg, 0.84 mmol, 1.5 equiv) was suspended in dry DCM (15 ml), and then (E)-(4-Formylstyryl)boronic acid (100 mg, 0.56 mmol) was added. After the suspension was stirred for 30 minutes, sodium triacetoxyborohydride (240 mg, 1.12 mmol, 2 equiv) was added portionwise, and the mixture was stirred at 30 °C overnight. After evaporation of DCM, the reaction was quenched with water (10 ml) and adjusted to pH 10 with 1 M NaOH. The solution was washed with DCM (10 mL×3), ethyl acetate (10 mL×3), and adjusted to pH 2 with 2 M HCl. The residue was freeze-dried and purified by HPLC (Agilent C18 column), and then freeze-dried again to obtain the title product as a white solid trifluoroacetate salt. Yield: 42%. 1 H NMR (500 MHz, DMSO-d6) δ 12.56 (s, 1H), 9.44 (s, 1H), 7.85 (s, 2H), 7.57 (d, J = 8.2 Hz, 2H), 7.48 (d, J = 8.2 Hz, 2H), 7.28 (d, J = 18.4 Hz, 1H), 6.19 (d, J = 18.4 Hz, 1H), 4.28 (d, J = 4.6 Hz, 2H), 3.40 (m, 2H), 3.26 - 3.28 (m, 1H), 2.91 - 2.98 (m, 2H), 2.04 - 2.07 (m, 2H), 1.66 - 1.74 (m, 2H). 13C NMR (125 MHz, DMSO-d6) δ 174.45, 158.16, 144.74, 138.74, 131.66, 129.30, 126.82, 124.84, 116.93, 58.76, 50.67, 37.74, 25.17. HRMS calculated for C 15 H20 BNO4 [M+H]+ 290.1519, observed value 290.1559.

[0339]

[0340] Synthesis route of SAB-MMAE(1D)

[0341] NH2-Val-Cit-PAB-MMAE(d) reference literature 4 Synthesized by the method.

[0342] Intermediate c (20.0 mg, 0.049 mmol, 6 equivalents), HOSu (7.4 mg, 0.064 mmol, 7.8 equivalents), DMAP (3 mg, 0.032 mmol, 3 equivalents) were dissolved in dry N,N-dimethylformamide (100 μL), then EDCI (12.3 mg, 0.064 mmol, 7.8 equivalents) was added, and the solution was stirred overnight at 30 °C. Subsequently, NH2-Val-Cit-PAB-MMAE (d, 10.0 mg, 8 μMol, 1 equivalent) was added. The reaction was stirred overnight at 30 °C and monitored by LC-MS. The final product was purified by HPLC (Agilent C18 column) to obtain a white powder (7.6 mg, 46%). HRMS calculated value C 73 H 112 BN 11 O 15 [M+Na] + 1416.8232, observed value 1416.8296.

[0343]

[0344] Synthesis route of H2N-Glu(OtBu)-Val-Cit-PAB-MMAE(f)

[0345] Fmoc-Glu(OtBu)-OH (28.0 mg, 0.066 mmol, 1 equiv), TSTU (23.3 mg, 0.079 mmol, 1.2 equiv), and DIEA (24 μL, 0.13 mmol, 2 equiv) were dissolved in anhydrous N,N-dimethylformamide (460 μL) and stirred at 30 °C for 1 h. Subsequently, the mixture (426 μL) was added to a 1 mL N,N-dimethylformamide solution of NH2-Val-Cit-PAB-MMAE (60 mg, 0.048 mmol, 0.72 equiv) and reacted for 3 - 4 h until HPLC-MS indicated completion of the reaction. 4-Methylpiperidine (9.5 μL, 0.1 mmol, 1.5 equiv) was added and stirred overnight at 30 °C. The product was purified by HPLC on an Agilent C18 column and lyophilized to give a white powder (56.4 mg, 89%). HRMS calculated [M+H]+ 1308.8105, observed 1308.8082.

[0346]

[0347] Synthetic route of DBCO-PEG3-NHS (g)

[0348] DBCO-PEG3-NHS refers to the literature 5 Synthesized by the method.

[0349] To a DCM (2 mL) mixture of DBCO-NHS (100 mg, 1 equiv) and 3-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)propanoic acid (66 mg, 1.2 equiv) was added DIEA (88 μL, 2 equiv), and stirred overnight at room temperature. After completion of the reaction, it was diluted with DCM, and the organic phase was washed successively with brine and 1M HCl, and dried over MgSO4. Concentrated under reduced pressure to obtain a white viscous liquid compound. This product (50 mg) was mixed with TSTU (38.5 mg, 1.3 equiv) and DIEA (26 μL, 1.5 equiv) in anhydrous dichloromethane / N,N-dimethylformamide (1.5 mL / 0.5 mL) and stirred overnight at room temperature. After completion of the reaction, it was diluted with DCM, and the organic phase was washed successively with brine and 1M HCl, and dried over magnesium sulfate. The solvent was removed under reduced pressure to obtain a viscous liquid compound (52 mg) without further purification.

[0350]

[0351] Synthetic route of DBCO-MMAE (h)

[0352] Dissolve H2N-Glu(OtBu)-Val-Cit-PAB-MMAE (30 mg, 0.023 mmol), DBCO-PEG3-NHS (16.3 mg, 0.027 mmol, 1.2 equiv), and DIEA (8 μL, 0.046 mmol, 2 equiv) in anhydrous N,N-dimethylformamide (600 μL), and stir at room temperature for 1 hour until LC-MS shows the reaction is complete. After concentration under reduced pressure, the crude product is precipitated with cold diethyl ether (4 mL), centrifuged at 2000×g for 3 minutes (repeated 3 times), and dried under reduced pressure. Add 30% TFA / DCM (2 mL) and react at 0 °C for 4 - 5 hours. The reaction solution is diluted with DCM (4 mL), washed with 5 mL of water (3 times), and concentrated under reduced pressure. The crude product is purified by HPLC on an Agilent C18 column and lyophilized to obtain a white powder (10 mg, 25%). HRMS calculated value [M+H] + 1742.9582, observed value 1742.9537.

[0353] Example 8: Protein Preparation

[0354] a. Expression and purification of recombinant proteins:

[0355] Construction of recombinant protein plasmids

[0356] The gene of the small ubiquitin-like modifier protein was synthesized by Genewiz Biotechnology Co., Ltd. in Suzhou, China. It was cloned into the vector pET22b. The genes of maltose-binding protein, sortase, and trigger factor were cloned into the vector pET28a. The gene of the nanobody was cloned into the vector pET26b. The amino acid sequences are shown below. DNA was amplified by PCR using KOD OneTM PCR MasterMix-Blue (ThermoFisher scientific). All gene fragments were assembled using the Gibson assembly kit (Cat.C115-01, Vazyme).

