Enhanced tumor control with a combination of APG-157 and immune checkpoint inhibitors for head and neck cancer.
Authors: Shin Daniel Sanghoon , Basak Saroj K. , Veena Mysore S. , Comin-Anduix Begoña , Bhattachrya Arjun , Dong Tien , Avila Luis Z. , Wang Marilene , Srivatsan Eri 
Published: June 2022

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Background: Natural botanical drugs containing flavonoids are under clinical investigation for the treatment of malignancies. A phase I clinical study for patients with locally advanced head and neck cancer with a new class of botanical drug APG-157 showed excellent tolerability, systemic absorption via sublingual administration, and enhanced immune cell infiltration into the tumor microenvironment and suppression of T cell inhibitory inflammatory cytokine production. We sought to evaluate the efficacy of tumor control for the combination of APG-157 and checkpoint blockade immunotherapy using a head and neck cancer mouse model. Methods: SCCVII (HPV-) and MEER (HPV+) head and neck cancer murine cell line models were utilized in in vivo tumor growth study. After optimization for route and cell number, we measured tumor growth after treating mice with following groups: i) control, ii) APG-157, iii) anti-PD-1, iv) anti-CTLA-4, v) APG-157 + anti-PD-1, vi) APG-157 + anti-CTLA-4, vii) anti-PD-1 and anti-CTLA-4, viii) APG-157 + anti-PD-1 + anti-CTLA-4. Tumors were collected after 2 injections of immune checkpoint inhibitors and characterized for immunophenotype by multi-color flow cytometry, immunohistochemistry, and RNA-sequence analysis. Fecal microbiome was analyzed by 16S rRNA sequence for SCCVII model to assess the impact of APG-157 on the microbiome as we observed the shift of oral microbiome species in the human trial. Results: We confirmed by serum concentration of APG-157 metabolites that the best route of administration is via oral route by mixing with APG-157 with mouse chow. Tumor growth optimization experiments determined that 100,000 cells of SCCVII is the ideal number of cells to inject and day 5-6 is the time to initiate treatment. Among 8 treatment groups, APG-157 + anti-CTLA-4 demonstrated best tumor control (p = 0.0065 compared to control), followed by anti-PD-1 + anti-CTLA-4 treatment group (p = 0.48 compared to control). The immunophenotype assessment by flow cytometry showed over 30% of CD8+ T cell in APG-157 + anti-CTLA-4 group compared to 4-5% of CD8+ T cell for the control group. GSEA analysis from the SCCVII tumors revealed that APG-157 + anti-CTLA-4 group showed an enriched set of genes for inflammatory response, apoptotic signaling pathways. The fecal microbiome analysis showed a substantial difference of lactobacillus genus among groups, highest in the group of APG-157 + anti-CTLA-4 treatment group. The MEER model (HPV positive) tested with same immunotherapy as single or in combination with APG-157 did not grow the tumor, thus we were unable to collect the tumor for immunophenotyping and other studies. Conclusions: These study results indicate that APG-157 is able to modulate the immune system and fecal microbial species that could potentially lead to improved anti-tumor immunity and warrants further studies to define mechanism and the potential for human clinical trial.