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US-12624063-B2 - Compositions and methods for modification of target molecules

US12624063B2US 12624063 B2US12624063 B2US 12624063B2US-12624063-B2

Abstract

The present disclosure provides a method for chemoselective modification of a target molecule. A subject method includes contacting a target molecule comprising a thiol moiety with a biomolecule comprising a reactive moiety, wherein the reactive moiety is generated by reaction of a biomolecule comprising a phenol moiety or a catechol with an enzyme capable of oxidizing the phenol or the catechol moiety. The contacting is carried out under conditions sufficient for conjugation of the target molecule to the biomolecule, thereby producing a modified target molecule. The present disclosure provides compositions comprising a subject target molecule comprising a thiol moiety, and a biomolecule comprising a phenol moiety or a catechol moiety. The present disclosure provides kits for carrying out a subject method. The present disclosure also provides modified target molecules and methods for using same.

Inventors

  • Matthew B. Francis
  • Marco Jackson Lobba
  • Johnathan Charles Maza
  • Alan M. Marmelstein
  • Jennifer A. DOUDNA
  • Christof Fellmann
  • Casey S. Mogilevsky

Assignees

  • THE REGENTS OF THE UNIVERSITY OF CALIFORNIA

Dates

Publication Date
20260512
Application Date
20200319

Claims (20)

  1. 1 . A method for chemoselective modification of a target molecule to produce a modified target molecule, the method comprising: contacting a target molecule comprising a thiol moiety with a first biomolecule comprising a reactive moiety; wherein the first biomolecule comprising the reactive moiety is generated by reaction of a first biomolecule comprising a phenol moiety or a catechol moiety with a tyrosinase polypeptide; and wherein said contacting is under conditions sufficient for conjugation of the target molecule to the first biomolecule comprising the reactive moiety, thereby producing a modified target molecule, and wherein the modified target molecule is of formula (IV) or (IVA): wherein: Y 1 is a first biomolecule, Y 2 is a second biomolecule; L is an optional linker; and n is an integer from 1 to 3, and wherein Y 1 and Y 2 can be the same or different biomolecule wherein the first biomolecule or the second biomolecule comprises an antibody or antibody fragment.
  2. 2 . The method of claim 1 , wherein the target molecule is a polypeptide or a polynucleotide.
  3. 3 . The method of claim 1 , wherein the tyrosinase polypeptide comprises an amino acid sequence having at least 75% amino acid sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs: 973-1020.
  4. 4 . The method of claim 1 , wherein the phenol moiety is present in a tyrosine residue and/or wherein the thiol moiety is present in a cysteine residue.
  5. 5 . The method of claim 1 , wherein the first biomolecule or the second biomolecule is a polypeptide selected from a fluorescent protein, an antibody or antibody fragment, an enzyme, a ligand for a receptor, and a receptor.
  6. 6 . The method of claim 1 , wherein the first biomolecule comprising a phenol moiety or a catechol moiety is of formula (I), and wherein the first biomolecule comprising a reactive moiety is of formula (II) or (IIA), or a combination thereof: wherein: Y 1 is a biomolecule, optionally comprising one or more moieties selected from, an active small molecule, an affinity tag, a fluorophore, and a metal-chelating agent; X 1 is selected from hydrogen and hydroxyl; and L is an optional linker.
  7. 7 . The method of claim 1 , wherein the modified target molecule is described by the formula (IVB) or (IVC), or a combination thereof: wherein: Y 1 is a biomolecule optionally comprising one or more groups selected from, an active small molecule, an affinity tag, a fluorophore, and a metal-chelating agent; each R 1 is independently selected from hydrogen, acyl, substituted acyl, alkyl, and substituted alkyl; Y 2 is a second biomolecule; L 1 is a linker selected from a straight or branched alkyl, a straight or branched substituted alkyl, a polyethylene glycol (PEG), a substituted PEG, and one or more peptides; and n is an integer from 1 to 3.
  8. 8 . The method of claim 7 , wherein the modified target molecule of formula (IVB) is of any of formulae (IVB1)-(IVB3): and the modified target molecule of formula (IVC) is of any of formulae (IVC1)-(IVC3):
  9. 9 . The method of claim 7 , wherein the modified target molecule of formula (IVB) is of any of formulae (IVB4)-(IVB5): and the modified target molecule of formula (IVC) is of any of formulae (IVC4)-(IVC5):
  10. 10 . The method of claim 1 , wherein the first biomolecule comprises a polypeptide.
  11. 11 . The method of claim 1 , wherein the first biomolecule comprises at least one of a fluorescent protein, an antibody or antibody fragment, a polynucleotide, a glycopolypeptide, a domain from a protein, a peptide, a cytokine, a chemokine, a peptide hormone, and an enzyme.
  12. 12 . The method of claim 1 , wherein the first biomolecule or the second biomolecule comprises a polypeptide, a nucleic acid, or a small molecule.
  13. 13 . The method of claim 12 , wherein the first biomolecule or the second biomolecule further comprises one or more moieties selected from a small molecule, an affinity tag, a fluorophore, and a metal-chelating agent.
  14. 14 . The method of claim 1 , wherein the antibody or antibody fragment is a single variable domain (VHH) antibody or a camelid antibody.
  15. 15 . The method of claim 1 , wherein the antibody or the antibody fragment comprises an anti-HER2 antibody.
  16. 16 . The method of claim 2 , wherein the polynucleotide comprises at least one of a single-stranded DNA molecule, double-stranded DNA molecule, a single-stranded RNA molecule, a small interfering RNA (siRNA), a short hairpin RNA (shRNA), a microRNA (miRNA), a ribozyme, an antisense oligonucleotide, a self-cleaving RNA, a microRNA mimic, a supermir, an aptamer, an antimir, an antagomir, a U1 adaptor, a triplex-forming oligonucleotide, an RNA activator, a long non-coding RNA, a short non-coding RNA, a piRNA, an immunomodulatory oligonucleotide, an antiviral oligonucleotide, or a decoy oligonucleotide.
  17. 17 . The method of claim 1 , wherein the first biomolecule comprises the antibody or antibody fragment.
  18. 18 . The method of claim 1 , wherein the second biomolecule comprises the antibody or antibody fragment.
  19. 19 . The method of claim 17 , wherein the antibody or antibody fragment comprises a VHH.
  20. 20 . The method of claim 18 , wherein the antibody or antibody fragment comprises a VHH.

