WO2024099368A1

WO2024099368A1 - Peptide tags, compositions and methods for site-specific protein conjugation

Info

Publication number: WO2024099368A1
Application number: PCT/CN2023/130506
Authority: WO
Inventors: Bobo DANG; Mengzhun GUO
Original assignee: Westlake University
Priority date: 2022-11-08
Filing date: 2023-11-08
Publication date: 2024-05-16

Abstract

It provides peptide tags and tag-modified protein that can be reacted with a chemical to specifically conjugating the chemical to the tag, to generate a protein-chemical conjugate, including antibody-drug conjugate. It also provides methods for preparing the tags, tag-modified proteins or protein-chemical conjugates. Further, it provides compositions and methods for treating diseases and cancers. It achieves significantly advantageous effect in conjugating a chemical to a desired position within a protein with high specificity and efficacy. The antibody-drug conjugates of it specifically target the target cells with significantly higher efficacy compared to antibody alone or drug alone.

Description

PEPTIDE TAGS, COMPOSITIONS AND METHODS FOR SITE-SPECIFIC PROTEIN CONJUGATION

Cross-Reference to Related Applications

This application claims priority from PCT international application PCT/CN 2022/130722 filed on November 8, 2022, which is incorporated herein by reference in its entirety.

Field of the Invention

The present invention relates to peptide tags, compositions and methods for site-specific protein conjugation.

Background of the Invention

Site-specific protein conjugations minimally affect protein functions and are highly desirable for intended applications. Current site-specific protein conjugations are mostly realized through unnatural amino acid incorporations or enzymatic transformations. Direct site-specific chemical conjugation through canonical amino acids might be more attractive because of its simplicity and convenience. However, to date, only a few cysteine-based site-specific chemical conjugations were reported. Methods beyond using cysteine are very rare, but could find wide applications in protein labeling and therapeutic conjugates preparations. Such conjugation methods development is one of the most challenging subjects in chemistry.

Transition metals catalyze a variety of chemoselective reactions on canonical amino acids. However, other than a few terminal selective reactions, transition metal catalyzed reactions generally lack site-specificity because same amino acid at different sites cannot be easily distinguished. In addition, it was unclear whether transition metal catalyzed site-specific conjugations can be achieved under biocompatible conditions.

We recently reported Ni (II) can bind a specific peptide motif (SNAC-tag) to function as a protease for site-specific protein cleavage. SNAC-tag serves as the metal ion ligand to activate the cleavage reaction while determining the cleavage site simultaneously.

We hypothesize site-specific protein conjugations may be achieved in the same manner: a peptide motifmay specifically bind a metal ion for the metal ion to catalyze a conjugation reaction with the peptide itself. We thus choose a few biocompatible, transition metal catalyzed reactions including Chan-Lam reaction to test our hypothesis.

Summary of the Invention

In one aspect, the present invention provides peptide tags that can be added to the C-or N-terminus of a protein, or inserted within a protein to produce a tag-modified protein. The tag-modified protein can then react with a chemical in the presence of a metal ion, to specifically conjugate the chemical to the tag, thereby generating a protein-chemical conjugate. The protein-chemical conjugate may be an antibody-drug conjugate. In another aspect, the present invention provides a DNA that encodes the tags or tag-modified proteins of the present invention. In another aspect, the present invention provides a composition comprising the tags, tag-modified proteins or protein-chemical conjugates of the present invention. In another aspect, the present invention provides methods for preparing the tags, tag-modified proteins or protein-chemical conjugates of the present invention. In another aspect, the present invention provides compositions and methods for treating diseases and cancers.

The present invention achieves significantly advantageous effect in conjugating a chemical to a desired position of a protein with high specificity and efficacy. The antibody-drug conjugates of the present invention specifically target the target cells with significantly higher efficacy compared to antibody alone or drug alone.

A first aspect of the present invention provides an peptide tag comprising the sequence: [Xaa₁] _a- [Xaa₂] _b- [Xaa₃] _c- [Xaa₄] _d-Xaa₅-Xaa₆-His- [Xaa₇] _e- [Xaa₈] _f;

wherein 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 wherein

Xaa₁=Phe, His, Arg, Asn, Pro, Gln, Trp, Asp, Glu, Lys, Ile, Met, Ser, Thr, or Tyr;

Xaa₂=Phe, Leu, His, Met, Asn, Ser, Thr, Pro, Tyr, Ala, Asp, Lys, Gln, Arg, Trp, or Ile;

Xaa₃=Lys, Ala, Asp, Glu, Phe, His, Ile, Asn, Thr, Ser, Pro, Arg, Gly, Gln, Leu, Trp, or Met;

Xaa₄=Asp, Lys, Gln, Gly, His, Asn, Thr, Ser, Met, Lys, Glu, Leu, Trp, Pro, Ala, Phe, Ile, Arg, or Tyr;

Xaa₅=Asp, Thr, Ser, or Asn;

Xaa₆=Asp, His, Ser, Thr, Glu, Ala, or Asn;

Xaa₇=Ala, Trp, Ile, Leu, Met, Tyr, Asn, Gln, Ser, Thr, Asp, Lys, Glu, His, Trp, Arg, Phe, Pro, or Gly; and

Xaa₈=Ala, Gly, Ile, Leu, Asn, Ser, Thr, Asp, Arg, Lys, Val, Phe, Trp, Tyr, Gln, Glu, or His.

A second aspect of the present invention provides a tagged protein comprising the peptide tag of claim 1 linked to a protein.

A third aspect of the present invention provides a protein-chemical conjugate, comprising a chemical, and the tagged protein of the present invention, wherein the chemical is conjugated to the peptide tag via a copper (II) catalyzed Chan-Lam reaction.

In some embodiments, the chemical is conjugated to Xaa₆of the peptide tag.

In some embodiments, the chemical is conjugated to the peptide tag via a linker.

In some embodiments, the copper (II) catalyzed Chan-Lam reaction is a reaction between a boronic acid group or a boronic acid derivative group and Xaa₆ of the peptide tag.

In some embodiments, the boronic acid is 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 boronic acid pinacol ester or a trifluoroborate salt.

In some embodiments, the boronic acid is substituted or unsubstituted vinyl boronic acid, cyclohexyl-vinyl boronic acid, or styryl boronic acid.

In some embodiments, the chemical comprises a drug, label, linker, reactive group, antigen, hapten, ligand of protein, or any combination thereof.

In some embodiments, the drug is a cytotoxic agent.

In some embodiments, the protein is an antibody.

A fourth aspect of the present invention provides a protein-protein complex, comprising any one of the above protein-chemical conjugates and a second protein; wherein the second protein associates with the chemical in the protein-chemical conjugate.

In some embodiments, the second protein is avidin, and the chemical is biotin, or the second protein is an antibody, and the chemical is an antigen specifically recognized by the antibody.

A fifth aspect of the present invention provides the above peptide tag, the above tagged protein, any one of the above protein-chemical conjugates, or any one of the protein-protein complexes, wherein the sequence of the tag is selected from the group consisting of SEQ ID NOs: 4-281 and DDH.

A sixth aspect of the present invention provides a DNA encoding the above peptide tag or the above tagged protein.

A seventh aspect of the present invention provides a method of preparing a tagged protein, comprising adding or inserting the above peptide tag to a protein to obtain the tagged protein.

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

In some embodiments, the method comprise contacting a chemical comprising or conjugated to boronic acid group or a boronic acid derivative group with the tagged protein under conditions suitable for a copper (II) catalyzed Chan-Lam reaction, thus generating a protein-chemical conjugate.

In some embodiments, the chemical is conjugated to boronic acid group or a boronic acid derivative group via a linker.

In some embodiments, the method comprises:

(i) contacting a first linker comprising or conjugated to boronic acid group or a boronic acid derivative group with the tagged protein under conditions suitable for a copper (II) catalyzed Chan-Lam reaction, thus generating a protein-linker conjugate; and

(ii) conjugating the chemical to the first linker of the protein-linker conjugate.

In some embodiments, the chemical is conjugated to a second linker, and step (ii) comprises conjugating a conjugate of the chemical and the second linker to the first linker.

In some embodiments, the chemical is a cytotoxic agent.

In some embodiments, the chemical is connected to the linker via a click reaction or a bioorthogonal reaction.

In some embodiments, the protein is an antibody.

A ninth aspect of the present invention provides a method of preparing a protein-protein complex, comprising:

(i) preparing a protein-chemical conjugate according to any one of the above methods, wherein the chemical of the protein-chemical conjugate is able to specifically associate with a second protein; and

(ii) reacting the protein-chemical conjugate with the second protein to form the protein-protein complex.

In some embodiments, the sequence of the tag is selected from the group consisting of SEQ ID NOs: 4-281 and DDH.

Brief Description of the Drawings

Figure 1: a) LC-MS analysis of the reaction mixture of peptide YFLHQSHHWG (SEQ ID NO: 308) and boronic acid 1. Starting material abbreviated as SM, modified product abbreviated as Mod. b) Q-Exactive MS/MS spectra of (E) -styrylboronic acid modified peptides YFLHQSHHWG. b ions andy ions fragments observed in the MS/MS spectrum are labeled accordingly.

Figure 2: LC-MS analysis of the conjugation reaction 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) .

Figure 3: LC-MS analysis of the conjugation reaction on a) peptide RWYFLKQSDHWG (SEQ ID NO: 314) at 37 ℃ for 3 hours, b) peptide RWYFLKQDDHWG (SEQ ID NO: 315) at 25 ℃ for 2 hours, c) peptide RWYFLKGDDHAG (SEQ ID NO: 316) at 37 ℃ for 1 hour, and d) peptide RWYFFKKDDHAA (SEQ ID NO: 317) at 25 ℃ for 15 minutes.

Figure 4: Development and characterization of CAST conjugation. a) Sequence optimization procedure. b) Chromatograms at 214 nm absorption from LC-MS analysis for the reaction between RWYFFKKDDHAA (SEQ ID NO: 317) and boronic acid 1. c) Reaction kinetics. d) Partial NOESY spectrum of the modified peptide. e) LC-MS/MS analysis of the modified peptide. f) Copper-bound ATCUN (amino terminal copper and nickel) motif.

Figure 5: a) Boronic acid reagents tested and the respective reaction yields. b) LC-MS analysis of the conjugation reaction between different boronic acid reagents and peptide RWYFFKKDDHAA (SEQ ID NO: 317) .

Figure 6: a) LC-MS analysis of the conjugation reaction between boronic acid 1 and peptide RWYFFKKDDHAA (SEQ ID NO: 317) in the presence of different metal ions. b) LC-MS analysis of the conjugation reaction between boronic acid 1 and peptide RWYFFKKDDHAA (SEQ ID NO: 317) in the presence of different molar equivalents of Cu (II) .

Figure 7: LC-MS analysis of the conjugation reaction between different modified styrylboronic acid molecules and peptide RWYFFKKDDHAA (SEQ ID NO: 317) .

Figure 8: a) LC-MS analysis of the conjugation reaction between boronic acid 1 and peptides having the tag FFKKDDHAA (SEQ ID NO: 192) at different positions. b) LC-MS analysis of the conjugation reaction between boronic acid 1 and Cys-protected RWCFFKKDDHAA (SEQ ID NO: 318) .

Figure 9: LC-MS analysis of the conjugation reaction between boronic acid 1 and peptides having different shortened versions of the CAST tag.

Figure 10: Stability comparison of boronic acid 1 modified RWYFFKKDDHAA (SEQ ID NO: 317) with maleimide modified DLAAEIAKHCG (SEQ ID NO: 324) at different conditions.

Figure 11: ESI ion series/deconvolution MS spectra: reactions between SMT3 and boronic acid 1, 1A, 1B, and 1C. a) SMT3. b) Reaction of SMT3 with boronic acid 1 (no modification observed) . c) SMT3-CAST. d) Reaction of SMT3-CAST with boronic acid 1. e) Reaction of SMT3-CAST with 1A. f) Reaction of SMT3-CAST with 1B. g) Reaction of SMT3-CASTwith 1C.

Figure 12: ESI ion series/deconvolution MS spectra: reactions between nanobody and boronic acid 1, 1A, 1B, and 1C. a) Nanobody. b) Reaction of nanobody 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.

Figure 13: ESI ion series/deconvolution MS spectra: reactions between MBP and boronic acid 1, 1A, 1B, and 1C. a) MBP. b) Reaction of MBP with boronic acid 1 (no modification observed) . c) MBP-CAST. d) Reaction of MBP-CAST with boronic acid 1. e) Reaction of MBP-CASTwith 1A. f) Reaction of MBP-CAST with 1B. g) Reaction of MBP-CAST with 1C.

Figure 14: ESI ion series/deconvolution MS spectra: reactions between trigger factor and boronic acid 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.

Figure 15: ESI ion series/deconvolution MS spectra: reactions between maleimide modified Sortase and boronic acid 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.

Figure 16: CAST fusion proteins are quantitatively modified with various styrylboronic acid derived reagents. Deconvoluted mass spectra of the protein peaks are shown for the starting, and 1, 1A, 1B, or 1C modified a) SMT3, b) nanobody, c) MBP, d) trigger factor, and e) Sortase.

Figure 17: ESI ion series/deconvolution MS spectra. a) Trastuzumab. b) Reaction of trastuzumab with boronic acid 1 (no modification observed either on light chain or on heavy chain) . c) Trastuzumab, treated with EndoS before ESI-MS analysis. d) Reaction of trastuzumab with boronic acid 1, treated with EndoS before ESI-MS analysis (no modification observed either on light chain or on heavy chain) .

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

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

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

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

Figure 18: CAST mediated site-specific antibody conjugation. a) Deconvoluted mass spectra of the light chain and the deglycosylated heavy chain peaks are shown for the starting, and 1A, 1B, 1C or SBA-MMAE modified Tra-CAST. b) Tra-CAST (K_D=0.43 nM) and Tra-CAST-MMAE (K_D=0.49 nM) binding affinities with HER2 characterizations using Bio-layer interferometry.

Figure 19: In vitro anti-tumor activity of Tra-CAST-MMAE.

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

Figure 21: Effects and properties of Tra-CASTi-MMAE. a) In vitro anti-tumor activity of Tra-CASTi-MMAE. b) Tra-CASTi-MMAE stability test in mouse plasma using anti-human IgG or anti-MMAE. c) SKOV-3 xenograft tumor models. Left: tumor volume measurement; right: percent survival (female Balb/c nude mice, n =5 for 12 mg/kg Fc isotype control, n=5 for 12 mg/kg Tra-CASTi, n=4 for 6 mg/kg Tra-CASTi-MMAE, n=6 for 12 mg/kg Tra-CASTi-MMAE) . Black arrow indicates drug administration, error bars represent s. e. m. d) Body weight after Tra-CASTi-MMAE administration (female Balb/c nude mice, n=5 for 12 mg/kg Fc isotype control, n=5 for 12 mg/kg Tra-CASTi, n=4 for 6 mg/kg Tra-CASTi-MMAE, n=6 for 12 mg/kg Tra-CASTi-MMAE) .

Figure 22: ESI ion series/deconvolution MS spectra. a) Reaction of MBP-CAST with 1E. b) Reaction of Tra-CAST with 1E, treated with EndoS before ESI-MS analysis.

Figure 23: LC-MS analysis of the conjugation reaction between boronic acid 1 and further shortened peptides.

