Abstract 3104: Synthesis and optimization of small molecules designed to stimulate the immune system by inducing DNA damage and blocking PD-1/PD-L1
Authors: Timiraos Ana Belen Fraga , Facchin Caterina , Babaa Nadia , Larroque-Lombard Anne-Laure , Jean-Claude Bertrand 
Published: June 2023

Abstract
Introduction. Activating the immune system against cancer is becoming an increasingly effective therapy option that can result in dramatic and durable responses in several cancer types. One approach to achieve the reactivation of endogenous antitumor T cells is by blocking PD-1/PD-L1 immune checkpoints expressed on T cells and other leukocytes. However, only limited cancer patients (15-25%) respond to anti-PD-1/PD-L1 immunotherapy. One of the most pressing current clinical challenges is to convert nonresponsive, “cold” tumors to responsive, “hot” tumors. Interestingly, after DNA-damaging chemotherapy, the immune environment may be changed from “cold” tumors to “hot” tumors by increasing the tumor mutation burden and the generation of neoantigens on the surface of cancer cells. Therefore, we surmised that a molecule capable of inducing promutagenic DNA and block PD-1/PD-L1 could not only induce neoantigens but also synergistically enhance immune response against the targeted tumor. Using an approach developed in our laboratory termed the “combi-targeting” strategy, we designed and synthesized a series of “combi-molecules” programmed to generate the promutagenic species and a small molecule capable of blocking PD-1/PD-L1.
Material and methods. Melanoma cell line B16-F10 was used to determine IC50 of the new molecules with SRB assay. Homogenous time-resolved fluorescence (HTRF) binding assay was used to determine the IC50 inhibition of PD-1/PD-L1. Drug metabolism in extracted cells was measured by LC-MS.
Results. We discovered a structure activity relationship of the combi-molecules based on the substitution of the side chain of the alkylating agent and the PD-1/PD-L1 scaffold. By altering the scaffolds of the combi-molecules from sulfonamides to biphenyl derivatives, we optimized binding to PD-1/PD-L1 from millimolar to micromolar levels. In vitro growth inhibitory analysis showed that the combi-molecules with biphenyl scaffold were 24-fold more potent in B16-F10. Importantly, analysis of intracellular metabolites of the combi-molecules revealed three main metabolites that can only result from the release of the shortlived promutagenic species.
Conclusions. The biphenyl scaffold is optimal for maintaining strong PD-1/PD-L1 binding potency and enough potent to contribute to cell death. Hydrolytic cleavage of the alkylating agent is an indirect evidence of the formation of the alkylating species required to induce promutagenic lesions.
Citation Format: Ana Belen Fraga Timiraos, Caterina Facchin, Nadia Babaa, Anne-Laure Larroque-Lombard, Bertrand Jean-Claude. Synthesis and optimization of small molecules designed to stimulate the immune system by inducing DNA damage and blocking PD-1/PD-L1 [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 3104.