[0357] Protein expression and purification

[0358] Escherichia coli BL21(DE3) cells transformed with plasmids of His-TEV-small ubiquitin-like modifier protein, His-TEV-maltose-binding protein, His-TEV-sortase, His-TEV-trigger factor, and His-TEV-nanobody were placed in 1 liter of LB medium containing kanamycin (50 mg / L) and cultured at 37 °C until OD 600= 0.6. Subsequently, the expression of His-TEV-Maltose Binding Protein, His-TEV-Sortase, His-TEV-Trigger Factor and His-TEV-Nanobody was induced overnight at 18 °C by adding 0.5 mM IPTG. The expression of His-TEV-Ubiquitin-like Small Molecule Protein was induced for 4 hours at 37 °C by adding 0.5 mM IPTG. After centrifuging to collect the cells (6000 rpm, 10 minutes), the cell pellet was lysed by sonication in 25 mL of 50 mM Tris buffer (pH 7.5) containing 150 mM NaCl. The suspension was centrifuged at 12000 rpm for 30 minutes to remove cell debris, and the supernatant was loaded onto 2 mL of nickel ion affinity resin (Genscript, catalog number L00666-100), first washed with 40 mL of 20 mM Tris buffer (pH 7.5) containing 150 mM NaCl, and then washed with 40 mL of the same buffer containing 20 mM imidazole. Finally, the target protein was eluted with 20 mM Tris, 150 mM NaCl buffer (pH 7.5) containing 250 mM imidazole. The Pur-A-LyzerTM Maxi dialysis kit (PURX12005, Sigma-Aldrich) was used to remove imidazole from the protein, and the Tris buffer was replaced with N-methylmorpholine buffer during dialysis. Protein purity and molecular weight were analyzed by LC-MS. For the TEV cleavage experiment, the enzyme and substrate were mixed at a ratio of 1:20 and reacted overnight at 4 °C.

[0359] His-TEV-Ubiquitin-like Small Molecule Protein

[0360] MGSHHHHHHENLYFQSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKI

[0361] KKTTPLRRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGG (Sequence No.: 282)

[0362] Ubiquitin-like Small Molecule Protein

[0363] SDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPLRRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGG (Sequence No.: 283)

[0364] His-TEV-Ubiquitin-like Small Molecule Protein-CAST

[0365] MGSHHHHHHENLYFQSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKI

[0366] KKTTPLRRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGGFFKKDDHAA(Sequence No.: 284)

[0367] Small ubiquitin-like modifier - CAST

[0368] SDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPLRRLMEAFAKR

[0369] QGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGGFFKKDDHAA(Sequence No.: 285)

[0370] His-TEV-Maltose Binding Protein

[0371] MHHHHHHHHGSENLYFQGGSKIEEGKLVIWINGDKGYNGLAEVGKKFEKDT

[0372] GIKVTVEHPDKLEEKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKA

[0373] FQDKLYPFTWDAVRYNGKLIAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDK

[0374] ELKAKGKSALMFNLQEPYFTWPLIAADGGYAFKYENGKYDIKDVGVDNAGA

[0375] KAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTINGPWAWSNIDTSK

[0376] VNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEA

[0377] VNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFWYAVRTAVINAASGRQTVDEALKDAQT(Sequence No.: 286)

[0378] Maltose-binding protein

[0379] GGSKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKFPQ

[0380] VAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYN

[0381] GKLIAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQE

[0382] PYFTWPLIAADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHM

[0383] NADTDYSIAEAAFNKGETAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSK

[0384] PFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEAVNKDKPLGAVALKSYE

[0385] EELAKDPRIAATMENAQKGEIMPNIPQMSAFWYAVRTAVINAASGRQTVDEALKDAQT(SEQ ID NO: 287)

[0386] His-TEV-Maltose-binding protein-CAST

[0387] MHHHHHHHHGSENLYFQGGSKIEEGKLVIWINGDKGYNGLAEVGKKFEKDT

[0388] GIKVTVEHPDKLEEKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKA

[0389] FQDKLYPFTWDAVRYNGKLIAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDK

[0390] ELKAKGKSALMFNLQEPYFTWPLIAADGGYAFKYENGKYDIKDVGVDNAGA

[0391] KAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTINGPWAWSNIDTSK

[0392] VNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEA

[0393] VNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFWYAVRTAVINAASGRQTVDEALKDAQTFFKKDDHAA (Sequence number: 288)

[0394] Maltose-binding protein-CAST

[0395] GGSKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKFPQ

[0396] VAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYN

[0397] GKLIAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQE

[0398] PYFTWPLIAADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHM

[0399] NADTDYSIAEAAFNKGETAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSK

[0400] PFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEAVNKDKPLGAVALKSYE

[0401] EELAKDPRIAATMENAQKGEIMPNIPQMSAFWYAVRTAVINAASGRQTVDEALKDAQTFFKKDDHAA (Sequence number: 289)

[0402] His-TEV-Nanobody

[0403] MAMHHHHHHHHGSENLYFQGGSDQVQLQESGGGLVQAGGSLRLSCAASGY

[0404] ISDAYYMGWYRQAPGKEREFVATITHGTNTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAVLETRSYSFRYWGQGTQVTVSSLE (SEQ ID NO: 290)

[0405] Nanobody

[0406] GGSDQVQLQESGGGLVQAGGSLRLSCAASGYISDAYYMGWYRQAPGKEREF

[0407] VATITHGTNTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAVLETRSYSFRYWGQGTQVTVSSLE (SEQ ID NO: 291)

[0408] His-TEV-Nanobody-CAST

[0409] MAMHHHHHHHHGSENLYFQGGSDQVQLQESGGGLVQAGGSLRLSCAASGY

[0410] ISDAYYMGWYRQAPGKEREFVATITHGTNTYYADSVKGRFTISRDNAKNTV

[0411] YLQMNSLKPEDTAVYYCAVLETRSYSFRYWGQGTQVTVSSLEFFKKDDHAA (SEQ ID NO: 292)

[0412] Nanobody-CAST

[0413] GGSDQVQLQESGGGLVQAGGSLRLSCAASGYISDAYYMGWYRQAPGKEREF

[0414] VATITHGTNTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAVLETRSYSFRYWGQGTQVTVSSLEFFKKDDHAA (SEQ ID NO: 293)

[0415] His-TEV-Trigger Factor

[0416] MHHHHHHHHGSENLYFQGGSQVSVETTQGLGRRVTITIAADSIETAVKSELV

[0417] NVAKKVRIDGFRKGKVPMNIVAQRYGASVRQDVLGDLMSRNFIDAIIKEKIN

[0418] PAGAPTYVPGEYKLGEDFTYSVEFEVYPEVELQGLEAIEVEKPIVEVTDADVD

[0419] GMLDTLRKQQATWKEKDGAVEAEDRVTIDFTGSVDGEEFEGGKASDFVLA

[0420] MGQGRMIPGFEDGIKGHKAGEEFTIDVTFPEEYHAENLKGKAAKFAINLKKV

[0421] EERELPELTAEFIKRFGVEDGSVEGLRAEVRKNMERELKSAIRNRVKSQAIEG

[0422] LVKANDIDVPAALIDSEIDVLRRQAAQRFGGNEKQALELPRELFEEQAKRRV

[0423] VVGLLLGEVIRTNELKADEERVKGLIEEMASAYEDPKEVIEFYSKNKELMDNMRNVALEEQAVEAVLAKAKVTEKETTFNELMNQQASAG (Sequence No.: 294) Trigger factor