Figure 24: LC-MS analysis of the conjugation reaction between boronic acid reagent and WLGFFKKDDHAA (a) and FFKKDDHAA (b) .

Detailed Description of the Invention

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of ordinary skill in the art to which this invention belongs. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples herein are illustrative only and not intended to be limiting.

The terms “about” and “approximate, ” when used along with a numerical variable, generally means the value of the variable and all the values of the variable within a measurement or an experimental error (e.g., 95%confidence interval for the mean) or within a specified value within a broader range (e.g., ±10%) .

As used herein, the singular forms “a” , “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise.

The term “comprise” and variations thereof, such as “comprises” and “comprising” , as well as “contain” , “containing” , “have” , “having” , “include” and “including” means including the recited steps or elements, but not excluding other steps or elements. “Consisting of” means excluding any step or element not specified. “Consisting essentially of” means not excluding steps or elements that do not materially affect the basic and novel characteristics of the claimed invention. The term “comprise" and its variants also include the cases of “consisting of......” and “consisting essentially of......” .

Where a range of values is provided, it is understood that the upper and lower limits, and each smaller range between the upper limit (or the lower limit) and any intervening value, or between any two intervening values in that range, shall be considered to be specifically disclosed. Any intervening range and all individual value in the stated range of value may be excluded from said range of value.

The term “and/or” refers to any one, several or all of the elements connected by the term.

Unless otherwise indicated, nucleic acids are written left to right in 5’ to 3’ orientation; amino acid sequences are written left to right in N-terminus to C-terminus orientation, respectively.

The term “conjugate” , “link” , “connect” , “couple” , “bond” or similar terms refer to that one element (such as a compound) is either directly joined to another element (such as another compound) , or else indirectly joined to another element (such as another compound) through an intervening moiety or moieties, in a covalent or non-covalent manner.

The terms “first” and “second” are used only to distinguish between elements or steps and do not imply a specific order of precedence or location relationship. It should be understood that when “second” element or step is mentioned, it is meant to be literally distinguished from other elements or steps and it is not necessary to have a corresponding “first” element or step.

In one aspect, the present invention provides a peptide tag comprising a sequence of [Xaa₁] _a- [Xaa₂] _b- [Xaa₃] _c- [Xaa₄] _d-Xaa₅-Xaa₆-His- [Xaa₇] _e- [Xaa₈] _f, wherein a, b, c, d, e, f is independently 0 or 1; , wherein Xaa₁ is an amino acid selected from the group consisting of Phe, His, Arg, Asn, Pro, Gln, Trp, Asp, Glu, Lys, Ile, Met, Ser, Thr, and Tyr; Xaa₂is an amino acid selected from the group consisting of Phe, Leu, His, Met, Asn, Ser, Thr, Pro, Tyr, Ala, Asp, Lys, Gln, Arg, Trp, and Ile; Xaa₃ is an amino acid selected from the group consisting of Lys, Ala, Asp, Glu, Phe, His, Ile, Asn, Thr, Ser, Pro, Arg, Gly, Gln, Leu, Trp, and Met; Xaa₄ is an amino acid selected from the group consisting of Asp, Lys, Gln, Gly, His, Asn, Thr, Ser, Met, Lys, Glu, Leu, Trp, Pro, Ala, Phe, Ile, Arg, and Tyr; Xaa₅ is an amino acid selected from the group consisting of Asp, Thr, Ser, and Asn; Xaa₆ is amino acid selected from the group consisting of Asp, His, Ser, Thr, Glu, Ala, and Asn; Xaa₇ is an amino acid selected from the group consisting of Ala, Trp, Ile, Leu, Met, Tyr, Asn, Gln, Ser, Thr, Asp, Lys, Glu, His, Trp, Arg, Phe, Pro, and Gly; and Xaa₈ is an amino acid selected from the group consisting of Ala, Gly, Ile, Leu, Asn, Ser, Thr, Asp, Arg, Lys, Val, Phe, Trp, Tyr, Gln, Glu, and His.

In some embodiments, the peptide tag comprises a sequence of Xaa₁-Xaa₂-Xaa₃-Xaa₄-Xaa₅-Xaa₆-His-Xaa₇-Xaa₈, wherein Xaa₁, Xaa₂, Xaa₃, Xaa₄, Xaa₅, Xaa₆, Xaa₇ and Xaa₈ are as defined as above. In some embodiments, the peptide tag comprises a sequence of Xaa₁-Xaa₂-Xaa₃-Xaa₄-Xaa₅-Xaa₆-His, wherein Xaa₁, Xaa₂, Xaa₃, Xaa₄, Xaa₅ and Xaa₆ are as defined as above. In some embodiments, the peptide tag comprises a sequence of Xaa₂-Xaa₃-Xaa₄-Xaa₅-Xaa₆-His-Xaa₇-Xaa₈, wherein Xaa₂, Xaa₃, Xaa₄, Xaa₅, Xaa₆, Xaa₇ and Xaa₈ are as defined as above. In some embodiments, the peptide tag comprises a sequence of Xaa₂-Xaa₃-Xaa₄-Xaa₅-Xaa₆-His, wherein Xaa₂, Xaa₃, Xaa₄, Xaa₅ and Xaa₆ are as defined as above. In some embodiments, the peptide tag comprises a sequence of Xaa₄-Xaa₅-Xaa₆-His-Xaa₇-Xaa₈, wherein Xaa₄, Xaa₅, Xaa₆, Xaa₇ and Xaa₈ are as defined as above. In some embodiments, the peptide tag comprises a sequence of Xaa₃-Xaa₄-Xaa₅-Xaa₆-His, wherein Xaa₃, Xaa₄, Xaa₅ and Xaa₆ are as defined as above. In some embodiments, the peptide tag comprises a sequence of Xaa₄-Xaa₅-Xaa₆-His, wherein Xaa₄, Xaa₅ and Xaa₆ are as defined as above. In some embodiments, the peptide tag comprises a sequence of Xaa₅-Xaa₆-His-Xaa₇-Xaa₈, wherein Xaa₅, Xaa₆, Xaa₇ and Xaa₈ are as defined as above. In some embodiments, the peptide tag comprises a sequence of Xaa₅-Xaa₆-His-Xaa₇, wherein Xaa₅, Xaa₆ and Xaa₇ are as defined as above. In some embodiments, the peptide tag comprises a sequence of Xaa₅-Xaa₆-His, wherein Xaa₅ and Xaa₆ are as defined as above. The peptide tag of the present invention can conjugate with the chemical via a copper (II) catalyzed Chan-Lam reaction.

In some embodiments, the peptide tag of the present invention does not comprise a sequence of 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) .

In some embodiments, the peptide tag comprises a sequence selected from the group consisting of SEQ ID NOs: 4-281 and DDH. In some embodiments, Xaa₅-Xaa₆-His- [Xaa₇] _e- [Xaa₈] _fis DDHAA.

The N-terminus of the peptide tag may be Xaa₁, Xaa₂, Xaa₃ or Xaa4, and/or the C-terminus of the peptide tag may be His, Xaa₇ or Xaa₈, which depends on the values of a, b, c, d, e and f.

In some embodiments, the sequence of the tag is selected from the group consisting of 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 peptide tag of the present invention linked to a protein. The tag and the protein can be covalently linked to form a fusion protein. The tag may be linked to the N-terminus or C-terminus of the protein, or be inserted inside the protein sequence. It should be understood that when the tag is inserted inside the protein sequence, it should be located at position that should not affect the activity of the protein. The protein, as used herein, includes a monomer consisting of one polypeptide chain or a multimer consisting of two or more polypeptide chains. When the protein is a multimer, the tag may be linked to any one, several or all of the two or more polypeptide chains. Each polypeptide may be linked to one, two or more tags. In some embodiments, the tag is heterologous for the protein. The protein modified by the tag may be an antibody, an enzyme, a receptor, a ligand, etc., including, but not limited to, SMT3, Maltose Binding Protein (MBP) , Nanobody, Trigger Factor, and Sortase. The protein of the present invention may have a molecular weight of about 1kDa to about 150kDa, e.g., about 5kDa to about 70kDa or about 10kDa to about 50kDa.

It should be understood that in the context of the tagged protein, the terms “linked to” and “inserted” are used only to indicate that the sequence of the tagged protein is identical to the sequence obtained by linking to the tag or being inserted by the tag, and do not imply that the linking and inserting are necessarily performed during the preparation of the tagged protein. The tagged protein can be prepared by any method known in the art, for example, by expressing the modified protein in a host cell by DNA recombinant technology and subsequently isolating and purifying it.

In some embodiments, the protein modified by the tag is an antibody, especially a monoclonal antibody, a scFv, or a nanobody. In some embodiments, the antibody is Trastuzumab. When the antibody comprises more than one peptide chain, such as heavy chain and light chain, the tag may be linked to the heavy chain or the light chain or both.

In another aspect, the present invention provides a protein-chemical conjugate, comprising the tagged protein of the present invention, and a chemical, wherein the chemical is connected to the tag of the tagged protein. In other words, the protein-chemical conjugate is formed by conjugating a chemical to the tag of the tagged protein of the present invention. In some embodiments, the chemical is conjugated to the Xaa₆ of the tag. In some embodiments, the chemical may be conjugated to the tag via a copper (II) catalyzed Chan-Lam reaction, e.g., between boronic acid or boronic acid derivative and the Xaa₆ of the tag (e.g., -NH, -OH, -SH of the Xaa₆) . The chemical may comprise or is conjugated to boronic acid group or boronic acid derivative group before the copper (II) catalyzed Chan-Lam reaction occurs. It should be understood that the boronic acid group or boronic acid derivative group is no longer present after copper (II) catalyzed Chan-Lam reaction with the peptide tag, and the protein-chemical conjugate comprises a product of copper (II) catalyzed Chan-Lam reaction, i.e., a product of the reaction between boronic acid or boronic acid derivative and the Xaa₆ of the tag (specifically, -NH, -OH, -SH of the Xaa₆) .

Boronic acid, as used herein, usually refers to organoboronic acid and has the general formula of R-B (OH) ₂, wherein R is an organic substituent comprising at least one carbon atom, and the boron atom is bonded directly to a carbon atom of the substituent. R may be an organic substituent, including, but not limited to, substituted or unsubstituted alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, heteroaryl, or cycloalkyl-alkenyl. In some embodiments, R may be substituted or unsubstituted vinyl or cyclohexyl-vinyl, styryl.

In some embodiments, the boronic acid may 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. In some embodiments, the alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, heteroaryl, or cycloalkyl-alkenyl may have 2-10 carbon atoms. In some embodiments, the alkenyl may be a vinyl. In some embodiments, the boronic acid may be a vinyl boronic acid, cyclohexyl-vinyl boronic acid, or styryl boronic acid. In some embodiments, the cycloalkenyl may be cyclohexenyl, cyclopentenyl, cyclobutenyl, or cyclopropenyl. In some embodiments, the boronic acid may be cyclohexenyl boronic acid, cyclopentenyl boronic acid, cyclobutenyl boronic acid, or cyclopropenyl boronic acid. In some embodiments, the cycloalkyl-alkenyl may be cyclohexyl-vinyl, cyclopentyl-vinyl, cyclobutyl-vinyl, or cyclopropyl-vinyl. In some embodiments, the boronic acid may be cyclohexyl-vinyl boronic acid, cyclopentyl-vinyl boronic acid, cyclobutyl-vinyl boronic acid, or cyclopropyl-vinyl boronic acid.

Boronic acid derivative, as used herein, includes but is not limited to boronic esters (such as boronic acid pinacol ester, and boronic acid trimethylene glycol ester) , and trifluoroborate salts (such as R-BF₃K) . In some embodiments, the boronic acid derivative may be a vinylboronic acid pinacol ester, potassium vinyltrifluoroborate, cyclohexyl-vinylboronic acid pinacol ester, potassium cyclohexyl-vinyltrifluoroborate, styrylboronic acid pinacol ester or potassium styryltrifluoroborate. In some embodiments, the boronic acid derivative group may be a pinacolboryl group or a trifluoroborate salt.

The chemical may be a small molecule, including, but not limited to, drugs, labels, linkers, reactive groups, antigens, haptens and ligands of protein. The chemical of the present invention may comprise more than one functional moiety. The functional moiety may be selected from, but is not limited to, drug, label, reactive group, antigen, hapten and ligand of protein. The chemical may comprise one or more drugs, labels, reactive groups, antigens, haptens or ligands of protein, or any combination thereof. In some embodiments, the drug may be a cytotoxic agent. In some embodiments, the label may be a detectable label or an affinity tag (or a purification tag) , e.g., a fluorescent label. In some embodiments, the ligand of protein may be biotin. In some embodiments, the reactive group may be a bioorthogonal reaction partner or click reaction partner.

In some embodiments, the chemical is conjugated to the peptide tag via a linker, wherein the chemical is connected to the linker and the linker is connected to the peptide tag of the tagged protein, the linker is between the chemical and the peptide tag. That is to say, the protein-chemical conjugate is formed by conjugating a chemical and a linker to the tag of the tagged protein of the present invention. In some embodiments, the linker is conjugated to the Xaa₆ of the tag. In some embodiments, the linker may be conjugated to the tag via a copper (II) catalyzed Chan-Lam reaction, e.g., between boronic acid or boronic acid derivative and the Xaa₆ of the tag (e.g., -NH, -OH, -SH of the Xaa₆) . The linker may comprise or be conjugated to additional boronic acid group or boronic acid derivative group before the copper (II) catalyzed Chan-Lam reaction occurs.

In some embodiments, the linker may be a cleavable linker (such as enzymatically-cleavable peptide linkers, acid sensitive hydrazone linkers, and glutathione-sensitive disulfide linkers, Cathepsin B sensitive linkers, etc. ) or a non-cleavable linker (such as SMCC) . The linker may be a linker commonly used in antibody drug conjugates (ADCs) . The linker may be a linker comprising PEG moiety. In some embodiments, the linker comprises Val-Cit-PAB. In some embodiments, the linker may comprise Val-Cit-PAB, Glu-Val-Cit-PAB, DBCO- (PEG) ₃-Val-Cit-PAB or DBCO-Glu-Val-Cit-PAB.

In some embodiments, the linker may comprise a reactive group that can react with the chemical. In some embodiments, the chemical may be connected to the linker via any chemical reaction. In some embodiments, the chemical may be connected to the linker via a click reaction or bioorthogonal reaction.

In some embodiments, the linker used to join the chemical to the tagged protein may comprises two or more linkers, which may be called a first linker, a second linker, and so on. In some embodiments, the first linker is used to conjugated to the peptide tag, and the second linker is used to conjugated to the chemical. The first linker and the second linker may be connected to each other via any chemical reaction, e.g., a click reaction or bioorthogonal reaction.

The chemical may be connected to the linker before or after the conjugation of the linker to the tag sequence. In some embodiments, a conjugate of the tagged protein and the first linker is connected to the conjugate of the chemical and the second linker to form the protein-chemical conjugate.

In some embodiments, the protein-chemical conjugate comprises a structure of protein-drug or protein-linker-drug. In some embodiments, the protein-chemical conjugate comprises a structure of protein-label or protein-linker-label.

