Tumor-Specific Chimeric Receptor-Mediated T Cell Activation Is Enhanced by 2B4 Signaling Via an NFκB-Independent Mechanism.

Genetic modification of polyclonal T cells with tumor antigen-specific chimeric receptors (chRec) specifically redirects their effector functions towards tumor cells. However, the therapeutic value of chRec-gene-modified T cells is limited, since T cell activation by chRec fails to mediate proliferative responses, probably due to the lack of costimulatory T cell signaling in response to tumor cells. The costimulatory signaling domain of CD28 has been shown to enhance chimeric-receptor mediated proliferation in polyclonal T cell populations but not in CD8+ effector memory CTL, which represent the primary mediators of effective antitumor immunity. Indeed, alternative costimulatory molecules are now recognised as having complementary roles in the activation of antigen-experienced effector T cells. The SLAM-related receptor 2B4 (CD244), which is expressed on NK cells and on CD8+ cytotoxic T cells, is known to positively regulate T-cell-mediated cytotoxicity. We hypothesized that inclusion of the 2B4 endodomain into the chRec would enhance tumor-specific activation and proliferation of in vitro expanded effector T cells. We focused our studies on a G D2 -specific scFv, which is currently being used in clinical trials for the treatment of neuroblastoma, and generated chRecs containing either the 2B4 signaling domain alone (14.G2a-2B4) or combined with TCR (14.G2a-2B4ζ). These chRecs were expressed in activated polyclonal human peripheral blood T cells using retroviral gene transfer. As controls, T cells were transduced with 14.G2aζ. High chRec surface expression was obtained for all three constructs, with 51±12% for 14.G2a-ζ, 70±15% for 14.G2a-2B4, and 53±7% for 14.G2a-2B4ζ. Immunphenotypes were dominated by a CD3+CD8+ population in all cell cultures. 51 Cr-release assays showed efficient and comparable lysis of G D2 + tumor targets by T cells transduced with 14.G2a-ζ (44±10%) and 14.G2a-2B4ζ (44±3%) at an effector-to-target ratio of 40:1, whereas 2B4 alone failed to mediate specific tumor cell lysis. Intracellular cytokine secretion by chRec+ T cells was induced in response to G D2 + tumor targets by 14.G2a-ζ (mean 18.3%, range 12.1–29.7% IFN-γ+ CD3+ T cells) and 14.G2a-2B4ζ (mean 7.7%, range 6.0–11.3%). In contrast, 14.G2a-2B4 transduced T cells failed to induce IFN-γ secretion (mean 0.2%, range 0.06–0.47%). Weekly stimulations with tumor cells resulted in substantially superior expansion of T cells transduced with 14.G2a-2B4ζ (8–45fold) compared to T cells transduced with either 14.G2a-ζ (3.5–18fold) or 14.G2a-2B4 (2–4fold) over a 5 week period. It is known that T cell costimulation by CD28 receptor family members is mediated by downstream signaling involving the NFκB pathway. However in our studies, luciferase reporter gene assays failed to show a significant difference in tumor antigen-specific NFκB recruitment between T cells transduced with 14.G2aζ (240,000±5,000 RLU) and 14.G2a-2B4ζ (180,000±7,000 RLU). Although the nature of the signaling pathway mediating 2B4-induced T cell stimulation is still to be defined, our data support a costimulatory role for the activating NK cell receptor 2B4 in peripheral blood T cells. Therefore, 2B4-containing chRec may be a powerful new tool for adoptive immunotherapy of cancer.