Upgrading cancer immunotherapy: Checkpoint blockade mAb-ODN conjugate

Programmed death 1 (PD-1) and its ligand PD-L1 provide inhibitory signals that affect the balance of T cell activation, playing major roles in tumor immune escape. Inhibition of negative regulators of T cell activation effectively remove signals that restrain T cell proliferation and gain of effector function. Hence, disruption of the PD-1/PD-L1 signaling pathway by the use of monoclonal antibodies (mAbs) can improve tumor control by enhancement of the endogenous antitumor immune responses. Toll-like receptors (TLR) are a specialized set of pathogen recognition receptors found in innate and adaptive immune system cells. Activation of these receptors by danger-associated molecules such as bacterial DNA or RNA, initiate signaling cascades leading to protective immune responses. Synthetic oligodeoxynucleotides (ODNs) containing unmethylated CpG motifs can mimic bacterial DNA and therefore function as ligands for intracellular TLR9, prompting consequential activation of immune responses. The purpose of this study was to assess the feasibility of using PD-1/PD-L1 mAbs conjugated to TLR9 agonists (ODNs) as a novel strategy for tumor-targeting immune checkpoint signaling and activation of immune cells. Antibodies recognizing PD-1 and PDL-1 were covalently modified with a 21mer ssDNA oligonucleotide linker sequence that served as an anchor site for the ODNs synthesized with a 39 extension. Annealing of complementary strands formed mAb-ODN conjugates. Quantification of ODNs per PD-1 or PDL-1 mAbs was verified by flow cytometry. Functionality of PD-1 and PDL-1-ODN conjugates was analyzed in vitro using splenocyte cultures and cell proliferation was evaluated by incorporation of radiolabeled H 3 Thymidine. In vivo experiments were carried out using Balb/c mice bearing 4T1 subcutaneous tumors. Flow cytometry analysis confirmed each modified mAb had 5 linker sequences available to bind ODNs, and also defined the binding capability of mAb portion of the conjugate to epitope target. Splenocytes proliferation at day 5 after treatment revealed that PD-1 and PDL-1 conjugated to ODNs have significantly increased proliferation in comparison to non-conjugated mAb and ODN controls. These results indicate that conjugation of mAb-ODN is feasible and that conjugation does not affect PD-1 and PDL-1 antibody functionality. Interestingly, in vivo experiments demonstrated PDL-1-ODN but not PD1-ODN conjugate affects 4T1 tumor growth. Use of this approach to improve the therapeutic index of immunotherapeutic mAbs requires further study and validation in different murine tumor models. Citation Format: Ana Paula Benaduce, Tal Gefen, Sergio Rodriguez, Brian Marples, Eli Gilboa, Adrian Ishkanian. Upgrading cancer immunotherapy: Checkpoint blockade mAb-ODN conjugate [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4702.