In some embodiments, the protein-chemical conjugate comprises any one of the following structures:

wherein Xaa₁, Xaa₂, Xaa₃, Xaa₄, Xaa₅, Xaa₆, Xaa₇ and Xaa₈ and a, b, c, d, e, f are as defined as above.

wherein the N-terminus of the structures (i.e., Xaa₁, Xaa₂, Xaa₃ or Xaa₄) or the C-terminus of the structures (i.e., the His before Xaa₇, Xaa₇, or Xaa₈) is at the terminus of the protein of the protein-chemical conjugate; or both the N-terminus and the C-terminus of the structures are connected to the amino acids within the protein of the protein-chemical conjugate, that is to say, the structure is inserted inside the protein of the protein-chemical conjugate;

wherein n is an integer between 0 and 12 inclusive.

In another aspect, the present invention provides a protein-protein complex comprising the protein-chemical conjugate as described previously and a second protein, wherein the second protein specifically associates with the chemical. The chemical and the second protein may be associated with each other by any interaction, including covalent binding or non-covalent binding. Examples of non-covalent binding includes electrostatic forces, hydrogen bonds, hydrophobic effects, van der Waals forces, etc., e.g., the interaction between a protein and its ligand or between antigen and antibody. In some embodiments, the chemical in the protein-protein complex is an antigen or a ligand of protein. In some embodiments, the second protein may be avidin, and the chemical may be biotin. In some embodiments, the second protein may be an antibody, and the chemical may be an antigen specifically recognized by the antibody.

In another aspect, the present invention provides a DNA that encodes the tag or tag-modified protein of the present invention.

In another aspect, the present invention provides a composition that comprises the tag, tagged 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 may be used to treat or prevent diseases, tumors or cancers.

In another aspect, the present invention provides a method of preparing the tagged protein of the present invention, comprising adding or inserting the peptide tag of the present invention to a protein to obtain the tagged protein. The sequence of the tag may be linked to the N-terminus or C-terminus of the protein, or inserted anywhere within the protein sequence.

In another aspect, the present invention provides a method of preparing the protein-chemical conjugate of the present invention, comprising conjugating the chemical to the tagged protein of the present invention via a copper (II) catalyzed Chan-Lam reaction to obtain the protein-chemical conjugate.

The method described can be achieved by a single reaction or a reaction with several steps, as long as the protein-chemical conjugate of the present application can be obtained. The reaction conditions for the copper (II) catalyzed Chan-Lam reaction are well known for those skilled in the art.

For example, in some embodiments, the method may comprise contacting a chemical comprising or conjugated to boronic acid group or a boronic acid derivative group with the tagged protein of the present invention under conditions suitable for a copper (II) catalyzed Chan-Lam reaction, thus generating a protein-chemical conjugate.

The chemical comprising or conjugated to boronic acid group or a boronic acid derivative group and/or the tagged protein of the present invention may be prepared by methods known in the art, such as chemical synthesis, DNA recombinant techniques, etc.

In some embodiments, the chemical is conjugated to boronic acid group or a boronic acid derivative group via a linker, and the method may comprise contacting a conjugate of the chemical and the linker with the tagged protein of the present invention under conditions suitable for a copper (II) catalyzed Chan-Lam reaction, thus generating a protein-chemical conjugate; wherein the chemical is connected to the linker and the linker comprises or is conjugated to a boronic acid group or a boronic acid derivative group.

The chemical may be connected to the linker via any chemical reaction, e.g., via a click reaction or bioorthogonal reaction.

In some embodiments, the method may comprise:

(i) contacting a linker comprising or conjugated to boronic acid group or a boronic acid derivative group with the tagged protein under conditions suitable for a copper (II) catalyzed Chan-Lam reaction, thus generating a protein-linker conjugate; and

(ii) conjugating the chemical to the linker of the protein-linker conjugate.

In some embodiments, the method may comprise:

(ii) conjugating a conjugate of the chemical and a second linker to the first linker of the protein-linker conjugate.

The second linker is connected to the first linker and is between the chemical and the linker of the protein-linker conjugate. The second linker may be connected to the first linker via any chemical reaction, e.g., via a click reaction or bioorthogonal reaction.

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

In another aspect, the present invention provides molecules, including the tagged proteins, protein-chemical conjugates, and protein-protein complex, that are prepared by the methods of the present invention.

In another aspect, the present invention provides a method of treating a disease, tumor or cancer, including administering to a subject a therapeutically effective amount of a protein-chemical conjugate of the present invention.

Definitions:

Peptide tag: The terms “peptide tag” and “tag” are used interchangeably to refer to a short amino acid sequence that is usable for conjugating a chemical to a polypeptide or a protein. As used herein, the term “tag” means a peptide sequence that may be attached to another peptide or protein to provide some functionality. The tag of the present invention may be at least about 3 amino acids in length, e.g., at least about 4 amino acids in length, at least about 5 amino acids in length, at least about 6 amino acids in length, at least about 7 amino acids in length, at least about 8 amino acids or at least about 9 amino acids in length. In some embodiments, the tag of the present invention may be about 3 to about 100 amino acids in length, e.g., about 3 to about 50 amino acids in length, about 3 to about 20 amino acids in length, or about 3 to about 10, 11, 12, 13, 14, 15, 16, 17, 18 or 19 amino acids in length. In some embodiments, the tag of the present invention may be about 3 to about 9 amino acids in length, about 4 to about 9 amino acids in length, about 5 to about 9 amino acids in length, about 6 to about 9 amino acids in length or about 7 or 8 amino acids in length.

Protein: The term “protein” refers to a polymeric form of amino acids of any length, which can include coded and non-coded amino acids, chemically or biochemically modified or derivatized amino acids, and proteins having modified peptide backbones. The protein of the present invention may be a monomer consisting of one polypeptide chain or a multimeric protein. A monomer protein may also be used interchangeably with the term “peptide” . The term “multimeric protein” refers to a protein that may exist as a multimer consisting of two or more polypeptide chains. In the multimer, the two or more polypeptide chains may be linked by covalent bonds such as disulfide bonds, linked by non-covalent bonds such as hydrogen bonds and hydrophobic interaction, or linked by a combination thereof. The multimer preferably comprises one or more intermolecular disulfide bonds. The multimer may be a homo-multimer consisting of a single kind of polypeptide chain, or may be a hetero-multimer consisting of two or more kinds of polypeptide chains.

Heterologous: The term “heterologous” is meant that a first entity and second entity are provided in an association that is not normally found in nature. A “heterologous” tag is meant that the sequence of the protein to which the tag is linked does not originally comprise the sequence the tag.

Antibody: The term “antibody” refers to any antigen-binding molecule or molecular complex comprising at least one complementarity determining region (CDR) that binds specifically to or interacts with a particular antigen. The term “antibody” encompasses various forms of antibodies including, without being limited to, monoclonal antibodies, polyclonal antibodies, antigen binding fragments, and engineered antibodies such as multispecific antibodies (e.g., bispecific antibodies or trispecific antibodies) . The antibodies of the present invention may also be chimeric antibodies, humanized antibodies or human antibodies. The terms “monoclonal antibody” , “complete antibody” and “immunoglobulin” are used interchangeably and refer to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds. Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region is comprised of three domains, CHI, CH2 and CH3. Each light chain is comprised of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region is comprised of one domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR) , interspersed with regions that are more conserved, termed framework regions (FR) . Each VH and VL is composed of three CDRs and four FRs arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant regions of the antibodies may 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 (Clq) of the classical complement system. Antibodies of the invention can be of any isotype {e.g., IgA, IgD, IgE, IgG or IgM, i.e., anα, δ, ε, γorμheavy chain) . Within the IgG isotype, antibodies may be lgG1, lgG2, lgG3 or lgG4 subclass. Antibodies of the invention may have aκor aλlight chain. The term “monoclonal antibody” as used herein refers to an antibody that is derived from a single clone, including any eukaryotic, prokaryotic, or phage clone, by any means available or known in the art, and is not limited to antibodies produced through hybridoma technology. Monoclonal antibodies useful with the present disclosure can be prepared using a wide variety of techniques known in the art including the use of hybridoma, genetic recombinant, and phage display technologies, or a combination thereof. The terms “antigen-binding fragment” refers to one or more fragments of a whole antibody that retain the ability to specifically bind to a given antigen. Antigen-binding fragments may be obtained by proteolytic digestion of whole antibody, recombinant DNA or phage display techniques. Examples of the term “antigen-binding fragment” include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) a Fab’ fragment, which is essentially a Fab with part of the hinge region; (iii) a F(ab’ ) 2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iv) a Fd fragment consisting of the VH and CH1 domains; (v) an Fv fragment consisting of the VL and VH domains of a single arm of an antibody; (vi) a single chain Fv fragment (scFv) , a single protein chain in which the VL and VH regions pair to form monovalent molecules; (vii) a disulfide stabilized Fv fragment (dsFv) , an Fv with an engineered intermolecular disulfide bond to stabilize the VH-VL pair; (viii) a single domain antibody (sdAb) which is also known as a nanobody, an antibody fragment consisting of a single monomeric variable antibody domain (e.g., a single heavy chain variable region, VHH) , etc.

Conjugate: The term “conjugate” refers to any compound resulting from the covalent attachment of two or more individual compounds, wherein one individual compound can be either directly covalently joined to another individual compound, or else indirectly covalently joined to another individual compound through an intervening moiety or moieties.

Copper (II) catalyzed Chan-Lam reaction: Chan-Lam reaction is a cross-coupling reaction between organoboronic acids and (thio) alcohols or amines, including an amide NH bond. Traditionally, Copper (II) (such as Cu (OAc) ₂) as catalyst in the presence of an amine base in chlorinated solvents at room temperature under air conditions is used in the Chan-Lam coupling.

Boronic acid: The term “boronic acid” , as used herein, is used interchangeably with “organoboronic acid” and has the general formula: R-B (OH) ₂, , wherein R is a substituent comprising at least one carbon atom, and the boron atom is bonded directly to a carbon atom of the substituent. R may be an organic substituent, including, but not limited to, substituted or unsubstituted alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, heteroaryl, or cycloalkyl-alkenyl. In some embodiments, R may be substituted or unsubstituted vinyl or cyclohexyl-vinyl, styryl.

Boronic acid derivative: The term “boronic acid derivative” refers to a compound resulting from replacing one or both of the two hydroxyl groups of a boronic acid with other functional groups. Boronic acid derivative includes boronic esters (such as boronic acid pinacol ester, and boronic acid trimethylene glycol ester) , and trifluoroborate salts (such as R-BF₃K) .

Chemical: The term “chemical” refers to any molecule having a specific chemical structure, including, but not limited to, drugs, small molecule labels, and reactive handles. Chemicals may comprise a boronic acid or boronic acid derivative group that is able to reacting with a peptide tag.

Linker: The term “linker” generally refers to a moiety linking other two functional moieties. The linker of the present invention may comprise at least one reactive group. The linker of the present invention comprises bifunctional linker.

Functional moiety: The term “functional moiety” refers to any moiety that exhibits one or more functions, which includes, but not limited to, being therapeutic, being detectable, being a linker, being reactive, being enzymatically active, being an affinity tag, an antigen or a hapten.

Bifunctional linker: The term “bifunctional linker” refers to a linker that comprises two reactive groups, where each reactive group can react with a corresponding reactive group on one of the function moieties to which the linker is linked.

Reactive group: The term “reactive group” as used herein refers to a group that is capable of reacting with another chemical group to form a covalent bond, i.e., is covalently reactive under suitable reaction conditions, and generally represents a point of attachment for another substance. Reactive group includes, but is not limited to, click reaction partners, and bioorthogonal reaction partners.

Ligand of protein: The term “ligand of protein” , as used herein, refers to a small molecule that can interact with a protein through non-covalent binding, such as electrostatic forces, hydrogen bonds, hydrophobic effects, van der Waals forces, etc. Examples of ligand of protein includes, but is not limited to biotin.

Antigen: The term “antigen” refers to a substance which can induce an immune response in an organism, particularly an animal, such as human. The antigen of the present invention includes small molecule antigen (e.g., hapten) , which includes, but is not limited to polysaccharides, lipids, nucleic acids and small molecule compounds and drugs.

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.

Drug: Drug refers to any chemical substance that causes a change in an organism’s physiology or psychology and exerts a therapeutic or prophylatic effect when consumed or applied.

Cytotoxic agent: Cytotoxic agent refers to compounds that are toxic to cells. Cytotoxic agents may be used as chemotherapy to treat tumors or cancers.

Small molecule: The term “small molecule” refers to a compound having a molecule weight of 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.

Label: Label refers to any small molecule that can assist in identification or purification of another chemical when attached to the chemical. The labels labels include fluorescent labels, and affinity tags for protein purification, etc.

Click reaction: Click reaction refers to any reaction that allows for attachment of a chemical to a biomolecule (such as protein, glycans, lipids and metabolites) . Click reactions include Copper (I) -catalyzed azide-alkyne cycloaddition, strain-promoted azide-alkyne cycloaddition, and strain-promoted alkyne-nitrone cycloaddition (SPANC) .

Click reaction partner: Click reaction partner refers to one of the two chemical groups that are reacted with each other in a click reaction and responsible for joining the chemical and biomolecule together.

Bioorthogonal reaction: Bioorthogonal reaction refers to any chemical reaction that can occur inside of living systems without interfering native biochemical processes. Bioorthogonal reaction includes nitrone dipole cycloaddition, norbornene cycloaddition, oxanorbornadiene cycloaddition, tetrazine ligation, [4+1] cycloaddition, quadricyclane ligation, and Staudinger reaction.

Bioorthogonal reaction partner: Bioorthogonal reaction partner refers to one of the two chemical groups that are reacted with each other in a bioorthogonal reaction and responsible for joining two molecules together.

Protein-linker: Protein-linker refers to the substance formed after the reaction of a linker and a tagged protein comprising a peptide tag and wherein the protein and linker are joined together.

Protein-chemical: Protein-chemical refers to the substance formed after the reaction of a protein and a chemical and wherein the protein and chemical are joined together.

Antibody drug conjugate (ADC) : ADC refers to any substance wherein an antibody is connected to a drug.

Therapeutically effective amount: The term “therapeutically effective amount” is an amount that is sufficient to provide the intended benefit of treatment. An effective amount of the active agents that can range from generally 0.1 mg active agent/kg body weight to about 50 mg active agent/kg body weight. However, dosage levels are based on a variety of factors, including the type of injury, the age, weight, sex, medical condition of the patient, the severity of the condition, the route of administration, and the particular active agent employed. Thus the dosage regimen may vary widely, but can be determined routinely by a surgeon using standard methods.

Pharmaceutically acceptable excipient: The term “pharmaceutically acceptable excipient” refers to a material, such as a carrier, diluent, stabilizer, dispersing agent, suspending agent, thickening agent, etc. which allows processing the active pharmaceutical ingredient (API) into a form suitable for administration. Pharmaceutically acceptable excipients refer to materials which do not substantially abrogate the desired biological activity or desired properties of the compound (i.e., API) , and is relatively nontoxic, i.e., the material is administered to an individual without causing undesirable biological effects or interacting in a deleterious manner with any of the components of the composition in which it is contained.

