Targeting a Cancer-Specific Epitope of the Epidermal Growth Factor Receptor in Triple-Negative Breast Cancer

Abstract

Background: Triple-negative breast cancers (TNBCs) are typically more aggressive and result in poorer outcomes than other breast cancers because treatment options are limited due to lack of hormone receptors or amplified human epidermal growth factor receptor 2 (HER2). Many TNBCs overexpress the epidermal growth factor receptor (EGFR) or manifest amplification of theEGFRgene, supporting EGFR as a therapeutic target. While EGFR-directed small molecule inhibitors have shown limited effectiveness in clinical settings, use of EGFR as a mechanism of delivering enzymatic cytotoxins to TNBC has not been demonstrated.

Methods: Using the single-chain variable fragment (scFv) of the 806 antibody that binds only cells with overexpressed, misfolded, or mutant variants of the EGFR, a recombinant immunotoxin was engineered through gene fusion withPseudomonas aeruginosaExotoxin A (806-PE38). The potency of 806-PE38 on reducing TNBC cell growth in vitro and in xenograft models (n ≥ 6) was examined for six TNBC cell lines. All statistical tests were two-sided.

Results: 806-PE38 statistically significantly reduced the viability of all tested TNBC lines, with IC50values below 10 ng/mL for three of six cell lines, while not affecting cells with wild-type EGFR (IC50>300 ng/mL). Systemic treatments with 806-PE38 vs vehicle resulted in statistically significantly reduced tumor burdens (806-PE38 mean = 128 mm(3)[SD = 46 mm(3)] vs vehicle mean = 749 mm(3)[SD = 395 mm(3)], P = .001) and increased median survival (806-PE38 median = 82 days vs vehicle median = 50 days,P= .01) in a MDA-MB-468 TNBC mouse xenograft. Deletion of the catalytic residue eliminated both cytotoxic activity in vitro and the reduction in tumor burden and survival (P= .52).

Conclusions: These data support the further development of the 806-PE38 immunotoxin as a therapeutic agent for the treatment of patients with EGFR-positive TNBC. Follow-up experiments with combination therapies will be attempted to achieve full remissions.

Figure 1.

Triple-negative breast cancer cells overexpress epidermal growth factor receptor (EGFR). Cell lysates from triple-negative breast cancer cell lines (BT-20, HCC70, MDA-MB-231, or MDA-MB-468), BRCA1-null (HCC1937, HCC1395) or the control WI-38 fibroblast cell line were probed with α-EGFR antibody to determine levels of EGFR protein. Actin was used as loading control. EGFR = epidermal growth factor receptor.

Figure 2.

The 806-PE38 immunotoxin binds to triple-negative breast cancer cells but not healthy cells. Assessment of immunotoxin binding was performed by flow cytometry. MDA-MB-468 (A), HCC70 (B), or WI-38 (C) cells were incubated with 10 µg/mL of either 806-PE38 (dark gray), 806-PE38Δ553 (light gray trace), or TGFα-PE40 (dashed trace). Bound immunotoxin was detected by the M40-1 anti-PE antibody. M40-1 in the absence of immunotoxin was used as a negative binding control (black trace). α–epidermal growth factor receptor (EGFR) antibody was used to determine total surface EGFR (empty trace). Data is representative of at least two independent replicates. EGFR = epidermal growth factor receptor.

Figure 3.

806-PE38, but not catalytically inactive 806-PE38Δ553, is cytotoxic to triple-negative breast cancer cells. A) BT-20, B) HCC70, C) MDA-MB-231, D) MDA-MB-468, E) HCC1937, F) HCC1395 were incubated with increasing concentrations of either 806-PE38 or 806-PE38Δ553 for 72 hours, at which point cell viability was measured. Data is from at least two independent experiments in triplicate. Error bars display SD value. Two tailed unpaired t tests were performed at each concentration point comparing 806-PE38 with 806-PE38Δ553. *P < .05, †P < .01, ‡P < .001.

Figure 4.

806-PE38 delays tumor growth and enhances survival time in triple-negative breast cancer xenograft models. A) HCC70 cells were implanted in the mammary fat pad of female athymic nude mice. Mice were treated with 806-PE38 (n = 7), 806-PE38Δ553 (n = 7), or vehicle alone (n = 6) 4 x qod, indicated by arrows. Tumor volume was calculated as (0.5 x LxW²). Tumor volumes were compared by unpaired two-tailed t test at each measurement. †P < .01, ‡P < .001. No statistically significant differences were noted between vehicle-treated and Δ553-treated mice. B) Kaplan-Meyer plot showing time to experimental endpoint for each HCC70 tumor-bearing mouse. Mice were killed once tumor volume was greater than 1200 mm³ or tumors became necrotic. Statistical significance was assessed by log-rank test. †P < .01 Number of mice at risk at weekly intervals is noted below. C) MDA-MB-468 cells were implanted in the mammary fat pad of female athymic nude mice. Mice were treated with 806-PE38 (n = 7), 806-PE38Δ553 (n = 7), or vehicle alone (circles) 4 x qod, indicated by arrows. Tumor volume was calculated as (0.5 x LxW²). Tumor volumes were compared by unpaired two-tailed t test at each measurement. *P < .05, †P < .01, ‡P < .001. No statistically significant differences were noted between vehicle-treated and Δ553-treated mice at any time point. D) Kaplan-Meyer plot showing time to experimental endpoint for each MDA-MB-468 tumor-bearing mouse. Mice were killed once tumor volume was greater than 1200 mm³ or tumors became necrotic. Statistical significance was assessed by log-rank test. ‡P < .001. Number of mice at risk at biweekly intervals is noted below the Kaplan-Meyer plot.