Chimeric form of tumor necrosis factor-alpha has enhanced surface expression and antitumor activity

Abstract

Tumor necrosis factor (TNF)-alpha is a type-II transmembrane protein that is cleaved by TNF-alpha-converting enzyme (TACE/ADAM-17) to release soluble TNF, a cytokine with potent antitumor properties whose use in clinical applications is limited by its severe systemic toxicity. We found that human cells transfected with vectors encoding TNF without the TACE cleavage site (DeltaTACE-TNF) still released functional cytokine at substantial levels that varied between transfected cell lines of different tissue types. Vectors encoding membrane-associated domains of CD154, another TNF-family protein, conjoined with the carboxyl-terminal domain of TNF, directed higher-level surface expression of a functional TNF that, in contrast to DeltaTACE-TNF, was resistant to cleavage in all cell types. Furthermore, adenovirus vectors encoding CD154-TNF had significantly greater in vivo biological activity in inducing regression of established, syngeneic tumors in mice than adenovirus vectors encoding TNF, and lacked toxicity associated with soluble TNF. As such, CD154-TNF is a novel TNF that appears superior for treatment of tumors in which high-level local expression of TNF is desired.

Figures

Figure 1

Chimeric TNF constructs. Scale representation of the chimeric constructs of CD154, TNF and CD70 indicating the membrane anchoring domains (MAD) and ligand binding domains (LBD). The boundaries of TNF-binding domain are identical in the chimeric constructs. The human and murine sequences display a high degree of similarity and differ in length by no more than two amino acids for homologous sequences.

Figure 2

Expression of TNF by transfected cells. (a) Secretion of soluble TNF from ΔTACE-TNF-transfected cells. HT1080 cells were transfected with pcDNA3 plasmids encoding vector alone (square), wtTNF (circle), or ΔTACE-TNF (triangle) and incubated for 2 days. Serial dilutions of cell supernatants were then incubated with L929 cells and soluble TNF bioactivity determined by the measurement of L929 apoptosis using the XTT colorimetric assay. (b) HT1080 cells were infected with adenovirus as indicated for two days. We examined the culture supernatants for soluble TNF by ELISA. (c) Cell-surface expression of TNF by Ad-infected HeLa cells. HeLa cells were infected with the indicated adenovirus and then analyzed for TNF surface expression by flow cytometry (open histograms). Shaded histograms represent cells stained with isotype control antibody. The percentage of surface-TNF-positive cells is indicated for each histogram. (d) The relationship of soluble TNF versus the mean fluorescence intensity ratio of TNF surface expression was plotted for HT1080 cells (left graph) and HeLa cells (right graph) infected at increasing MOI with adenovirus encoding wtTNF (closed squares), ΔTACE-TNF (open squares), CD70-TNF (open triangles) and CD154-TNF (closed circles). Data points from four different MOI for each TNF construct were used to determine the linear functions plotted on the graph.

Figure 3

In vitro and in vivo toxicity of soluble and cell-surface TNF. (a) Contact-dependent killing of L929 cells by Ad-TNF-infected HeLa cells. HeLa cells were infected with adenovirus encoding the indicated transgene (GFP, wtTNF or CD154-TNF) and then either stained with CFSE and plated out with an equal number of L929 cells, to allow cell–cell contact (coculture, filled bars) or HeLa cells were placed in the upper well of a transwell filter insert (transwell, empty bars) to prevent direct cell–cell contact with L929 cells. Following 18 h coculture, L929 cells were analyzed for viability by flow cytometry as described in Material and methods. (b) Hemorrhagic tumor necrosis after injection of Ad-TNF. BALB/c mice with WEHI-164 tumors were injected intratumorally with 109 PFU Ad-blank, Ad-wtTNF or Ad-CD154-TNF. Pictures were taken 1 week after injection and are representative for each group. (c) Toxicity after systemic injection of Ad-TNF. Groups of eight FVB/N mice were injected intraperitoneally twice in a 48 h interval with 109 PFU adenovirus encoding wtTNF (closed triangle), CD154-TNF (closed diamond) or adenovirus containing no transgene (open square). Survival of animals is plotted over time.

Figure 4

Intratumoral injection of WEHI-164 tumor nodules. BALB/c mice were injected subcutaneously with 3 × 106 WEHI-164 tumor cells at day 0. On days 8, 12 and 16, tumor nodules were injected with 109 PFU adenovirus encoding GFP (open triangles, n = 15), wtTNF (closed triangles, n = 28), CD154-TNF (closed diamonds, n = 27) or saline (open squares, n = 8). (a) Mean tumor size and (b) animal survival are plotted over time. Asterisks indicate statistically significant differences between CD154-TNF and wtTNF treatment (Student’s t-test or log-rank test, respectively).

Figure 5

Intratumoral injection of A20 lymphoma nodules. BALB/c mice were injected subcutaneously with 1 × 105 A20 tumor cells at day 0. On day 10, tumor nodules remained untreated (closed square, n = 10) or were injected with 5 × 108 PFU adenovirus carrying no transgene (Ad-blank, open square, n = 16), Ad-wtTNF (open triangle, n = 15) or Ad-CD154-TNF (closed diamond, n = 16). (a) The mean tumor size (±s.e.) and (b) animal survival are plotted over time. Asterisks indicate statistically significant difference between CD154-TNF and wtTNF treatment (Student’s t-test or log-rank test, respectively). (c) Immune response to A20 cells in treated mice analyzed by IFN-γ ELISPOT assay. Splenocytes were isolated from three mice in each treatment group approximately 2 weeks after injection of Ad. Splenocytes were then cocultured with an equal number of mitomycin C-treated A20 cells in a 96-well filter plate and the number of IFN-γ-secreting spleen cells was determined by ELISPOT assay. Bars depict the mean number (±s.d.) of IFN-γ-producing cells per 2 × 105 splenocytes. The asterisk indicates a statistically significant difference (P<0.05, Bonferroni t-test) in the mean number of spots between the Ad-CD154-TNF group and all other treatment groups. (d) Mice with complete tumor regression after treatment with Ad-wt-TNF (open circle, n = 4) or Ad-CD154-TNF (cross, n = 7) were rechallenged with 2 × 105 A20 cells in the contralateral flank. A group of naïve mice (closed squares, n = 5) also received 2 × 105 A20 cells. The mean tumor size (± s.e.) for each group is depicted over time.