Abstract 4892: MMAE drives immunomodulatory changes in a preclinical xenograft model that are distinct from other clinical-stage ADC payloads
Authors: Ulrich Michelle , Klussman Kerry , Gosink John J. , Allred Sean , Hensley Kelly , Treuting Piper M. , Parekh Nikhil J. , Gray Elizabeth E. , Gardai Shyra J. 
Published: June 2023

Abstract
Monomethyl auristatin E (MMAE), the payload delivered by vedotin ADCs, exerts its cytotoxic effects via microtubule disruption and induction of ER stress, leading to apoptosis and tumor cell death. In addition, MMAE also has the ability to induce immunogenic cell death (ICD) and immune modulation in the tumor microenvironment (TME) (Liu, 2020; Gray, 2020). Other clinical-stage and approved ADCs incorporate payloads that also cause microtubule disruption (DM1, DM4), or that drive DNA damage through topoisomerase I inhibition (exatecan) as the primary mechanism of action (MOA). While ADCs with different linker payloads produce clinical benefit, their long-term impact on survival and how they pair with PD(L)1 agents may differ based on their ability to elicit immune modulation. In this study we have investigated how these payloads compare to MMAE in their ability to drive immune changes in the TME and describe how these changes could support anti-tumor immunity and improve outcomes in the clinic.Building upon an initial in vitro assessment in which we observed superior induction of ICD markers and underlying ER stress with an MMAE ADC, we utilized a B7H4-expressing MDA-MB-468 xenograft model of triple-negative breast cancer to further characterize the ICD properties of ADC payloads. Tumor-bearing mice were treated with a single dose of B7H4-targeted antibody conjugated to either MMAE, DM1, DM4 or exatecan. Tumors were harvested seven days after treatment and processed for immunohistochemistry (IHC) to assess changes in immune cell infiltration and analyzed by RNA-seq to determine alterations in gene expression. Payload-driven anti-tumor activity was observed with all ADC treatments, each leading to a similar reduction in tumor volume compared to vehicle or unconjugated antibody. IHC staining of tumor sections for F4/80, a mouse macrophage marker, revealed that of the microtubule-disrupting agents, only MMAE significantly increased F4/80+ macrophage infiltration in both the tumor nests and stroma. Increased F4/80+ macrophages in tumor nests and stroma was also observed as a result of B7H4-exatecan ADC treatment. RNA-seq analysis revealed a unique profile for the MMAE conjugate, with an increase in human transcripts encoding cytokine and type I interferon response genes. In summary, the anti-tumor activity of the B7H4-MMAE ADC resulted in recruitment of mouse innate immune cells to the xenograft tumors. Importantly, immune response genes related to ICD were increased following B7H4-MMAE treatment compared to DM1, DM4, and exatecan conjugates. Together, these data suggest that treatment with vedotin ADCs results in robust immunomodulatory changes in vivo that are distinct from other clinical ADC payloads.
Citation Format: Michelle Ulrich, Kerry Klussman, John J. Gosink, Sean Allred, Kelly Hensley, Piper M. Treuting, Nikhil J. Parekh, Elizabeth E. Gray, Shyra J. Gardai. MMAE drives immunomodulatory changes in a preclinical xenograft model that are distinct from other clinical-stage ADC payloads. [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 4892.