Abstract
Providing a proliferative and survival advantage to tumor-specific cytotoxic T lymphocytes (CTLs) remains a challenge in the adoptive therapy of cancer patients. It is now evident that the in vivo expansion of T cells after adoptive transfer is best accomplished in the lymphodepleted host due to the increased production of endogenous IL15 and IL7, which help restore lymphopoiesis. We have found that antigen activated cytotoxic T lymphocytes (CTLs) directed to tumor associated epitopes (for example derived from EBV, or from cancer testis antigens such as PRAME) down regulate a chain of IL7R, a common γ chain cytokine receptor, impairing their capacity to respond to IL7. We hypothesized that despite receptor downregulation, the signal transduction pathway for IL7R would remain intact in the CTLs so that forced expression of IL7Rα would restore IL7 responsiveness and improve in vivo expansion and survival of CTLs. We used EBV-specific CTLs as our model, and showed in vitro that a functional IL-7Ra molecule can be expressed in CTLs using retroviral gene transfer so that the percentage of receptor + cells increased from 2.4%±0.5% to 50%±20. This modification restored the in vitro proliferation of genetically modified CTLs in response to IL7 so that cell numbers increased from 1×106 cells to 0.1×109 (range, 0.6×108 to 0.3×109)] comparable with the effects of IL2 [from 1×106 cells to 0.7×109 (range, 0.7×107 to 1.6×109)] In contrast, control EBV-CTL with IL7 progressively declined in number (p<0.001) These effects were accomplished without alteration of antigen specificity or responsiveness to other common γ chain cytokines, and cell survival remained antigen dependent. In a xenogeneic mouse model, CTLs expressing IL7Ra significantly expanded in vivo in response to EBV-tumor antigen and the administration of IL7. By day 15, both control CTLs and IL7Ra+ CTLs had modestly proliferated in response to IL-2 (2.3 fold, range 1.1–5.1 for control CTLs, and 2.67 fold, range 0.6 to 8.15 for IL7Ra+ CTLs). In contrast, only IL7Ra+ CTLs significantly expanded in the presence of IL7, showing a 6.09 fold increase (range 0.7 to 25.2) compared to mice that received control CTLs and IL7 (0.9 fold, range 0.5–1.7) (p<0.0001). Modified CTLs also provided enhanced anti-tumor activity. SCID mice engrafted i.p with 3×106 tumor cells marked with Firefly luciferase, showed a rapid increase in signal in the absence of CTLs (Fold increase in luminance = 29.8 median, range 4.4 to 103) by day 14 after tumor engraftment. Similar tumor growth was observed in mice receiving IL7Ra+ CTLs without cytokines (luminance increase14.4 fold, range 1 to 90). In contrast, mice receiving IL7Ra+ CTLs and either IL2 or IL7, had a decline in tumor luminance (fold expansion 0.7, range 0.08 to 2.9, and 0.8, range 0.004 to 3.5, respectively p<0.0001). Although growth of the transgenic T cells remained antigen dependent, as a further safety measure, we incorporated an inducible suicide gene based on icaspase9 that can be activated by exposure to a small chemical inducer of dimerization (CID) (AP20187). Incorporation of this suicide gene did not affect the in vitro or in vivo anti-tumor activity of the CTL’s but allowed them to be rapidly eliminated. So that after a single dose of CID (50 nM) the transgenic population were decreased by >98.5% We conclude that forced expression of the IL-7Ra by CTLs can be used to recapitulate the response of these cells to this cytokine and thereby promote their in vivo anti-tumor activity after adoptive transfer either in a lymphodepleted host or after the administration of the recombinant protein.
Targeting necroptosis in muscle fibers ameliorates inflammatory myopathies