Examples

Example 1: Tag Sequence Optimization

a. Initial Screen

We started our investigation by screening a few metal ion binding peptides. The sequences of different petides used are shown in in Table 1. In order to be better identified in HPLC and mass spectrometry, amino acid Y was added to the N-terminus of the sequences shown in Table 1. The boronic acid reagents used are shown below:

Standard reaction conditions in Examples 1a and 1b: to a solution of peptides (8.6 μL of 5 mM stock solution in water, 0.43 mM final concentration) in NMM buffer (82.4 μL of 5 mM stock solution, pH 7.4) , a boronic acid reagent (2 μL of 50 mM stock solution in DMSO, 1.0 mM final concentration) and CuCl₂·2H₂O (7 μL of 5 mM stock solution in water, 0.35 mM final concentration) were added subsequently. The mixture was vortexed and shaken at 37 ℃ for a certain time as specified in the experiments. Ethylenediaminetetraacetic acid tetrasodium salt (Na₄-EDTA, dihydrate) (2 μL of 500 mM stock solution in H₂O, 20 mM final concentration) was added to quench the reactions, then the crude reaction mixture was centrifuged to remove any particulates, and the supernatant was analyzed.

The different metal ion binding peptides were each reacted with different boronic acid reagents under the standard reaction conditions for 12 hours at 37 ℃. The reaction mixture was analyzed by LC-MS to determine the reaction yield. The reaction yield was analyzed using HPLC and calculated by dividing the peak area of the reaction product by the sum of the starting material and reaction product peak area. The results are shown in Table 1.

The LC-MS results of the reaction mixture of boronic acid 1 and YFLHQSHHWG (SEQ ID NO: 308) are shown in Fig. 1a (starting material is abbreviated as “SM” , modified product as “Mod” ) .

These initial results of Chan-Lam reactions were encouraging as very similar peptide sequences gave quite different reaction yields. These results indicate Cu (II) mediated Chan-Lam conjugation reaction does have sequence specificity. The reactions were slow though, with the best sequence (FLHQSHHWG at 0.43 mM) gave～55%product after 12 hours (Table 1 and Fig. 1a) .

The Q-Exactive MS/MS spectra of (E) -styrylboronic acid modified YFLH₁QSH₂H₃WG (wherein H₁, H₂ and H₃ all denote the amino acid H, and the subscript 1, 2 or 3 are used to differentiate the H at different positions) is shown in Figure 1b. This mass-spectrometry analysis revealed the reaction site is at the residue H₂ which means residue H₃ is the anchoring residue based on previous studies (Fig. 1b) .

Table 1. Reaction yield of different metal ion binding peptides with different boronic acid reagents.

b. Mixed Peptide Screen

We then individually randomized each residue around H₃ in FLH₁QSH₂H₃WG to identify the best residues that can increase reaction rates. In order to be better identified in HPLC and mass spectrometry, amino acids RWY was added to the N-terminus of each sequence shown in Tables 2a, 3, 4, and 5.

First, different amino acids are screened for the X₁ position in FLHQX₁HHWG (SEQ ID NO: 319) . The sequences tested are listed in Table 2a.

Table 2a.

Table 2b.

A mixture of all the peptides listed in Table 2a were prepared by using an automated parallel peptide synthesizer (SyroⅡ, Biotage) . During the cycle for synthesizing X₁ in RWYFLHQX₁HHWG (SEQ ID NO: 309) , a mixture of amino acids A, D, E, H, L, K, M, P, S, N, Q, Y, W, I and R in certain proportions (as shown in Table 2b) was used to ensure equal distribution of the synthesized peptides.

The peptide mixture and boronic acid 1 were reacted under standard reaction conditions for 4 hours at 37 ℃. The reaction mixture was analyzed by LC-MS.

As shown by the LC-MS results in Fig. 2a, amino acids Asp, His, and Ser are preferred at the X₁ position.

Second, different amino acids are screened for the X₂ position in FLHQSHHX₂G (SEQ ID NO: 320) . The sequences used are listed in Table 3.

Table 3.

A mixture of all the peptides listed in Table 3 were prepared by the method described above. The peptide mixture and boronic acid 1 were reacted under standard reaction conditions for 2 hours at 37 ℃. The reaction mixture was analyzed by LC-MS.

As shown by the LC-MS results in Fig. 2b, amino acids Asp, Glu, His, Leu, Ser, Tyr, Trp, and Arg are preferred at the X₂ position.

Third, different amino acids are screened for the X₃ position in FLHQSX₃HWG (SEQ ID NO: 321) . The sequences used are listed in Table 4.

Table 4.

A mixture of all the peptides listed in Table 4 were prepared by the method described above. The peptide mixture and boronic acid 1 were reacted under standard reaction conditions for 2 hours at 37 ℃. The reaction mixture was analyzed by LC-MS.

As shown by the LC-MS results in Fig. 2c, amino acids Asp, Asn, and Ser, are preferred at the X₃ position.

Fourth, different amino acids are screened for the X₄ position in FLHX₄SHHWG (SEQ ID NO: 322) . The sequences used are listed in Table 5.

Table 5.

A mixture of all the peptides listed in Table 5 were prepared by the method described above. The peptide mixture and boronic acid 1 were reacted under standard reaction conditions for 2 hours at 37 ℃. The reaction mixture was analyzed by LC-MS.

As shown by the LC-MS results in Fig. 2d, amino acids Gln, Asp, and Ser are preferred at the X₄ position.

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

Table 6.

A mixture of all the peptides listed in Table 6 were prepared by the method described above. The peptide mixture and boronic acid 1 were reacted under standard reaction conditions for 2 hours at 37 ℃. The reaction mixture was analyzed by LC-MS.

As shown by the LC-MS results in Fig. 2e, amino acids Asp, Lys, and His are preferred at the X₅ position.

c. Combining Preferred Amino Acids and Further Optimization

The results showed Asp, Asn and Ser are often preferred at many sites. We then combined the preferred amino acids at different sites aiming for synergistic effects. In order to be better identified in HPLC and mass spectrometry, amino acids RWY was added to the N-terminus of each sequence shown in Tables 7 and 9-13.

Standard reaction conditions in Example 1c: to a solution of peptide (1 μL of 5 mM stock solution in water, 0.05 mM final concentration) in NMM buffer (97 μL of 5 mM stock solution, pH 7.4) , boronic acid 1 (1 μL of 50 mM stock solution in DMSO, 0.5 mM final concentration) and CuCl₂·2H₂O (1 μL of 5 mM stock solution in water, 0.05 mM final concentration) were added subsequently. The mixture was vortexed and shaken for a certain time and at a temperature as specified in the experiments. Ethylenediaminetetraacetic acid tetrasodium salt (Na₄-EDTA, dihydrate) (2 μL of 500 mM stock solution in H₂O, 20 mM final concentration) was added to quench the reactions, then the crude reaction mixture was centrifuged to remove any particulates, and the supernatant was analyzed by LC-MS to determine the reaction yield.

Sequences shown in Table 7 were first tested. Each peptide was reacted with boronic acid 1 under the standard reaction conditions at 37 ℃. Reaction yields at 3, 6, and 18 hours of reaction time were determined.

Table 7

Further experiments were performed on the sequence giving rise to the best reaction yield shown in Table 7.

Peptide RWYFLKQSDHWG (SEQ ID NO: 314) was reacted with boronic acid 1 under the standard reaction conditions at 4 ℃, room temperature, and 37 ℃. Reaction yields at 0.5, 1, 2, 3, and 18 hours of reaction time are shown in Table 8. The LC-MS data for the 3 hours 37 ℃reaction mixture are shown in Fig. 3a. The peptide gave 72%yield within 3 hours at 50 μM concentration.

Table 8.

We then performed further rounds of sequence optimizations by point mutations based on FLKQSDHWG (SEQ ID NO: 85) . The sequences tested and the reaction yields are shown in Table 9.

Each peptide was reacted with boronic acid 1 under the standard reaction conditions at 25 ℃ for 2 hours. The reaction yields were then determined. The LC-MS data for peptide RWYFLKQDDHWG (SEQ ID NO: 315) are shown in Fig. 3b.

Table 9.

We then performed another round of sequence optimization by replacing the Q in FLKQDDHWG (SEQ ID NO: 111) with other amino acids. The sequences tested and the reaction yields are shown in Table 10.

To a solution of a peptide (2 μL of 5 mM stock solution in water, 0.1 mM final concentration) in NMM buffer (94 μL of 5 mM stock solution, pH 7.4) , boronic acid 1 (2 μL of 50 mM stock solution in DMSO, 1mM final concentration) and CuCl₂·2H₂O (2 μL of 5 mM stock solution in water, 0.1 mM final concentration) were added subsequently. The mixture was vortexed and shaken for 1 hour at 25 ℃. Reaction yields were then determined.

Table 10.

We then performed another round of sequence optimization by replacing amino acids in FLKGDDHWG with Alanine (A) . The sequences tested and the reaction yields are shown in Table 11.

Each peptide was reacted with boronic acid 1 under the standard reaction conditions at 37 ℃ for 1 hour. The reaction yields were then determined. The LC-MS data for peptide RWYFLKGDDHAG (SEQ ID NO: 316) are shown in Fig. 3c.

Table 11.

We then performed another round of sequence optimization by replacing the A or the terminal G in FLKGDDHAG (SEQ ID NO: 143) with other amino acids. The sequences tested and the reaction yields are shown in Table 12.

Each peptide was reacted with boronic acid 1 under the standard reaction conditions at 37 ℃ for 1 hour. The reaction yields were then determined.

Table 12.

We then performed another round of sequence optimization by point mutations based on FLKKDDHAA. The sequences tested and the reaction yields are shown in Table 13.

Each peptide was reacted with boronic acid 1 under the standard reaction conditions at 25 ℃ for 30 minutes. The reaction yields were then determined.

Table 13.

In addition, to a solution of the peptide of the sequence RWYFFKKDDHAA (SEQ ID NO: 317) (1 μL of 5 mM stock solution in water, 0.05 mM final concentration) in NMM buffer (97 μL of 5 mM stock solution, pH 7.4) , boronic acid 1 (1 μL of 50 mM stock solution in DMSO, 0.5 mM final concentration) and CuCl₂·2H₂O (1 μL of 15 mM stock solution in water, 0.15 mM final concentration) were added subsequently. The mixture was vortexed and shaken for 15 minutes at 25 ℃. The mixture was then analyzed by LC-MS, and the results are shown in Fig. 3d.

d. Superior Reaction Kinetics of the Present Tags

The above efforts resulted in the peptide FFKKDDHAA (SEQ ID NO: 192) , possessing the highest reactivity with styrylboronic acid (Fig. 4a-b) . The peptide FFKKDDHAA (SEQ ID NO: 192) is referred to as CAST (copper assisted sequence-specific conjugation tag) . Trace amount (<2%) of the 2^nd boronic acid conjugation could be observed on CAST during the reaction (Fig. 4b and Fig. 3d) , MS/MS analysis indicates the reaction site for the 2^nd conjugation was on the anchoring His residue.

The reaction kinetics of CAST was further studied. To a solution of peptide RWYFFKKDDHAA (SEQ ID NO: 317) (1 μL of 5 mM stock solution in water, 0.05 mM final concentration) in NMM buffer (97 μL of 50 mM stock solution, pH 7.4) , boronic acid 1 (1 μL of 50 mM stock solution in DMSO, 0.5 mM final concentration) and CuCl₂·2H₂O (1 μL of 15 mM stock solution in water, 0.15 mM final concentration) were added subsequently. The mixture was vortexed and shaken at 37 ℃. The mixture was analyzed by LC-MS at 0 minute, 2 minutes, 5 minutes, 8 minutes, 10 minutes, and 15 minutes.

The reaction kinetics study showed that the rate constant is 8.1 M^-1 s^-1, and t_1/2 is less than 2 minutes at 50 μM CAST peptide concentration (Fig. 4c) .

MS/MS and 2D NMR experiments confirmed the reaction site is on the Asp amide nitrogen before the anchoring His of the CAST peptide (Figure 4d-e) . As previously proposed, Cu (II) probably binds to the His containing CAST peptide as the “ACTUN” motif to activate the backbone N-H of the i-1 residue (residue before His) (Fig. 4f) .

Based on our sequence optimization data, we generated 15 more peptide sequences which are similar to the CAST peptide. These peptides all have comparable reaction kinetics as the CAST peptide (Table 14) . The sequences and the reaction yields are shown in Table 14. In order to be better identified in HPLC and mass spectrometry, Phenyl-PEG₂-SPG was added to the N-terminus of each sequence shown in Table 14.

To a solution of a peptide (1 μL of 5 mM stock solution in water, 0.05 mM final concentration) in NMM buffer (97 μL of 50 mM stock solution, pH 7.4) , boronic acid 1 (1 μL of 50 mM stock solution in DMSO, 0.5 mM final concentration) and CuCl₂·2H₂O (1 μL of 15 mM stock solution in water, 0.15 mM final concentration) were added subsequently. The mixture was vortexed and shaken for 5 min at 37 ℃, and analyzed by LC-MS to calculate the reaction yield.

Table 14.

e. Further Improving Conjugation Yield by Changing Conjugation Conditions.

For the same tag sequence, we compared the yield at two different conjugation conditions. In order to be better identified in HPLC and mass spectrometry, amino acids RWY was added to the N-terminus of each sequence shown in Table 15.

Conditions a: To a solution of peptide (1 μL of 5 mM stock solution in water, 0.05 mM final concentration) in NMM buffer (97 μL of 5 mM stock solution, pH 7.4) , boronic acid reagent (1 μL of 50 mM stock solution in DMSO, 0.5 mM final concentration) and CuCl₂·2H₂O (1 μL of 5 mM stock solution in water, 0.05 mM final concentration) were added subsequently. The mixture was vortexed and shaken for 30 minutes at 25 ℃, and analyzed by LC-MS to determine the reaction yield.

Conditions b: To a solution of peptide (4 μL of 5 mM stock solution in water, 0.2 mM final concentration) in NMM buffer (92.7 μL of 5 mM stock solution, pH 7.4) , boronic acid reagent (2 μL of 100 mM stock solution in DMSO, 2 mM final concentration) and CuCl₂·2H₂O (1.3 μL of 75 mM stock solution in water, 1 mM final concentration) were added subsequently. The mixture was vortexed and shaken for 2 h at 37 ℃, and analyzed by LC-MS to determine the reaction yield.

The results are shown in Table 15.

Table 15.

The results demonstrated that the reaction yields can be further improved by extending the reaction time, increasing the concentrations of the reagents, and/or increasing the temperature.

f. Conjugation of FFKKDDHAA (SEQ ID NO: 192) and WLGFFKKDDHAA (SEQ ID NO: 346) to Boronic Acid reagent.

To a solution of peptide WLGFFKKDDHAA (1 μL of 5 mM stock solution in water, 0.05 mM final concentration) in NMM buffer (97 μL of 5 mM stock solution, pH 7.4) , styrylboronic acid (1 μL of 50 mM stock solution in DMSO, 0.5 mM final concentration) and CuCl₂·2H₂O (1 μL of 25 mM stock solution in water, 0.25 mM final concentration) were added subsequently. The mixture was vortexed and shaken for 15 minutes at 37 ℃. LC-MS analysis of the conjugation reaction between the peptide WLGFFKKDDHAA and the boronic acid reagent is shown in Fig. 24a.

