Prime-Boost Immunization Eliminates Metastatic Colorectal Cancer by Producing High-Avidity Effector CD8+ T Cells

Abstract

Heterologous prime-boost immunization with plasmid DNA and viral vector vaccines is an emerging approach to elicit CD8⁺ T cell-mediated immunity targeting pathogens and tumor Ags that is superior to either monotherapy. Yet, the mechanisms underlying the synergy of prime-boost strategies remain incompletely defined. In this study, we examine a DNA and adenovirus (Ad5) combination regimen targeting guanylyl cyclase C (GUCY2C), a receptor expressed by intestinal mucosa and universally expressed by metastatic colorectal cancer. DNA immunization efficacy was optimized by i.m. delivery via electroporation, yet it remained modest compared with Ad5. Sequential immunization with DNA and Ad5 produced superior antitumor efficacy associated with increased TCR avidity, whereas targeted disruption of TCR avidity enhancement eliminated GUCY2C-specific antitumor efficacy, without affecting responding T cell number or cytokine profile. Indeed, functional TCR avidity of responding GUCY2C-specific CD8⁺ T cells induced by various prime or prime-boost regimens correlated with antitumor efficacy, whereas T cell number and cytokine profile were not. Importantly, although sequential immunization with DNA and Ad5 maximized antitumor efficacy through TCR avidity enhancement, it produced no autoimmunity, reflecting sequestration of GUCY2C to intestinal apical membranes and segregation of mucosal and systemic immunity. Together, TCR avidity enhancement may be leveraged by prime-boost immunization to improve GUCY2C-targeted colorectal cancer immunotherapeutic efficacy and patient outcomes without concomitant autoimmune toxicity.

Figure 1. Route and Electroporation Enhance Transgene Expression

A–C, control or GFP-expressing plasmids were injected ID or IM with or without electroporation. Skin (ID) or leg (IM) tissues were harvested 3, 6, or 9 days later and imaged by fluorescence microscopy. Fluorescence images were overlaid onto bright-field images of dissected tissues. B, time course of protein expression by electroporation of IM or ID administered control or GFP-expressing plasmids. C, EP is required for efficient transfection of control or GFP-expressing plasmids (analysis on day 6). Data are representative >4 independent experiments.

Figure 2. Adenoviral vaccination is superior to DNA vaccination for GUCY2C

A, GUCY2C-specific DNA vaccinations were administered IM or ID with EP, followed 14 days later by quantification of T-cell responses to the dominant GUCY2C_254–262 epitope by IFNγ ELISpot. B–C, a comparison of the optimal IM DNA vaccination and Ad5 vaccination revealed the superiority of Ad5 by GUCY2C-specific ELISpot 14 days after immunization (B) and by antitumor immunity following intravenous challenge with GUCY2C-expressing CT26 colorectal cancer cells and monitoring of survival (C). *** P < 0.001, **** P < 0.0001, T test (A and B) and Mantel-Cox Log-Rank Test (C). Data are shown as means +/− SD, representative of 4–6 independent experiments containing n=4–5 mice/group/experiment (A–B). N=10 mice/group (C).

Figure 3. DNA+Ad5 Prime-Boost Vaccination Maximizes GUCY2C-Specific Antitumor Immunity

A–D, mice were immunized with control vaccine (Control) or GUCY2C-specific Ad5, DNA, or homologous and heterologous combinations of Ad5 and DNA (Ad5+Ad5, Ad5+DNA, DNA+Ad5). A, following immunization, mice were challenged with GUCY2C-expressing CT26 colorectal cancer cells to establish lung metastases and survival was monitored. B–D, T cells were collected from immunized mice 14 days after the final immunization to quantify GUCY2C_254–262-specific T-cell number by IFNγ ELISpot (B), cytokine polyfunctionality by IFNγ/TNFα/MIP1α FACS (C) and TCR avidity by IFNγ ELISpot (D). NS P > 0.05, * P < 0.05, ** P < 0.01, **** P < 0.0001, One-way ANOVA (B), Two-way ANOVA (C), Sum-of-Squares F test (D). N=10 mice/group (A). Data are shown as means +/− SEM of 3–6 independent experiments with N=5 mice/group/experiment (B–C) or non-linear regression (line) with 95% confidence intervals (cloud) computed from results obtained in 3–6 independent experiments with N=5 mice/group/experiment (D). Statistical comparisons were made to Ad5 alone (B–C) or as indicated.

Figure 4. DNA+Ad5 Prime-Boost Synergy Reflects TCR Avidity Enhancement

A–D, mice were immunized with control vaccine (Control), Ad5-GUCY2C_254–262 or GUCY2C-specific DNA+Ad5. A–C, T cells were collected from immunized mice to quantify GUCY2C-specific T-cell number by IFNγ ELISpot (A), cytokine polyfunctionality by IFNγ/TNFα/MIP1α FACS (B) and TCR avidity by IFNγ ELISpot (C). D, following immunization, mice were challenged with GUCY2C-expressing CT26 colorectal cancer cells to establish lung metastases and survival was monitored. NS P > 0.05, **** P < 0.0001, T test (A), Two-way ANOVA (B), Sum-of-Squares F Test (C) and Mantel-Cox Log-Rank Test (D). Data are shown as means +/− SEM of 3–6 independent experiments with N=5 mice/group/experiment (A–B) or non-linear regression (line) with 95% confidence intervals (cloud) computed from results obtained in 3 independent experiments with N=5 mice/group/experiment (C). N=10 mice/group (D).

Figure 5. Functional TCR Avidity Predicts Antitumor Efficacy

ELISpot number (A), 1, 2, or 3 cytokine polyfunctionality (B), and TCR functional avidity (C) produced by the tested vaccine combinations were correlated with improvement in median survival beyond control vaccination in mice with metastatic GUCY2C-expressing colorectal cancer. P values obtained from F test of linear regression analysis. T-cell measurements are shown as means +/− SEM of 3–6 independent experiments with N=5 mice/group/experiment.

Figure 6. Safety of TCR Avidity Enhancing DNA+Ad5 Immunization

A–B, mice were untreated (Control) or immunized with GUCY2C-specific DNA or DNA+Ad5 vaccines and tissues were collected 14 or 180 days later for histopathologic scoring. A, representative small and large intestine images for each vaccine regimen are shown. B, no statistically significant differences in histopathology were observed in any tissue between any of the immunizations groups (Two-way ANOVA; N=4–5 mice/group). C-E, mice immunized with control or GUCY2C-specific DNA+Ad5 vaccines with treated with an 8-day course of 5% DSS in their drinking water (N=10 mice/group). C, body weights were measured daily for 4 weeks and used to calculate disease severity as area under the curve (D; T test). E, histology scores of colon tissues collected on day 9 indicate equivalent DSS-induced inflammation (T test).