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 antigens that is superior to either monotherapy. Yet, mechanisms underlying the synergy of prime-boost strategies remain incompletely defined. Here, 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 intramuscular delivery via electroporation, yet it remained modest compared to Ad5. Sequential delivery of DNA and Ad5 (DNA+Ad5) produced superior antitumor efficacy associated with increased T-cell receptor (TCR) avidity, while 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 was correlated with antitumor efficacy, while T-cell number and cytokine profile were not. Importantly, while DNA+Ad5 immunization 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.

Introduction

Colorectal cancer is the fourth most commonly diagnosed cancer, and second leading cause of cancer-related deaths in the United States (1). Current standard of care employs surgical removal of primary tumor and adjuvant chemotherapy to treat metastatic disease. In the context of low efficacy and high toxicity of existing adjuvant therapies, immunotherapies, particularly vaccines, for colon cancer are an emerging alternative reflecting their potential specificity and resulting low toxicity (2). However, cancer vaccine platforms with utility in patients are limited. Protein and peptide vaccines have been largely ineffective, though next-generation peptide vaccines may prove more efficacious (3). In contrast, while dendritic cell vaccines are generally immunogenic, including the FDA-approved Sipuleucel-T vaccine for prostate cancer (4), those treatments require expensive, personalized vaccines, limiting their use to a small subset of patients. Recombinant viral vectors offer great promise and have been approved as vaccine vectors for infectious diseases (5). Unfortunately, few viral vectors are suitable for humans (typically adenovirus and poxviruses) and these are limited by pre-existing and/or vaccine-induced neutralizing immunity (6). Thus, novel vaccine platforms are needed to optimally treat cancer in humans.

DNA vaccines provide a safe and inexpensive alternative vaccine strategy that is not limited by vector immunity. DNA vaccines have been examined for cancers and infectious diseases with their primary advantages being stability, safety, and absent vector immunity, permitting multiple administrations (7, 8). Indeed, DNA vaccines have been approved for animal use to treat canine melanoma and infectious diseases (9, 10). However, low transfection rates and immunogenicity have limited the utility of DNA vaccine monotherapy in humans. These have been partially overcome by electroporation (EP), gene gun, nanoparticle, and ultrasound delivery and by the inclusion of adjuvants (7, 8), but DNA vaccine success has been limited to combinations with other vaccine platforms in heterologous prime-boost regimens (11). Guanylyl cyclase C (GUCY2C) is an immunotherapeutic target in colorectal cancer reflecting its limited expression in normal tissues and persistent expression in colorectal cancers (12–17). GUCY2C is confined to the apical surfaces of intestinal epithelial cells (18–21) and a subset of hypothalamic neurons (22, 23), areas that are structurally segregated from the systemic immune compartment (12). Further, its expression is maintained throughout colorectal tumorigenesis and GUCY2C is found in >95% of colorectal cancer metastases (24, 25). Previously, we demonstrated that recombinant replication-deficient adenoviral vaccines (Ad5) expressing the extracellular domain of GUCY2C induce protective GUCY2C-specific CD8⁺ T-cell responses, without toxicity, in a mouse model of metastatic colorectal cancer (13–15, 26).

Here, we examined a prime-boost strategy comprising GUCY2C-expressing DNA and adenoviral vaccines to enhance antitumor efficacy, identifying a heterologous strategy of DNA vaccine priming and adenovirus vaccine boosting (DNA+Ad5) that induces superior antitumor immunity, preventing disease progression in the majority of animals, without collateral autoimmunity. Moreover, the enhanced antitumor immune responses produced by DNA+Ad5 vaccination reflected the production of high-avidity effector CD8⁺ T cells. In the context of enhanced immune responses, superior antitumor immunity, and absence of toxicity, the DNA+Ad5 vaccination strategy identified here is poised for clinical translation to prevent recurrent disease in early stage colorectal cancer patients.

Materials and Methods

Mice

BALB/c mice were obtained from Charles River. Animal protocols were approved by the Thomas Jefferson University Institutional Animal Care and Use Committee.

