Tumor vaccines expressing flt3 ligand synergize with ctla-4 blockade to reject preimplanted tumors

Abstract

The transformation of a healthy cell into a malignant neoplasm involves numerous genetic mutations and aberrations in gene expression. As few of these changes are shared between individuals or types of cancer, the best source for eliciting broad-spectrum tumor immunity remains each patient's own tumor. Previously, we have shown that combining blockade of the T-cell-negative costimulatory molecule CTL-associated antigen 4 (CTLA-4) and vaccination with irradiated B16 tumor expressing granulocyte macrophage colony-stimulating factor (GM-CSF; Gvax) promotes rejection of established murine melanomas. Here we show that, like GM-CSF, the cytokine Flt3 ligand (Flt3L) expressed in B16 and coupled with CTLA-4 blockade promotes both prophylactic and therapeutic rejection of B16. When administered at the site of growing tumor, Gvax fails to prevent tumor outgrowth in any mice, whereas the B16-Flt3L vaccine (Fl3vax) induces the rejection of 75% of melanomas implanted 3 days before vaccination. Relative to Gvax, Fl3vax promotes greater infiltration of both the vaccine site and the tumor site by CD8+ T cells and "sentinel" and plasmacytoid dendritic cells. Gvax and Fl3vax did not synergize when used in combination in treating B16 melanoma even in the context of CD25+ regulatory T-cell depletion. Further, we show that a combination of Flt3L expression and CTLA-4 blockade can also promote the rejection of established TRAMP prostate adenocarcinomas, proving that the utility of this treatment extends beyond melanoma. Engineering Flt3L to be constitutively secreted and attaching an IgG2a tail yielded a B16 vaccine that, when combined with CTLA-4 blockade, prevented the outgrowth of significantly more 5-day implanted B16-BL6 tumors than did Gvax.

Figure 1. Fl3vax, like Gvax, synergizes with CTLA-4 blockade to treat 3-day pre-implanted B16 melanomas

A) Percent tumor free mice following vaccination on Days −4, −7 (n=4, 5–15 mice/group). * denotes statistical significance (p<0.05) by Fisher’s exact test compared to B16-YFP. B) Tumor growth is shown for montherapy treatment of 5 mice/group on Days 3,6,9. C) Kaplan-Meier survival curves are shown for vaccine w/CTLA-4 blockade on Days 3,6,9 where the end-point was defined as tumor burden ≥ 1000mm³ (n-3, 10mice/group). Surviving mice were tumor free. D) Tumor growth is shown for a representative experiment of 3.

Figure 2. Fl3vax with CTLA-4 blockade prevents tumor outgrowth in nearly all mice when given at the tumor site where Gvax proves ineffective, while B16-IP10 is effective locally but not distally

A) Percent tumor free mice following vaccination on Days 3,6,9 (n=2, 10 mice/group except PBS vaccination was only distal to 5 mice/group). B) Mean tumor size at the first measurement for one representative experiment of 2 independent experiments (10 mice/group) is shown. Growth curves could not be generated in these studies as no measurements could be made for 3–4 weeks due to post-vaccination granulomas at the tumor site.

Figure 3. Fl3vax and Gvax fail to synergize when used in combination to treat B16 melanomas implanted 5 days prior even in mice pre-depleted of Tregs

A) Percent tumor free mice following vaccination on Days 3,6,9 (n=3, 10 mice/group). B) Tumor growth is shown for a representative experiment of 3. C,D are replicates of A,B except that mice were pre-depleted of CD25+ cells by PC-61 injection on Day-4 (n=2, 10 mice/group).

Figure 4. Fl3vax elicits strong infiltration of the vaccine and tumor sites by CD8+ T-cells, “sentinel” DCs, and plasmacytoid DCs

A) The percent of the total vaccine site infiltrating lymphocytes for each subset and the indicated vaccine is shown (n=2, 5 individually analyzed mice/group). The percent infiltration relative to Gvax is also shown. B) The percent composition of total TIL for each subset and the indicated vaccine is shown (n=3, 5 individually analyzed mice/group). The percent infiltration relative to Gvax is also shown. C) Changes in intra-tumoral CD8+ T-cell, CD4+ Treg, and the resulting intra-tumoral CD8+ T-cell to Treg ratios are shown. D) The percent of TIL composed of CD11b+GR1+ cells is shown for each treatment group.

Figure 5. Flt3L converts irradiated TRAMP-C2 prostatic adenocarcinoma cells into an effective vaccine which synergizes with CTLA-4 blockade to protect against outgrowth of pre-implanted TRAMP tumors

A) Percent tumor free mice following vaccination on Days 2,5,8 (n=2, 10 mice/group). B) Tumor growth is shown for a representative experiment of 2.

Figure 6. A secreted, Ig-tailed FL3vax synergizes with CTLA-4 blockade more effectively treats mice of 5-day pre-implanted B16 melanomas than does Gvax

A) Percent tumor free mice following vaccination on Days 5,8,11 (n=3, 10 mice/group). B) Tumor growth is shown for a representative experiment of 3.