To a solution of peptide FFKKDDHAA (1 μL of 5 mMstock solution in water, 0.05 mM final concentration) in NMM buffer (97 μL of 5 mM stock solution, pH 7.4) , styrylboronic acid (1 μL of 50 mM stock solution in DMSO, 0.5 mM final concentration) and CuCl₂·2H₂O (1 μL of 25 mM stock solution in water, 0.25 mM final concentration) were added subsequently. The mixture was vortexed and shaken for 15 minutes at 37 ℃. LC-MS analysis of the conjugation reaction between the peptide FFKKDDHAA and the boronic acid reagent is shown in Fig. 24a.

Example 2: Reagents Screen

a. Screen of Boronic Acid Reagents

Different boronic acid reagents were screened by reaction with the peptide RWYFFKKDDHAA (SEQ ID NO: 317) .

To a solution of the peptide (1 μL of 5 mM stock solution in water, 0.05 mM final concentration) in NMM buffer (97 μL of 50 mM stock solution, pH 7.4) , a boronic acid reagent shown in Fig. 5a (1 μL of 50 mM stock solution in DMSO, 0.5 mM final concentration) and CuCl₂·2H₂O (1 μL of 15 mM stock solution in water, 0.15 mM final concentration) were added subsequently. The mixture was vortexed and shaken for 15 minutes at 37 ℃. Na₄-EDTA (2 μL of 500 mM stock solution in H₂O, 20 mM final concentration) was added to quench the reactions, then the crude reaction mixture was centrifuged to remove any precipitates, and the supernatant was analyzed by LC-MS to determine the reaction yield.

The reaction yields are shown in Fig. 5a below each boronic acid reagent. Selected LC-MS results are shown in Fig. 5b.

Vinyl boronic acids and derivatives thereof showed better activity with styrylboronic acid being the best.

b. Screen of Metal Salts

Different metal salts were screened by reaction with the peptide RWYFFKKDDHAA (SEQ ID NO: 317) and boronic acid 1.

To a solution of the peptide (1μL of 5 mM stock solution in water, 0.05 mM final concentration) in NMM buffer (97 μL of 50 mM stock solution, pH 7.4) , boronic acid 1 (1 μL of 50 mM stock solution in DMSO, 0.5 mM final concentration) and different metals ions (CuSO₄·5H₂O, Cu (OAc) ₂·H₂O, CuCl₂·2H₂O NiCl₂·6H₂O, FeCl₃·6H₂O, FeCl₂·4H₂O, or CoCl₄·6H₂O) (1 μL of 15 mM stock solution in water, 0.15 mM final concentration) were added subsequently. The mixture was vortexed and shaken for 15 minutes at 37 ℃. Na₄-EDTA (2 μL of 500 mM stock solution in H₂O, 20 mM final concentration) was added to quench the reactions, then the crude reaction mixture was centrifuged to remove any precipitates, and the supernatant was analyzed by LC-MS to determine the reaction yield. The results are shown in Fig. 6a.

Cu(II) at different molar equivalents to the peptide RWYFFKKDDHAA (SEQ ID NO: 317) was studied.

To a solution of the peptide (1 μL of 5 mM stock solution in water, 0.05 mM final concentration) in NMM buffer (97 μL of 50 mM stock solution, pH 7.4) , boronic acid 1 (1 μL of 50 mM stock solution in DMSO, 0.5 mM final concentration) and CuCl₂·2H₂O (0.05 mM, 0.15 mM, 0.25 mM, or 0.5 mM final concentration) were added subsequently. The mixture was vortexed and shaken for 15 minutes at 37 ℃. Na₄-EDTA (2 μL of 500 mM stock solution in H₂O, 20 mM final concentration) was added to quench the reactions, then the crude reaction mixture was centrifuged to remove any precipitates, and the supernatant was analyzed by LC-MS. The results are shown in Fig. 6b.

The results demonstrated that the reaction rate can be increased by increasing the molar equivalent of Cu (II) . The reaction equilibrium was reached within 10 minutes when 3 equivalents of Cu (II) was used. If more time is allowed, then less Cu(II) can be added to achieve the same yield.

Example 3: Reactions with Modified Styrylboronic Acid

Alkyne, azide, or biotin modified styrylboronic acid molecules were then synthesized.

To a solution of peptide RWYFFKKDDHAA (SEQ ID NO: 317) (1μL of 5 mM stock solution in water, 0.05 mM final concentration) in NMM buffer (96 μL of 50 mM stock solution, pH 7.4, ) , modified styrylboronic acid (2 μL of 25 mM stock solution in DMSO, 0.5 mM final concentration) and CuCl₂·2H₂O (1 μL of 15 mM stock solution in water, 0.15 mM final concentration) were added subsequently. The mixture was vortexed and shaken for 15 minutes at 37 ℃. Na₄-EDTA (2 μL of 500 mM stock solution in H₂O, 20 mM final concentration) was added to quench the reactions, then the crude reaction mixture was centrifuged to remove any precipitates, and the supernatant was analyzed by LC-MS. The results are shown in Fig. 7.

The results demonstrated that different functional handles connected to styrylboronic acid are all compatible with the conjugation without impeding the reaction rates.

Example 4: Putting the Tag at Different Positions

The peptide tags identified above can be put at any position within, or at the C-or N-terminus of, another peptide to react with boronic acid-containing molecules.

For example, the peptide FFKKDDHAA (SEQ ID NO: 192) was inserted at the N-terminus or C-terminus or in the middle of another peptide to create a longer peptide. Then, to a solution of the longer peptide (1μL of 5 mM stock solution in water, 0.05 mM final concentration) in NMM buffer (97 μL of 50 mM stock solution, pH 7.4) , boronic acid 1 (1 μL of 50 mM stock solution in DMSO, 0.5 mM final concentration) and CuCl₂·2H₂O (1 μL of 15 mM stock solution in water, 0.15 mM final concentration) were added subsequently. The mixture was vortexed and shaken for 15 minutes at 37 ℃. Na₄-EDTA (2 μL of 500 mM stock solution in H₂O, 20 mM final concentration) was added to quench the reactions, then the crude reaction mixture was centrifuged to remove any precipitates, and the supernatant was analyzed by LC-MS. The results are shown in Fig. 8a.

The results demonstrated that the tag achieves the same high level of reactivity and reaction yield towards boronic acid reagents irrespective of the position of the tag in the peptide.

Moreover, the disulfide bond compatibility of the conjugation reaction was studied.

To a solution of the Cys-protected peptide shown in Fig. 8b (1μL of 5 mM stock solution in water, 0.05 mM final concentration) in NMM buffer (97 μL of 50 mM stock solution, pH 7.4) , boronic acid 1 (1 μL of 50 mM stock solution in DMSO, 0.5 mM final concentration) and CuCl₂·2H₂O (1 μL of 25 mM stock solution in water, 0.25 mM final concentration) were added subsequently. The mixture was vortexed and shaken for 15 minutes at 37 ℃. Na₄-EDTA (2 μL of 500 mM stock solution in H₂O, 20 mM final concentration) was added to quench the reactions, then the crude reaction mixture was centrifuged to remove any precipitates, and the supernatant was analyzed by LC-MS. The results are shown in Fig. 8b.

The results demonstrated that disulfide bond is compatible with the conjugation reaction between the tag and the boronic acid. Therefore, solvent exposed Cys can be temporarily protected with disulfide bond while performing conjugation if desired.

Example 5: Shortening the Tag

This experiment was conducted to see whether the peptide FFKKDDHAA (SEQ ID NO: 192) can be further shortened while still maintaining its specific reactivity with boronic acid containing molecules.

Specifically, different shortened versions of FFKKDDHAA (SEQ ID NO: 192) were tested. In order to be better identified in HPLC for analysis of reaction yields, the amino acid sequence WLG was added to the N-terminus of each peptide. To a solution of the peptide shown in Figure 9 (1 μL of 5 mM stock solution in water, 0.05 mM final concentration) in NMM buffer (97 μL of 50 mM stock solution, pH 7.4) , boronic acid 1 (1 μL of 50 mM stock solution in DMSO, 0.5 mM final concentration) and CuCl₂·2H₂O (1 μL of 15 mM stock solution in water, 0.15 mM final concentration) were added subsequently. The mixture was vortexed and shaken for 15 minutes at 37 ℃. Na₄-EDTA (2 μL of 500 mM stock solution in H₂O, 20 mM final concentration) was added to quench the reactions, then the crude reaction mixture was centrifuged to remove any precipitates, and the supernatant was analyzed by LC-MS. The results are shown in Fig. 9.

The results demonstrated that the tag can be trimmed down to six amino acids, FKKDDH (SEQ ID NO: 276) , without affecting the reactivity.

Different further shortened versions of the peptide tag were tested. In order to be better identified in HPLC for analysis of reaction yields, naphthalene was linked to the N-terminus of each peptide. To a solution of peptide (1 μL of 5 mM stock solution in water, 0.05 mM final concentration) in NMM buffer (97 μL of 50 mM stock solution, pH 7.4) , boronic acid 1 (1 μL of 50 mM stock solution in DMF, 0.5 mM final concentration) and CuCl₂·2H₂O (1 μL of 25 mM stock solution in water, 0.25 mM final concentration) were subsequently added. The mixture was vortexed and shaken for 3 hours at 37 ℃. Na₄-EDTA (2 μL of 500 mM stock solution in H₂O, 20 mM final concentration) was added to quench the reactions, then the crude reaction mixture was centrifuged to remove any precipitates, and the supernatant was analyzed by LC-MS. The results are shown in Fig. 23. The results demonstrated that the tag can be trimmed down to six, five, four or three 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, without affecting the reactivity.

Example 6: Stability of the Conjugation Product

This experiment was conducted to study the stability of the conjugation product resulted from the reaction between the tag peptide and boronic acid reagents.

Specifically, to a solution of RWYFFKKDDHAA (SEQ ID NO: 317) (1μL of 5 mM stock solution in water, 0.05 mM final concentration) in NMM buffer (97 μL of 50 mM stock solution, pH 7.4) , boronic acid 1 (1 μL of 50 mM stock solution in DMSO, 0.5 mM final concentration) and CuCl₂·2H₂O (1 μL of 15 mM stock solution in water, 0.15 mM final concentration) were added subsequently. The mixture was vortexed and shaken for 15 minutes at 37 ℃. Then the modified RWYFFKKDDHAA (SEQ ID NO: 317) was incubated at 95 ℃ for 1 hour or incubated in 10 mM cysteine at 95 ℃ for 1 hour, and analyzed by LC-MS. The results are shown in Fig. 10b.

For comparison, to a solution of peptide DLAAEIAKHCG (1μL of 5 mM stock solution in water, 0.05 mM final concentration) in PBS buffer (89 μL of 10 mM stock solution, pH 7.4) , maleimide (10 μL of 50 mM stock solution in DMSO, 5 mM final concentration) were added subsequently. The mixture was vortexed and shaken for 30 minutes at room temperature. After removing excessive maleimide, the product was incubated at 95 ℃ for 1h or incubated in 10 mM cysteine at 95 ℃ for 1 hour, and analyzed by LC-MS. The results are shown in Fig. 10a.

The results demonstrated that the conjugation product resulting from the present tag remains stable under elevated temperature and in the presence of excessive thiol nucleophile (cysteine) . On the other hand, the product resulting from the prior art tag hydrolyzes in the high-temperature and/or excessive-thiol nucleophile environment.

Example 7: Preparation of boronic acid derivatives

a. Synthesis of boronic acid derivatives

tert-butyl (E) - (4- (2- (4, 4, 5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) vinyl) benzyl) carbamate (S1) . Starting material tert-butyl (E) - (4- (2- (4, 4, 5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) vinyl) benzyl) carbamate was synthesized following literature procedures³. Tert-butyl (4-ethynylbenzyl) carbamate (4.00 mmol) , pinacolborane (4.40 mmol, 0.640 mL) and H₃B·THF (0.40 mmol, 1 M in THF, 0.40 mL) were added sequentially to a 25 mL sealed reaction vial flushed with nitrogen, reaction mixture was stirred at 60 ℃ for 4 hours. Upon completion, the solvents were removed under reduced pressure. The crude reaction mixture was purified by basified flash column chromatography (SiO₂: hexane/EtOAc: 15: 1～10: 1, yellow oil, yield: 70%) .

¹H NMR (600 MHz, Chloroform-d) δ7.37 (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) . ¹³C 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 C₂₀H₃₁BNO₄ [M+H] ⁺360.2341, observed 361.2568.

(E) - (4- ( ( (tert-butoxycarbonyl) amino) methyl) styryl) boronic acid (S2) . 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 reaction vial, then acetone (32 mL) and H₂O (16 mL) were added. The reaction mixture was stirred at room temperature for 12 hours, then quenched with brine and extracted with EtOAc (3×40 mL) . The organic phase was collected, Na₂SO₄ was added and solvents were removed under reduced pressure. The product was used without further purification (White solid, yield: 90%) .

¹H NMR (500 MHz, DMSO-d₆) δ7.75 (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) . ¹³C NMR(125 MHz, DMSO-d₆) δ155.69, 145.48, 140.43, 136.06, 127.16, 126.42, 122.58, 77.69, 43.04, 28.14. HRMS calculated for C₁₄H₂₀BNO₄ [M+Na] ⁺300.1383, observed 300.1377.

(E) - (4- (aminomethyl) styryl) boronic acid Hydrochloride (S3) . (E) - (4- ( ( (tert-butoxycarbonyl) amino) methyl) styryl) boronic acid was added to reaction vial and HCl (4.0 M in 1, 4-Dioxane) was added to fully dissolve the boronic acid substrate. The reaction mixture was stirred at room temperature for 1-2 hours. Upon completion, the solvents were removed under reduced pressure. (E) - (4- (aminomethyl) styryl) boronic acid hydrochloride was obtained quantitatively (White solid) .

¹H NMR (500 MHz, DMSO-d₆) δ8.51 (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) . ¹³C NMR (125 MHz, DMSO-d₆) δ144.99, 137.63, 134.03, 129.22, 126.56, 123.94, 41.74. HRMS calculated for C₉H₁₀BO₂ [M-NH₂] ⁺161.0768, observed 161.0757.

(E) - (4- (hex-5-ynamidomethyl) styryl) boronic acid (1A) .

5-Hexynoic acid (20.2 mg, 0.18 mmol) and HATU (60.8 mg, 0.16 mmol) were dissolved in DMF (1.0 mL) . N, N-Diisopropylethylamine (31.3 uL, 0.18 mmol) was added at 0 ℃, and the mixture was stirred at room temperature for 10 minutes. The reaction was cooled by ice cold bath and S3 (21.3 mg, 0.1 mmol) was added. The mixture was stirred at room temperature overnight, and all of the volatiles were removed by vacuum pump. The crude product was purified by reverse-phase HPLC (5.0–65%MeCN over 40 minutes) . Lyophilization of collected fractions gave the product.

¹H NMR (500 MHz, DMSO-d₆) δ8.35 (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) . ¹³C NMR(125 MHz, DMSO-d₆) δ171.36, 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 C₁₅H₁₈BNO₃ [M+H] ⁺272.1413, observed 272.1474.

(E) - (4- ( (2-azidoacetamido) methyl) styryl) boronic acid (1B) .

