Site-specifically versus randomly THP-functionalized hPD-L1 single domain antibody; preliminary data toward kit development

Introduction. Single-domain antibodies (sdAbs) have ideal characteristics for imaging. NOTA-sdAbs and necessary reagents can be lyophilized into clinical-grade kits for easy gallium-68 (68Ga)-labeling.[1] However, the requirement of NaOAc as buffer (pH = 4.5—5) for labeling hampers the lyophilization, resulting in a two-step reconstitution process of the lyophilized kit. Utilizing the THP-chelator for 68Ga-labeling proved more efficient at milder pH with other small proteins.[2] We aim to apply this strategy to investigate other conditions for 68Ga-labeling, and to develop a one-pot radiolabeling of sdAbs. Methods. Human programmed death-ligand 1 (hPD-L1) sdAbs were coupled to maleimide-THP (site-specifically at the C-terminal) or NCS-THP (randomly on lysines) following existing procedures[3,4] in presence of 3—7% DMSO. Quality controls were performed by mass spectrometry (MS), SEC and SDS-PAGE. Radiolabelings were performed at room temperature for 10 min, variating sdAb concentration or buffers. Radiochemical purity (RCP) was measured by radio-SEC, radio-instant thin layer chromatography and radio-SDS-PAGE. For the [68Ga]Ga-THP-mal-cys-sdAb, stability at 37°C was assessed in human serum. Specificity was assessed on hPD-L1 positive (POS) MEL624 cells. Negative cells and POS cells blocked by an excess of non-radioactive competitor were used as controls.  Results. The THP-mal-cys-sdAb and THP-NHCS-sdAb were obtained in 52±4% (N = 2) and 46±4% (N = 2) yield respectively. MS results for the THP-mal-cys-sdAb only showed presence of the THP-sdAb (16072 Da) and deamidated product (16055 Da) and the product remained stable over 4 months at -20°C. For both strategies, SDS-PAGE analysis showed presence of starting sdAb and dimerized/aggregated sdAb (faint bands) (Fig. 1). [68Ga]Ga-THP-mal-cys-sdAb was obtained in RCP > 90% using 4 µM sdAb and 0.5 M Na-Carbonate buffer pH = 8.5 (0.065 M in total volume, final pH = 6.5±0.2), while [68Ga]Ga-THP-NHCS-sdAb was obtained in RCP < 70%. The main impurities were radiolabeled sdAb-aggregates, as confirmed by the faint signals seen at ±55 kDa and ±25 kDa on radio-SDS-PAGE (Fig. 1) and radio-SEC. At 37°C, [68Ga]Ga-THP-mal-cys-sdAb remained stable in serum (RCP > 90% over 1 h) (Fig. 2.A). The [68Ga]Ga-THP-mal-cys-sdAb showed specificity towards hPD-L1POS cells (Fig. 2.B). Conclusion. The THP chelator allowed for radiolabeling under promising conditions especially for the site-specific strategy. Before further investigating the lyophilization, it will be necessary to optimize the THP-coupling to avoid aggregation, which was surprising as not observed in the case of maleimide-NOTA coupling,[3] and then assess the in vivo stability of the radiopharmaceutical.  Acknowledgments. We thank Annelies Neukermans for technical assistance. Financial disclosure. This project is funded by the Wetenschappelijke Fonds Willy Gepts of the UZ Brussel (WFWG) and by the Strategic Research Programme (SRP50) of the Vrije Universiteit Brussel (VUB). Henri Baudhuin is an employee of Abscint SA. I or one of my co-authors have the following patent to disclose regarding the subject matter of this presentation: Broos K., Van Ginderachter J., Raes G., Devoogdt N., Keyaerts M., Lecocq Q., Xavier C., K. Breckpot K., Bridoux J. Human PD-L1 binding immunoglobulins, WO2019166622A1, priority date 01/03/2018 References [1]Baudhuin, H. et al., Eur J Pharm Biopharm. (2021). [2]Nawaz, S. et al., EJNMMI Research (2017). [3]Moguli, D. et al., Pharmaceuticals (2021). [4]Bridoux, J. et al., Biomolecules (2020)