Abstract 3955: Tumor macrophage-mediated antibody dependent cell phagocytosis (ADCP) is theprimary mechanism mediating HER2 mAb (Trastuzumab) anti-tumor responses which can be synergistically enhanced by CD47 innate immune checkpoint blockade
Authors: Tsao Li-Chung , Wei Jun-Ping , Lei Gang-jun , Wang Tao , Yang Xiao-Yi , Liu Cong-Xiao , Lyerly H. Kim , Hartman Zachary C. 
Published: August 2019

Abstract
Approximately 20% of breast cancers (BC) are defined by HER2 overexpression and treated using HER2-specific monoclonal antibodies (mAb), such as Trastuzumab. Previous studies have demonstrated Trastuzumab can limit signaling, elicit antibody dependent cell cytotoxicity (ADCC), antibody dependent phagocytosis (ADCP), and adaptive immune responses to HER2, however its primary therapeutic mechanism of action (MOA) remains unclear. As HER2 mAb efficacy is subverted in advanced cancers, understanding and boosting their MOA is of critical clinical interest and scientific significance for HER2+ BC as well as for other targeted mAbs.
Using a series of fully murine Trastuzumab mAbs of different isotypes, we found that the IgG2A (high A/I) isotype was essential for tumor growth suppression, suggesting an innate immune MOA. In contrast to previous reports, our studies revealed that NK cells, neutrophils, T-cell, or B-cell populations were unnecessary for this anti-tumor MOA. However, we found that a murine IgG2A Trastuzumab (mTras) altered the immune microenvironment in vivo through neutrophil suppression (~4 fold, p&lt;.001) but stimulated a significant expansion (~7 fold, p&lt;.001) and activation of resident tumor associated-macrophages (TAMs). Using DiD dye-labelled tumors, we confirmed that mTras elicited striking HER2-specific ADCP in ~50% of resident TAMs (versus ~16% in control), while in vitro studies confirmed that mTras-mediated macrophage anti-tumor efficacy was dependent upon ADCP, but not ADCC, through activation of FCGR4. Critically, we also found BC CD47 expression suppressed ADCP and resulted in suboptimal mTras anti-tumor efficacy. To explore this innate resistance pathway, we utilized CD47 KO BC lines and CD47 mAbs and found that both significantly enhanced mTras-mediated ADCP in vitro (&gt;4 fold, p&lt;.01) without altering ADCC activity. Moreover, we also found that this combination significantly enhanced TAM expansion and activation in vivo, which significantly enhanced human HER2+ BC anti-tumor efficacy (88% complete regression vs. 0% for single agent) and prolonged survival in multiple murine HER2+ BC models, including an endogenous treatment-resistant HER2+ BC mouse model.
Thus, our study demonstrates the primary MOA of a clinically relevant HER2 mAb requires engagement with TAMs to stimulate their expansion and elicit ADCP, which could be significantly enhanced by CD47-SIRPa blockade. This suggests that the strategic use of CD47-SIRPa innate blockade may allow for significant enhancement of anti-tumor immunity in combination with other cancer antigen targeting mAb therapies (targeting EGFR, CD20, etc) as a more effective strategy to stimulate anti-tumor immune responses in advanced immunosuppressive cancers.
Citation Format: Li-Chung Tsao, Jun-Ping Wei, Gang-jun Lei, Tao Wang, Xiao-Yi Yang, Cong-Xiao Liu, H. Kim Lyerly, Zachary C. Hartman. Tumor macrophage-mediated antibody dependent cell phagocytosis (ADCP) is theprimary mechanism mediating HER2 mAb (Trastuzumab) anti-tumor responses which can be synergistically enhanced by CD47 innate immune checkpoint blockade [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3955.