CCL2 blockade augments cancer immunotherapy

Abstract

Altering the immunosuppressive microenvironment that exists within a tumor will likely be necessary for cancer vaccines to trigger an effective antitumor response. Monocyte chemoattractant proteins (such as CCL2) are produced by many tumors and have both direct and indirect immunoinhibitory effects. We hypothesized that CCL2 blockade would reduce immunosuppression and augment vaccine immunotherapy. Anti-murine CCL2/CCL12 monoclonal antibodies were administered in three immunotherapy models: one aimed at the human papillomavirus E7 antigen expressed by a non-small cell lung cancer (NSCLC) line, one targeted to mesothelin expressed by a mesothelioma cell line, and one using an adenovirus-expressing IFN-alpha to treat a nonimmunogenic NSCLC line. We evaluated the effect of the combination treatment on tumor growth and assessed the mechanism of these changes by evaluating cytotoxic T cells, immunosuppressive cells, and the tumor microenvironment. Administration of anti-CCL2/CCL12 antibodies along with the vaccines markedly augmented efficacy with enhanced reduction in tumor volume and cures of approximately half of the tumors. The combined treatment generated more total intratumoral CD8+ T cells that were more activated and more antitumor antigen-specific, as measured by tetramer evaluation. Another important potential mechanism was reduction in intratumoral T regulatory cells. CCL2 seems to be a key proximal cytokine mediating immunosuppression in tumors. Its blockade augments CD8+ T-cell immune response to tumors elicited by vaccines via multifactorial mechanisms. These observations suggest that combining CCL2 neutralization with vaccines should be considered in future immunotherapy trials.

Figure 1. CCL2 blockade significantly augments tumor immunotherapy

Panel A – Effects of α-CCL2 and α-CCL12 blockade. Mice bearing large TC1 tumors were treated in one of four ways: 1) No treatment (control); 2) α-CCL2 mAb twice per week starting at day 13 (a-CCL2); 3) α-CCL12 mAb twice per week starting at day 13 (a-CCL12); and 4) Combination of α-CCL2 and α-CCL12 mAb (a-CCL2/12). α-CCL2 or α-CCL12 had a trend to slow tumor growth, but only the combination of both mAbs significantly reduced tumor growth (* = p<0.05 vs. control). Panels B–D – Combination of Immunotherapy and CCL2 blockade. Mice bearing large flank tumors, were treated in one of four ways: 1) No treatment (control); 2) I.P. α-CCL2/CCL12 mAb twice per week starting at day 13 (a-CCL2); 3) Immunotherapy; and 4) Combination of immunotherapy and α-CCL2 mAb (Combo). In all 3 models only combination therapies led to clear tumor regression and cures. Three different models of immunotherapy are shown: AdE7 in the NSCLC cell line TC-1 (Panel B, *=p<0.01 for Combo vs. Ad.E7); A listeria-mesothelin vaccine (Lm-meso) in the mesothelin-expressing mesothelioma cell line AE17 (Panel C, *=p<0.05 vs. control for each); and Ad.IFNα in the NSCLC cell line LKR (Panel D, *=p<0.05 for combo vs. each group).

Figure 2. CCL2 blockade increases the activity and antigen-specificity of splenic CD8

⁺ T-cells and reduces splenic T-regs. Flow cytometry was performed on spleens from control tumor-bearing mice (TC1), and mice treated with α-CCL2 mAb, Ad.E7 or the combination of both, 2 days after the second (booster) Ad.E7 vaccine (n=5 per group). Panel A summarizes the percentage of CD8⁺ T cells expressing the activation marker 4-1BB out of all splenocytes, showing increased activity in the combination therapy (*=p<0.05). Panels B–C show splenic CD8⁺/tetramer-E7⁺. Panel B shows representative FACS tracings of splenic CD8 versus E7 tetramer⁺ cells in each group. The number in each quadrant is the percentage of the CD8⁺ cells. Panel C summarizes the mean number of positive cells per spleen in the four groups, showing increased numbers in the combination group (n=5, *=p<0.05). Panel D shows the mean number of splenic T-regs (defined as CD4⁺/CD25⁺) in each group. T-regs were significantly lower in the combination group (n=5, *=p<0.01).

Figure 3. CCL2 blockade in mice treated with immunotherapy increases the number and activity of intra-tumoral CD8

⁺ T Cells Mice (n = 4–6 for each group) bearing large TC-1 tumors, were treated in one of four ways: 1) No treatment (Control); 2) I.P. α-CCL2 mAb (a-CCL2); 3) S.Q. vaccine with Ad.E7 (Ad.E7); and 4) combination of Ad.E7 and α-CCL2. Two days after the second (booster) Ad.E7 vaccine, tumors were harvested. Panels A–B summarize the number of intratumoral CD8⁺ cells by IHC (Panel A), and their percentage of total tumor by flow cytometry (Panel B). The combination of vaccine plus CCL2/CCL12 blockade significantly increased intra-tumoral CD8⁺ cells (*=p<0.05). Each dot represents one mouse. Panel C summarizes the percentage of intratumoral CD8⁺ T cells expressing the activation marker 4-1BB, showing increased activity in the combination therapy (*=p<0.05). Panel D summarizes the percentage of intratumoral CD8⁺/tetramer-E7⁺ cells, showing increased antigen-specific cells in the combination therapy (*=p<0.05).

Figure 4. CCL2 blockade in mice treated with immunotherapy decreases the percentage of intratumoral T-regs, but does not change macrophages phenotype

Mice (n = 4–5 for each group) bearing large TC-1 tumors, were treated as in Figure 3. Two days after the booster vaccine, tumors were and subjected to flow cytometry. Panel A summarizes the fold-change in the percentage of FoxP3⁺ cells out of intratumoral CD4⁺ T-cells, normalized to control, in 5 different experiments (3–5 mice pooled in each subgroup), showing a significant decrease in T-regs in the combination group. (*=p<0.05). Panel B shows representative FACS tracings of CD4 versus FoxP3 in each group. The number in each quadrant is the percentage of FoxP3⁺ cells out of CD4⁺ cells. Panel C summarizes the percentage of CD4⁺/FoxP3⁻ cells out of intratumoral CD4⁺ T-cells, in 5 different experiments (3–5 mice pooled in each subgroup), showing a significant increase in activated CD4⁺ T cells in the combination group. (*=p<0.05 vs. AdE7). Panel D summarizes the percentage of classically and alternatively activated macrophages (defined as CD11b⁺/F480⁺ and CD206⁻ or CD206⁺, respectively). Immunotherapy mildly increased CD206⁺ macrophages, but induced a stronger increase in CD206⁻ macrophages. CCL2 blockade did not further alter these changes (n=15–20, *=p<0.05).