Abstract 2973: NI-3201,a PD-L1xCD28 bispecific antibody for immune checkpoint-dependent CD28 co-stimulation
Authors: Majocchi Sara , Lloveras Pauline , Burnet-Merlin Coline , Nouveau Lise , Malinge Pauline , Moine Valery , Magistrelli Giovanni , Shang Limin , Fischer Nicolas , Masternak Krzysztof , Ferlin Walter 
Published: April 2023

Abstract
CD28 agonism on T cells delivers a critical second signal alongside TCR ligation for complete T cell activation. In the tumor microenvironment (TME), signal 2 can be provided by bispecific antibodies (bsAbs) targeting CD28 and a tumor associated antigen (TAA), enabling CD28 clustering on T cells in a TAA-dependent manner. The inhibitory PD-L1/PD-1 axis is described to repress T cell activation by directly inhibiting CD28 driven costimulation, affecting mostly activated and/or exhausted T cells. Preclinical studies have shown the benefit of combining blocking anti-PD-1/-L1 antibodies and costimulatory CD28 bsAbs for the treatment of solid tumors. We reasoned that the effect of such a combination could be integrated into a single molecule, by associating a blocking anti-PD-L1 antibody arm with an agonist anti-CD28 antibody arm. To this end, a PD-L1xCD28 bsAb (NI-3201) was generated on the κλ body platform to block the PD-L1/PD-1 immune checkpoint, hence release the brake on CD28 signaling and provide signal 2 when co-ligating PD-L1+ tumor and immune cells (e.g., antigen-presenting cells). The capacity of NI-3201 to provide signal 2 to T cells following co-engagement of PD-L1 on tumor or immune cells was assessed in a variety of assays and models. Using a sensitive reporter cell bioassay, NI-3201 was shown to efficiently block the PD-L1/PD-1 axis. The pharmacological activity of NI-3201 was then assessed through in vitro assays of cancer cell lines co-cultured with human peripheral blood mononuclear cells (PBMCs). NI-3201 synergized with CD3 bsAbs to enhance activation and proliferation of both CD4 and CD8 T cells, ultimately leading to strengthened T cell-dependent cellular cytotoxicity (TDCC) against PD-L1-positive cancer cell lines. In immunocompetent huCD28-transgenic mice engrafted with huPD-L1-expressing murine colon adenocarcinoma MC38 cells, NI-3201 displayed superior single-agent activity as compared to the anti-PD-L1 antibody Atezolizumab, with tumor regression being observed in all treated mice. At study termination, most of the NI-3201-treated mice rejected MC38-huPD-L1 tumors. All surviving mice rechallenged with wild type MC38 cells rejected the tumors, thus demonstrating a durable anti-tumor immunological memory response induced by NI-3201. Finally, as the anti-PD-L1 arm of NI-3201 is cross-reactive to mouse PD-L1, it was noteworthy that NI-3201-treated huCD28-transgenic mice displayed no clinical signs of toxicity. NI-3201 has demonstrated potent activity in a plethora of assays and single agent anti-tumor in vivo activity superior to a clinically approved PD-L1 benchmark. The mechanism of action of NI-3201 and the data presented herein support the use of this molecule as a universal combination partner for CD3-bispecifics. Pre-clinical development of NI-3201 is ongoing, with pharmacokinetic and tolerability studies in non-human primates planned for early 2023.
Citation Format: Sara Majocchi, Pauline Lloveras, Coline Burnet-Merlin, Lise Nouveau, Pauline Malinge, Valery Moine, Giovanni Magistrelli, Limin Shang, Nicolas Fischer, Krzysztof Masternak, Walter Ferlin. NI-3201,a PD-L1xCD28 bispecific antibody for immune checkpoint-dependent CD28 co-stimulation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2973.