[0424] GGSQVSVETTQGLGRRVTITIAADSIETAVKSELVNVAKKVRIDGFRKGKVPM

[0425] NIVAQRYGASVRQDVLGDLMSRNFIDAIIKEKINPAGAPTYVPGEYKLGEDFT

[0426] YSVEFEVYPEVELQGLEAIEVEKPIVEVTDADVDGMLDTLRKQQATWKEKD

[0427] GAVEAEDRVTIDFTGSVDGEEFEGGKASDFVLAMGQGRMIPGFEDGIKGHKA

[0428] GEEFTIDVTFPEEYHAENLKGKAAKFAINLKKVEERELPELTAEFIKRFGVEDG

[0429] SVEGLRAEVRKNMERELKSAIRNRVKSQAIEGLVKANDIDVPAALIDSEIDVL

[0430] RRQAAQRFGGNEKQALELPRELFEEQAKRRVVVGLLLGEVIRTNELKADEER

[0431] VKGLIEEMASAYEDPKEVIEFYSKNKELMDNMRNVALEEQAVEAVLAKAKVTEKETTFNELMNQQASAG(SEQ ID NO:295)

[0432] His-TEV-Trigger Factor-CAST

[0433] MHHHHHHHHGSENLYFQGGSQVSVETTQGLGRRVTITIAADSIETAVKSELV

[0434] NVAKKVRIDGFRKGKVPMNIVAQRYGASVRQDVLGDLMSRNFIDAIIKEKIN

[0435] PAGAPTYVPGEYKLGEDFTYSVEFEVYPEVELQGLEAIEVEKPIVEVTDADVD

[0436] GMLDTLRKQQATWKEKDGAVEAEDRVTIDFTGSVDGEEFEGGKASDFVLA

[0437] MGQGRMIPGFEDGIKGHKAGEEFTIDVTFPEEYHAENLKGKAAKFAINLKKV

[0438] EERELPELTAEFIKRFGVEDGSVEGLRAEVRKNMERELKSAIRNRVKSQAIEG

[0439] LVKANDIDVPAALIDSEIDVLRRQAAQRFGGNEKQALELPRELFEEQAKRRV

[0440] VVGLLLGEVIRTNELKADEERVKGLIEEMASAYEDPKEVIEFYSKNKELMDNMRNVALEEQAVEAVLAKAKVTEKETTFNELMNQQASAGFFKKDDHAA(SEQ ID NO:296)

[0441] Trigger Factor-CAST

[0442] GGSQVSVETTQGLGRRVTITIAADSIETAVKSELVNVAKKVRIDGFRKGKVPM

[0443] NIVAQRYGASVRQDVLGDLMSRNFIDAIIKEKINPAGAPTYVPGEYKLGEDFT

[0444] YSVEFEVYPEVELQGLEAIEVEKPIVEVTDADVDGMLDTLRKQQATWKEKD

[0445] GAVEAEDRVTIDFTGSVDGEEFEGGKASDFVLAMGQGRMIPGFEDGIKGHKA

[0446] GEEFTIDVTFPEEYHAENLKGKAAKFAINLKKVEERELPELTAEFIKRFGVEDG

[0447] SVEGLRAEVRKNMERELKSAIRNRVKSQAIEGLVKANDIDVPAALIDSEIDVL

[0448] RRQAAQRFGGNEKQALELPRELFEEQAKRRVVVGLLLGEVIRTNELKADEER

[0449] VKGLIEEMASAYEDPKEVIEFYSKNKELMDNMRNVALEEQAVEAVLAKAKVTEKETTFNELMNQQASAGFFKKDDHAA(Sequence No.: 297)

[0450] His-TEV-Sortase

[0451] MHHHHHHHHGSENLYFQGGSQAKPQIPKDKSKVAGYIEIPDADIKEPVYPGP

[0452] ATREQLNRGVSFAEENESLDDQNISIAGHTFIDRPNYQFTNLKAAKKGSMVYF

[0453] KVGNETRKYKMTSIRNVKPTAVEVLDEQKGKDKQLTLITCDDYNEETGVWETRKIFVATEVKLE(Sequence No.: 298)

[0454] Sortase

[0455] GGSQAKPQIPKDKSKVAGYIEIPDADIKEPVYPGPATREQLNRGVSFAEENESL

[0456] DDQNISIAGHTFIDRPNYQFTNLKAAKKGSMVYFKVGNETRKYKMTSIRNVKPTAVEVLDEQKGKDKQLTLITCDDYNEETGVWETRKIFVATEVKLE (Sequence No.: 299)

[0457] His-TEV-Sortase-CAST

[0458] MHHHHHHHHGSENLYFQGGSQAKPQIPKDKSKVAGYIEIPDADIKEPVYPGP

[0459] ATREQLNRGVSFAEENESLDDQNISIAGHTFIDRPNYQFTNLKAAKKGSMVYF

[0460] KVGNETRKYKMTSIRNVKPTAVEVLDEQKGKDKQLTLITCDDYNEETGVWETRKIFVATEVKLEFFKKDDHAA (Sequence No.: 300)

[0461] Sortase-CAST

[0462] GGSQAKPQIPKDKSKVAGYIEIPDADIKEPVYPGPATREQLNRGVSFAEENESL

[0463] DDQNISIAGHTFIDRPNYQFTNLKAAKKGSMVYFKVGNETRKYKMTSIRNVK

[0464] PTAVEVLDEQKGKDKQLTLITCDDYNEETGVWETRKIFVATEVKLEFFKKDDH (Sequence No.: 301)

[0465] b. Expression and Purification of Antibodies

[0466] The pVITRO1-Trastuzumab plasmid was purchased from Addgene (#61883). The pVITRO1-Tra-CAST plasmid was constructed by inserting the CAST polypeptide into the C-terminus of the Trastuzumab heavy chain using the ClonExpress Ultra One Step Cloning Kit from Vazyme Co., Ltd. (C115-01). The light and heavy chain sequences of Trastuzumab, Tra-CAST, and Tra-CASTi are as follows:

[0467] Trastuzumab - light chain

[0468] MLPSQLIGFLLLWVPASRGDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVA

[0469] WYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYC

[0470] QQHYTTPPTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPR

[0471] EAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC*(Sequence No.: 302)

[0472] Trastuzumab - heavy chain

[0473] MDWTWRILFLVAAATGAHSEVQLVESGGGLVQPGGSLRLSCAASGFNIKDT

[0474] YIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQM

[0475] NSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPS

[0476] SKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSS

[0477] VVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGG

[0478] PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKT

[0479] KPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG

[0480] QPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTT

[0481] PPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG*(Sequence No.: 303)

[0482] Tra-CAST light chain

[0483] MLPSQLIGFLLLWVPASRGDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVA

[0484] WYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYC

[0485] QQHYTTPPTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPR

[0486] EAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC*(Sequence No.: 304)

[0487] Tra-CAST heavy chain

[0488] MDWTWRILFLVAAATGAHSEVQLVESGGGLVQPGGSLRLSCAASGFNIKDT

[0489] YIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQM

[0490] NSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPS

[0491] SKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSS

[0492] VVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGG

[0493] PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKT

[0494] KPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG

[0495] QPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTT

[0496] PPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGFFKKDDHAA*(Sequence No.: 305)

