Combination of Alphavirus Replicon Particle–Based Vaccination with Immunomodulatory Antibodies: Therapeutic Activity in the B16 Melanoma Mouse Model and Immune Correlates

Anti–CTLA-4 and anti-GITR mAbs comparably enhance tumor-specific CD8⁺ T-cell response induced by the VRP–TRP2 vaccine. A, ELISPOT analysis of IFN-γ–secreting CD8⁺ T cells purified from spleens of non–tumor-bearing mice immunized with VRP–GFP or VRP–TRP2 in combination with the indicated mAbs or isotype control and restimulated with TRP2_181–189 or SIINFEKL peptide as negative control. Each dot represents an individual mouse. B, ex vivo cytotoxicity assay of the indicated titration of effector CD8⁺ and CD4⁺Foxp3⁻ T cells purified from tumors of mice treated with VRP–TRP2 in combination with the indicated mAbs or isotype control, and B16F10 as target cells. Each point of the Effector:Target (Eff:Target) titration curve represents the average of a duplicate culture (i). Representative histogram plots showing the percentage of live B16F10 and B78H1 cells after incubation with the indicated T cells at Eff:Target = 1:1 (ii). C, ex vivo cytotoxicity assay in three-dimensional fibrin–collagen gel with effector CD8⁺ (left) or CD4⁺Foxp3⁻ (right) T cells purified from spleens (SP) of mice treated with VRP–GFP as negative control, or tumors of mice treated with VRP–TRP2 in combination with the indicated mAbs or isotype control, and B16F10 cells as target (Eff:Target = 1:1). Each bar represents the average of a triplicate culture ± SEM. Dotted lines indicate the background level, calculated as the average percentage killing + (3×SD) using effector T cells isolated from naïve spleens. Each of the above experiments was repeated at least two times and showed similar results. αGITR, anti-GITR; αCTLA-4, anti–CTLA-4. **, P < 0.01; *, P < 0.05; ns, not statistically significant.

Anti–CTLA-4 and anti-GITR mAbs comparably enhance the frequency of vaccine-induced TRP2-specific Abs. Analysis of circulating anti-TRP2 Abs in mice immunized with VRP–TRP2 in combination with the indicated mAb or isotype control, or with VRP–GFP + isotype (VRP–GFP) as negative control. A, anti-TRP2 total IgG, or IgG1, IgG2a or IgG2c were measured by ELISA using the specific secondary Abs. Each dot represents an individual mouse. B, ELISA results of total circulating IgG recognizing the indicated peptides (1–10) of TRP2 in mice (9–10/group) treated with VRP–TRP2 plus isotype control (top), anti–CTLA-4 (αCTLA-4, middle), or anti-GITR (αGITR, bottom) mAb. Each color of the bar graph indicates an individual mouse. Each of the above experiments was repeated at least two times and showed similar results. **, P < 0.01; *, P < 0.05; ns, not statistically significant.

Anti–CTLA-4 and anti-GITR mAbs differently affect the accumulation of intratumor CD4⁺ T cells. B16F10 melanoma-bearing mice were treated with VRP–TRP2 in combinations with anti–CTLA-4 (αCTLA-4) or anti-GITR (αGITR) mAb or isotype control as shown in Fig. 1. A, weight of Matrigel plugs when explanted 16 days after tumor injection. B, percentage of live CD45⁺ immune cells infiltrating the tumor. C, flow cytometry analysis of CD3⁺CD8⁺ (CD8), CD3⁺CD4⁺ (CD4), NK1.1⁺CD3⁺ (NKT), NK1.1⁺CD3⁻ (NK), CD3⁻NK1.1⁻CD11c⁺ (CD11c), CD3⁻NK1.1⁻CD11c⁻ (other) cells infiltrating B16F10-Matrigel plugs. D, CD4⁺Foxp3⁺ Treg/CD45⁺ cell frequency in tumors from mice treated as indicated. E, ratio between frequencies of CD8⁺ (i) or CD4⁺Foxp3⁻ T cells (ii) and CD4⁺Foxp3⁺ Tregs in tumors from mice treated as indicated. Data represent mean ratios ± SEM of four to seven tumors analyzed individually per group. **, P < 0.01; *, P < 0.05; ns, not statistically significant.

Treatment with anti–CTLA-4 mAb favors the accumulation of CD4⁺Foxp3⁻PD-1⁺ T cells at the tumor site. B16F10 melanoma-bearing mice were treated with VRP–TRP2 plus isotype control, anti–CTLA-4 (αCTLA-4), or anti-GITR (αGITR) mAb, as shown in Fig. 1. A, representative dot plots (left) and percentage of PD-1⁺/CD4⁺ TILs (right). B, representative dot plots and percentage of PD-1–expressing CD4⁺Foxp3⁻ (left) and CD4⁺Foxp3⁺ (right) TILs. Each dot represents an individual mouse. *, P < 0.05; **, P < 0.01; ns, not statistically significant.