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WO-2026096757-A2 - INTRODUCTION OF BIOORTHOGONAL CONJUGATION SITES USING NONCANONICAL AMINO ACIDS FOR RAPID ASSEMBLY AND SCREENING OF THERAPEUTICALLY LOADED NANOBODY ASSEMBLIES

WO2026096757A2WO 2026096757 A2WO2026096757 A2WO 2026096757A2WO-2026096757-A2

Abstract

The present disclosure includes bioorthogonal conjugation systems and methods of producing same. The systems include at least one ncAA that participates in an orthogonal click reaction owing to its reactive group. The system further includes at least one plasmid having a gene for one or more of: an aminoacyl-tRNA synthetase, a tRNA, and a nanobody. Each nanobody includes suppression stop codons that define locations for conjugation of a ncAA, such that reactive groups of the ncAAs are incorporated at each defined location of the nanobody. Locations are not restricted to N and C terminals of the peptide, facilitating nanobody functionality post-conjugation. Resulting nanobodies are modified with the reactive groups of the ncAA in one or more locations, such that multiple cargos can be conjugated to each nanobody. Additionally, nanobodies can be conjugated to other nanobodies having corresponding reactive groups to form assemblies, which may be used as therapeutic nanobody libraries.

Inventors

  • ADOMANIS, Roman
  • KIMMEL, Blaise
  • PHAN, Nathan

Assignees

  • OHIO STATE INNOVATION FOUNDATION

Dates

Publication Date
20260507
Application Date
20251030
Priority Date
20241030

Claims (20)

  1. 1. A bioorthogonal conjugation system, comprising: at least one non-canonical amino acid (ncAA) configured for participating in an orthogonal click reaction; and at least one plasmid comprising a gene for one or more of: an aminoacyl-tRNA synthetase (aaRS), a tRNA, and a nanobody having at least one suppression stop codon that defines a location for conjugation of the at least one ncAA.
  2. 2. The bioorthogonal conjugation system of claim 1, wherein the at least one ncAA is a phenylalanine derived azide containing amino acid or a tetrazine containing amino acid.
  3. 3. The bioorthogonal conjugation system of claim 2, wherein the phenylalanine derived azide containing amino acid is 4-Azido-L-phenylalanine (pAzF).
  4. 4. The bioorthogonal conjugation system of claim 2, wherein the tetrazine containing amino acid is 3-(6-methyl-s-tetrazin-3-yl)phenylalanine (Tet-3.0-Me) or (S)-2-amino-3-(6-phenyl- l,2,4,5-tetrazin-3-yl)propanoic acid (Tet-4.0-Ph).
  5. 5. The bioorthogonal conjugation system of claim 1, wherein the at least one ncAA is capable of participating in an orthogonal click chemistry reaction.
  6. 6. The bioorthogonal conjugation system of claim 5, wherein the at least one ncAA is capable of participating in spontaneous copper-free Diels- Alder cycloaddition reactions with trans-cyclooctene (TCO) and dibenzo cyclooctyne (DBCO) groups.
  7. 7. The bioorthogonal conjugation system of claim 5, wherein the at least one ncAA is capable of participating in spontaneous copper-free azide-alkyne cycloaddition reactions with bicyclo[6.1.0]non-4-yne (BCN) and dibenzo cyclooctyne (DBCO) groups.
  8. 8. The bioorthogonal conjugation system of claim 1 , wherein the location for conjugation of the at least one ncAA is surface-exposed on an expressed nanobody of the bioorthogonal conjugation system.
  9. 9. The bioorthogonal conjugation system of claim 1, comprising a first plasmid having genes for a first aaRS and a first tRNA, and a second plasmid having a gene for the nanobody with a first suppression stop codon.
  10. 10. The biorthogonal conjugation system of claim 9, wherein the first tRNA is configured to incorporate a first ncAA at the first suppression stop codon.
  11. 11. The biorthogonal conjugation system of claim 10, wherein the nanobody further includes a second suppression stop codon, and further comprising a third plasmid having genes for a second aaRS and a second tRNA.
  12. 12. The biorthogonal conjugation system of claim 11, wherein the second tRNA is configured to incorporate a second ncAA at the second suppression stop codon, such that an expressed nanobody includes dual, bioorthogonal conjugation sites at the first ncAA and second ncAA.
  13. 13. The bioorthogonal conjugation system of claim 1, comprising one plasmid having genes for the aaRS, the tRNA, and the nanobody with the suppression stop codon.
  14. 14. The bioorthogonal conjugation system of claim 13, wherein the tRNA is configured to incorporate the at least one ncAA at the suppression stop codon such that an expressed nanobody includes a biorthogonal conjugation site at the ncAA.
  15. 15. A modified protein for modular conjugation, the modified protein having at least one bioorthogonal conjugation site where a noncanonical amino acid (ncAA) is located, the ncAA capable of participating in an orthogonal click chemistry reaction.
  16. 16. The modified protein of claim 15, having at least two bioorthogonal conjugation sites, each where a different ncAA is located.
  17. 17. The modified protein of claim 16, wherein each bioorthogonal conjugation site is configured to conjugate a different cargo.
  18. 18. A protein assembly comprising one or more modified protein of claim 15.
  19. 19. The protein assembly of claim 18, wherein each bioorthogonal conjugation site is configured to conjugate a different cargo.
  20. 20. The protein assembly of claim 18, wherein the one or modified proteins are assembled with a linker selected from the group consisting of TCO-PEG-TCO, DBCO-PEG-DBCO, and TCO-PEG-DBCO.

