Cancer vaccine formulation dictates synergy with CTLA-4 and PD-L1 checkpoint blockade therapy

Abstract

Anticancer vaccination is a promising approach to increase the efficacy of cytotoxic T lymphocyte-associated protein 4 (CTLA-4) and programmed death ligand 1 (PD-L1) checkpoint blockade therapies. However, the landmark FDA registration trial for anti-CTLA-4 therapy (ipilimumab) revealed a complete lack of benefit of adding vaccination with gp100 peptide formulated in incomplete Freund's adjuvant (IFA). Here, using a mouse model of melanoma, we found that gp100 vaccination induced gp100-specific effector T cells (Teffs), which dominantly forced trafficking of anti-CTLA-4-induced, non-gp100-specific Teffs away from the tumor, reducing tumor control. The inflamed vaccination site subsequently also sequestered and destroyed anti-CTLA-4-induced Teffs with specificities for tumor antigens other than gp100, reducing the antitumor efficacy of anti-CTLA-4 therapy. Mechanistically, Teffs at the vaccination site recruited inflammatory monocytes, which in turn attracted additional Teffs in a vicious cycle mediated by IFN-γ, CXCR3, ICAM-1, and CCL2, dependent on IFA formulation. In contrast, nonpersistent vaccine formulations based on dendritic cells, viral vectors, or water-soluble peptides potently synergized with checkpoint blockade of both CTLA-4 and PD-L1 and induced complete tumor regression, including in settings of primary resistance to dual checkpoint blockade. We conclude that cancer vaccine formulation can dominantly determine synergy, or lack thereof, with CTLA-4 and PD-L1 checkpoint blockade therapy for cancer.

Keywords: Cancer immunotherapy; Immunology.

Figure 1. Anti–CTLA-4 therapeutic activity is impaired by the addition of gp100/IFA vaccination.

(A) Experimental scheme. (B) Mice bearing 3-day-old B16 tumors received naive gp100-specific CD90.1⁺ pmel-1 T cells i.v., followed by s.c. vaccination with hgp100/IFA or control/IFA on day 0 and/or anti–CTLA-4 plus Gvax therapy on days 0, 3, and 6, or were left untreated. Tumor size in individual mice is shown. Numbers in parentheses represent the proportion of mice with tumor-free survival. (C) pmel-1 T cells in PBMCs 7 days after vaccination. Data represent mean ± SEM, n = 5. (D) Kaplan-Meier survival curves. Data are pooled from 2 independent experiments, each with 8 to 15 mice per group.

Figure 2. CD8⁺ Teffs localize to sites of vaccination with noncognate antigen.

(A) Mice bearing 7-day-old, s.c. B16 tumors received 6-day-cultured, gp100-specific pmel-1 or OVA-specific OT-1 Teffs i.v., followed by vaccination with hgp100/IFA (right flank, s.c.) or OVA/IFA (right flank, s.c.). v-effLuc–transduced OT-1 T cells (left panel) and v-effLuc–transduced pmel-1 T cells (right panel) were visualized by whole-mouse imaging 4 days after vaccination. Color bars represent mean ± SEM photons/second (n = 5, *P < 0.05) determined by 1-way ANOVA with a post hoc test. Statistical differences between the 2 groups were determined by the unpaired 2-tailed t test. (B–E) Mice bearing 3-day-old, s.c. B16 melanoma received naive CD90.1⁺ pmel-1 T cells, early anti–CTLA-4 therapy, and vaccination with control/IFA or hgp100/IFA. Tumor and vaccination sites were harvested 9 days after vaccination. (B) Non–pmel-1 Teffs (CD44^hiCD11a^hiCD8⁺CD90.1^–). (C) TRP-2–specific Teffs (TRP-2_181–188 pentamer–specific Teffs). (D) pmel-1 Teffs (CD44^hiCD11a^hiCD8⁺CD90.1⁺). Absolute number counts were adjusted per tissue weight resection from tumor and vaccination sites. Data (B–D) represent mean ± SEM of 3 independent experiments (n = 5, *P < 0.05 unpaired 2-tailed t test). (E) Experimental scheme (top) showing Kaplan-Meier survival curve data pooled from 3 independent experiments (n = 5 mice per group, bottom). *P < 0.05, log-rank test.

Figure 3. Exhaustion and apoptosis of sequestered anti–CTLA-4 activated CD8⁺ Teffs.

