RT Journal Article SR Electronic T1 Nuts and Bolts of Antibody Radiolabeling from a Small Molecule Chemist’s Perspective JF Journal of Nuclear Medicine JO J Nucl Med FD Society of Nuclear Medicine SP 241974 OP 241974 VO 65 IS supplement 2 A1 Pakula, Ryan A1 Shao, Xia A1 Scott, Peter YR 2024 UL http://jnm.snmjournals.org/content/65/supplement_2/241974.abstract AB 241974 Introduction: Antibodies use in nuclear medicine is growing due to their numerous benefits, including exquisite selectivity and strong binding to targets.[1] While literature reports of radiolabeling of antibodies are abundant, there are various levels of detail on a wide variety of methods for their production, labeling, and analysis, and we found some methods in our hands were easier, more informative, or more reproducible than others. Entering this field from our background as small molecule chemists, we arrived at an approach that worked for us through trial and error of methods from the literature and ideas from knowledgeable experts. As the field grows, other small molecular chemists will enter this area with similar perspective, knowledge, and skills as we have and may benefit from what we’ve learned. Thus, herein we share our method as well as pitfalls we have faced in learning how to generate radiolabeled antibodies for positron emission tomography.Methods: Monospecific IgG antibodies and bispecific antibodies were recombinantly expressed in HEK or CHO cells under standard conditions. Purification approaches included binding to Protein A resin, binding to myc tag resin, and/or size exclusion chromatography, as well as buffer exchange and sterilization steps. Antibody conjugation with NOTA-based chelators utilized N-hydroxysuccinimidyl (NHS) ester or isothiocyanate (SCN) chemistry, and purification methods evaluated included passage through desalting resins and size-based centrifugal filters. The resulting NOTA-conjugated antibodies were analyzed for chelator/antibody ratio using MALDI-TOF mass spectrometry and a colorimetric plate-based assay.[2] Conditions for radiolabeling and purification of radiolabeled products were evaluated using [68Ga]GaCl3 due to in-house access to Ge/Ga generators. Analytical methods and uses of the final radiolabeled antibodies discussed here include radio-TLC, HPLC, autoradiography, microPET scans in rodents.Results: Conjugation of NOTA to antibodies with SCN chemistry was <s>much</s> more successful and consistent in our hands than attempts at utilizing NHS chemistry. The structural MALDI-TOF evaluation and the functional colorimetric assay gave different results for chelator/antibody ratio, and the colorimetric assay’s results were a more useful tool for guiding towards successful subsequent experiments. By the colorimetric assay, antibodies treated with EDTA prior to NOTA conjugation had slightly higher available NOTA than those not.[3] Use of metal-free solutions in all steps from the NOTA conjugation onward proved critical and could be demonstrated using the colorimetric assay or radiolabeling. Purification of NOTA conjugated antibodies was most successful when using size-based centrifugal filters (30 kDa MWCO). These same filters were also useful in purification of the final radiolabeled antibodies, though, with Ga-68 labeled antibodies, use of desalting resins in addition to the centrifugal filters was required for obtaining samples free of solid radiometal biproducts and suitable for autoradiography and microPET scans.Conclusions: The use of antibodies in nuclear medicine is expected to only grow in the coming years, and this will bring new minds to this research space. Innumerable aspects of working with antibodies arise as one delves into the literature, and we hope our "beginner’s guide" may help focus efforts and smooth the path for new colleagues.References: [1] Pakula, R. J.; Scott, P. J. H. J. Labelled Comp. Radiopharm. 2023, 66 (9), 269. [2] Al-Ejeh, F.; Darby, J. M.; Thierry, B.; Brown, M. P. Nucl. Med. Biol. 2009, 36, 295. [3] Reyes, S. T.; Azevedo, E.C.; Cropper, H.C.; et al. JoVE 2023, e64133.