Carcinoembryonic antigen (CEA) recognition by T lymphocytes after peptide or heteroclitic peptide stimulation

Abstract

4705

Introduction: The carcinoembryonic antigen (CEA)-derived heteroclitic peptide CAP1-6D has been proposed as a more efficient stimulator of HLA-A2 restricted CEA-specific T cells in vivo than the native CAP1 peptide. We investigated the relative prevalence, peptide/heteroclitic peptide cross-reactivity, and avidity of T cells from normal donors following in vitro stimulation (IVS) with CAP1 or CAP1-6D peptide. Methods: Peripheral blood mononuclear cells (PBMC) from normal donors were stimulated in complete media, IL-2 (30 iu/ml) and IL-7 (10ng/ml) with peptide (10 mcg/ml). At culture day 7, 5x10e3 cell aliquots (semi-clones) were re-stimulated with 1x10e5 irradiated autologous peptide-loaded PBMC and expanded in IL-2 (30iu/ml). Semi-clones were tested for reactivity after 4, 5,and 6 stimulations by cytokine release assays using peptide-loaded T2 cell targets. Reactive semi-clones were re-stimulated 4 additional times, then rapidly expanded in complete media using anti-CD3 antibody (30 ng/ml), IL-2 (600 iu/ml), and irradiated allogeneic PBMC at 500:1. The avidity of reactive cultures was measured by cytokine release assays using T2 cell targets loaded with limiting concentrations of peptide (10 pg –10 mcg/ml). Results: CAP1 and CAP1-6D reactive semi-clone cultures were identified from 4/5 normal donors after 6 stimulations. The number of CAP1 reactive semi-clones among 96 cultures was 2, 8, 16, and 41 for the 4 donors. The number of CAP1-6D reactive semi-clones among 96 cultures was 7, 1, 17, and 5 for the same donors. Cross-reactivity was tested in 3 semi-clone populations: reactivity against CAP1-6D was seen in 6/11 and 0/24 CAP1 reactive cultures from 2 donors. Reactivity against CAP1 was seen in 0/24 CAP1-6D reactive cultures from 1 donor. Avidity determined by half-maximum stimulation of cultures was achieved at target-loading peptide concentrations of 1-10 ng/ml (n=2), 10-100 ng/ml (n=1), or 100-1000 ng/ml (n=3). Summary and Conclusions: T cell cultures recognizing CAP1 and CAP1-6D can be generated by IVS from normal donors. When stimulated by either antigen in vitro, many of the reactive cultures do not recognize the other antigen. The avidity of these cells for the self-peptide CAP1 is broad, ranging from moderate to weak. These data suggest that 1) CAP1-6D based immune therapy may not be optimal in some patients with CAP1 specific precursors and / or 2) the T cell precursor population may differ between patients and normal donors due to the presence of the antigen-bearing tumor.