Immunotherapy seeks to enhance or manipulate host antitumor immunity by various approaches, among them, in situ vaccination. In situ vaccination is a local intervention in which immune enhancing agents are injected locally into one site of tumor, triggering a T-cell immune response locally that then travels to attack cancer throughout the body. We have employed a preclinical strategy whereby the same syngeneic tumor is implanted at two separate sites in the body. One tumor is then injected with the test agents and the resulting systemic immune response, if any, is detected by the regression of the distant, untreated tumor. In this test for abscopal therapeutic effects, the combination of unmethylated CG-enriched oligodeoxynucleotide (CpG)—a TLR9 ligand—and agonist anti-OX40 antibody has provided the most impressive results. This combination lead to durable disease control and long-term treatment-free survival in multiple mouse models of cancer. CpG induced myeloid cells to secrete cytokines, which subsequently induced OX40 expression on T-cells. Thus, we hypothesized that administration sequence and timing may affect the antitumor responses of in situ vaccination. In order to screen for the best sequence and timing we implanted A20 lymphoma tumors bilaterally in opposite sides of the abdomen of Balb/C mice. After tumors were established, one tumor was injected at the selected sequence and timing with the test agents and the resulting immune response was monitored by the measuring growth of the distant, untreated tumor. The systemic antitumor response required the presence of both CD4+ and CD8+ T-cells, as mice treated with the corresponding depleting antibodies were unable to control tumor growth. Even a single injection of low-dose CpG (50µg) and anti-OX40 (8µg) resulted in a fully protective systemic immune response. In addition, the cured animals were protected from rechallenge with the same A20 tumor but not unrelated tumors. Decreasing the dose even further to 10µg CpG and 1µg anti-OX40 partially preserved the therapeutic response with a long-term survival of 60%. Concurrent administration of CpG and anti-OX40 resulted in eradication of both local and distant disease. Sequential administration of CpG followed by anti-OX40 preserved the therapeutic efficacy. However, sequential administration of anti-OX40 followed by CpG significantly attenuated the therapeutic effect. While CpG followed by a 24- or 48-hour-delayed anti-OX40 treatment preserved the therapeutic efficacy of concurrent therapy, 72h delay in anti-OX40 administration resulted in reduced therapeutic effect. These data demonstrate the importance of the administration sequence for fully protective and curative antitumor immune responses. Our data suggest that the anti-OX40 antibody should be administered at the same time as CpG or somewhat delayed but not the other way around. The combination of anti-OX40 and CpG is currently studied in a phase I trial. Our results here impact the planning of future clinical trials of in situ vaccination with these two agents.

Citation Format: Idit Sagiv-Barfi, Debra K. Czerwinski, Ronald Levy. Timing and sequence of CpG and anti-OX40 is critical for in situ vaccination [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B128.