Trastuzumab/pertuzumab combination therapy stimulates antitumor responses through complement-dependent cytotoxicity and phagocytosis
Li-Chung Tsao, Erika J. Crosby, Timothy N. Trotter, Junping Wei, Tao Wang, Xiao Yang, Amanda N. Summers, Gangjun Lei, Christopher A. Rabiola, Lewis A. Chodosh, William J. Muller, Herbert Kim Lyerly, Zachary C. Hartman
Li-Chung Tsao, Erika J. Crosby, Timothy N. Trotter, Junping Wei, Tao Wang, Xiao Yang, Amanda N. Summers, Gangjun Lei, Christopher A. Rabiola, Lewis A. Chodosh, William J. Muller, Herbert Kim Lyerly, Zachary C. Hartman
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Research Article Oncology Therapeutics

Trastuzumab/pertuzumab combination therapy stimulates antitumor responses through complement-dependent cytotoxicity and phagocytosis

Abstract

Two HER2-specific mAbs, trastuzumab and pertuzumab (T+P), combined with chemotherapy comprise standard-of-care treatment for advanced HER2+ breast cancers (BC). While this antibody combination is highly effective, its synergistic mechanism-of-action (MOA) remains incompletely understood. Past studies have suggested that the synergy underlying this combination occurs through the different mechanisms elicited by these antibodies, with pertuzumab suppressing HER2 heterodimerization and trastuzumab inducing antitumor immunity. However, in vivo evidence for this synergy is lacking. In this study, we found that the therapeutic efficacy elicited by their combination occurs through their joint ability to activate the classical complement pathway, resulting in both complement-dependent cytotoxicity and complement-dependent cellular phagocytosis of HER2+ tumors. We also demonstrate that tumor C1q expression is positively associated with survival outcome in HER2+ BC patients and that complement regulators CD55 and CD59 were inversely correlated with outcome, suggesting the clinical importance of complement activity. Accordingly, inhibition of C1q in mice abolished the synergistic therapeutic activity of T+P therapy, whereas knockdown of CD55 and CD59 expression enhanced T+P efficacy. In summary, our study identifies classical complement activation as a significant antitumor MOA for T+P therapy that may be functionally enhanced to potentially augment clinical therapeutic efficacy.

Authors

Li-Chung Tsao, Erika J. Crosby, Timothy N. Trotter, Junping Wei, Tao Wang, Xiao Yang, Amanda N. Summers, Gangjun Lei, Christopher A. Rabiola, Lewis A. Chodosh, William J. Muller, Herbert Kim Lyerly, Zachary C. Hartman

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Figure 5

T+P combination therapy boosts tumor phagocytosis through complement activation and tumor opsonization.

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T+P combination therapy boosts tumor phagocytosis through complement act...
(A) HER2 mAb–induced antibody-dependent cellular phagocytosis (ADCP) of HER2+ BC cells by mouse BM-derived macrophages (BMDM) in vitro. KPL-4 or BT-474 cells were labeled with Brilliant Violet 450 Dye (BV450), and cocultured with BMDM with control or HER2 mAbs (10 μg/mL). ADCP rates were measured by percentage of BMDM uptake of labeled tumor cells (CD45+ and BV450+). n = 3. (B) In vivo ADCP experiment. KPL-4 cells were labeled with Vybrant DiD dye and implanted (1 × 106 cells) into mammary fat pads of SCID-beige mice. Once tumor volume reached ~300 mm3, mice were treated with HER2 mAbs (200 μg each) or control IgG. The next day, tumors were harvested and analyzed by FACS. TAMs that had phagocytosed DiD-labeled tumor cells were quantified for ADCP. (C) Tumor-associated macrophages (TAMs), identified as CD11b+F480+LY6G–LY6C–, were quantified. n = 7–8; 1-way ANOVA with Tukey’s multiple-comparison post hoc test. (D) HER2 mAb–induced C3b deposition is dependent on C1q activation. KPL-4 cells were incubated with HER2 mAbs and NHS to promote C3b deposition as described in Figure 3. Where indicated, cells were treated with or without C1 inhibitor (C1-INH) to inhibit C1 activation and C3b deposition. n = 3. (E) C3b-opsonized antibody-bound KPL-4 in D were cocultured with human macrophages to assess complement-enhanced ADCP. n = 3. (F) KPL-4 cells were cultured with BMDMs derived from WT or C1q-deficient mice, and ADCP activity with HER2 mAbs were quantified as before. n = 3. (A and D–F) Data are shown as mean ± SEM, 2-way ANOVA with Tukey’s multiple-comparison post hoc test.