Vaccine Constructs and DNA Electroporation

Except where indicated, all DNA and Ad5 GUCY2C-specific vaccine constructs employed mouse GUCY2C fused at the C-terminus to the influenza HA_107–119 epitope (known as Site 1 or S1) previously described (14). The Ad5 vaccine composed of mouse GUCY2C-S1 (Ad5-GUCY2C-S1) was described previously (14). The DNA vaccine construct employed mouse GUCY2C-S1 cloned into pcDNA-DEST47 (Life Technologies). Plasmids pCCL20 (pEF1-mCCL20), pCCL21 (pCUB-mCCL21), and pMaxGFP were previously described (27). The control plasmid pcDNA 3.1 is from Life Technologies. For intramuscular (IM) and intradermal (ID) electroporation, 50 μg plasmid DNA in 50 μL water was delivered into gastrocnemius muscle of each leg or two distant skin sites per mouse, respectively. DNA delivery was followed by 10 electric pulses (field strength = 100 V/cm; pulse length = 20 ms; pulse interval=1s) delivered by an ECM830 Square Wave Electroporation System (BTX Harvard Apparatus).

Immunizations

1×10⁸ IFU of Ad5-GUCY2C-S1 was administrated to each gastrocnemius muscle by IM injection. For DNA vaccinations, the pCCL20 (25 μg) and pCCL21 (25 μg) plasmids in water were combined and injected with EP, followed 12 days later by immunization with 50 μg of GUCY2C-expressing or control DNA via EP. Prime-boost strategies were administered as described with 21 days between vaccinations. For therapeutic studies (Supplementary Fig. 3), control or GUCY2C-expressing DNA vaccinations were administered without chemokines on day 3 and control or GUCY2C Ad5 was administered on day 10. Radiotherapy employed a PanTak, 310 kVe x-ray machine to deliver 10 Gy irradiation on day 3 to only the chest, protecting other organs with lead shielding. The checkpoint inhibitor antibodies anti-CTLA-4 (Clone UC10-4F10-11; BioXCell) and anti-PD-L1 (Clone 10F.9G2; BioXCell) were administered IP (100 ug each) in PBS on days 1, 4, 7, 11 and 15.

Immunoassays

Immune responses to the dominant GUCY2C-specific CD8⁺ T cell epitope GUCY2C_254–262 (26) were measured in splenocytes 14 days after the final immunization.

ELISpot

Multiscreen filtration plates (Millipore) are coated with 10 μg/ml anti-mouse interferon gamma (IFNγ)-capture antibody (BD Biosciences) overnight. Splenocytes (1×10⁶ per well) were stimulated with DMSO or 10 μg/ml GUCY2C_254–262 peptide for 24 hours. Spots were developed with 2 μg/ml biotinylated anti-IFNγ detection antibody (BD Biosciences) and 2 μg/ml alkaline phosphatase-conjugated streptavidin (Pierce), followed by NBT/BCIP substrate (Pierce). Spot forming cells were enumerated using computer-assisted video imaging analysis (ImmunoSpot v5, Cellular Technology).

Intracellular cytokine staining (ICS)

Splenocytes were stimulated for 6 hours with DMSO or GUCY2C_254–262 and Protein Transport Inhibitor Cocktail (eBioscience). Cells were stained with Live/Dead fixable Aqua Dead Cell stain kit (Invitrogen) and αCD8α-PerCP-Cy5.5 (clone 53-6.7, BD Pharmingen) and ICS staining was performed using the BD Cytofix/Cytoperm Kit (BD Biosciences) and the following antibodies: αIFNγ-APC-Cy7 (XMG1.2) and αTNFα-PE-Cy7 (MP6-XT22) from BD Biosciences, and αMIP1α-PE (clone 39624, R&D Systems). Cells were fixed in 2% PFA and analyzed on a BD LSR II flow cytometer. Analyses were performed using FlowJo software (Tree Star).

TCR avidity

1×10⁶ splenocytes were plated/well in ELISpot plates with varying concentrations of GUCY2C_254–262 peptide for 24 hours. ELISpot plates were developed as described above.

Tumor Immunity

Mice were immunized as described above and challenged intravenously with 5×10⁵ CT26 cells expressing mouse GUCY2C (15) in PBS 6 days later, and survival was measured longitudinally. Intravenous administration of CT26 cells is a well-established model of metastatic colorectal cancer, preferentially forming metastases in the lungs (13–15, 26, 28, 29).

Autoimmunity

Tissues were collected from mice 14 or 180 days after no treatment or treatment with GUCY2C-expressing DNA or DNA+Ad5 prime-boost vaccination. Tissues were fixed in formalin and embedded in paraffin. Sections were stained with hematoxylin and eosin and scored for toxicity by a blinded pathologist (L.B.B.). Scoring criteria are described in Supplementary Table 1. DSS studies were performed as previously described with some modification for the BALB/c mouse model (13). Three weeks after completing control or GUCY2C DNA+Ad5 immunization regimens, DSS (Sigma-Aldrich) was administered ad libitum in the drinking water at 5% (w/v) for 8 days, followed by normal drinking water for the remainder of the experiment. Mice were weighed daily throughout the experiment and 3 representative mice were euthanized on day 9 following DSS initiation for histopathologic analysis (L.B.B.). Negative area under the curve (AUC) peaks were calculated on weight curves using GraphPad Prism v6 and absolute values were plotted.

