Abstract 2261: Understanding the mechanism underlying resistance to immunotherapy in TNBC
Authors: Anselme Ann C. , Qian Wei , Kozielski Anthony , Rosato Roberto R. , Chang Jenny C. 
Published: August 2019

Abstract
Triple Negative Breast Cancer (TNBC) accounts for 15-20% of all breast cancer cases. TNBC patients have the worst outcome amongst all breast cancers; and do not respond well to conventional therapies. Although TNBC patients have a high amount of tumor infiltrating lymphocytes, and express higher level of PD-L1 compared to other breast cancer subtypes, they do not all respond to PD-1 blockade therapy. IL-12 is a cytokine produced by dendritic cells (DCs), and other APCs, known to induce the activation of natural killer cells, cytotoxic T-cells, and induce a Th1 phenotype. Although the immune activating potential of Il-12 are known, its systemic uses have been limited due to high toxicity. Using three different tumor models (PDXs: BCM 4913, MC1 in humanized mice and E0771 syngeneic model C57/Bl), we are investigating the response of combining intratumoral injection of Adenovirus IL-12 (ad.Il-12) and anti-PD1 therapy.
Materials/Methods: E0771, a mice TNBC cell line was injected in the mammary fat pad of C57BL/6 mice. The same procedure was followed using MC1 and BCM 4913, in humanized NSG-MGM3 mice. The mice received weekly intratumoral injection of ad.IL-12; a replication defective adenoviral vector containing mIL-12 (mouse) and hIL-12 (PDX) cDNA under the transcriptional control of Rous sarcoma virus long terminal repeat (received from Dr. Chen). Anti-PD1 (InVivoMab anti-mouse PD-1 CD279) was administered 3 times a week, Pembrolizumab (once a week). Post treatment, INF-gamma level were measured from blood and tumor samples, in addition, tumor sizes were compared between treatment groups (Control/ad.IL-12/anti-PD1/ad.IL-12 + anti-PD1), as well as survival curves. Immunohistochemistry was used to assess levels on TILs in the tumors.
Results: Ad.Il-12 and PD-1 blockade therapy combination was more effective at reducing tumor growth in BCM4913, while in E00771 and MC1 combination treatment showed no difference compared to single agent. In all the models, levels of INF-gamma in the tumor were significantly upregulated in the combination group compared to control and single agents, while no difference was found between treatment groups from blood samples. Tumor growth in the combination group and the treated group were slower compared to the controls in BCM 4913. IHC data indicate a higher level of TILs in the treatment groups, with a statistically significant difference in the combination group compared to the others.
Conclusion: Our preliminary data suggest that intratumoral injection of IL-12 confine the release of INF-gamma in the tumor microenvironment, and mitigate the toxicity associated with systemic admission of ad.IL-12. Treatment response is not achieved in every model, suggesting an underlying mechanism which confers resistance to the treatment. We are currently investigating different models in the hope to identify markers of responders versus non responders.
Citation Format: Ann C. Anselme, Wei Qian, Anthony Kozielski, Roberto R. Rosato, Jenny C. Chang. Understanding the mechanism underlying resistance to immunotherapy in TNBC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2261.