[0497] Tra-CASTi - Light Chain

[0498] MLPSQLIGFLLLWVPASRGDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVA

[0499] WYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYC

[0500] QQHYTTPPTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPR

[0501] EAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC*(Sequence No.: 306)

[0502] Tra-CASTi - Heavy Chain

[0503] MDWTWRILFLVAAATGAHSEVQLVESGGGLVQPGGSLRLSCAASGFNIKDT

[0504] YIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQM

[0505] NSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPS

[0506] SKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSS

[0507] VVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGG

[0508] PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKT

[0509] KPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG

[0510] QPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTT

[0511] PPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGIAPDDHAA*(Sequence number: 307)

[0512] HEK 293F cells (Invitrogen) were cultured in a CRYSTAL shaker (140 rpm) at 37 °C and 6% CO2 using Freestyle medium (Gibco, lot number 2164683). When the cell density reached approximately 1.5×10 6 / mL, transient transfection was performed with trastuzumab plasmid and polyethyleneimine (PEI) (Polysciences, catalog number 24765-1). 1 mg of plasmid was premixed with 2.6 mg of PEI in 50 mL of fresh medium for 15 minutes and then added to 1 liter of cell culture medium. After culturing for 72 hours, the transfected cells were obtained.

[0513] The culture supernatant was collected by centrifugation at 1000×g for 5 minutes. Subsequently, the supernatant was loaded onto Protein A Sepharose beads (GenScript, catalog number L00210-50) and washed with PBS buffer. The protein was eluted with 0.1 M glycine (pH 3.0). The eluted protein was concentrated and then purified by size exclusion chromatography (Superdex 200 Increase 10 / 300 GL, GE Healthcare) in N-methylmorpholine buffer (50 mM, 0.2 M NaCl, pH 7.4). The peak fractions were collected and concentrated. The purified IgGs were confirmed for molecular weight and purity by LC-MS analysis and stored at -20 °C.

[0514] Example 9: Protein Reaction

[0515] a. Conjugation of Small Ubiquitin-like Modifier Proteins

[0516] Conjugation with Boronic Acid 1: To a solution of Small Ubiquitin-like Modifier Protein-CAST or Small Ubiquitin-like Modifier Protein (final concentration 10 μM) in N-methylmorpholine buffer (50 mM, 0.2 M NaCl, pH 7.4), boronic acid 1 (1 μL of 5 mM stock solution, final concentration 0.1 mM) and CuCl2·2H2O (1 μL of 1.5 mM stock solution, final concentration 30 μM) were added sequentially. The total reaction volume was 50 μL. The mixture was incubated at 37 °C for 10 minutes. After the reaction was completed, Na2-EDTA (2 μL of 500 mM stock solution, final concentration 20 mM) was added, and the crude reaction mixture was directly injected for ESI-MS analysis.

[0517] Conjugation with Boronic Acids 1A, 1B, or 1C: To a solution of Small Ubiquitin-like Modifier Protein-CAST (final concentration 10 μM) in N-methylmorpholine buffer (50 mM, 0.2 M NaCl, pH 7.4), boronic acids 1A, 1B, or 1C (1 μL of 25 mM stock solution, final concentration 0.5 mM) and CuCl2·2H2O (1 μL of 1.5 mM stock solution, final concentration 30 μM) were added sequentially. The total reaction volume was 50 μL. The mixture was incubated at 37 °C for 30 minutes. After the reaction was completed, Na2-EDTA (2 μL of 500 mM stock solution, final concentration 20 mM) was added, and the crude reaction mixture was directly injected for ESI-MS analysis. The ESI ion series / deconvoluted mass spectra are shown in Figure 11 .

[0518] b. Conjugation of Nanobodies

[0519] Coupling with Boric Acid 1: To a solution of Nanobody-CAST or Nanobody (final concentration 10 μM) in N-methylmorpholine buffer (50 mM, 0.2 M NaCl, pH 7.4), boric acid 1 (1 μL of 5 mM stock solution, final concentration 0.1 mM) and CuCl2·2H2O (1.7 μL of 1.5 mM stock solution, final concentration 50 μM) were added successively. The total reaction volume was 50 μL. The mixture was incubated at 37 °C for 1.5 h. After the reaction was completed, Na2-EDTA (2 μL of 500 mM stock solution, final concentration 20 mM) was added, and the crude reaction mixture was directly injected for ESI-MS analysis.

[0520] Coupling with Boric Acids 1A, 1B or 1C: To a solution of Nanobody-CAST (final concentration 10 μM) in N-methylmorpholine buffer (50 mM, 0.2 M NaCl, pH 7.4), boric acids 1A, 1B or 1C (1 μL of 25 mM stock solution, final concentration 0.5 mM) and CuCl2·2H2O (1.7 μL of 1.5 mM stock solution, final concentration 50 μM) were added successively. The total reaction volume was 50 μL. The mixture was incubated at 37 °C for 1.5 h. After the reaction was completed, Na2-EDTA (2 μL of 500 mM stock solution, final concentration 20 mM) was added, and the crude reaction mixture was directly injected for ESI-MS analysis. The ESI ion series / deconvoluted mass spectra are shown in Figure 12 .

[0521] c. Coupling of Maltose-Binding Protein

[0522] Binding with Boric Acid 1: To N-methylmorpholine buffer (50 mM, 0.2 M NaCl, pH 7.4) containing Maltose-Binding Protein-CAST or Maltose-Binding Protein (final concentration 10 μM), boric acid 1 (1 μL of 5 mM stock solution in N,N-dimethylformamide, final concentration 0.1 mM) and CuCl2·2H2O (1.7 μL of 1.5 mM stock solution in water, final concentration 50 μM) were subsequently added. The total reaction volume was 50 μL. The mixture was incubated at 37 °C for 1 h. After the reaction was completed, Na2-EDTA (2 μL of 500 mM stock solution in H2O, final concentration 20 mM) was added, and the crude reaction mixture was directly injected into ESI-MS for analysis.

[0523] Coupling reaction with boric acid 1A, 1B or 1C: To the solution of maltose-binding protein-CAST in N-methylmorpholine buffer (50 mM, 0.2 M NaCl, pH 7.4) (final concentration 10 μM), boric acid 1A, 1B or 1C (1 μL of 25 mM N,N-dimethylformamide stock solution, final concentration 0.5 mM) and CuCl2·2H2O (1.7 μL of 1.5 mM aqueous stock solution, final concentration 50 μM) were added successively. The total reaction volume was 50 μL, and the mixture was incubated at 37 °C for 2 hours. After the reaction was completed, Na2-EDTA (2 μL of 500 mM aqueous stock solution, final concentration 20 mM) was added, and the crude reaction mixture was directly subjected to ESI-MS analysis. Figure 13 Shows the ESI ion series / deconvoluted mass spectra.

[0524] d. Coupling reaction of trigger factor

[0525] Coupling reaction with boric acid 1: To the solution of trigger factor-CAST or trigger factor in N-methylmorpholine buffer (50 mM, 0.2 M NaCl, pH 7.4) (final concentration 5 μM), boric acid 1 (1 μL of 5 mM N,N-dimethylformamide stock solution, final concentration 0.1 mM) and CuCl2·2H2O (1.7 μL of 1.5 mM aqueous stock solution, final concentration 50 μM) were added successively. The total reaction volume was 50 μL, and the mixture was incubated at 37 °C for 2 hours. After the reaction was completed, Na2-EDTA (2 μL of 500 mM aqueous stock solution, final concentration 20 mM) was added, and the crude reaction mixture was directly subjected to ESI-MS analysis.