2-azidoacetic acid (18.2 mg, 0.18 mmol) and HATU (60.8 mg, 0.16 mmol) were dissolved in DMF (1.0 mL) . N, N-Diisopropylethylamine (31.3 μL, 0.18 mmol) was added at 0 ℃, and the mixture was stirred at room temperature for 10 minutes. The reaction was cooled by ice cold bath and S3 (21.3 mg, 0.1 mmol) was added. The mixture was stirred at room temperature overnight, and all of the volatiles were removed by vacuum pump. The crude product was purified by reverse-phase HPLC (5.0–65%MeCN over 40 minutes) . Lyophilization of collected fractions gave the product.

¹H NMR (500 MHz, DMSO-d₆) δ8.62 (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) . ¹³C NMR (125 MHz, DMSO-d₆) δ167.24, 145.37, 139.11, 136.34, 127.62, 126.49, 122.82, 50.68, 41.88. HRMS calculated for C₁₁H₁₃BN₄O₃ [M+H] ⁺261.1114, observed 261.1170.

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

Biotin (44.0 mg, 0.18 mmol) and HATU (60.8 mg, 0.16 mmol) were dissolved in DMF (1.0 mL) . N, N-Diisopropylethylamine (31.3 uL, 0.18 mmol) was added at 0 ℃, and the mixture was stirred at room temperature for 10 minutes. The reaction was cooled by ice cold bath and S3 (21.3 mg, 0.1 mmol) was added. The mixture was stirred at room temperature overnight, and all of the volatiles were removed by vacuum pump. The crude product was purified by reverse-phase HPLC (5.0–65%MeCN over 40 minutes) . Lyophilization of collected fractions gave the product.

¹H NMR (500 MHz, DMSO-d₆) δ8.30 (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) . ¹³C NMR (125 MHz, DMSO-d₆) δ171.94, 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 C₁₉H₂₆BN₃O₄S [M+H] ⁺404.1771, observed 404.1836.

Reagents and conditions: (i) Pd (P^tBu₃) ₄, Pinacol vinylboronate, TEA, toluene, 90 ℃, 16 h; (ii) NaIO₄, HCl, THF/H₂O, r. t., 2 h; (iii) piperidine-4-carboxylic acid, NaBH (OAc) ₃, DCE, 30 ℃, overnight.

(E) -4- (2- (4, 4, 5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) vinyl) benzaldehyde (a) To a mixture of 4-bromobenzaldehyde (2 g, 10.9 mmol) and4, 4, 5, 5-tetramethyl-2-vinyl-1, 3, 2-dioxaborolane (2.2 mL, 13 mmol, 1.2 equiv. ) was added toluene (20 mL) , Et₃N (3 mL, 21.8 mmol, 2 equiv. ) , followed by Pd (P^tBu₃) ₄ (0.28 g, 1.1 mmol, 10 mol%) . The resulting mixture was purged with nitrogen, then heated at 90 ℃ for 18 hours. After cooling to room temperature, it was quenched with sat. NaHCO₃ (20 mL) , extracted with EtOAc (30 mL×3) . After evaporation of the solvents, the residue was purified by Biotage column system (EtOAc/Petroleum ether: 0-10%) to afford a white solid. Yield: 60%. ¹H NMR (500 MHz, DMSO-d₆) δ10.01 (s, 1H) , 7.90 (d, J=8. 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) . ¹³C NMR (125 MHz, DMSO-d₆) δ192.52, 147.68, 142.31, 136.11, 129.78, 127.57, 120.47, 83.19, 24.53. HRMS calculated for C₁₅H₁₉BO₃ [M+H] ⁺259.1461, observed 259.1489.

(E) - (4-formylstyryl) boronic acid (b)

(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 mixed solvent of THF: water (4: 1) . To this solution, NaIO₄ (1.2 g, 3.0 equiv. ) was added and stirred for 5 minutes. Then an aqueous solution of HCl (2.0 M, 2.0 mL) was added and stirred for 1 hour until the boronic esters were completely consumed as monitored by TLC. The reaction mixture was extracted with EtOAc (20 mL×3) , the combined organic layers were washed with H₂O. After evaporation of the solvents, the residue was purified by Biotage column system (EtOAc/Petroleum ether: 0-20%) to afford a pale-yellow solid. Yield: 88%. ¹H NMR (500 MHz, DMSO-d₆) δ10.00 (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) . ¹³C NMR (125 MHz, DMSO-d₆) δ192.48, 144.31, 143.18, 135.65, 129.91, 127.38, 127.09. HRMS calculated for C₉H₉BO₃ [M+H] ⁺177.0678, observed 177.0703.

(E) -1- (4- (2-boronovinyl) benzyl) piperidine-4-carboxylic acid (c)

Piperidine-4-carboxylic acid (100 mg, 0.84 mmol, 1.5 eq) was suspended in dry DCM(15 ml) , then (E) - (4-formylstyryl) boronic acid (100 mg, 0.56 mmol) was added. The suspension was stirred for 30 minutes and then sodium triacetoxyborohydride (240 mg, 1.12 mmol, 2 equiv. ) was added portionwise and the suspension was stirred overnight at 30 ℃. After evaporation of DCM, water (10 ml) was added to quench the reaction and the solution was adjusted to pH 10 by 1 M NaOH. The solution was washed with DCM (10 mL×3) , EtOAc (10 mL×3) and adjusted to pH 2 by 2 M HCl. The residue was lyophilized and purified by HPLC with Agilent C18 column. Then the residue was lyophilized to afford the title product trifluoroacetate as a white solid. Yield: 42%. ¹H NMR (500 MHz, DMSO-d₆) δ12.56 (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) δ174.45, 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 C₁₅H₂₀BNO₄ [M+H] ⁺290.1519, observed 290.1559.

Synthetic scheme of SAB-MMAE (1D)

NH₂-Val-Cit-PAB-MMAE (d) was synthesized according to the reported literature⁴. Intermediate c (20.0 mg, 0.049 mmol, 6 equiv. ) , HOSu (7.4 mg, 0.064 mmol, 7.8 equiv. ) , DMAP (3 mg, 0.032 mmol, 3 equiv. ) were dissolved in dry DMF (100 μL) and then EDCI (12.3 mg, 0.064 mmol, 7.8 equiv. ) was added and the solution was stirred at 30 ℃overnight. Then NH₂-Val-Cit-PAB-MMAE (d, 10.0 mg, 8μmol, 1 equiv. ) was added to the solution. The reaction was stirred at 30 ℃overnight and monitored by LC-MS. Finally, the product was purified by HPLC with Agilent C18 column to give a white powder (7.6 mg, 46%) . HRMS calculated for C₇₃H₁₁₂BN₁₁O₁₅ [M+Na] ⁺1416.8232, observed 1416.8296.

Synthetic scheme of H₂N-Glu (O^tBu) -Val-Cit-PAB-MMAE (f)

Fmoc-Glu (O^tBu) -OH (28.0 mg, 0.066 mmol, 1 equiv. ) , TSTU (23.3 mg, 0.079 mmol, 1.2 equiv. ) , DIEA (24 μL, 0.13 mmol, 2 equiv. ) were dissolved in dry DMF (460 μL) and the solution was stirred at 30 ℃for 1 hour. Then the mixture (426 μL) was added to the solution of NH₂-Val-Cit-PAB-MMAE (60 mg, 0.048 mmol, 0.72 equiv. ) in 1mL DMF for 3-4 h until HPLC-MS indicated that the reaction was completed. Then 4-Methylpiperidine (9.5 μL, 0.1 mmol, 1.5 equiv. ) was added to the mixture and stirred at 30 ℃overnight. The product was purified by HPLC with agilent C18 column and lyophilized to get a white powder (56.4 mg, 89%) . HRMS Calcd for [M+H] ⁺1308.8105, observed 1308.8082.

Synthetic scheme of DBCO-PEG₃-NHS (g)

DBCO-PEG₃-NHS was synthesized according to the reported literature⁵. DIEA (88 μL, 2 equiv. ) was added to a mixture of DBCO-NHS (100 mg, 1 equiv. ) and 3- (2- (2- (2-aminoethoxy) edioxy) ethoxy) propanoic acid (66 mg, 1.2 equiv. ) in DCM(2 mL) , then reaction mixture was stirred overnight at room temperature. After the reaction was completed, the reaction mixture was diluted with DCM. Then the organic layer was washed with brine, 1 M HCl and dried over MgSO₄. The residue was concentrated in vacuo to afford a white viscous liquid compound. Then the product (50 mg) was added to a mixture of TSTU (38.5 mg, 1.3 equiv. ) and DIEA (26 μL, 1.5 equiv. ) in dry DCM/DMF (1.5 mL/0.5 mL) . The reaction mixture was stirred overnight at room temperature. After the reaction was completed, the reaction mixture was diluted with DCM. Then, the organic layer was washed with brine, 1 M HCl and dried over MgSO₄. The organic solvent was removed under reduced pressure to afford a viscous liquid compound (52 mg) without further purification.

Synthetic scheme of DBCO-MMAE (h)

H₂N-Glu (O^tBu) -Val-Cit-PAB-MMAE (30 mg, 0.023 mmol) , DBCO-PEG₃-NHS (16.3 mg, 0.027 mmol, 1.2 equiv. ) , DIEA (8 μL, 0.46 mmol, 2 equiv. ) were dissolved in dry DMF (600 μL) and the solution was stirred at room temperature for 1 hour until LC-MS indicated that the reaction was completed. Then the solution was concentrated in vacuo and the crude product was precipitated with cold diethyl ether (4 mL) followed by centrifugation at 2, 000×g for 3 minutes (3 times) and dried in vacuo. 30%TFA/DCM (2 mL) was added to the product at 0 ℃ for 4-5 hours. Then the reaction mixture was diluted with DCM (4ml) , washed with 5 mL water (3 times) and concentrated in vacuo. The resulting crude product was purified by HPLC with agilent C18 column and lyophilized to get a white powder (10 mg, 25%) . HRMS Calcd for [M+H] ⁺1742.9582, observed 1742.9537.

Example 8: Preparation of Proteins

a. Expression and purification of recombinant proteins:

Construction of plasmids for recombinant proteins.

The gene of SMT3 was synthesized by Genewiz, Suzhou, China. It was cloned into the vector pET22b. The genes of MBP, Sortase and Trigger factor were cloned into the vector pET28a. The gene of Nanobody was cloned into the vector pET26b. The amino acid sequences are shown below. KOD One^TM PCR Master Mix-Blue (ThermoFisher scientific) was used to PCR amplify the DNA. All the gene fragments were assembled by the Gibson assembly kit (Cat. C115-01, Vazyme) .

Protein expression and purification.

E. coli BL21 (DE3) cells transformed with His-TEV-SMT3, His-TEV-MBP, His-TEV-Sortase, His-TEV-Trigger factor and His-TEV-Nanobody plasmids were grown in 1 L of LB medium containing kanamycin (50 mg/L) at 37 ℃until OD600=0.6. Then, expression of His-TEV-MBP, His-TEV-Sortase, His-TEV-Trigger factor and His-TEV-Nanobody were induced by addition of 0.5 mM IPTG overnight at 18 ℃. Expression of His-TEV-SMT3 was induced by addition of 0.5 mM IPTG at 37 ℃for 4 hours. After harvesting the cells by centrifugation (6, 000 rpm for 10 minutes) , the cell pellet was lysed by sonication in 25 mL of 50 mM Tris with 150 mM NaCl (pH 7.5) buffer. The suspension was centrifuged at 12,000 rpm for 30 minutes to remove cell debris. The supernatant was loaded onto 2 mL Ni-Charged Resin (Genscript, Cat. NO. L00666-100) , first washed with 40 mL of 20 mM Tris with 150 mM NaCl (pH 7.5) , and then washed with 40 mL of 20 mM imidazole in 20 mM Tris with 150 mM NaCl (pH 7.5) . The protein was eluted from the column with buffer containing 250 mM imidazole, 20 mM Tris, 150 mM NaCl (pH 7.5) . Imidazole was removed from protein using Pur-A-Lyzer^TM Maxi Dialysis Kit (PURX12005, Sigma-Aldrich) , Tris buffer was changed to NMM buffer during the dialysis. The protein was analyzed by LC-MS to confirm its purity and molecular weight. TEV enzyme digestion experiments were carried out by mixing the enzyme and substrate in the ratio of 1: 20 at 4 ℃ overnight.

His-TEV-SMT3

SMT3

His-TEV-SMT3-CAST

SMT3-CAST

His-TEV-MBP

MBP

His-TEV-MBP-CAST

MBP-CAST

His-TEV-Nanobady

Nanobody

His-TEV-Nanobody-CAST

Nanobody-CAST

His-TEV-Trigger Factor

Trigger Factor

His-TEV-Trigger Factor-CAST

Trigger Factor-CAST

His-TEV-Sortase

Sortase

His-TEV-Sortase-CAST

Sortase-CAST

b. Expression and purification of antibodies

The pVITRO1-trastuzumab plasmid was purchased from Addgene (#61883) . The pVITRO1-Tra-CAST plasmids were constructed by inserting the CAST peptide at the C-terminus of the trastuzumab heavy chain, using the ClonExpress Ultra One Step Cloning Kit which was purchased from Vazyme (C115-01) . The light chain and heavy chain sequences for the trastuzumab, Tra-CAST, Tra-CASTi are listed below:

Trastuzumab-Light Chain

Trastuzumab-Heavy Chain

Tra-CAST-Light Chain

Tra-CAST-Heavy Chain

Tra-CASTi-Light Chain

Tra-CASTi-Heavy Chain

HEK 293F cells (Invitrogen) were cultured in Freestyle medium (Gibco, Lot. 2164683) at 37 ℃ under 6%CO₂ in a CRYSTAL shaker (140 rpm) . The cells were transiently transfected with trastuzumab plasmids and polyethylenimine (PEI) (Polysciences, Cat. 24765-1) when the cell density reached approximately 1.5×10⁶/mL. 1 mg of plasmid was premixed with 2.6 mg PEI in 50 mL of fresh medium for 15 minutes before adding to one liter of cell culture. The transfected cells were cultured for 72 hours before harvesting.

The supernatant of medium was harvested by centrifugation at 1000×g for 5 minutes. Then the supernatants were loaded on Protein A beads (GenScript, Cat. L00210-50) and washed with PBS buffer. Proteins were then eluted with 0.1 M glycine (pH 3.0) . The eluted proteins were concentrated and subjected to size-exclusion chromatography (Superdex 200 Increase 10/300 GL, GE Healthcare) in NMM buffer (50 mM, 0.2 M NaCl, pH 7.4) . The peak fractions were collected and concentrated. The purified IgGs were analyzed by LC-MS to confirm their molecular weight and purity, and stored at-20 ℃.

Example 9: Reactions on proteins

a. Conjugation of SMT3

Conjugation with Boronic Acid 1: to a solution of SMT3-CAST or SMT3 (10 μM final concentration) in NMM buffer (50 mM, 0.2 M NaCl, pH 7.4) , boronic acid 1 (1 μL of 5 mM stock solution in DMF, 0.1 mM final concentration) and CuCl₂·2H₂O (1 μL of 1.5 mM stock solution in water, 30 μM final concentration) were added subsequently. The total reaction volume is 50 μL. The mixture was incubated at 37 ℃for 10 minutes. After reaction is completed, Na₂-EDTA (2 μL of 500 mM stock solution in H₂O, 20 mM final concentration) was added, the crude reaction mixture was directly injected onto ESI-MS for analysis.