(A) Gating strategy for naive, non–pmel-1 Teffs and pmel-1 Teffs from PBMCs (top). Mice bearing 3-day-old, s.c. B16 melanoma received CD90.1⁺ pmel-1 T cells, early anti–CTLA-4 therapy, and vaccination with control/IFA or hgp100/IFA. (B) Ki67 protein expression by naive CD8⁺ T cells, non–pmel-1 CD8⁺ Teffs, and pmel-1 CD8⁺ Teffs in PBMCs, VdLNs, and at the vaccination site 9 days after the start of therapy. pmel-1 histograms show T cells in a gp100/IFA setting. (C) PD-1/LAG-3 expression in Teffs from PBMCs, VdLNs, tumor site, and vaccination site. (D) TRP-2–specific CD8⁺ Teffs in VdLNs (top panel) and quantitation of IFN-γ⁺TNF-α⁺ TRP-2–specific CD8⁺ Teffs in PBMCs, tumor site, and vaccination site (bottom panel). (E) Fas expression on naive T cells, non–pmel-1 Teffs, and pmel-1 Teffs from VdLNs, spleen, tumor site, and at the vaccination site 9 days after the start of therapy. (F) Apoptotic cell death from VdLNs, spleen, and at the vaccination site of naive CD8⁺ T cells, non–pmel-1 CD8⁺ Teffs, and pmel-1 CD8⁺ Teffs 9 days after the start of therapy, as measured by flow cytometry of annexin V and 7-AAD staining. Plots are shown as mean ± SEM (n = 5, *P < 0.05 unpaired 2-tailed t test). Data shown are representative of 2 experiments.

Figure 4. CXCR3 is required for CD8⁺ Teff localization to tumor.

(A) Experimental scheme. Mice bearing 3-day-old, s.c. B16-BL6 melanomas received naive pmel-1 T cells and early anti–CTLA-4 therapy or vaccination with hgp100 in IFA. (B) Chemokine receptor expression on naive CD8⁺ T cells, pmel-1 CD8⁺ Teffs, and non–pmel-1 CD8⁺ Teffs in blood 9 days after vaccination. (C) Mice bearing 3-day-old, s.c. B16-BL6 melanomas received pmel-1 T cells and early anti–CTLA-4 therapy or vaccination with hgp100/IFA or control/IFA. Cytokine and chemokine concentrations in supernatant from tumor and vaccination site homogenates 9 days after vaccination. All data shown are mean ± SEM and are representative of 3 experiments (n = 5 mice per group, *P < 0.05 determined by unpaired 2-tailed t test). (D and E) Mice bearing 3-day-old, s.c. B16-BL6 melanomas received naive pmel-1 T cells and early anti–CTLA-4 therapy or vaccination with hgp100 in IFA (s.c.) or anti-CXCR3 (i.p.) or IgG therapy (i.p.) on days 3, 5, 7, 9, and 11 after tumor injection. (D) Kaplan-Meier survival curves. (E) Absolute number of non–pmel-1 and pmel-1 T cells (mean ± SEM, n = 5) at the tumor and vaccination sites (mean ± SEM, n = 5) analyzed 9 days after the start of CXCR3 blockade. Data shown are representative of 3 experiments. (F) CXCL9 and CXCL10 mRNA expressions by RNA sequencing and overall patient survival obtained from public TCGA repositories (https://tcga-data.nci.nih.gov and http://gdac.broadinstitute.org/). Bars show mean ± SEM CD8 mRNA level as determined by paired 2-tailed t test.

Figure 5. Impact of vaccination on the ICAM-1/VCAM-1 expression on tumor vasculature.

(A) Experimental scheme. (B) Expression of adhesion surface markers on naive, pmel-1, and non–pmel-1 CD8⁺ T cells in PBMCs 9 days after vaccination, anti–CTLA-4 therapy, or left untreated. (C) Mice bearing B16-BL6 melanomas were injected i.v. with pmel-1 T cells. Mice were immunized with hgp100 or control in IFA on day 3; and/or received anti–CTLA-4 therapy on days 3, 6, and 9; and/or were neutralized with IFN-γ on days 3, 5, 7, 9, and 11 after tumor injection; or they were left untreated. Graphs show ICAM-1 or VCAM-1 expression on CD31⁺ vasculature at tumor site 9 days after vaccination. Data are mean ± SEM and are representative of 2 experiments (n = 5 mice per group, *P < 0.05 determined by nonparametric Kruskal-Wallis test).

Figure 6. CD8⁺ Teff accumulation at vaccination and tumor sites is dependent on LFA-1/ICAM-1 interaction.

(A) Mice bearing 3-day-old, s.c. B16-BL6 melanomas received naive pmel-1 T cells, early anti–CTLA-4 therapy, and vaccination with hgp100 in IFA. Presence of ICAM-1⁺VCAM-1⁺ inflammatory monocytes and granulocytes in blood and at the vaccination site 9 days after gp100/IFA vaccination. (B–D) Mice bearing 3-day-old, s.c. B16-BL6 tumors received i.v. pmel-1 T cells and vaccination with hgp100/IFA and/or anti–CTLA-4 therapy, followed by anti–ICAM-1 or IgG (i.p.) on days 3, 5, 7, 9, and 11 after tumor injection. Tumor and vaccination sites were analyzed 9 days after the start of ICAM-1 blockade (B) Kaplan-Meier survival curves, 2 independent experiments each with n = 10 mice per group. *P < 0.05,log-rank test. (C) Non–pmel-1 and pmel-1 CD8⁺ Teffs at the tumor and vaccination sites. Data are mean ± SEM, n = 5, *P < 0.05 as determined by unpaired 2-tailed t test. (D) iMos and Grans at the tumor and vaccination sites. Plots are mean ± SEM, n = 5, *P < 0.05 as determined by unpaired 2-tailed t test. Data shown (B–D) are representative of 2 experiments. (E) ICAM-1 mRNA expression by RNA sequencing and overall patient survival obtained from public TCGA repositories (https://tcga-data.nci.nih.gov and http://gdac.broadinstitute.org/).