[0526] Coupling reaction with boric acid 1A, 1B or 1C: To the solution of trigger factor-CAST in N-methylmorpholine buffer (50 mM, 0.2 M NaCl, pH 7.4) (final concentration 5 μM), boric acid 1A, 1B or 1C (1 μL of 25 mM N,N-dimethylformamide stock solution, final concentration 0.5 mM) and CuCl2·2H2O (1.7 μL of 1.5 mM aqueous stock solution, final concentration 50 μM) were added successively. The total reaction volume was 50 μL, and the mixture was incubated at 37 °C for 2 hours. After the reaction was completed, Na2-EDTA (2 μL of 500 mM aqueous stock solution, final concentration 20 mM) was added, and the crude reaction mixture was directly subjected to ESI-MS analysis. Figure 14 Shows the ESI ion series / deconvoluted mass spectra.

[0527] e. Coupling reaction of sortase

[0528] In the sortase reaction, we observed that unprotected cysteine could react with styrylboronic acid. Therefore, before performing the CAST coupling reaction on sortase, this cysteine was blocked with maleimide.

[0529] The sortase-CAST (10 μM) was incubated with maleimide (5 mM) in PBS at room temperature for 40 minutes, and then the PBS buffer was replaced with N-methylmorpholine buffer (50 mM, pH 7.4, 0.2 M NaCl).

[0530] Coupling reaction with boric acid 1: To the maleimide-modified sortase-CAST or maleimide-modified sortase solution (final concentration 10 μM) in N-methylmorpholine buffer (50 mM, 0.2 M NaCl, pH 7.4), boric acid 1 (1 μL of 5 mM N,N-dimethylformamide stock solution, final concentration 0.1 mM) and CuCl2·2H2O (1.7 μL of 1.5 mM aqueous stock solution, final concentration 50 μM) were added sequentially. The total reaction volume was 50 μL, and the mixture was incubated at 37 °C for 30 minutes. After the reaction was completed, Na2-EDTA (2 μL of 500 mM aqueous stock solution, final concentration 20 mM) was added, and the crude reaction mixture was directly subjected to ESI-MS analysis.

[0531] Coupling reaction with boric acid 1A, 1B or 1C: To the maleimide-modified sortase-CAST solution (final concentration 10 μM) in N-methylmorpholine buffer (50 mM, 0.2 M NaCl, pH 7.4), boric acid 1A, 1B or 1C (1 μL of 25 mM N,N-dimethylformamide stock solution, final concentration 0.5 mM) and CuCl2·2H2O (1.7 μL of 1.5 mM aqueous stock solution, final concentration 50 μM) were added sequentially. The total reaction volume was 50 μL, and the mixture was incubated at 37 °C for 30 minutes. After the reaction was completed, Na2-EDTA (2 μL of 500 mM aqueous stock solution, final concentration 20 mM) was added, and the crude reaction mixture was directly subjected to ESI-MS analysis. Figure 15 The ESI ion series / deconvoluted mass spectra are shown.

[0532] f. Coupling reaction of Tra-CAST

[0533] Coupling with Boric Acid Reagent 1: To an N-methylmorpholine buffer (50 mM, 0.2 M NaCl, pH 7.4) containing Tra-CAST or trastuzumab (final concentration 4 μM), boric acid 1 (1 μL of 12.5 mM N,N-dimethylformamide stock solution, final concentration 0.25 mM) and CuCl2·2H2O (1 μL of 1.0 mM aqueous stock solution, final concentration 20 μM) were added successively. The total reaction volume was 50 μL. The mixture was incubated at 37 °C for 2 hours. After the reaction was completed, (2 μL of 500 mM aqueous stock solution, final concentration 20 mM) was added to the crude reaction mixture, and ESI-MS analysis was carried out directly. In some experiments, the reaction mixture was treated with endoglycosidase (EndoS) before ESI-MS analysis to remove N-linked glycans.

[0534] Coupling with Boric Acids 1A, 1B or 1C: To an N-methylmorpholine buffer (50 mM, 0.2 M NaCl, pH 7.4) containing Tra-CAST (final concentration 4 μM), boric acids 1A, 1B or 1C (1 μL of 12.5 mM N,N-dimethylformamide stock solution, final concentration 0.25 mM) and CuCl2·2H2O (1 μL of 1.0 mM aqueous stock solution, final concentration 20 μM) were added successively. The total reaction volume was 50 μL. The mixture was incubated at 37 °C for 4 hours. After the reaction was completed, (2 μL of 500 mM aqueous stock solution, final concentration 20 mM) was added to the crude reaction mixture, and ESI-MS analysis was carried out directly. In some experiments, the reaction mixture was treated with endoglycosidase (EndoS) before ESI-MS analysis to remove N-linked glycans.

[0535] Coupling with SBA-MMAE (Preparation of Tra-CAST-MMAE): To an N-methylmorpholine buffer (50 mM, 0.2 M NaCl, pH 7.4) containing Tra-CAST (final concentration 4 μM), SBA-MMAE (1 μL of 25 mM N,N-dimethylformamide stock solution, final concentration 0.5 mM), CuCl2·2H2O (1.5 μL of 1.0 mM aqueous stock solution, final concentration 30 μM) and 5 μL of N,N-dimethylformamide were added successively. The total reaction volume was 50 μL. The mixture was incubated at 37 °C for 7 hours. After the reaction was completed, Na2-EDTA (2 μL of 500 mM aqueous stock solution, final concentration 20 mM) was added to the crude reaction mixture, and ESI-MS analysis was carried out directly. In some experiments, the reaction mixture was treated with endoglycosidase (EndoS) before ESI-MS analysis to remove N-linked glycans.

[0536] Tra-CAST-MMAE

[0537]

[0538] Conjugation of g.CTra-CASTi (Preparation of Tra-CASTi-MMAE)

[0539] To the N-methylmorpholine buffer solution (50 mM, 0.2 M NaCl, pH 7.4) containing Tra-CASTi (final concentration 4 μM), boric acid 1B (20 μL of 12.5 mM N,N-dimethylformamide mother liquor, final concentration 0.25 mM) and CuCl2·2H2O (20 μL of 1.0 mM aqueous solution mother liquor, final concentration 20 μM) were added successively. The total reaction volume was 1 mL. The mixture was incubated at 37 °C for 4 hours. After the reaction was completed, Na2-EDTA (40 μL of 500 mM aqueous solution mother liquor, final concentration 20 mM) was added, and the excess 1B was removed by dialysis.

[0540] Click reaction for MMAE conjugation: To the PBS solution of 1B-modified Tra-CASTi (4 μM), DBCO-MMAE (4 μL of 20 mM DMSO mother liquor, 20-fold equivalent) was added, and the mixture was incubated at 37 °C for 2 hours. The reaction was monitored by LC-MS, and after completion, the excess DBCO-MMAE was removed by dialysis.

