Method for conjugating antibody and physiologically active substance
Inventors
Yoo, Tae Hyeon • PARK, Jisoo • Lee, Yumi
Assignees
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Publication Number
US-11998612-B2
Publication Date
2024-06-04
Expiration Date
Abstract
An antibody conjugating peptide including an amino acid having a photoreactive functional group, a physiologically active substance modified with the conjugating peptide, and an antibody conjugate having an antibody linked to the physiologically active substance are disclosed. When the physiologically active substance modified with the conjugating peptide is linked to the antibody, the conjugation efficiency between the antibody and the physiologically active substance is remarkably improved as compared to that of the conventional art, and thus, the drug may be firmly bound without impairing the specificity of the antibody, thereby making it possible to accelerate commercialization of the antibody conjugate.
Core Innovation
The invention relates to an Fc site-specific conjugating peptide mutant derived from SEQ ID NO: 1, used to modify a physiologically active substance for site-specific conjugation to an Fc-domain-containing antibody. The Fc site-specific conjugating peptide mutant comprises the amino acid sequence of SEQ ID NO: 1 and further comprises a valine to p-benzoyl phenylalanine substitution at position 10.
A photoreactive functional group is introduced into the Fc site-specific conjugating peptide mutant by substitution at position 5, 10, or 11, preferably by p-benzoyl phenylalanine (pBpa). Upon UV irradiation, the conjugate generation forms covalent bonds while maintaining antibody specificity, thereby improving covalent, site-specific antibody-substance conjugation.
The modified physiologically active substance can be provided directly or via a linker, and the antibody conjugate is produced by photoreaction between a substance-peptide conjugate and an Fc-domain-containing molecule. The disclosure includes example conjugation constructs in which Fc binding is achieved via pBpa at Val10 with covalent attachment to an antibody residue (Met252, as reported by LC-MS/MS).
Claims Coverage
The independent claim covers one inventive feature centered on Fc site-specific conjugation using an SEQ ID NO: 1-based peptide mutant with a Val10 to p-benzoyl phenylalanine substitution. The claim emphasizes higher binding efficiency to the Fc site of an antibody than pBpa substitutions at position 5 or 11.
Substance linked to Fc site-specific conjugating peptide mutant with Val10 p-benzoyl phenylalanine
A substance modified with an Fc site-specific conjugating peptide mutant, wherein the substance is linked to the Fc site-specific conjugating peptide mutant either directly or via a linker; the Fc site-specific conjugating peptide mutant comprises the amino acid sequence of SEQ ID NO: 1 and further comprises a valine to p-benzoyl phenylalanine substitution at position 10; and the Fc site-specific conjugating peptide mutant comprising p-benzoyl phenylalanine substitution at position 10 shows higher binding efficiency to Fc site of an antibody than a mutant comprising p-benzoyl phenylalanine substitution at position 5 or 11.
Across the independent claim and its refinements, the core coverage centers on Fc site-specific conjugation using an SEQ ID NO: 1-based peptide mutant with a Val10 to p-benzoyl phenylalanine substitution, emphasizing improved Fc-site binding efficiency versus analogous mutants at positions 5 or 11.
Stated Advantages
Higher binding efficiency to the Fc site of an antibody for the p-benzoyl phenylalanine substitution at position 10 than for substitutions at position 5 or 11.
Improved covalent, site-specific antibody-substance conjugation upon UV irradiation while maintaining antibody specificity.
Retention of functional activity/efficacy as demonstrated by an EF2 activity assay and cell growth inhibition.
Documented Applications
Immunoconjugates involving cetuximab and trastuzumab, including cetuximab-FcIII-PE24 and trastuzumab-FcIII-PE24.
Conjugates involving β-lactamase and β-lactamase zymogen, including FcIII-β-lactamase and FcIII-β-lactamase zymogen constructs.
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