Background: Folate receptor α is a GPI-anchored cell surface protein that mediates the intracellular transport of folates. It is over-expressed in a number of malignancies, including ovarian carcinoma and mesothelioma and has limited normal tissue expression. More recently it has been found to be over-expressed in breast cancer, and particularly a less favourable prognostic form, known as ‘triple-negative’ breast cancer (TNBC). We examined a therapeutic antibody candidate recognising FRα; which we are preparing for Phase 1 clinical trials for its potential to target human immune effector cells against breast cancer cells. As part of this effort, we endeavoured to identify a breast cancer cell line model in which the ability for this antibody to induce Antibody-dependent Cell-mediated breast tumour cell death could be evaluated in vitro.
Methods: Twenty-two breast cancer cell lines were evaluated by flow cytometry for surface expression of FRα using the anti-FRα antibody compared with IGROV1, a well characterised ovarian cancer cell line used as a positive control. We confirmed these findings with mRNA levels for cell lines available from the publically accessible Cancer Cell Line Encyclopaedia (CCLE). The two highest expressing breast cancer cell lines, T47D and CAL51 were then studied in a functional flow cytometric assay which can simultaneously measure antibody-dependent cell-mediated cytotoxicity (ADCC) and phagocytosis (ADCP). The human monocytic U937 cells which express Fc receptors for IgG and IgE antibodies, served as effectors. We compared the capacity of the the anti-FRα antibodies (IgG1 and IgE isotypes) to activate monocytic cells against the FRα-expressing ovarian carcinoma IGROV1 cells, the breast carcinoma T47D and CAL51 cells and the FRα non-expressing breast cancer cell line, HCC1428.
Results: There was wide variety in the levels of cell surface expression of FRα; by breast cancer cell lines, expressed as Mean Fluorescent Intensity (>than an isotype control). The highest expressing breast cancer cell lines, T47D and CAL51 had values of 1273 (SEM 13.6) and 1638 (SEM 24.7) respectively in comparison with the non-expressing cell line, HCC1428 with a value of 8 (SEM 0.54). Expression levels in the ovarian cancer cell line, IGROV1 were 16580 (SEM 2534). These values correlated well with mRNA levels from the CCLE database (Spearman's rank, r=0.6011, p=0.0065) which varied from 1x104-1x1011 amongst breast cancer cell lines but were still below those detected with IGROV1 ovarian cancer cells with an mRNA level of 7x1012. We detected no clear evidence of expressing/non-expressing cell lines clustering amongst any known molecular subtype of TNBC.
In vitro functional assay assessments of breast carcinoma cell killing in the non-FRα expressing cell line, HCC1428, revealed that neither IgG nor IgE induced breast cancer cell cytotoxicity above that induced by a non-targeting isotype antibody control. In contrast anti-FRα IgG1 and IgE antibodies could induce 20-30% more ADCP and ADCC, respectively, of the FRα+ IGROV1 tumour cells than the isotype control. The breast carcinoma T47D cells, likewise, were killed by ADCP when exposed to anti-FRα IgG, while the anti-FRα IgE antibody induced high levels of ADCC of ~40% above isotype control. Furthermore, treatment of breast carcinoma cells with anti-FRα antibodies induced direct signalling effects that may lead to reduced growth and survival cascades.
Conclusions: We have identified two breast cancer cell line models which could be candidates for further in vitro and in vivo assessment of the efficacy and mechanisms of the novel anti-FRα; IgE against breast carcinomas. We have demonstrated similar levels of human immune effector cell-mediated breast cancer cell killing in one of these cell lines, T47D, to a well characterised ovarian cancer cell line, IGROV1, not seen in the FRα; non-expressing cell line, HCC1428. This tumour antigen warrants further characterisation as a potential new immune therapy in TNBC, where immune infiltration is already known to be favourably prognostic.
Citation Format: Matthew W. Fittall, Anthony Cheung, Gyula M. Petranyi, Diana Rodriguez-Dominguez, Heather J. Bax, Panagiotis Karagiannis, Kristina M. Ilieva, Andrew Tutt, Sophia N. Karagiannis. Exploring folate receptor α immunotherapy of breast carcinomas: Human monocytic cell-mediated killing triggered by IgG1 and IgE antibodies. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A090.