Description

CROSS-REFERENCE This application is a national stage filing under 35 U.S.C. § 371 of PCT/US2020/023634, filed Mar. 19, 2020, which claims the benefit of U.S. Provisional Patent Application No. 62/822,616, filed Mar. 22, 2019, and U.S. Provisional Patent Application No. 62/910,836, filed Oct. 4, 2019, which applications are incorporated herein by reference in their entirety. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH This invention was made with government support under Grant Nos. 1059083 and 1808189 awarded by the National Science Foundation. The government has certain rights in the invention. INCORPORATION BY REFERENCE OF SEQUENCE LISTING PROVIDED AS A TEXT FILE A Sequence Listing is provided herewith as a text file, “BERK-405WO_SEQ_LISTING_ST25.txt” created on Mar. 17, 2020 and having a size of 8,056 KB. The contents of the text file are incorporated by reference herein in their entirety. INTRODUCTION Coupling biomolecules to target molecules, to generate conjugates, while preserving the function of the biomolecule and the target molecule, has long been a goal of chemical biology and biopharmaceutical research. Examples of conjugates include protein-peptide conjugates for vaccine development, antibody-drug, and antibody-protein conjugates for immunotherapeutics. While many techniques have been developed to allow for attachment of moderately sized molecules to proteins, it has been challenging to develop a simple biomolecule modification procedure that can attach proteins or biomolecules in a site-specific manner to any position on a protein's surface. There is a need for improved target molecule modification procedures that can modify a target molecule in a simple yet site specific manner. SUMMARY The present disclosure provides a method for chemoselective modification of a target molecule. A subject method includes contacting a target molecule comprising a thiol moiety with a biomolecule comprising a reactive moiety, wherein the reactive moiety is generated by reaction of a biomolecule comprising a phenol moiety or a catechol with an enzyme capable of oxidizing the phenol or the catechol moiety. The contacting is carried out under conditions sufficient for conjugation of the target molecule to the biomolecule, thereby producing a modified target molecule. The present disclosure provides compositions comprising a subject target molecule comprising a thiol moiety, and a biomolecule comprising a phenol moiety or a catechol moiety. The present disclosure provides kits for carrying out a subject method. The present disclosure also provides modified target molecules and methods for using same. BRIEF DESCRIPTION OF THE DRAWINGS The invention is best understood from the following detailed description when read in conjunction with the accompanying figures. It is emphasized that, according to common practice, the various features of the figures are not to-scale. On the contrary, the dimensions of the various features are arbitrarily expanded or reduced for clarity. Included in the drawings are the following figures. It is understood that the figures, described below, are for illustration purposes only. The figures are not intended to limit the scope of the present teachings in any way. FIG. 1A illustrates activation of phenol and catechol moieties with a tyrosinase enzyme to provide a quinone intermediate, and subsequent reaction of the quinone intermediate with potential nucleophiles. FIG. 1B illustrates an exemplary subject chemoselective modification reaction of a target protein with solvent exposed thiol (A) with a tyrosine/phenol containing coupling partner (B) to provide a covalently bound conjugation product (C). FIG. 2, panel A depicts ESI-TOF data showing MS2 N87C modified with alpha-endorphin peptide, as well as maleimide blocking experiments, illustrating that capping the thiols via a maleimide on the protein blocks addition via tyrosinase catalyzed reaction, and that where tyrosinase is performed first this also blocks the reaction of maleimide. This Figure demonstrates that the surface cysteines are the residues being modified. FIG. 2, panel B depicts stability studies for protein-peptide conjugates under various conditions. All samples were stored in 50 mM phosphate buffer at the stated conditions. FIG. 3 illustrates exemplary examples of biomolecules comprising a phenol moiety compatible in the subject methods. FIG. 4 illustrates ESI-TOF data showing coupling of various peptides to cysteine-containing mutants of the MS2 viral capsid. The peptides consisted of the following sequences with acylated N-termini: 2NLS: Ac-YGPKKKRKVGGSPKKKRKV (SEQ ID NO: 943); IL13: Ac-GYACGEMGWVRCGGSK (SEQ ID NO: 944); R8: Ac-YGRRRRRRRR (SEQ ID NO: 945); and HIV-Tat: Ac-YGRKKRRQRRRPPQ (SEQ ID NO: 946). FIG. 5, panel A illustrates ESI-TOF data showing Cas9 (C80, C574) is modified twice by endorphin. FIG. 5, panel B depicts an in vitro DNA cleavage assay, demonstrating that Cas9 (RNP) modified with peptide (End) retai