Abstract 5078: Immune response markers following combination treatment with oncolytic adenovirus AMUN-003 and immune checkpoint inhibitors in a murine model of triple negative breast cancer
Authors: Shin Soon Cheon , Filimon Beniamin , Yang Yuefeng , Hu Zebin , Gadi Vijayakrishna K , Xu Weidong 
Published: April 2023

Abstract
Background: Accounting for about 10-15% of all breast cancer diagnoses, triple negative breast cancer (TNBC) has the highest mortality rate partly attributable to few effective treatment options. We evaluated a novel oncolytic adenovirus (rAd.sT.GM; named as AMUN-003 by AmunBio, Inc) encoding sTGFβRIIFc (a TGF-β protein decoy) and GM-CSF transgenes in combination with ICI treatments in the mouse TNBC 4T1 subcutaneous model. We previously reported that infection of target cancer cells with AMUN-003 and co-treatment with ICI led to potent inhibition of tumor progression and lung metastasis without off-target organ or systemic toxicity. Here, we provide details on the specific immune markers associated with responses.
Methods: Immune competent BALB/C mice harboring 4T1 tumors were treated with combinations of anti-PD1 antibody, anti-CTLA antibody, AMUN-003 or sham injections. We measured inflammatory serum cytokines/chemokine levels by Meso Scale immunoassays and immune modulator gene expression in lung, spleen and tumor by qRT-PCR.
Results: The “Triplet” combination of AMUN-003, anti-PD1 and anti-CTLA-4 antibodies was associated with decrease of poor prognosis biomarkers in serum: TGF-β1: Triplet p&lt; 0.001, ICIs p&lt;0.01, AMUN-003 p&lt; 0.05; IL-27p28/IL-30: Triplet and ICIs p&lt; 0.05; IL-1β: Triplet, AMUN-003, ICIs, and AMUN-003+anti-CTLA-4 p&lt; 0.05; TNF-α: Triplet p&lt; 0.01, ICIs p&lt;0.05, and increase of immune stimulatory IFN-γ: Triplet p&lt;0.01 (all compared to the buffer). In lung tissue, no significant changes in cytokine/chemokine expression were detected, however Granzyme B levels were significantly increased in Triplet: p&lt; 0.05. In spleen, we detected increased cytokine expression (IL-2: Triplet, anti-CTLA-4, AMUN-003+anti-PD-1 p&lt; 0.05; IL-4: Triplet p&lt; 0.001, AMUN-003+anti-PD-1, AMUN-003+anti-CTLA-4: p&lt; 0.01). In tumor, expression of TGF-β1 was significantly decreased by triplet and ICIs treatments (both p&lt;0.05), but increases in IFN-γ, IL-4, CXCR4, and Perforin were observed in several treatment groups.
Conclusions and Summary: Our data support the interpretation that the most potent intratumoral and systemic anti-tumor response took place with triplet therapy. Specifically, triplet therapy led to reduction of markers of systemic pathogenic inflammation but activation of anti-tumor cellular immunity (e.g., IFN-γ). In lung (site of 4T1 metastases), Granzyme B expression indicated activated NK cell and cytotoxic T cell infiltration was greatest in Triplet. Tumor expression data for IFN-γ, IL-4, CXCR4, and Perforin have limited interpretability because several animals had complete remissions at the primary tumor. Collectively, quantitative protein and expression immune response data presented here further support advancement of combination testing of AMUN-003 and ICIs in human clinical trials.
Citation Format: Soon Cheon Shin, Beniamin Filimon, Yuefeng Yang, Zebin Hu, Vijayakrishna K Gadi, Weidong Xu. Immune response markers following combination treatment with oncolytic adenovirus AMUN-003 and immune checkpoint inhibitors in a murine model of triple negative breast cancer. [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 5078.