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

Abstract

Purpose: 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.

Experimental design: 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.

Results: 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.

Conclusions: 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.

Figure 1.

A. Total HER2 and EGFR, HER3, HER4 expression by Western blot in HER2-amplified SKBR3 and HCC1954, and HER2-low MCF-7, T47D, CAMA-1 and CAL-51 breast cancer cell lines. All samples at 30μg except SKBR3 and HCC1954 (2.5μg). Relative HER2 expression determined by densitometry on triplicate blots. B. Proliferation for trastuzumab (T), pertuzumab (P) and combination (T/P) in the cell line panel. All data determined in triplicate ± standard deviation (StdDev). * p<0.05 vs. untreated control (100%). C. IC₅₀ values for lapatinib (LAP), afatinib (AFAT) and neratinib (NER) ordered by ascending LAP resistance. CAMA-1 IC₅₀ LAP > 10,000 nM, SKBR3 IC₅₀ NER < 0.01nM. All data in triplicate ± StdDev. D. Total HER2 expression by Western blot following 48 hour treatment with 2μM LAP, AFAT, NER, 0.2% DMSO or medium (MED). Blots shown are representative of three independent experiments.

Figure 2

A. Frequency bar charts displaying matched pre- and post-treatment tumor samples from LPT109096 showing an increase or decrease in protein levels post-two weeks of treatment with trastuzumab, lapatinib or both. Readouts for each target were not available for every sample leading to the variation in total numbers. The difference between treatment arms did not reach significance (Fisher’s Exact test). B. Impact of 48 hour treatment with 10μg/ml trastuzumab (T), 2μM lapatinib (L) or the combination on levels of total and phosphorylated (p) HER2/EGFR expression in SKBR3 and HCC1954. Densitometry for HER2 shown. C. High content analysis of membrane HER2 expression in SKBR3, HCC1954, T47D and MCF-7 following 48 hour TKI treatments using an ECD-targeted antibody. Triplicate data, normalised to untreated control +/− StdDev. *p< 0.05 relative to DMSO control.

Figure 3

Trastuzumab (T)-, pertuzumab (P)- and trastuzumab + pertuzumab (T+P) – mediated ADCC in SKBR3, HCC1954, T47D, MCF-7, CAL-51 and CAMA-1 following 48 hour TKI (2 μM) treatments. Results shown represent PBMC:target ratios of 5:1. Results of three separate experiments +/− StdDev.*p< 0.05 relative to the 0.2% DMSO control, † p<0.05 relative to lapatinib-treated cells.

Figure 4.

A. Activated NK cell gene signature fractions (Cibersort) from matched pre- and post-treatment (two weeks of trastuzumab (H), lapatinib (L) or both) TRIO-B07 tumor samples overall, and by treatment arm B. Post-treatment samples from Figure 4A broken down by pathological complete response (pCR), partial response (PR) or non-response (NR) to subsequent docetaxel/carboplatin (TC)H, TCL or TCHL therapy C. MCP-counter analysis of NK cell gene signature levels in ICORG 10–05 patient samples (n=8) by pre-treatment and post-cycle 1 of therapy (TCH/TCL/TCHL). Each individual sample examined showed an increase in NK cell score on-treatment. p< 0.05 is significant and corrected.

Figure 5.

A. Direct NK cell cytotoxicity against HER2-positive (SKBR3, HCC1954) and HER2-low (MCF-7, T47D) cell lines following treatment with lapatinib, afatinib and neratinib (2μM) for 48 hours. Three ratios of NK cells to target cells were examined. B. NK cell-mediated ADCC induced by trastuzumab (T), pertuzumab (P) or trastuzumab plus pertuzumab (T+P) following treatment with lapatinib, afatinib or neratinib (2μM) for 48 hours. Three ratios of NK cells to target cells were examined with the 1:1 ratio shown for SKBR3, HCC1954 and MCF-7. Only the 0.5:1 ratio was available for the T47D cell line due to sensitivity to direct NK cell cytotoxicity. * p< 0.05 is significant vs. DMSO-treated control, † p<0.05 significant relative to lapatinib-treated cells.

Figure 6.

A. The average % cell number staining positive for trastuzumab (T), pertuzumab (P), trastuzumab and pertuzumab (T+P) or those remaining unlabeled immediately after pre-treatment (T0) with TKIs (2μM) for 48 hours. Results show the average of three independent experiments. Original data for co-exposure and each individual mAb +/− StdDev and p values can be found in Supplementary Table 1. B. pHER2, HER3, pHER3 and EGFR expression following treatment with medium (M), DMSO (D), or 2μM lapatinib (L), afatinib (A) or neratinib (N). All samples at 30μg. C. MHC Class I, HLA-E and PD-L1 expression by Western blot in all cell lines examined in this study. All samples at 30μg. MHC Class I, HLA-E and PD-L1 expression levels following treatment with TKIs (2μM for 48 hours) in SKBR3 and HCC1954 (D) and MCF-7 and T47D (E). All blots carried out in triplicate.