Gerald Wallweber

A novel proximity assay for the detection of proteins and protein complexes: quantitation of HER1 and HER2 total protein expression and homodimerization in formalin-fixed, paraffin-embedded cell lines and breast cancer tissue.

The availability of drugs targeting the EGFR/HER/erbB signaling pathway has created a need for diagnostics that accurately predict treatment responses. We have developed and characterized a novel assay to provide sensitive and quantitative measures of HER proteins and homodimers in formalin-fixed, paraffin-embedded (FFPE) cell lines and breast tumor tissues, to test these variables. In the VeraTag assay, HER proteins and homodimers are detected through the release of fluorescent tags conjugated to specific HER antibodies, requiring proximity to a second HER antibody. HER2 protein quantification was normalized to tumor area, and compared to receptor numbers in 12 human tumor cell lines determined by fluorescence-activated cell sorting (FACS), and with HER immunohistochemistry (IHC) test categories and histoscores in cell lines and 170 breast tumors. HER1 and HER2 expression levels determined by the VeraTag assay are proportional to receptor number over more than a 2 log10 range, and HER homodimer levels are consistent with crosslinking and immunoprecipitation results. VeraTag HER2 measurements of breast tumor tissue and cell lines correlate with standard IHC test categories (P<0.001). VeraTag HER2 levels also agree with IHC histoscores at lower HER2 protein levels, but are continuous and overlapping between IHC test categories, extending the dynamic range 5-fold to 10-fold at higher HER2 levels. The VeraTag assay specifically and reproducibly measures HER1 and HER2 protein and homodimers in FFPE tissues. The continuous measure of HER2 protein levels over a broad dynamic range, and the novel HER2 homodimer measure, are presently being assessed as predictive markers for responses to targeted HER2 therapy.

Increased expression of HER2, HER3, and HER2:HER3 heterodimers in HPV-positive HNSCC using a novel proximity-based assay: Implications for targeted therapies

Purpose: In other cancer types, HPV infection has been reported to coincide with overexpression of HER2 (ERBB2) and HER3 (ERBB3); however, the association between HER2 or HER3 expression and dimer formation in HNSCC has not been reported. Overexpression of HER2 and HER3 may contribute to resistance to EGFR inhibitors, including cetuximab, although the contribution of HPV in modulating cetuximab response remains unknown. Determination of heterodimerization of HER receptors is challenging and has not been reported in HNSCC. The present study aimed to determine the expression of HER proteins in HPV+ versus HPV− HNSCC tumors using a proximity-based protein expression assay (VeraTag), and to determine the efficacy of HER-targeting agents in HPV+ and HPV− HNSCC cell lines. Experimental Design: Expression of total HER1, HER2, and HER3, p95HER2, p-HER3, HER1:HER1 homodimers, HER2:HER3 heterodimers, and the HER3–PI3K complex in 88 HNSCC was determined using VeraTag, including 33 baseline tumors from individuals treated in a trial including cetuximab. Inhibition of cell growth and protein activation with cetuximab and afatinib was compared in HPV+ and HPV− cetuximab-resistant cell lines. Results: Expression of total HER2, total HER3, HER2:HER3 heterodimers, and the HER3:PI3K complex were significantly elevated in HPV+ HNSCC. Total EGFR was significantly increased in HPV− HNSCC where VeraTag assay results correlated with IHC. Afatinib significantly inhibited cell growth when compared with cetuximab in the HPV+ and HPV− cetuximab-resistant HNSCC cell lines. Conclusions: These findings suggest that agents targeting multiple HER proteins may be effective in the setting of HPV+ HNSCC and/or cetuximab resistance. Clin Cancer Res; 21(20); 4597–606. ©2015 AACR.

Abstract 3225: Development of a proximity-based immunoassay to measure the PD1:PD-L1 complex in fixed samples

