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Site-specific chelator-antibody conjugation for PET and SPECT imaging with radiometals
摘要： Antibodies and their derivatives radiolabelled with positron- and gamma-emitting radiometals enable sensitive and quantitative molecular Positron Emission Tomography (PET) and Single Photon Emission Computed Tomography (SPECT) imaging of antibody distribution in vivo. Chelators that are covalently attached to antibodies allow radiolabelling with metallic PET and SPECT radioisotopes. Conventional strategies for chelator-protein conjugation generate heterogeneous mixtures of bioconjugates that can exhibit reduced affinity for their targets, and undesirable biodistribution and pharmacokinetics. Recent advances in bioconjugation technology enable site-specific modification to generate well-defined constructs with superior properties. Herein we survey existing site-specific chelator-protein conjugation methods. These include chelator attachment to cysteines/disulfide bonds or the glycan region of the antibody, enzyme-mediated chelator conjugation, and incorporation of sequences of amino acids that chelate the radiometal. Such technology will allow better use of PET and SPECT imaging in the development of antibody-based therapies.
关键词： antibody,radiometals,PET,chelator,site-specific conjugation,SPECT,molecular imaging
更新于2025-09-23 15:21:21
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Development, optimization and structural characterization of an efficient peptide-based photoaffinity crosslinking reaction for generation of homogeneous conjugates from wild-type antibodies
摘要： Site-specific conjugation of small molecules to antibodies represents an attractive goal for the development of more homogeneous targeted therapies and diagnostics. Most site-specific conjugation strategies require modification or removal of antibody glycans or interchain disulfide bonds or engineering of an antibody mutant that bears a reactive handle. While such methods are effective, they complicate the process of preparing antibody conjugates and can negatively impact biological activity. Herein, we report the development and detailed characterization of a robust photoaffinity crosslinking method for site-specific conjugation to fully-glycosylated wild-type antibodies. The method employs a benzoylphenylalanine (Bpa) mutant of a previously-described 13-residue peptide derived from phage display to bind tightly to the Fc domain; upon UV irradiation, the Bpa residue forms a diradical that reacts with the bound antibody. First, we describe the initial discovery of an effective Bpa mutant peptide and optimization of reaction conditions to enable efficient conjugation without concomitant UV-induced photodamage of the antibody. Second, we assessed the scope of the photoconjugation reaction across different human and non-human antibodies and antibody mutants. Third, the specific site of conjugation on a human antibody was characterized in detail by mass spectrometry experiments and at atomic resolution by X-ray crystallography. Finally, we adapted the photoconjugation method to attach a cytotoxic payload site-specifically to a wild-type antibody and show that the resulting conjugate is both stable in plasma and as potent as a conventional antibody drug conjugate in cells, portending well for future biological applications.
关键词： wild-type antibodies,Fc domain,photoaffinity crosslinking,site-specific conjugation,antibody-drug conjugates
更新于2025-09-23 15:19:57