Monitoring of immune responses to CA125 with an IFN-γ ELISPOT assay

Abstract

The CA125 (carcinoma antigen 125)-specific antibody MAb-B43.13 (OvaRex® MAb-B43.13) is in clinical trials for patients with ovarian carcinoma as an immunotherapeutic agent. To develop an assay for monitoring CA125-specific as well as tumor-specific T cells in the peripheral circulation of ovarian cancer patients treated with this antibody, the IFN (interferon)-γ enzyme-linked immuno SPOT (ELISPOT) assay was optimized. Various assay formats and experimental conditions were evaluated, using peripheral blood mononuclear cells (PBMC) obtained from normal donors and patients with ovarian cancer. T cell proliferation assays were performed in parallel. Conditions tested included different types of antigen presenting cells (APC), the need for and length of in vitro sensitization to enrich in T cell precursors, titration of the antigen and comparison of pulsing dendritic cells (DC) with immune complexes (IC) of CA125 and monoclonal antibody (MAb)-B43.13 or CA125 antigen alone. Proliferation assays were found not to be sufficiently sensitive for detection of CA125-specific T cells in the peripheral circulation of normal donors. A more sensitive 24-h direct ELISPOT assay was also negative. However, ELISPOT assays preceded by a 7-day in vitro sensitization (IVS) step allowed the detection of CA125 antigen-specific T cells in the circulation of normal donors and patients with ovarian cancer. The use of DC as APC was compared to whole PBMC or immortalized B cells. Most donors and ovarian cancer patients responded best to DC pulsed with CA125 in the form of immune complexes. Optimal responses were seen using CA125 concentrations of 500 U/ml and 5 μg/ml of MAb-B43.13. The data indicate that precursor cells specific for CA125 antigen are present at low frequencies in the peripheral circulation of normal donors and patients with ovarian carcinoma and need to be expanded ex vivo for frequency determinations. The optimized assay is able to detect increases in T cell precursor frequencies to CA125 in ovarian cancer patients after immunization with MAb-B43.13.