Introduction: The binding of tumor cells expressing PD-L1 to PD1 receptors on activated T-cells inhibits T-cell activation, thereby evading an immune response against tumor progression. Currently, therapeutic monoclonal antibodies directed against either PD1 or PD-L1 block this interaction and restore T-cell function, leading to tumor lysis. PD-L1 immunohistochemistry (IHC) is the most common biomarker assay for these therapies, but disagreement exists on the antibodies used for IHC, which cell types to score, and the criteria for positivity. Quantitative immunofluorescence identified a significant correlation between the proximity of PD1 and PD-L1 protein expression and response to anti-PD1 therapy (Tumeh et al., Nature. 2014; 515:568-71). We have developed a direct quantitative measurement of the PD1:PD-L1 complex for the stratification of patients for anti-PD1 or anti-PD-L1 therapy. The VeraTag PD1:PD-L1 complex immunoassay utilizes two antibody pairs for the proximity-dependent release of a fluorescent reporter (VeraTag), which is measured with high sensitivity via capillary electrophoresis to accurately quantify the amount of complex in fixed samples. Methods: Anti-PD-L1 rabbit mAb E1L3N was selected from our previously established PD-L1 VeraTag immunoassay. Mouse anti-PD1 mAbs were screened against FFPE human tonsil and PBMCs for development of a PD1 VeraTag assay. The VeraTag assay for PD1:PD-L1 complex combined the anti-PD-L1 rabbit mAb E1L3N and the anti-PD1 mouse mAb NAT105 together with a goat anti-rabbit secondary Ab conjugated to the VeraTag reporter and a goat anti-mouse secondary Ab conjugated to biotin. Fixed cell preparations used in the VeraTag PD1:PDL1 complex assay were prepared by co-culturing MB453, MB231 or RKO cancer cell lines expressing varying amounts of PD-L1 with Jurkat T cells. After 48-hours, non-adherent cells were removed by washing and remaining cells were fixed overnight in formalin. Results: Following phytohemagglutinin (PHA) stimulation of human PMBCs and Jurkat cells, VeraTag measurements of PD1 protein expression increased 8- to 10-fold, whereas PD-L1 protein expression varied Conclusions: We have developed sensitive and quantitative assays for PD1, PD-L1 and the PD1:PD-L1 complex, which may provide a more direct measurement of PD1/PD-L1 pathway interaction. Citation Format: Gerald Wallweber, Roy Ravanera, Ahmed Chenna, David Stathas, Weidong Huang, John Winslow, Christos Petropoulos. Development of a proximity-based immunoassay to measure the PD1:PD-L1 complex in fixed samples. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3225.

Abstract LB-281: Development of a sensitive and quantitative PD-L1 immunoassay superior to IHC with application in human FFPE tissue samples

Introduction: Cancer immunotherapy approaches and targets are rapidly expanding for the treatment of many different types of cancer. The programmed cell death-1 receptor (PD-1) and its ligand PD-L1 have garnered a great deal of interest lately, partially due to current therapeutic agents demonstrating a long and durable clinical response with low toxicities in several cancer types. Binding of the PD-1 receptor on activated T-cells to PD-L1 expressing tumor cells reduces T-cell activation, thereby evading an immune response against tumor progression. PD-L1 expression by immunohistochemistry (IHC) has been shown to be a prognostic and potential predictive biomarker for response to both anti-PD-L1 and anti-PD-1 therapy, however, currently the IHC assay is not standardized and the definitions used for positivity are variable and subjective due to a visual scoring system. A lack of sensitivity of the IHC assay may partially explain the observed response to therapy in patients whose tumors were identified as IHC = 0/PD-L1 negative. In an attempt to offer a more sensitive and quantitative assay, we developed a PD-L1 protein expression assay using the VeraTag technology. The PD-L1 VeraTag assay utilizes the release of a unique fluorescent reporter (VeraTag), which is measured with high sensitivity via capillary electrophoresis to accurately and objectively quantify the amount of PD-L1 protein expression in FFPE samples. Methods: The anti-PD-L1 rabbit monoclonal antibody E1L3N (Cell Signaling Technologies) was utilized in the VeraTag assay together with a goat anti-rabbit secondary antibody conjugated to the VeraTag reporter. FFPE cancer cell line lines were used to optimize antigen retrieval, primary antibody concentration and signal/background ratio, with emphasis on the lower end of the dynamic range. Results: VeraTag measurements of PD-L1 protein expression correlated to both IHC and PD-L1 gene expression (CCLE, R squared = 0.7212) in FFPE cancer cell lines. The PD-L1 protein expression by VeraTag and IHC was compared across a group of FFPE squamous cell carcinoma of the head and neck (SCCHN) and HER2- and HER2+ breast samples. VeraTag assays for the measurement of the HER-family of receptors (HER1, HER2 and HER3 total, HER1-HER1 homodimer, HER2-HER3 heterodimer, phospho-HER3 and HER3-PI3 kinase complex) were evaluated for correlation to PD-L1 protein expression in these two cancer types. There was good agreement between the VeraTag and IHC measurements of PD-L1 protein expression, with the added advantage of the VeraTag assay providing an ∼5-fold range of PD-L1 expression within the IHC = 0 category. Conclusions: We have developed a sensitive and quantitative measurement of PD-L1 protein expression in FFPE human SCCHN and breast cancer samples utilizing the VeraTag technology. Measurement of PD-L1 expression from clinical samples with the VeraTag assay is warranted. Citation Format: Gerald Wallweber, Ahmed Chenna, Roy Ravanera, David Stathas, Weidong Huang, Christos Petropoulos. Development of a sensitive and quantitative PD-L1 immunoassay superior to IHC with application in human FFPE tissue samples. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-281. doi:10.1158/1538-7445.AM2015-LB-281