Figure 7. CCL2 and CXCR3 mediate a feed-forward loop of CD8⁺ Teffs and inflammatory monocyte accumulation at the vaccination site.

(A and B) Mice bearing 3-day-old, s.c. B16-BL6 melanomas were injected with naive pmel-1 T cells, vaccinated with hgp100/IFA, and received early anti–CTLA-4 therapy. (A) Distribution of CD11b⁺ leukocyte subsets at tumor and vaccination sites. (B) CCR2 expression on leukocyte subsets as in A and CD8⁺ non–pmel-1 and pmel-1 Teffs in blood observed 9 days after vaccination. (C) Mice bearing 3-day-old, s.c. B16-BL6 melanomas received pmel-1 T cells and early anti–CTLA-4 therapy or vaccination with hgp100/IFA or control/IFA. CCL2 and IFN-γ chemokine concentrations (mean ± SEM) in supernatant from tumor and vaccination site homogenates 9 days after vaccination. Data shown are representative of 3 experiments, each with n = 5 mice per group. *P < 0.05 as determined by unpaired 2-tailed t test. (D and E) Mice treated the same as in C underwent CCL2 depletion or received IgG on days 3, 5, 7, 9, and 11 after tumor injection. (D) Absolute number of iMos and Grans at the tumor and vaccination sites. (E) Absolute number of CD8⁺ Teffs at the tumor and vaccination sites. (F and G) Mice treated as in C received anti-CXCR3 mAb therapy or IgG on days 3, 5, 7, 9, and 11 after tumor injection. Plots shown are absolute number of (F) iMos and (G) Grans at the tumor and vaccination sites. Data shown are mean ± SEM, representative of 3 experiments, each with n = 5 mice per group. *P < 0.05 as determined by unpaired 2-tailed t test.

Figure 8. Nonpersistent viral vaccine synergizes with therapeutic CTLA-4 and PD-L1 checkpoint blockades.

(A) Experimental scheme. Kaplan-Meier survival curves in mice with 3-day-old, s.c. B16 melanomas after receiving naive pmel-1 T cells (i.v.). (B) Early anti–CTLA-4 therapy and vaccination with hgp100/IFA (s.c.), VSV.gp100 (i.v.), VSV.OVA, or combination. (C) Early anti–PD-L1 (i.p.) plus Gvax therapy and vaccination with hgp100/IFA (s.c.), VSV.gp100 (i.v.), or combination. Data were pooled from 2 independent experiments, each with n = 10 mice per group. *P < 0.05, log-rank test.

Figure 9. Nonpersistent vaccine formulations overcome primary resistance to CTLA-4 and PD-L1 checkpoint blockade therapy.

(A) Experimental scheme. Kaplan-Meier survival curves of mice bearing 7-day-old, s.c. B16 tumors after receiving naive pmel-1 T cells (i.v.). (B) Late anti–CTLA-4 therapy and/or vaccination with hgp100/IFA (s.c.) or hgp100 peptide in saline (s.c.) with covax. (C) Covax or late anti–CTLA-4 or anti–PD-L1 therapy and/or hgp100 peptide in saline (s.c.) and covax, or no treatment. (D) Late anti–CTLA-4 or anti–PD-L1 therapy and/or vaccination with hgp100 peptide in IFA or VSV.gp100, or left untreated. (E) Late dual checkpoint blockade therapy with anti–CTLA-4 and anti–PD-L1 combined with vaccination with hgp100/IFA (s.c.) and/or VSV.gp100 (i.v.). Data are pooled from 2 independent experiments, each with n = 15 mice per group. *P < 0.05, log-rank test.

Figure 10. Mechanism of vaccine antigen–specific and non–vaccine antigen–specific Teff recruitment to the inflamed vaccination site.

Left: Vaccine antigen–specific Teffs respond to vaccine antigen at the persistent vaccination site by releasing IFN-γ, which induces CCL2-dependent recruitment of ICAM⁺ iMos. IFN-γ also induces expression of CXCL9 and CXCL10 chemokines. CTLA-4 blockade–induced CXCR3⁺LFA-1^hi Teffs, regardless of antigen specificity, are attracted by CXCL9 and CXCL10 and engage with ICAM⁺ iMos in a feed-forward loop maintained by iMos, IFN-γ, CCL2, CXCL9, CXCL10, and ICAM-1. Teffs at the vaccination site do not reach the tumor and undergo apoptosis, resulting in loss of tumor control. Right: Without a persistent, inflamed vaccination site, CTLA-4 blockade–induced Teffs are free to localize to the tumor site, resulting in tumor control.