Conjugation with Boronic Acid 1A, 1B, or 1C: to a solution of SMT3-CAST (10 μM final concentration) in NMM buffer (50 mM, 0.2 M NaCl, pH 7.4) , boronic acid 1A, 1B or 1C (1 μL of 25 mM stock solution in DMF, 0.5 mM final concentration) and CuCl₂·2H₂O (1 μL of 1.5 mM stock solution in water, 30 μM final concentration) were added subsequently. The total reaction volume is 50 μL. The mixture was incubated at 37 ℃ for 30 minutes. After reaction is completed, Na₂-EDTA (2 μL of 500 mM stock solution in H₂O, 20 mM final concentration) was added, the crude reaction mixture was directly injected onto ESI-MS for analysis. The ESI ion series/deconvolution MS spectra are shown in Fig. 11.

b. Conjugation of Nanobody

Conjugation with Boronic Acid 1: to a solution of nanobody-CAST or nanobody (10 μM final concentration) in NMM buffer (50 mM, 0.2 M NaCl, pH 7.4) , boronic acid 1 (1 μL of 5 mM stock solution in DMF, 0.1 mM final concentration) and CuCl₂·2H₂O (1.7 μL of 1.5 mM stock solution in water, 50 μM final concentration) were added subsequently. The total reaction volume is 50 μL. The mixture was incubated at 37 ℃ for 1.5 hours. After reaction is completed, Na₂-EDTA (2 μL of 500 mM stock solution in H₂O, 20 mM final concentration) was added, the crude reaction mixture was directly injected onto ESI-MS for analysis.

Conjugation with Boronic Acid 1A, 1B or 1C: To a solution of nanobody-CAST (10 μM final concentration) in NMM buffer (50 mM, 0.2 M NaCl, pH 7.4) , boronic acid 1A, 1B or 1C (1 μL of 25 mM stock solution in DMF, 0.5 mM final concentration) and CuCl₂·2H₂O (1.7 μL of 1.5 mM stock solution in water, 50 μM final concentration) were added subsequently. The total reaction volume is 50 μL. The mixture was incubated at 37 ℃ for 1.5 hours. After reaction is completed, Na₂-EDTA (2 μL of 500 mM stock solution in H₂O, 20 mM final concentration) was added, The crude reaction mixture was directly injected onto ESI-MS for analysis. The ESI ion series/deconvolution MS spectra are shown in Fig. 12.

c. Conjugation of MBP

Conjugation with Boronic Acid 1: to a solution of MBP-CAST or MBP (10 μM final concentration) in NMM buffer (50 mM, 0.2 M NaCl, pH 7.4) , boronic acid 1 (1 μL of 5 mM stock solution in DMF, 0.1 mM final concentration) and CuCl₂·2H₂O (1.7 μL of 1.5 mM stock solution in water, 50 μM final concentration) were added subsequently. The total reaction volume is 50 μL. The mixture was incubated at 37 ℃for 1 hour. After reaction is completed, Na₂-EDTA (2 μL of 500 mM stock solution in H₂O, 20 mM final concentration) was added, the crude reaction mixture was directly injected onto ESI-MS for analysis.

Conjugation with Boronic Acid 1A, 1B, or 1C: to a solution of MBP-CAST (10 μM final concentration) in NMM buffer (50 mM, 0.2 M NaCl, pH 7.4) , boronic acid 1A, 1B or 1C (1 μL of 25 mM stock solution in DMF, 0.5 mM final concentration) and CuCl₂·2H₂O (1.7 μL of 1.5 mM stock solution in water, 50 μM final concentration) were added subsequently. The total reaction volume is 50 μL, the mixture was incubated at 37 ℃ for 2 hours. After reaction is completed, Na₂-EDTA (2 μL of 500 mM stock solution in H₂O, 20 mM final concentration) was added, The crude reaction mixture was directly injected onto ESI-MS for analysis. The ESI ion series/deconvolution MS spectra are shown in Fig. 13.

d. Conjugation of Trigger Factor

Conjugation with Boronic Acid 1: to a solution of trigger factor-CAST or trigger factor (5 μM final concentration) in NMM buffer (50 mM, 0.2 M NaCl, pH 7.4) , boronic acid 1 (1 μL of 5 mM stock solution in DMF, 0.1 mM final concentration) and CuCl₂·2H₂O (1.7 μL of 1.5 mM stock solution in water, 50 μM final concentration) were added subsequently. The total reaction volume is 50 μL. The mixture was incubated at 37 ℃ for 2 hours. After reaction is completed, Na₂-EDTA (2 μL of 500 mM stock solution in H₂O, 20 mM final concentration) was added, the crude reaction mixture was directly injected onto ESI-MS for analysis.

Conjugation with Boronic Acid 1A, 1B, or 1C: To a solution of trigger factor-CAST (5 μM final concentration) in NMM buffer (50 mM, 0.2 M NaCl, pH 7.4) , boronic acid 1A, 1B or 1C (1 μL of 25 mM stock solution in DMF, 0.5 mM final concentration) and CuCl₂·2H₂O (1.7 μL of 1.5 mM stock solution in water, 50 μM final concentration) were added subsequently. The total reaction volume is 50 μL. The mixture was incubated at 37 ℃ for 2 hours. After reaction is completed, Na₂-EDTA (2 μL of 500 mM stock solution in H₂O, 20 mM final concentration) was added, the crude reaction mixture was directly injected onto ESI-MS for analysis. The ESI ion series/deconvolution MS spectra are shown in Fig. 14.

e. Conjugation of Sortase

For the reactions with Sortase, we observed the single unprotected Cys could react with styrylboronic acid. We thus blocked this single Cys with maleimide before performing CAST conjugation reaction on Sortase.

Sortase-CAST (10 μM) was incubated with Maleimide (5 mM) in PBS at r. t. for 40 min, Then PBS buffer was exchanged into NMM buffer (50 mM, pH 7.4, 0.2 M NaCl) .

Conjugation with Boronic Acid 1: to a solution of maleimide modified Sortase-CAST (10 μM final concentration) or maleimide modified Sortase in NMM buffer (50 mM, 0.2 M NaCl, pH 7.4) , boronic acid 1 (1 μL of 5 mM stock solution in DMF, 0.1 mM final concentration) and CuCl₂·2H₂O (1.7 μL of 1.5 mM stock solution in water, 50 μM final concentration) were added subsequently. The total reaction volume is 50 μL. The mixture was incubated at 37 ℃ for 30 minutes. After reaction is completed, Na₂-EDTA (2 μL of 500 mM stock solution in H₂O, 20 mM final concentration) was added, the crude reaction mixture was directly injected onto ESI-MS for analysis.

Conjugation with Boronic Acid 1A, 1B, or 1C: to a solution of maleimide modified Sortase-CAST (10 μM final concentration) in NMM buffer (50 mM, 0.2 M NaCl, pH 7.4) , boronic acid 1A, 1B or 1C (1 μL of 25 mM stock solution in DMF, 0.5 mM final concentration) and CuCl₂·2H₂O (1.7 μL of 1.5 mM stock solution in water, 50 μM final concentration) were added subsequently. The total reaction volume is 50 μL. The mixture was incubated at 37 ℃ for 30 minutes. After reaction is completed, Na₂-EDTA (2 μL of 500 mM stock solution in H₂O, 20 mM final concentration) was added, the crude reaction mixture was directly injected onto ESI-MS for analysis. The ESI ion series/deconvolution MS spectra are shown in Fig. 15.

f.Conjugation of Tra-CAST

Conjugation with Boronic Acid Reagent 1: to a solution of Tra-CAST or trastuzumab (4 μM final concentration) in NMM buffer (50 mM, 0.2 M NaCl, pH 7.4) , boronic acid 1 (1 μL of 12.5 mM stock solution in DMF, 0.25 mM final concentration) and CuCl₂·2H₂O (1 μL of 1.0 mM stock solution in water, 20 μM final concentration) were added subsequently. The total reaction volume is 50 μL. The mixture was incubated at 37 ℃ for 2 hours. After reaction is completed, Na₂-EDTA (2 μL of 500 mM stock solution in H₂O, 20 mM final concentration) was added the crude reaction mixture was directly injected onto ESI-MS for analysis. In some experiments, before the ESI-MS analysis, the reaction mixture was treated with endoglycosidase (EndoS) to remove the N-linked glycans.

Conjugation with Boronic Acid 1A, 1B, or 1C: to a solution of Tra-CAST (4 μM final concentration) in NMM buffer (50 mM, 0.2 M NaCl, pH 7.4) , boronic acid 1A, 1B or 1C (1 μL of 12.5 mM stock solution in DMF, 0.25 mM final concentration) and CuCl₂·2H₂O (1 μL of 1.0 mM stock solution in water, 20 μM final concentration) were added subsequently. The total reaction volume is 50 μL. The mixture was incubated at 37 ℃ for 4 hours. After reaction is completed, Na₂-EDTA (2 μL of 500 mM stock solution in H₂O, 20 mM final concentration) was added the crude reaction mixture was directly injected onto ESI-MS for analysis. In some experiments, before the ESI-MS analysis, the reaction mixture was treated with endoglycosidase (EndoS) to remove the N-linked glycans.

Conjugation with SBA-MMAE (preparation of Tra-CAST-MMAE) : to a solution of Tra-CAST (4 μM final concentration) in NMM buffer (50 mM, 0.2 M NaCl, pH 7.4) , SBA-MMAE (1 μL of 25 mM stock solution in DMF, 0.5 mM final concentration) and CuCl₂·2H₂O (1.5 μL of 1.0 mM stock solution in water, 30 μM final concentration) , 5 μL DMF were added subsequently. The total reaction volume is 50 μL. The mixture was incubated at 37 ℃ for 7 hours. After reaction is completed, Na₂-EDTA (2 μL of 500 mM stock solution in H₂O, 20 mM final concentration) was added the crude reaction mixture was directly injected onto ESI-MS for analysis. In some experiments, before the ESI-MS analysis, the reaction mixture was treated with endoglycosidase (EndoS) to remove the N-linked glycans.

Tra-CAST-MMAE

g. Conjugation of Tra-CASTi (preparation of Tra-CASTi-MMAE)

To a solution of Tra-CASTi (4 μM final concentration) in NMM buffer (50 mM, 0.2 M NaCl, pH 7.4) , boronic acid 1B (20 μL of 12.5 mM stock solution in DMF, 0.25 mM final concentration) and CuCl₂·2H₂O (20 μL of 1.0 mM stock solution in water, 20 μM final concentration) were added subsequently. The total reaction volume is 1 mL. The mixture was incubated at 37 ℃ for 4 hours. After reaction is completed, Na₂-EDTA (40 μL of 500 mM stock solution in H₂O, 20 mM final concentration) was added. Excessive 1B is removed through dialysis.

Click reactions for MMAE attachment: DBCO-MMAE (4 μL of 20 mM stock solution in DMSO, 20 equivalents) was added to a solution of the 1B modified Tra-CASTi (4 μM) in PBS, and the mixture was incubated at 37 ℃ for 2 hours. The reaction was monitored using LC-MS. After reaction is completed, excessive DBCO-MMAE is removed through dialysis.

Example 10: Conjugations between Boronic Acids and Proteins Comprising the Tag

The tags of the present invention were attached to the C-termini of different proteins (including: SMT3 (11 kDa) , MBP (41 kDa) , Nanobody (15 kDa) , Trigger Factor (48 kDa) , and Sortase (17kDa) ) , and the reactions between these proteins and boronic acid reagents (Boronic acid 1, 1A, 1B, 1C, 1D) were studied.

For reactions on proteins, protein concentrations were kept at 10 μM, the reaction rates were slower than those observed in peptides due to the lower concentrations. Nonetheless, reactions with styrylboronic acid in general could still afford>95%conjugated products within 2 hours while control proteins lacking CAST peptide shown no observable activities (Figs. 11-16) .

Reactivities for alkyne, azide or biotin modified styrylboronic acid are all similarly high for the same protein comprising the tag. In addition, for all the reactions we did on proteins, second boronic acid conjugation were observed only at a minimal level (<5%) .

Example 11: Properties and Effects of Antibody Drug Conjugates of the Present Invention

Antibody drug conjugate (ADC) has emerged to be a major class of therapeutic format in recent years. The preparation of antibody drug conjugates requires a chemical crosslinking reaction between antibody and the cytotoxic agent. Currently, cysteine based chemical conjugation is widely adopted for attaching drug payloads because of its distinct reactivity. However, disrupting disulfides could affect antibody stability, site-specific Cys conjugation might be hard to achieve given that many disulfides are present on antibodies resulting in heterogeneous products. Cysteine maleimide conjugation product also exhibits stability problems in plasma. Pai-clamp and DBCO-tag could achieve site-specificity, but still rely on Cys-based reactions.

Conjugation based on the tags of the present invention is entirely a different reaction system. It avoids manipulating antibody disulfides to provide an alternative method for antibody drug conjugate preparations. The excellent conjugation results in the above described recombinant proteins suggest the present tags could be fused to antibodies to prepare homogeneous antibody drug conjugate.

a. CAST-based ADCs

1) Conjugation Reactions

We inserted the CAST peptide to the C-terminus of trastuzumab heavy chain through mutagenesis to produce Tra-CAST (with heavy chain shown by SEQ ID NO: 305, light chain shown by SEQ ID NO: 304) and reacted the Tra-CASTwith boronic acid 1, 1A, 1B, 1C or 1D. For controls, trastuzumab without any tags were reacted with boronic acid 1 under the same conditions.

While trastuzumab antibody shows no observable activity with styrylboronic acid (Fig. 17a-d) , Tra-CAST efficiently reacts with styrylboronic acid (Fig. 17e-h) . Tra-CAST could also react with 1A, 1B and 1C with essentially the same efficiency as styrylboronic acid (Fig. 17i-n) .

To prepare the antibody drug conjugate, we synthesized styrylboronic acid-Val-Cit-PAB-MMAE (SBA-MMAE) conjugate and then performed a single step conjugation with Tra-CAST to produce Tra-CAST-MMAE. The conjugation reaction took 7 hours to complete due to lower concentrations and larger molecular sizes (Fig. 17o) . No reaction was observed on the trastuzumab light chain and only single modification was observed on the heavy chain.

2) Properties and Effects

The below experiment was conducted to see ifthe antibody drug conjugates of the present invention retain the same level of affinity as the unconjugated antibody.

In vitro binding assays were performed using ForteBio Octet BioLayer Interferometry system at the room temperature. Briefly, AHC (Anti-Human Fc Capture) tips were dipped into 200 μL of antibody solution (10μg/ml Tra-CAST or-Tra-CAST-MMAE in PBS with 0.1%BSA and 0.02%tween) for the loading of the antibodies. The tips loaded with antibody were dipped into PBS containing 0.1%BSA, 0.02%tween and recombinant HER2 (Sino biological 10004-H08H1-50) at various HER2 concentrations, or dipped into PBS with 0.1%BSA and 0.02%tween only, to obtain the association curve, with the buffer only data serving as the reference. After association, the tips were dipped into PBS with 0.1%BSA and 0.02%tween to obtain the dissociation curve. Following the protocols provided by Fortebio Biosystems, the association and dissociation curves of each sample were manually fitted using Solver in excel to obtain the K_D. The final K_D was reported as the average of the K_D obtained from experiments with serially diluted HER2.

The binding affinity of Tra-CAST-MMAE to HER2 (Kd 0.49 nM) is nearly the same as trastuzumab affinity to HER2 (Kd 0.43 nM) (Fig. 18b) .

