Figure 1.
Optimizing the SA-4-1BBL/E7 vaccine formulation for the most effective therapy in the TC-1 cervical cancer mouse model.
C57BL/6 mice were inoculated s.c. with 1×105 live TC-1 tumor cells and vaccinated with the indicated doses of E7 and SA-4-1BBL proteins on day 6 post-tumor challenge. Mice vaccinated with PBS were used as controls. (A) Schematic depiction of the experimental procedure. (B) Optimizing the dose of E7 antigen. Mice were vaccinated with the indicated doses of E7 in µg with a fixed amount of SA-4-1BBL (25 μg). *P<0.05 vs. all other groups, except E7 100 µg. (C) Optimizing the dose of SA-4-1BBL. Mice were vaccinated with the indicated doses of SA-4-1BBL with the optimum 50 µg dose of E7 as determined in (B). The 25-μg SA-4-1BBL treatment group was significant (*P<0.05) from all other groups and found to be the optimal dose. The SA-4-1BLL/E7 (25 /50 µg) group from panel A is re-graphed for direct comparison with the other groups.
Figure 2.
The therapeutic efficacy of SA-4-1BBL/E7 vaccine is improved by multiple vaccinations in the TC-1 cervical cancer model.
(A) Schematic depiction of the experimental procedure. (B) C57BL/6 mice were immunized s.c. with the optimized SA-4-1BBL/E7 (25 µg/50 µg) vaccine formulation on day 6 post-challenge with 1×105 live TC-1 tumor cells. A group of mice received two more vaccinations administered on days 15 and 24 post-tumor challenge. Mice vaccinated with PBS were used as controls. P<0.05 vs. PBS group, but not SA-4-1BBL/E7 x 1 group.
Figure 3.
Long-term immune memory induced by SA-4-1BBL/E7 vaccine is systemic and tumor-specific.
C57BL/6 mice that had eradicated TC-1 tumors in response to vaccination with SA-4-1BBL/E7 were given a second dose of TC-1 tumor cells (1×105) in either left or right flank 60 days after primary tumor challenge to evaluate long-term immune memory and if the memory is systemic. (A) Schematic depiction of the experimental procedure. (B) Survival. None of the mice developed TC-1 tumors in left or right flanks within the 30-day observation period at which time the animals were inoculated with live 1×105 heterologous 3LL tumor cells into the opposite flanks to the last TC-1 challenge. All long-term mice developed 3LL tumors in a similar tempo to naïve (data not shown) mice without recurrence of TC-1 tumor growth, demonstrating that anti-tumor immune responses are systemic and tumor type-specific.
Figure 4.
CD4+ T cells play a critical role in the SA-4-1BBL/TAAs-induced therapeutic primary responses against tumors.
(A) Schematic depiction of the experimental procedure. (B) Assessing the role of CD4+ and CD8+ T cells in the SA-4-1BBL/E7 vaccine induced therapeutic immunity against TC-1 tumor. C57BL/6 mice were treated i.p. with depleting doses of Abs against CD4 (500 μg/mouse) or CD8 (500 μg/mouse) either one day before vaccination (CD4 or CD8-prevac) with an optimized formulation of SA-4-1BBL/E7 (25/50 µg) administered on day 6 post-tumor challenge or one day before tumor challenge (CD4-prechal) Controls included mice without T cell depletion vaccinated with SA-4-1BBL/E7 or PBS. (C) Assessing the role of CD4+ T cells in the SA-4-1BBL/SVN vaccine induced therapeutic immunity against 3LL tumor. C57BL/6 mice were inoculated with 1×105 3LL tumor cells s.c. and vaccinated with SA-4-1BBL/SVN (25/50 µg) on day 6 post-tumor challenge. One group of mice received the depleting dose of anti-CD4 Ab one day before tumor challenge (CD4-prechal) while another group received the Ab one day before vaccination (CD4-prevacc) *P<0.01; ns, not significant. Some of the mice in SA-4-1BBL/SVN and CD4-prevac groups have recently been published (ref. 31) and pooled with more mice and graphed for comparison purposes.
Figure 5.
CD4+ T cells are required for SA-4-1BBL/TAAs vaccine-induced long-term immune memory against 3LL, but not against TC-1 tumor.
(A) Schematic depiction of the experimental procedure. (B) Long-term C57BL/6 mice that had eradicated TC-1 tumors irrespective of CD4+ T cell depletion one day before vaccination with SA-4-1BBL/E7 had long-term protective immune response as assessed by a secondary challenge with TC-1 tumor cells 60 days after the initial tumor inoculation. Naïve mice injected with PBS and challenged with 1×105 TC-1 cells served as controls and expired from tumor burden within 40 days. (C) Long-term C57BL/6 mice that had eradicated 3LL tumors irrespective of CD4+ T cell depletion one day before vaccination with SA-4-1BBL/SVN had significantly compromised memory immune response as assessed by a secondary challenge with 3LL tumor cells 60 days after the initial tumor inoculation. Naïve mice injected with PBS and challenged with 1×105 3LL cells served as controls and expired from tumor burden within 50 days.