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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.

Background: ZR2002 is a dual EGFR-DNA-targeting combi-molecule that carries a chloroethyl group at the six-position of the quinazoline ring designed to alkylate DNA. Despite its good pharmacokinetics, ZR2002 is metabolized in vivo into dechlorinated metabolites, losing the DNA-alkylating function required to damage DNA. To increase the DNA damage activity in tumor cells in vivo, we compared ZR2002 with two of its 6-N,N-disubstituted analogs: “JS61”, with a nitrogen mustard function at the six-position of the quinazoline ring, and “JS84”, with an N-methyl group. Methods: Tumor xenografts were performed with the human Saos-2 osteosarcoma cell line expressing EGFR. Mice were treated with ZR2002, JS84 or JS61, and the tumor burden was measured with a caliper and CT/PET imaging. Drug metabolism was analyzed with LC-MS. EGFR and ɣ-H2AX phosphorylation were quantified via Western blot analysis and immunohistochemistry. Results: In vivo analysis showed that significant tumor growth inhibition was only achieved when ZR2002 was administered in its naked form. The metabolic dealkylation of JS61 and JS84 did not release sufficient concentrations of ZR2002 for the intratumoral inhibition of P-EGFR or enhanced levels of P-H2AX. Conclusions: The results in toto suggest that intratumoral concentrations of intact ZR2002 are correlated with the highest inhibition of P-EGFR and induction of DNA damage in vivo. ZR2002 may well represent a good drug candidate for the treatment of EGFR-expressing osteosarcoma.

Introduction. Glioblastoma multiforme (GBM) is the most aggressive form of primary brain tumours with an average survival rate of 12-18 months. The existing treatment (chemotherapy with temozolomide (TMZ)+radiotherapy) increases the median survival by 2-3 months. One protein, the epidermal growth factor receptor (EGFR) is expressed in 57.4% of GBM and half of them expresses its aggressive mutant, EGFRvIII, whose expression is associated with resistance to apoptosis, activation of DNA repair and ultimately resistance to chemotherapy. In the context of a strategy termed “combi-targeting”, we developed molecules designed to block EGFRvIII while being capable of inducing DNA damage. In vivo analysis suggests that the first prototype of such a class ZR2002 loses its DNA damaging function through metabolic oxidation. Recently, we surmised that a molecule designed to be metabolically stable may require a lower dose to be active in vivo. To test this hypothesis, we measured the cytotoxicity of the new combi-molecule, called "AF143", in a panel of glioblastoma cell lines and then we determined its efficacy and drug metabolism in an in vivo model of glioblastoma derived from human glioblastoma stem cells (GSC) with known resistance to TMZ. Material and Methods. Three glioblastoma cells line (U87-wt, U87-EGFR and U87-EGFRvIII) and the human GSC (1123IC7R) were used to determine IC50 of AF143 and ZR2002 with SRB or alamarBlue assay. Xenograft of tumors were obtained by intracranial injection of 1123IC7R-Luc GSC into the head of NSG female mice. One group of mice was treated with ZR2002 (N=8), the other with AF143 (N=7), the last with vehicle (N=8). Mice received 10 total doses: 2 cycles of 1 dose (100mg/kg) daily for 5 consecutive days followed by 2 days off, by oral gavage. The tumour burden was measured by bioluminescent imaging and drug metabolism by LC-MS. Body weights, mortality and toxicity were monitored. Survival curves and statistical analysis calculated. Results. In vitro growth inhibitory analysis showed that AF143 was 2-fold more potent than ZR2002 in U87-EGFR and 1123IC7R GSC and 3-fold more potent in U87-EGFRvIII. In vivo analysis of AF143 given alone showed significant antitumor activity (p<0.05) after 5 doses treatment, while ZR2002 induced an apparent tumour delay (p>0.05). Remarkably, AF143 and ZR2002 treatment significantly prolonged the overall survival of mice compared with the control group (p= 0.0004 for AF143 and p=0.04 for ZR2002). Analysis of drug metabolism revealed that in contrast to ZR2002, AF143 remained intact after 2h treatment, with moderate levels of N-acetyl and N6-dealkylated metabolites. Conclusion. The results in toto suggest that the intact structure of AF143 is stable to metabolism, can cross the blood brain barrier and induce antitumour activity in a EGFRvIII expressing glioblastoma stem cell model. Citation Format: Caterina Facchin, Ana Belen Fraga Timiraos, Brian Meehan, Nadia Babaa, Lata Adnani, Anne-Laure Larroque, Janusz Rak, Bertrand J Jean-Claude. In vivo efficacy and metabolism of a new synthetized dual EGFR-DNA targeting combi-molecule in a human stem cells-derived model of glioblastoma implanted intracranially in mice [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 1647.