[0541] Example 10: Conjugation of Boric Acid with Tagged Proteins

[0542] The tag of the present invention was linked to the C-terminus of different proteins (including: small ubiquitin-like modifier protein (11 kDa), maltose-binding protein (41 kDa), nanobody (15 kDa), trigger factor (48 kDa) and sortase (17 kDa)), and the reactions of these proteins with boric acid reagents (boric acid 1, 1A, 1B, 1C, 1D) were studied.

[0543] The concentration in the protein reaction was maintained at 10 μM. Due to the low concentration, the reaction rate was lower than that of polypeptides. However, the stilbeneboric acid reaction could still obtain >95% conjugate products within 2 hours, while the control protein lacking the CAST polypeptide did not show activity ( Figures 11 - 16 )

[0544] For the same protein containing the tag, the reactivity of alkynyl-, azide- or biotin-modified stilbeneboric acid was equally high. In addition, in all the reactions we carried out on proteins, the binding of the second boric acid was only observed at the lowest level (<5%).

[0545] Example 11: Characteristics and Effects of Antibody-Drug Conjugates in the Present Invention

[0546] In recent years, antibody-drug conjugates (ADCs) have become an important therapeutic dosage form. Their preparation requires a chemical cross-linking reaction between an antibody and a cytotoxic drug. Currently, cysteine-based chemical conjugation is widely used for drug loading due to its unique reactivity, but disrupting disulfide bonds may affect antibody stability, and product heterogeneity results from the presence of multiple disulfide bonds in the antibody, making site-specific cysteine conjugation difficult to achieve. The plasma stability of maleimide-cysteine conjugates is also a problem. Although Pai-clamp and DBCO-tag can achieve site-specificity, they still rely on the cysteine reaction system.

[0547] The conjugation based on the tag of the present invention is a completely different reaction system that does not require manipulation of antibody disulfide bonds, providing a new method for the preparation of antibody-drug conjugates. The excellent conjugation effect of the aforementioned recombinant protein indicates that this tag can be fused to an antibody to prepare homogeneous antibody-drug conjugates.

[0548] a. Antibody-drug conjugate based on CAST

[0549] 1) Conjugation reaction

[0550] We inserted the CAST polypeptide segment into the C-terminus of the heavy chain of trastuzumab by mutation to prepare Tra-CAST (the heavy chain sequence is shown in SEQ ID NO: 305, and the light chain is shown in SEQ ID NO: 304), and reacted it with boric acid 1, 1A, 1B, 1C, or 1D. In the control experiment, untagged trastuzumab was reacted with boric acid 1 under the same conditions.

[0551] Although trastuzumab has no visible reactivity with styrene boric acid ( Figure 17 a-d), Tra-CAST can react efficiently with styrene boric acid ( Figure 17 e-h). The reaction efficiency of Tra-CAST with 1A, 1B, and 1C is basically equivalent to that of styrene boric acid ( Figure 17 i-n).

[0552] To prepare the antibody-drug conjugate, we synthesized the styrene boric acid-Val-Cit-PAB-MMAE (SBA-MMAE) complex and obtained Tra-CAST-MMAE by one-step conjugation of Tra-CAST. Due to the low concentration and large molecular size, the conjugation reaction took 7 hours to complete ( Figure 17 o). No reaction was observed in the light chain of trastuzumab, and only a single modification was detected in the heavy chain.

[0553] 2) Characteristics and effects

[0554] The following experiments were designed to verify whether the antibody-drug conjugate of the present invention maintains the same affinity level as the un-conjugated antibody.

[0555] The in vitro binding experiments were performed at room temperature using a ForteBio Octet biolayer interferometry system. The AHC (anti-human Fc capture) probe was immersed in 200 μL of antibody solution (10 μg / ml Tra-CAST or Tra-CAST-MMAE, dissolved in PBS containing 0.1% BSA and 0.02% Tween) to load the antibody. The antibody-loaded probe was then immersed in PBS (containing 0.1% BSA and 0.02% Tween) with different concentrations of recombinant HER2 (Sinobiological 10004-H08H1-50), or only immersed in PBS containing 0.1% BSA and 0.02% Tween, to obtain the binding curve (buffer data as reference). After the binding phase, the probe was transferred to PBS with the same composition to obtain the dissociation curve. According to the ForteBio operation manual, the binding / dissociation curves of each sample were manually fitted using Excel Solver to calculate the K D value, and the final K D value was the average of the KD values obtained from the HER2 serial dilution experiments.

[0556] The binding affinity (K D 0.49 nM) of Tra-CAST-MMAE for HER2 was almost the same as that of trastuzumab for HER2 (K D 0.43 nM) ( Figure 18 b).

[0557] The following experiments were designed to study the in vitro cytotoxicity of the antibody-drug conjugate of the present invention.

[0558] In a 96-well white opaque plate, CHO cells were seeded at a density of 5×10 3 / well, and BT474, MCF7, and SK-BR-3 cells were seeded at a density of 1×10 4 / well. The cells were allowed to adhere for 24 hours in a humidified environment at 37 °C with 5% CO2. Subsequently, BT474, MCF7, and SK-BR-3 cells were treated with serial dilutions of Tra-CAST, Tra-CAST-MMAE, and SBA-MMAE for 96 hours, and CHO cells were treated for 72 hours (shorter treatment time to prevent overgrowth). Cell viability was detected using CellTiter Glo reagent (G7571) according to the instructions, and the data were normalized to the untreated group. Curves were plotted using Graphpad software, and the half-maximal effective concentration (EC 50 ) was calculated using sigmoidal Boltzmann fitting.

[0559] As Figure 19As shown, Tra-CAST showed extremely low toxicity to all cells at the tested concentrations; SBA-MMAE had low toxicity and could not effectively distinguish between HER2-negative and positive cells; Tra-CAST-MMAE was non-toxic to HER2-negative CHO cells and MCF-7 cells, but had significant killing effects on HER2-positive SK-BR-3 and BT-474 cells (EC 50 were 0.2 nM and 0.6 nM, respectively).

[0560] b. Antibody-drug conjugates based on CASTi

[0561] 1) Plasma stability of the tag

[0562] We found that the IAPDDHAA sequence (Sequence No.: 269, abbreviated as CASTi) was highly stable in plasma and serum. The polypeptide remained essentially intact after incubation in plasma for 144 hours ( Figure 20 ).

[0563] In vitro serum / plasma stability detection of the polypeptide: Fresh blood of 8-week-old male BALB / c mice was obtained from the Experimental Animal Center of Westlake University. Serum preparation: Centrifuge at 1500 g for 10 minutes after standing at room temperature for 30 minutes; Plasma preparation: Centrifuge at 1500 g for 20 minutes. The polypeptide (2 mM) was co-incubated with fresh serum at 37 °C, and samples were taken at 0, 18, 48, 96, and 144 hours. Acetonitrile with a final concentration of 75% was added to precipitate plasma proteins, and the precipitate was removed by centrifugation at 12000 g for 5 minutes. The supernatant was diluted 20-fold with 0.1% TFA / water (v / v) and then subjected to LC / MS analysis.

