Abstract P041: Simultaneous checkpoint inhibition and immune cell activation that is safely localized to solid tumors
Authors: Richieri Richard A. , Narula Navneet , Loomis Cynthia A. , Mezzano Valeria , Billimek John , Reynolds Glenn T. , Reutelingsperger Chris , Zijlstra Andries , Parseghian Missag H. 
Published: January 2022

Abstract
Unlike other checkpoint inhibitors, our targeted immunotherapeutic localizes to any solid tumor and simultaneously shields an agent of immunosuppression while presenting a signal for immunostimulation. Phosphatidylserine (PS) exposure on the extracellular surface of living tumor cells and their vasculatures provides one avenue by which the tumor microenvironment promotes immunosuppression. Extracellular surface PS is inherent to a tumor and its vasculature, even for inoperable tumors, and its expression cannot be mutated nor affected by acquired drug resistance. Annexin A5 (AnxA5) is a direct, high-affinity PS-binding protein that localizes to cells with PS exposed on the outer plasma membrane. In our studies, we conjugated a proprietary modified AnxA5, lacking cellular internalization, to TNFα (AnxA5MOD-TNFα) to convert the immunosuppresive environs of a murine 4T1 triple negative breast cancer (TNBC) into an immunostimulated one. This strategy localized the immune response to the tumor and minimized side effects, as evidenced by a lack of toxicity for up to 7 days in non-tumor bearing Balb/c female mice given up to 1 mg/kg. Proper assembly and functionality of AnxA5MOD-TNFα was verified simultaneously by ellipsometry, an optical technique similar to plasmon resonance. Fully assembled constructs were tested for binding to PS coated slides. The degree of light polarization is proportional to the amount of PS bound by the AnxA5 complex. Samples could be further incubated with TNF receptors to verify TNFα activity. Based on dose escalation studies in 4T1 tumor-bearing mice where the TNBC tumors were grown in the mammary fat pads, optimal dosages were determined for AnxA5MOD-TNFα (18 µg) and AnxA5MOD alone as a control (180 µg). These doses were further tested in a 4T1 growth inhibition study. Tumor size was tracked by caliper in two groups of mice (n=5/group) receiving drug treatment on days 12, 14 and 16 and a repeated measures ANOVA was conducted on measurements taken before, during and post-treatment. While median tumor size did not differ between control and drug treatment groups during the pre-treatment interval (p=0.84), there was a significant difference post-treatment (p&lt;0.001) with mice receiving AnxA5MOD-TNFα having much smaller TNBC tumors. Tumors from the study were embedded in paraffin, sectioned (5 µm) and the overall immune cell content determined by H&E staining. Once it was evident there was a greater quantity of immune cells in AnxA5MOD-TNFα treated tumors vs. controls, sections were stained with validated antibodies to identify and count the immunoactivated T-cells, NK-cells and macrophages. There was a 3X greater mean percentage of CD8 and CD4 T-cells in mice receiving drug vs. control (p=0.03) along with 2.5X and 5X increases in NK-cells and M1 immunoactive macrophages, respectively. Conclusion: Our AnxA5MOD-TNFα inhibits the PS inhibitor while simultaneously activating TNF activators!
Citation Format: Richard A. Richieri, Navneet Narula, Cynthia A. Loomis, Valeria Mezzano, John Billimek, Glenn T. Reynolds, Chris Reutelingsperger, Andries Zijlstra, Missag H. Parseghian. Simultaneous checkpoint inhibition and immune cell activation that is safely localized to solid tumors [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2021 Oct 5-6. Philadelphia (PA): AACR; Cancer Immunol Res 2022;10(1 Suppl):Abstract nr P041.