Abstract 1285: Tumor associated macrophages can process antibody-drug conjugates and contribute to antitumor activity in preclinical xenograft models
Authors: Li Fu , Ulrich Michelle , Jonas Mechthild , Linares Germein , Zhang Xinqun , Westendorf Lori , Benjamin Dennis , Law Che-Leung 
Published: July 2016

Abstract
The primary mechanism of antibody-drug conjugates (ADCs) is the targeted delivery of a cytotoxic payload via cancer antigen mediated internalization. However, stromal components in the tumor microenvironment may also play a role in ADC penetration, distribution, and processing. Here, we study the potential roles of Fc-FcγR interaction between tumor-associated macrophages (TAMs) and ADCs in the antitumor activity observed in xenograft models.
In the CD30+ L428 Hodgkin lymphoma (HL) model, the anti-CD30 ADC (cAC10-vcMMAE) and a non-binding control ADC (h00-vcMMAE) showed similar antitumor activity over a two-week period post treatment initiation. The antitumor activity was correlated with payload release, as h00-vcMMAE produced intratumoral MMAE concentration comparable to cAC10-vcMMAE in this time frame. Histopathology analysis of L-428 xenografts revealed high abundance of TAMs, leading to the hypothesis that TAMs can internalize and process ADCs for payload release. Using immunohistochemistry and flow cytometry, we confirmed that h00-vcMMAE bound to macrophage cell lines and TAMs. We further examined the presence of TAMs in additional xenograft models and correlated that to the antitumor activity of non-binding h00-vcMMAE. High levels of TAMs were observed in the KM-H2 HL and BR620 breast cancer models that were sensitive to h00-vcMMAE. In contrast, xenograft models with much fewer TAMs (DOHH2, SU-DHL8, and Karpas-299) did not respond to h00-vcMMAE treatment. Interestingly, h00-vcMMAF, releasing a membrane non-permeable payload, had no activity on the L-428 tumor model. These data suggest TAM-processed drug can mediate bystander tumor killing when the released payload is membrane permeable.
To evaluate whether Fc-FcγR interaction plays a role in the ADC uptake by TAMs, we mutated the Fc region of h00-vcMMAE to decrease FcγR binding affinity (E233P:L234V:L235, G1V1). h00G1V1-vcMMAE lost its cytotoxicity activity in FcγR+ THP-1 monocytes. Furthermore, h00G1V1-vcMMAE could no longer mediate tumor regression or growth delay in three xenograft models that are sensitive to h00-vcMMAE treatment. These results suggest ADC-FcγR interaction is required for ADC processing by TAMs in these xenografts.
These results suggest that TAMs can contribute to ADC processing through FcγR interaction in preclinical tumor models. Moreover, TAM-processed membrane permeable payload can mediate bystander tumor cell killing and contribute to ADC activity. Although this phenomenon may be an additional mechanism of ADCs in vivo, whether TAMs play a role in patients’ response to ADCs requires further correlated studies in clinical trials.
Citation Format: Fu Li, Michelle Ulrich, Mechthild Jonas, Germein Linares, Xinqun Zhang, Lori Westendorf, Dennis Benjamin, Che-Leung Law. Tumor associated macrophages can process antibody-drug conjugates and contribute to antitumor activity in preclinical xenograft models. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1285.