[0564] 2) Conjugation reaction

[0565] We then inserted -CASTi at the C-terminus of the Fc domain to generate Tra-CASTi (the heavy chain was shown by Sequence No.: 307, and the light chain was shown by Sequence No.: 306). To produce large amounts of antibody-drug conjugates, we reacted Tra-CASTi with 1B and then with DBCO-(PEG)3-Glu-Val-Cit-PAB-MMAE (DBCO-MMAE) to generate Tra-CASTi-MMAE ( Figure 17 p-r) for further in vitro and in vivo studies.

[0566] 3) Characteristics and effects

[0567] We confirmed that Tra-CASTi-MMAE could effectively kill HER2-positive SK-BR-3 cells (EC50 0.2 nM), BT-474 cells (EC50 0.2 nM), and SKOV-3 cells (EC50 0.2 nM), while showing extremely low toxicity to HER2-negative MCF-7 cells( Figure 21 a). Tra-CASTi-MMAE also showed excellent plasma stability( Figure 21 b).

[0568] Plasma stability detection of ADC: Tra-CASTi-MMAE (100 μg / mL, 1.2 μL PBS solution) was added to undiluted BALB / c mouse plasma (118.8 μL) to a final concentration of 1 μg / mL. After incubation at 37 °C for different times, aliquots (15 μL each time) were stored at -80 °C for later measurement. Analysis was performed using a sandwich ELISA method: High-binding 96-well plates (Corning) were coated with self-made Her2 protein (100 ng / well) overnight at 4 °C. After coating, 200 μL of 2% BSA-PBS solution (PBS-T) containing 0.05% Tween 20 was added, and the plates were shaken at room temperature for 2 hours for blocking. After discarding the blocking solution, ADC samples (100 μL of PBS-T solution containing 2% BSA) were added to each well and incubated overnight at 4 °C. After washing the plates four times, 100 μL of rabbit anti-human IgG antibody (1:5000) was added and incubated at room temperature for 2 hours. After washing the plates again, the detection antibody was added: 100 μL of goat anti-rabbit IgG-HRP (1:5000). After washing the plates, 100 μL of TMB substrate was added. After color development for 10 - 30 minutes, 100 μL of 2 M HCl was added to each well to terminate the reaction, and the absorbance at 450 nm was read using a microplate reader (Thermo Varioskan LUX). Concentrations were calculated based on the standard curve. The detection method for human plasma stability was the same, except that self-made human HER2 protein (100 ng / well) was used for coating, and the detection antibodies were mouse anti-MMAE antibody (1:5,000) and goat anti-mouse IgG-HRP conjugate (1:5,000) respectively.

[0569] The in vivo efficacy of Tra-CASTi-MMAE was evaluated in a SKOV-3 xenograft tumor model. Tra-CASTi-MMAE (12 mg / kg or 6 mg / kg), Tra-CASTi (12 mg / kg), or Fc isotype control (12 mg / kg) was intravenously injected once a week for a total of four times. No significant toxicity was observed in all groups during the experiment. Tumor growth in the 12 mg / kg Tra-CASTi-MMAE group was significantly inhibited, while tumors in the control group grew rapidly to 1500 mm 3 (study endpoint, Figure 21 c and 22). These results demonstrate that the present invention is indeed suitable for in vivo therapeutic conjugate applications.

[0570] Animal model preparation and in vivo anti-tumor experiment: All operations were approved by the Animal Experiment Ethics Committee of Zhejiang University. Female Balb / c nude mice (4 weeks old) were purchased from Zhejiang Vital River Laboratory Animal Technology Co., Ltd. They were adaptively fed for 1 week before the experiment and had free access to food and water. A HER2-positive human ovarian cancer xenograft model was established to evaluate the anti-tumor effect of the ADC compound: SKOV-3 cells were suspended in serum-free and antibiotic-free DMEM medium, and 100 μL of the cell suspension (1×107 / 100 μL) was subcutaneously injected into each mouse. The formula for calculating the tumor volume: Tumor volume = 0.52 × length × width2. When the average volume of the xenograft reached 100 - 150 mm 3 at this time, they were randomly divided into five groups (n = 5), and Tra-CASTi-MMAE (12 or 6 mg / kg) and the control (Tra-CASTi 12 mg / kg; Fc 12 mg / kg) were injected into the tail vein on days 0, 7, 14, and 28, respectively. The tumor volume and body weight were monitored three times a week, and the mice were sacrificed when the xenograft reached 1500 mm 3 at this time.

[0571] Example 12: Protein fluorescence labeling

[0572] We performed fluorescence labeling on maltose-binding protein and trastuzumab using the label of the present invention.

[0573] (1) Coupling conditions

[0574] Labeling of maltose-binding protein: To the N-methylmorpholine buffer (50 mM, 0.2 M NaCl, pH 7.4) containing maltose-binding protein-CAST (final concentration 10 μM), boric acid 1E (1 μL of 25 mM N,N-dimethylformamide stock solution, final concentration 0.5 mM) and CuCl2·2H2O (1.7 μL of 15 mM aqueous solution, final concentration 50 μM) were added in sequence, with a total volume of 50 μL. After reacting at 37 °C for 2 hours, sodium ethylenediaminetetraacetate (Na4-EDTA·2H2O) was added, and the crude reaction solution was directly analyzed by ESI-MS.

[0575] Trastuzumab: To a 10 μM Tra-CAST solution in N-methylmorpholine buffer (50 mM, 0.2 M NaCl, pH 7.4), 1E boric acid (1 μL of a 12.5 mM stock solution in N,N-dimethylformamide, final concentration 0.5 mM) and copper(II) chloride dihydrate (1 μL of a 10 mM stock solution in water, final concentration 20 μM) were added successively. The total volume of the reaction was 50 μL. The mixture was incubated at 37 °C for 5 hours. Then sodium ethylenediaminetetraacetate (Na4-EDTA·2H2O) was added. The reaction mixture was then treated with EndoS to remove N-linked glycans and directly subjected to ESI-MS.

[0576]

[0577] 1E

[0578] The results are as Figure 22 shown. The reaction of maltose-binding protein-CAST with 1E resulted in 95% conversion to fluorophore-attached maltose-binding protein; the reaction of Tra-CAST with 1E similarly resulted in a high conversion rate to fluorophore-attached Tra-CAST.

[0579] Discussion

[0580] Direct site-specific chemical protein conjugation through natural amino acids has been widely used in basic research and therapeutic development. However, due to the similar chemical environments of the same amino acid at different protein sites, this conjugation is usually difficult to achieve. Here, we successfully developed a polypeptide tag and demonstrated that transition metal-catalyzed reactions can indeed achieve protein conjugation specificity through typical amino acids. The polypeptide tag of the present invention has superior reaction kinetics, exceeding most chemical conjugations and enzyme conjugations, and can be fused with different proteins to achieve site-specific conjugation of various small molecules. We also successfully prepared homogeneous antibody-drug conjugates through this tag and showed that these conjugates can selectively kill antigen-positive cells while being non-toxic to antigen-negative cells. In addition, the antibody-drug conjugates of the present invention remain stable in plasma and achieve significant therapeutic and anti-tumor effects in vivo.

