Comparative In Vitro and In Vivo Evaluation of Anti-CCR8 Full-Sized IgG and Its Fab Fragments in Murine Colorectal...

CCR8 chemokine receptor is a selective marker of tumor-infiltrating regulatory T cells (ti-Tregs) which interfere with the efficacy of checkpoint inhibitor immunotherapy (ICI) in many types of cancer. Eliminating CCR8+ ti-Tregs dramatically improves the results of subsequent ICI. We have recently reported using 225Actinium-labeled anti-CCR8 IgG for killing CCR8+ ti-Tregs in murine colorectal tumors which synergized with subsequent anti-CTLA4 ICI. Here, we aimed to compare the in vivo behavior of anti-CCR8 full-sized IgG and its Fab fragments to select the best antibody format for the pre-clinical development of this combination modality. Anti-CCR8 Fab fragments were generated by papain digest of the whole IgG. The whole IgG and Fab were conjugated to bifunctional chelating agent DOTA and labeled with 111Indium (¹¹¹In). MC8 and CT6 murine colorectal tumor-bearing C57Bl6 and Balb/c mice, respectively, were administered ¹¹¹In-DOTA-IgG or ¹¹¹In-DOTA-Fab and imaged with microSPECT/CT at 2-72 h post-injection. A biodistribution was performed after the last imaging time point. Both ¹¹¹In-DOTA-IgG and ¹¹¹In-DOTA-Fab demonstrated high tumor uptake in both MC38 and CT26 tumors, with ¹¹¹In-DOTA-IgG uptake being significantly higher from the 24 h time point and onwards. ¹¹¹In-DOTA-Fab also exhibited pronounced kidney uptake which persisted even at 72 h. The kidney clearance and retention of ¹¹¹In-DOTA-Fab might represent a problem during therapy employing 225Actimium or other long-lived therapeutic radionuclides by potentially causing a dose-limiting kidney toxicity. This imaging/biodistribution evaluation not only determined that full-size anti-CCR8 IgG is the optimal antibody format for pre-clinical development but also informed on the timing of immunotherapy administration in future radioimmunotherapy and immunotherapy combination studies.