Description

INTRODUCTION OF BIOORTHOGONAL CONJUGATION SITES USING NONCANONICAL AMINO ACIDS FOR RAPID ASSEMBLY AND SCREENING OF THERAPEUTICALLY LOADED NANOBODY ASSEMBLIES CROSS REFERENCE TO RELATED APPLICATIONS [0001] This application claims priority to United States Provisional Patent Application Number 63/713,855 filed on October 30, 2024, the entire contents of which are hereby incorporated by reference. SEQUENCE LISTING [0002] An electronic sequence listing (069596-00103.xml; size 24.0 KB; date of creation October 30, 2025) submitted herewith is incorporated by reference in its entirety. FIELD [0003] The present disclosure relates to systems and methods of introducing conjugation sites using noncanonical amino acids for generation of nanobody assemblies. BACKGROUND [0004] Bispecific antibodies and antibody fragments have become increasingly relevant therapeutic molecules in immunotherapy. However, these dual treatments are more expensive and associated with a higher occurrence of adverse events compared to single-agent therapies. Thus, over the past two decades, recombinant variable domains of heavy-chain only antibodies - referred to as nanobodies - have become an increasingly valuable diagnostic and therapeutic tool in cancer and other rare diseases as alternative bispecific therapeutics. However, a challenge in the clinical adoption of these nanobody therapies has been the creation of stable, chemically defined, and highly specific conjugation sites within the nanobody. Stable nanobody-protein conjugates formed through genetic fusion are restricted to linkages on the N and C terminals, which can be deleterious to their binding affinity and activity in vivo. Exploration of the nanobody-protein conjugate combinatorial design space is also limited by the cost and labor of genetic fusion, which requires the generation of an individual plasmid per new nanobody-protein combination. SUMMARY [0005] In an aspect of the present disclosure, a bioorthogonal conjugation system is provided. The system includes at least one non-canonical amino acid (ncAA) configured for participating in an orthogonal click reaction and at least one plasmid comprising a gene for one or more of: an aminoacyl-tRNA synthetase (aaRS), a tRNA, and a nanobody having at least one suppression stop codon that defines a location for conjugation of at least one ncAA. [0006] In some embodiments, at least one ncAA is a phenylalanine derived azide containing amino acid or a tetrazine containing amino acid. In some instances, the phenylalanine derived azide containing amino acid is 4-Azido-L-phenyl alanine (pAzF). In some instances, the tetrazine containing amino acid is 3-(6-methyl-s-tetrazin-3-yl)phenylalanine (Tet-3.0-Me) or (S)-2-amino- 3-(6-phenyl-l,2,4,5-tetrazin-3-yl)propanoic acid (Tet-4.0-Ph). [0007] In some embodiments, at least one ncAA is capable of participating in an orthogonal click chemistry reaction. In some instances, the at least one ncAA is capable of participating in spontaneous copper-free Diels- Alder cycloaddition reactions with trans-cyclooctene (TCO) and dibenzo cyclooctyne (DBCO) groups. In some instances, at least one ncAA is capable of participating in spontaneous copper-free azide-alkyne cycloaddition reactions with bicyclo[6.1.0]non-4-yne (BCN) and dibenzo cyclooctyne (DBCO) groups. [0008] In some embodiments, the location for conjugation of at least one ncAA is surface- exposed on an expressed nanobody of the bioorthogonal conjugation system. In some embodiments, the system includes a first plasmid having genes for a first aaRS and a first tRNA, and a second plasmid having a gene for the nanobody with a first suppression stop codon. In some instances, the first tRNA is configured to incorporate a first ncAA at the first suppression stop codon. In some cases, the nanobody further includes a second suppression stop codon, and further includes a third plasmid having genes for a second aaRS and a second tRNA. In some cases, the second tRNA is configured to incorporate a second ncAA at the second suppression stop codon, such that an expressed nanobody includes dual, bioorthogonal conjugation sites at the first ncAA and second ncAA. [0009] In some embodiments, the system further includes one plasmid having genes for the aaRS, the tRNA, and the nanobody with the suppression stop codon. In some instances, the tRNA is configured to incorporate at least one ncAA at the suppression stop codon such that an expressed nanobody includes a biorthogonal conjugation site at the ncAA. [0010] In another aspect of the present disclosure, there is presented a modified protein for modular conjugation, the modified protein having at least one bioorthogonal conjugation site where a ncAA is located, the ncAA capable of participating in an orthogonal click chemistry reaction. [0011] In some embodiments, the modified protein has at least two bioorthogonal conjugation sites, each where a different ncAA is located. In some instances, each bioorthogonal