Claims

1. A polypeptide tag comprising the sequence: [Xaa1] a -[Xaa2] b -[Xaa3] c -[Xaa4] d -Xaa5-Xaa6-His-[Xaa7] e -[Xaa8]f; where a = 0 or 1, b = 0 or 1, c = 0 or 1, d = 0 or 1, e = 0 or 1, and / or f = 0 or 1; and Xaa1 = phenylalanine, histidine, arginine, asparagine, proline, glutamine, tryptophan, aspartic acid, glutamic acid, lysine, isoleucine, methionine, serine, threonine or tyrosine; Xaa2 = phenylalanine, leucine, histidine, methionine, asparagine, serine, threonine, proline, tyrosine, alanine, aspartic acid, lysine, glutamine, arginine, tryptophan or isoleucine; Xaa3 = lysine, alanine, aspartic acid, glutamic acid, phenylalanine, histidine, isoleucine, asparagine, threonine, serine, proline, arginine, glycine, glutamine, leucine, tryptophan or methionine; Xaa4 = aspartic acid, lysine, glutamine, glycine, histidine, asparagine, threonine, serine, methionine, lysine, glutamic acid, leucine, tryptophan, proline, alanine, phenylalanine, isoleucine, arginine or tyrosine; Xaa5 = aspartic acid, threonine, serine or asparagine; Xaa6 = aspartic acid, histidine, serine, glutamic acid, alanine or asparagine; Xaa7 = alanine, tryptophan, isoleucine, leucine, methionine, tyrosine, aspartic acid, glutamine, serine, threonine, aspartic acid, lysine, glutamic acid, histidine, tryptophan, arginine, phenylalanine, proline or glycine; and Xaa8 = alanine, glycine, isoleucine, leucine, aspartic acid, serine, threonine, aspartic acid, arginine, lysine, valine, phenylalanine, tryptophan, tyrosine, glutamine, glutamic acid or histidine.

2. A labeled protein comprising the polypeptide tag according to claim 1 linked to a protein.

3. A protein - chemical conjugate comprising a chemical substance and the labeled protein according to claim 2, wherein the chemical substance is conjugated to the polypeptide tag through a copper(II)-catalyzed Chan - Lam reaction.

4. The protein - chemical conjugate according to claim 3, wherein the chemical substance is conjugated to the Xaa6 site of the polypeptide tag.

5. The protein-chemical conjugate according to claim 3 or 4, wherein the chemical substance is conjugated to the polypeptide tag through a linker.

6. The protein-chemical conjugate according to any one of claims 3-5, wherein the copper(II)-catalyzed Chan-Lam reaction is a reaction between a boronic acid group or a boronic acid derivative group and the Xaa6 site of the polypeptide tag.

7. The protein-chemical conjugate according to claim 6, wherein the boronic acid is a substituted or unsubstituted alkyl boronic acid, cycloalkyl boronic acid, alkenyl boronic acid, cycloalkenyl boronic acid, alkynyl boronic acid, cycloalkynyl boronic acid, aryl boronic acid, heteroaryl boronic acid or cycloalkyl-alkenyl boronic acid, and the boronic acid derivative group is a pinacol boronic ester or a trifluoroborate.

8. The protein-chemical conjugate according to claim 6, wherein the boronic acid is a substituted or unsubstituted vinyl boronic acid, cyclohexyl-vinyl boronic acid or styryl boronic acid.

9. The protein-chemical conjugate according to any one of claims 3-8, wherein the chemical substance comprises a drug, a marker, a linker, a reactive group, an antigen, a hapten, a protein ligand or any combination thereof.

10. The protein-chemical conjugate according to any one of claims 3-9, wherein the drug is a cytotoxic agent.

11. The protein-chemical conjugate according to any one of claims 3-10, wherein the protein is an antibody.

12. A protein-protein complex comprising the protein-chemical conjugate according to any one of claims 3-11 and a second protein; wherein the second protein binds to the chemical substance in the protein-chemical conjugate.

13. The complex according to claim 12, wherein the second protein is avidin and the chemical substance is biotin, or the second protein is an antibody and the chemical substance is an antigen specifically recognized by the antibody.

14. The polypeptide tag according to claim 1, the labeled protein according to claim 2, the protein-chemical conjugate according to any one of claims 3-11 or the complex according to any one of claims 12-13, wherein the sequence of the tag is selected from the group consisting of SEQ ID NOs: 4-281 and DDH.

15. A DNA encoding the polypeptide tag according to claim 1 or the labeled protein according to claim 2.

16. A method for preparing a labeled protein, comprising adding or inserting the polypeptide tag according to claim 1 to a protein to obtain the labeled protein.

17. A method for preparing a protein-chemical conjugate, comprising conjugating a chemical substance with the labeled protein according to claim 2 through a copper(II)-catalyzed Chan-Lam reaction.

18. The method according to claim 17, comprising: Under conditions suitable for copper(II)-catalyzed Chan-Lam reaction, a chemical substance containing a boronic acid group or a boronic acid derivative group or conjugated thereto is contacted with the labeled protein to generate a protein-chemical conjugate.

19. The method according to claim 18, wherein the chemical substance is conjugated with a boronic acid group or a boronic acid derivative group through a linker.

20. The method according to claim 17, comprising: (i) Under conditions suitable for copper(II)-catalyzed Chan-Lam reaction, a first linker containing a boronic acid group or a boronic acid derivative group is contacted with a labeled protein to generate a protein-linker conjugate; (ii) A chemical substance is conjugated to the first linker in the protein-linker conjugate.

21. The method according to claim 20, wherein the chemical substance is conjugated with a second linker, and step (ii) comprises conjugating the conjugate of the chemical substance and the second linker with a first linker.

22. The method according to any one of claims 16-21, wherein the boronic acid is a substituted or unsubstituted alkyl boronic acid, cycloalkyl boronic acid, alkenyl boronic acid, cycloalkenyl boronic acid, alkynyl boronic acid, cycloalkynyl boronic acid, aryl boronic acid, heteroaryl boronic acid or cycloalkyl-alkenyl boronic acid, and the boronic acid derivative group is a pinacol borate or a trifluoroborate.

23. The method according to any one of claims 16-18, wherein the boronic acid is a substituted or unsubstituted vinyl boronic acid, cyclohexyl-vinyl boronic acid or styryl boronic acid.

24. The method according to any one of claims 17-23, wherein the chemical substance comprises a drug, a label, a linker, a reactive group, an antigen, a hapten, a protein ligand or any combination thereof.

25. The method according to any one of claims 17-24, wherein the chemical substance is a cytotoxic agent.

26. The method according to any one of claims 19-25, wherein the chemical substance is connected to the linker through a click reaction or a bioorthogonal reaction.

27. The method according to any one of claims 17-26, wherein the protein is an antibody.

28. A method for preparing a protein-protein complex, comprising: (i) A protein-chemical conjugate is prepared by the method according to any one of claims 17-27, wherein the chemical substance in the protein-chemical conjugate is capable of specifically binding to a second protein; (ii) The protein-chemical conjugate is reacted with the second protein to form a protein-protein complex.

29. The method according to claim 28, wherein the second protein is avidin and the chemical substance is biotin, or the second protein is an antibody and the chemical substance is an antigen specifically recognized by the antibody.

30. The method according to any one of claims 16-29, wherein the sequence of the label is selected from the group consisting of sequence numbers: 4-281 and DDH.

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