Impact of site-specific conjugation strategies on the pharmacokinetics of antibody conjugated radiotherapeutics

Antibody radionuclide conjugates are an emerging modality for targeted imaging and potent therapy of disseminated disease. Coupling of radionuclides to monoclonal antibodies (mAbs) is typically achieved by applying non-site-specific labelling techniques. With the ambition of reducing variability, increasing labelling efficacy and stability, several site-specific conjugation strategies have been developed in recent years for toxin- and fluorophore-mAb conjugates. In this study, we studied two site-specific labelling strategies for the conjugation of the macrocyclic chelating agent, DOTA, to the anti-Leucine Rich Repeat Containing 15 (LRRC15) mAb DUNP19. Specifically, one approach utilized a DOTA-bearing peptide (FcIII) with a strong affinity for the fragment crystallizable (Fc) domain of the human IgG₁ of DUNP19 (DUNP19^LF-FcIII-DOTA^SS), while the other leveraged a chemo-enzymatic technique to substitute the N-linked bi-antennary oligosaccharides in the human IgG₁ Fc domain with DOTA (DUNP19^LF-gly-DOTA^SS). To assess if these methods impact the antibody's binding properties and targeting efficacy, comparative in vitro and in vivo studies of the generated DUNP19-conjugates were performed. While the LRRC15 binding of both radioimmunoconjugates remained intact, the conjugation methods had different impacts on their abilities to interact with FcRn and FcγRs. In vitro assessments of DUNP19^LF-FcIII-DOTA^SS and DUNP19^LF-gly-DOTA^SS demonstrated markedly decreased affinity for FcRn and FcγRIIIa (CD16), respectively. DUNP19^LF-FcIII-DOTA^SS demonstrated increased blood and tissue kinetics in vivo, confirming loss of FcRn binding. While the ablated FcγR interaction of DUNP19^LF-gly-DOTA^SS had no immediate impact on in vivo biodistribution, reduced immunotherapeutic effect can be expected in future studies as a result of reduced NK-cells interaction. In conclusion, our findings underscore the necessity for meticulous consideration and evaluation of mAb labelling strategies, extending beyond mere conjugation efficiency and radiolabeling yields. Notably, site-specific labelling methods were found to significantly influence the immunological impact of Fc interactions. Therefore, it is of paramount importance to consider the intended diagnostic or therapeutic application of the construct and to adopt conjugation strategies that ensure the preservation of critical pharmacological properties and functionality of the antibody in use.