American Association for Cancer Research
10780432ccr202007-sup-243973_3_supp_6649713_qz7w5z.pdf (674.2 kB)

Supplementary Figure 2 from Effects of HER Family–targeting Tyrosine Kinase Inhibitors on Antibody-dependent Cell-mediated Cytotoxicity in HER2-expressing Breast Cancer

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journal contribution
posted on 2023-03-31, 22:30 authored by Denis M. Collins, Stephen F. Madden, Nicola Gaynor, Dalal AlSultan, Marion Le Gal, Alex J. Eustace, Kathy A. Gately, Clare Hughes, Anthony M. Davies, Thamir Mahgoub, Jo Ballot, Sinead Toomey, Darran P. O'Connor, William M. Gallagher, Frankie A. Holmes, Virginia Espina, Lance Liotta, Bryan T. Hennessy, Kenneth J. O'Byrne, Max Hasmann, Birgit Bossenmaier, Norma O'Donovan, John Crown

Flow cytometry control scatter plots for trastuzumab-488 and pertuzumab-550


Caroline Foundation

Cancer Clinical Research Trust

Irish Research Council Enterprise Partnership Scheme

Health Research Board

Science Foundation Ireland-funded Molecular Therapeutics for Cancer Ireland

Irish Cancer Society Collaborative Cancer Research Centre BREAST-PREDICT

Roche Products Ireland Ltd



Antibody-dependent cell-mediated cytotoxicity (ADCC) is one mechanism of action of the monoclonal antibody (mAb) therapies trastuzumab and pertuzumab. Tyrosine kinase inhibitors (TKIs), like lapatinib, may have added therapeutic value in combination with mAbs through enhanced ADCC activity. Using clinical data, we examined the impact of lapatinib on HER2/EGFR expression levels and natural killer (NK) cell gene signatures. We investigated the ability of three TKIs (lapatinib, afatinib, and neratinib) to alter HER2/immune-related protein levels in preclinical models of HER2-positive (HER2+) and HER2-low breast cancer, and the subsequent effects on trastuzumab/pertuzumab-mediated ADCC. Preclinical studies (proliferation assays, Western blotting, high content analysis, and flow cytometry) employed HER2+ (SKBR3 and HCC1954) and HER2-low (MCF-7, T47D, CAMA-1, and CAL-51) breast cancer cell lines. NCT00524303 provided reverse phase protein array–determined protein levels of HER2/pHER2/EGFR/pEGFR. RNA-based NK cell gene signatures (CIBERSORT/MCP-counter) post-neoadjuvant anti-HER2 therapy were assessed (NCT00769470/NCT01485926). ADCC assays utilized flow cytometry–based protocols. Lapatinib significantly increased membrane HER2 levels, while afatinib and neratinib significantly decreased levels in all preclinical models. Single-agent lapatinib increased HER2 or EGFR levels in 10 of 11 (91%) tumor samples. NK cell signatures increased posttherapy (P = 0.03) and associated with trastuzumab response (P = 0.01). TKI treatment altered mAb-induced NK cell–mediated ADCC in vitro, but it did not consistently correlate with HER2 expression in HER2+ or HER2-low models. The ADCC response to trastuzumab and pertuzumab combined did not exceed either mAb alone. TKIs differentially alter tumor cell phenotype which can impact NK cell–mediated response to coadministered antibody therapies. mAb-induced ADCC response is relevant when rationalizing combinations for clinical investigation.