The below experiment was conducted to study the in vitro cytotoxicity of the antibody drug conjugates of the present invention.

In a 96-well white opaque plate, CHO cells were seeded at a density of 5 × 10³/well, and BT474, MCF7 or SK-BR-3 cells were seeded at a density of 1 × 10⁴/well. Cells were allowed to attach for 24 h at 37 ℃ and 5%CO₂ in humidified atmosphere. BT474, MCF7, and SK-BR-3 cells were then treated with serial dilutions of Tra-CAST, Tra-CAST-MMAE and SBA-MMAE for 96 h; CHO cells were similarly treated for 72 h. Treatment time was shortened for CHO cells to prevent overgrowth. The viability of cells was measured using CellTiter Glo reagents (G7571) following the manufacturer’s protocol and was normalized to the viability of cells without any treatment. The data were plotted using Graphpad software, and the half-maximal effective concentration (EC₅₀) values were obtained by fitting the viability curves with a sigmoidal Boltzmann fit.

As shown in Fig. 19, Tra-CAST shows minimal toxicity towards all the cells in the tested concentrations. SBA-MMAE shows low toxicity and couldn’ t effectively distinguish HER2-negative cells from HER2-positive cells. Tra-CAST-MMAE exhibits no toxicity towards HER2-negative CHO cells and MCF-7 cells, while it effectively kills HER2-positive SK-BR-3 cells and BT-474 cells with EC₅₀ of0.2 nM and 0.6 nM respectively.

b. CASTi-based ADCs

1) Plasma Stability of the Tag

We found IAPDDHAA sequence (SEQ ID NO: 269) (referred to as CASTi) is very stable in plasma and serum. This peptide remains mostly intact after incubation in plasma for 144 hours (Fig. 20) .

Peptide in vitro serum/plasma stability determination: Fresh blood was obtained from Male BALB/c mice (8 weeks old) from the Laboratory Animal Resources Center of Westlake University. The serum was prepared by centrifugation at 1500 g for 10 min after standing at room temperature for 30 min. The plasma was prepared by centrifugation at 1500 g for 20 min. Peptide was individually incubated with fresh serum at 2 mM at 37 ℃. Samples were taken at 0, 18, 48, 96, and 144 hours. Then acetonitrile at 75%final concentration was added to serum samples to precipitate plasma proteins, precipitates were removed by centrifugation at 12000 g for 5 min. The supernatant was diluted 20 times with 0.1%TFA/H₂O (v/v) and analyzed by LC/MS.

2)Conjugation Reactions

We then inserted-CASTi at the C-terminus of Fc domain to produce Tra-CASTi (with heavy chain shown by SEQ ID NO: 307, light chain shown by SEQ ID NO: 306) . To produce the antibody drug conjugate in large quantity, we reacted Tra-CASTi with 1B, followed by DBCO- (PEG) 3-Glu-Val-Cit-PAB-MMAE (DBCO-MMAE) to produce Tra-CASTi-MMAE (Fig. 17p-r) for further in vitro and in vivo studies.

3) Properties and Effects

We confirmed Tra-CASTi-MMAE can effectively kill HER2-positive SK-BR-3 cells (EC₅₀0.2 nM) , BT-474 cells (EC₅₀0.2 nM) and SKOV-3 cells (EC₅₀0.2 nM)while it exhibits very low toxicity to HER2-negative MCF-7 cells (Fig. 21a) . Tra-CASTi-MMAE also exhibited excellent plasma stability (Fig. 21b) .

ADC plasma stability: Tra-CASTi-MMAE (100μg/mL, 1.2 μL in PBS) was added to undiluted BALB/c mouse plasma (118.8 μL) to a final concentration of 1 μg/mL. After incubation at 37 ℃ for varying time, aliquots (15 μL each) were taken and stored at-80 ℃ until use. Samples were analyzed by sandwich ELISA assay. A high-binding 96-well plate (Corning) was coated with homemade Her2 protein (100 ng per well) . After overnight coating at 4 ℃, the plate was blocked with 200 μL of 2%BSA in PBS containing 0.05%Tween 20 (PBS-T) with agitation at room temperature for 2 hours. Subsequently, the solution was removed and ADC sample (100 μL in PBS-T containing 2%BSA) was added to each well, and the plate was incubated at 4 ℃ overnight. After sample incubation, each well was washed four times with 200 μL of PBS-T. For Trastuzumab detection, 100 μL of rabbit anti-human IgG antibody (1: 5000) was added to each well at room temperature for two hours. Then each well was washed four times with 200 μL of PBS-T followed by adding detection antibody. 100 μL goat anti-rabbit (1: 5000) was added to each well, then washed four times with 200 μL PBS-T before adding 100 μL the TMB substrate. After color was developed for 10-30 minutes, 100 μL of 2 M HCl was added to each well to stop the reaction and then the absorbance at 450 nm was recorded using a plate reader (Thermo Varioskan LUX) . Concentrations were calculated based on a standard curve. For stability in human plasma, assays were performed in the same manner using homemade human HER2 (100 ng per well) for plate coating, mouse anti-MMAE antibody (1: 5, 000) , and goat anti-mouse IgG–HRP conjugate (1: 5, 000) as secondary detection antibodies, respectively.

The in vivo efficacy of Tra-CASTi-MMAE was evaluated on the SKOV-3 xenograft tumor models. Tra-CASTi-MMAE (12 mg/kg or 6 mg/kg) , Tra-CASTi (12 mg/kg) , or Fc isotype control (12 mg/kg) were i. v. administered into tumor bearing mice weekly for four times. No significant toxicity was observed in all groups during the course of the study. Tumors in mice receiving Tra-CASTi-MMAE (12 mg/kg) were greatly suppressed, while tumors in the control group mice grow rapidly to reach 1500 mm³ which is the end point of the mice study (Figs. 21c and 22) . These results demonstrate the present invention is indeed compatible with in vivo therapeutic conjugate applications.

Animal model preparation and in vivo antitumor experiment: All procedures were approved by the Institutional Animal Care and Use Committee ofZhejiang University. Female Balb/c nude mice (age, 4 weeks) were purchased from Ziyuan Laboratory Animal lnc. (Hangzhou, China) . Mice were acclimated for 1 week before the experiment and kept under standard laboratory conditions with food and water ad libitum. HER2-positive human ovarian xenograft tumor model was used to evaluate the anti-tumor effect of ADC compounds. Briefly, cultured SKOV-3 cells were suspended in DMEM medium without serum and antibiotics. Mice received 100 μL subcutaneous injection of SKOV-3 cells suspension (1 x 10⁷/100μL) . Tumor volume was calculated by using the following formula: Tumor volume=0.52 x Length x Width². When the volume of xenograft tumor reached average of 100-150 mm³, the mice were randomly divided into five groups (n=5) . Tra-CASTi-MMAE (12 or 6 mg/kg) and control (Tra-CASTi, 12 mg/kg; Fc, 12 mg/kg) were administrated to mice via tail vein on days 0, 7, 14 and 28. Tumor volume and body weight were monitored three times a week. When xenografts tumor grew to 1500 mm³, the mice were killed.

Example 12: Fluorescent Labeling of Proteins

We attached a fluorescent label to MBP protein and trastuzumab by using the tags of the present invention.

(1) Conjugation Conditions

MBP: To a solution of MBP-CAST (10 μM final concentration) in NMM buffer (50 mM, 0.2 M NaCl, pH 7.4) , boronic acid 1E (1 μL of 25 mM stock solution in DMF, 0.5 mM final concentration) and CuCl₂·2H₂O (1.7 μL of 15 mM stock solution in water, 50 μM final concentration) were added subsequently. The total reaction volume is 50 μL. The mixture was incubated at 37 ℃ for 2 h. Ethylenediaminetetraacetic acid tetrasodium salt (Na₄-EDTA, dihydrate) was then added, and the crude reaction mixture was directly injected onto ESI-MS.

Trastuzumab: To a solution of Tra-CAST (10 μM final concentration) in NMM buffer (50 mM, 0.2 M NaCl, pH 7.4) , boronic acid 1E (1 μL of 12.5 mM stock solution in DMF, 0.5 mM final concentration) and CuCl₂·2H₂O (1 μL of 10 mM stock solution in water, 20 μM final concentration) were added subsequently. The total reaction volume is 50 μL. The mixture was incubated at 37 ℃ for 5h. Ethylenediaminetetraacetic acid tetrasodium salt (Na₄-EDTA, dihydrate) was then added. The reaction mixture was then treated with EndoS to remove the N-linked glycans and directly injected onto ESI-MS.

The results are shown in Figure 22. The reaction between MBP-CAST and 1E resulted in 95%conversion to fluorophore attached MBP; the reaction between Tra-CAST and 1E resulted in similarly high conversion conversion to fluorophore attached Tra-CAST.

Discussion

Direct site-specific chemical protein conjugations through canonical amino acids have wide applications in fundamental research and therapeutics development. However, such conjugations are generally difficult to achieve because of the similar chemical environment of same amino acid at different protein sites. Herein, we have successfully developed peptide tags and demonstrated transition metal catalyzed reactions can indeed achieve site-specificity for protein conjugations through canonical amino acids. The peptide tags of the present invention have superior reaction kinetics exceeding that of most chemical conjugations and enzymatic conjugations, and can be fused to different proteins to achieve site-specific conjugation of a variety of small molecules. We also successfully prepared homogeneous antibody drug conjugates through the present tags, and showed the conjugates can selectively kill antigen-positive cells while exhibiting no toxicity to antigen-negative cells. Furthermore, the antibody drug conjugates remain stable in plasma and achieve significantly advantageous therapeutic and anti-tumor results in vivo.

Claims

An peptide tag comprising the sequence: [Xaa₁] _a- [Xaa₂] _b- [Xaa₃] _c- [Xaa₄] _d-Xaa₅-Xaa₆-His- [Xaa₇] _e- [Xaa₈] _f;

wherein 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

wherein

Xaa₁=Phe, His, Arg, Asn, Pro, Gln, Trp, Asp, Glu, Lys, Ile, Met, Ser, Thr, or Tyr;

Xaa₂=Phe, Leu, His, Met, Asn, Ser, Thr, Pro, Tyr, Ala, Asp, Lys, Gln, Arg, Trp, or Ile;

Xaa₃=Lys, Ala, Asp, Glu, Phe, His, Ile, Asn, Thr, Ser, Pro, Arg, Gly, Gln, Leu, Trp, or Met;

Xaa₄=Asp, Lys, Gln, Gly, His, Asn, Thr, Ser, Met, Lys, Glu, Leu, Trp, Pro, Ala, Phe, Ile, Arg, or Tyr;

Xaa₅=Asp, Thr, Ser, or Asn;

Xaa₆=Asp, His, Ser, Thr, Glu, Ala, or Asn;

Xaa₇=Ala, Trp, Ile, Leu, Met, Tyr, Asn, Gln, Ser, Thr, Asp, Lys, Glu, His, Trp, Arg, Phe, Pro, or Gly; and

Xaa₈=Ala, Gly, Ile, Leu, Asn, Ser, Thr, Asp, Arg, Lys, Val, Phe, Trp, Tyr, Gln, Glu, or His.
A tagged protein comprising the peptide tag of claim 1 linked to a protein.
A protein-chemical conjugate, comprising a chemical, and the tagged protein of claim 2, wherein the chemical is conjugated to the peptide tag via a copper (II) catalyzed Chan-Lam reaction.
The protein-chemical conjugate of claim 3, wherein the chemical is conjugated to Xaa₆ of the peptide tag.
The protein-chemical conjugate of claim 3 or 4, wherein the chemical is conjugated to the peptide tag via a linker.
The protein-chemical conjugate of 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 Xaa₆ of the peptide tag.
The protein-chemical conjugate of claim 6, wherein the boronic acid is 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 boronic acid pinacol ester or a trifluoroborate salt.
The protein-chemical conjugate of claim 6, wherein the boronic acid is substituted or unsubstituted vinyl boronic acid, cyclohexyl-vinyl boronic acid, or styryl boronic acid.
The protein-chemical conjugate of any one of claims 3-8, wherein the chemical comprises a drug, label, linker, reactive group, antigen, hapten, ligand of protein, or any combination thereof.
The protein-chemical conjugate of any one of claims 3-9, wherein the drug is a cytotoxic agent.
The protein-chemical conjugate of any one of claims 3-10, wherein the protein is an antibody.
A protein-protein complex, comprising the protein-chemical conjugate of any one of claims 3-11 and a second protein; wherein the second protein associates with the chemical in the protein-chemical conjugate.
The complex of claim 12, wherein the second protein is avidin, and the chemical is biotin, or the second protein is an antibody, and the chemical is an antigen specifically recognized by the antibody.
The peptide tag of claim 1, the tagged protein of claim 2, the protein-chemical conjugate of any one of claims 3-11, or the complex of 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.
A DNA encoding the peptide tag of claim 1, or the tagged protein of claim 2.
A method of preparing a tagged protein, comprising adding or inserting the peptide tag of claim 1 to a protein to obtain the tagged protein.
A method of preparing a protein-chemical conjugate, comprising conjugating a chemical to the tagged protein of claim 2 via a copper (II) catalyzed Chan-Lam reaction.
The method of claim 17, comprising:

contacting a chemical comprising or conjugated to boronic acid group or a boronic acid derivative group with the tagged protein under conditions suitable for a copper (II) catalyzed Chan-Lam reaction, thus generating a protein-chemical conjugate.
The method of claim 18, wherein the chemical is conjugated to boronic acid group or a boronic acid derivative group via a linker.
The method of claim 17, comprising:

(i) contacting a first linker comprising or conjugated to boronic acid group or a boronic acid derivative group with the tagged protein under conditions suitable for a copper (II) catalyzed Chan-Lam reaction, thus generating a protein-linker conjugate; and

(ii) conjugating the chemical to the first linker of the protein-linker conjugate.
The method of claim 20, wherein the chemical is conjugated to a second linker, and step (ii) comprises conjugating a conjugate of the chemical and the second linker to the first linker.
The method of any one of claims 16-21, wherein the boronic acid is 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 boronic acid pinacol ester or a trifluoroborate salt.
The method of any one of claims 16-18, wherein the boronic acid is substituted or unsubstituted vinyl boronic acid, cyclohexyl-vinyl boronic acid, or styryl boronic acid.
The method any one of claims 17-23, wherein the chemical comprises a drug, label, linker, reactive group, antigen, hapten, ligand of protein, or any combination thereof.
The method of any one of claims 17-24, wherein the chemical is a cytotoxic agent.
The method of any one of claims 19-25, wherein the chemical is connected to the linker via a click reaction or a bioorthogonal reaction.
The method of any one of claims 17-26, wherein the protein is an antibody.
A method of preparing a protein-protein complex, comprising:

(i) preparing a protein-chemical conjugate according to the method of any one of claims 17-27, wherein the chemical of the protein-chemical conjugate is able to specifically associate with a second protein; and

(ii) reacting the protein-chemical conjugate with the second protein to form the protein-protein complex.
The method of claim 28, wherein the second protein is avidin, and the chemical is biotin, or the second protein is an antibody, and the chemical is an antigen specifically recognized by the antibody.
The method of any one of claims 16-29, wherein the sequence of the tag is selected from the group consisting of SEQ ID NOs: 4-281 and DDH.