Statistics

All analyses employed GraphPad Prism Software v6. T-cell enumeration by ELISpot was analyzed by One-way ANOVA (multiple comparisons) or T test (single comparisons). Cytokine polyfunctionality employed Two-way ANOVA. TCR avidity measurements were analyzed by non-linear regression [log(agonist) vs. normalized response] and comparisons were made by exact sum-of-squares F test (GraphPad v6). Non-linear regression plots depict the regression results (line) with 95% confidence intervals (cloud) computed from results calculated from multiple independent experiments. Survival comparisons employed Mantel-Cox log-rank test. The relationship between each immunoassay and tumor immunity was analyzed by F test of linear regression analysis. Autoimmunity comparisons employed One-way ANOVA and DSS comparisons employed T test.

Results

Route of administration effects transgene expression

We previously employed intradermal (ID) administration of DNA vaccines combined with CCL20 and CCL21 chemokine adjuvanation co-delivered by in vivo electroporation (EP) to enhance melanoma-specific immune response and local immunotherapeutic efficacy targeting dermal melamoma tumors (27). Here, we examined DNA vaccine immunogenicity following intramuscular (IM) administration to treat systemic colorectal cancer metastases, characterized by dissemination to lung, liver and brain. As expected, antigen expression assayed by GFP fluorescence was superior following IM, compared to ID, administration, in a time-dependent fashion (Fig. 1A–B). Increased antigen expression following IM administration suggests this route may be favorable for DNA vaccine-induced immune responses. Moreover, DNA vaccine administration in the absence of EP eliminated antigen expression (Fig. 1C), consistent with literature suggesting a 100–1000 fold increase in transgene expression with EP (30, 31). Thus, transgene expression was optimized by IM, rather than ID, administration of DNA plasmid in the context of EP.

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.

Ad5 vaccination is superior to DNA vaccination

In the context of robust transgene expression following IM, rather than ID, DNA delivery (Fig. 1), we compared the immunogenicity of IM and ID vaccinations (Fig. 2A). IM DNA vaccination with the syngeneic mouse colorectal cancer antigen GUCY2C produced systemic GUCY2C-specific CD8⁺ T-cell responses quantified by ELISpot that were >10-fold greater than ID immunization (Fig. 2A), consistent with greater transgene expression in muscle (Fig. 1). However, adenoviral vector immunization against GUCY2C (Ad5) produced GUCY2C-specific CD8⁺ T-cell response that were >10-fold greater than those produced by IM DNA vaccination (Fig. 2B). Moreover, Ad5 vaccination produced an 18-fold increase in median survival time of mice with GUCY2C-expressing colorectal cancer metastases in lung compared to DNA vaccination (Fig. 2C). Moreover, repeated administrations of DNA vaccination failed to improve GUCY2C-specific CD8⁺ T-cell responses (Supplementary Fig. 1). Taken together, IM DNA vaccination produces GUCY2C-specific CD8⁺ T-cell responses and antitumor responses, but those are not comparable to Ad5 vaccination, suggesting that DNA vaccination alone is not a viable strategy for GUCY2C-directed tumor immunotherapy.

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).

Heterologous DNA+Ad5 prime-boost enhances CD8⁺ T cell avidity and antitumor immunity

While GUCY2C-specific DNA vaccination alone was poorly immunogenic, we hypothesized that it could be combined with Ad5 in prime-boost regimens to improve antitumor immunity. To maximize GUCY2C-specific immune responses and antitumor immunity, different heterologous prime-boost strategies were explored. Combining GUCY2C-specific DNA and Ad5 vaccinations in either order (DNA+Ad5 or Ad5+DNA) increased survival of mice with GUCY2C-expressing CT26 colorectal cancer metastases (Fig. 3A). While both heterologous prime-boost strategies improved median survival time compared to Ad5 alone (43.5 days), DNA+Ad5 was superior to Ad5+DNA (111 vs. 77.5 days, P<0.05) and produced GUCY2C-specific memory responses in surviving mice (Supplementary Fig. 2). A truncated version of the DNA+Ad5 regimen (no chemokines; 7 days between boosting) also extended survival in a therapeutic setting when combined with local radiotherapy (Supplementary Fig. 3). Examination of GUCY2C-specific CD8⁺ T-cell responses quantified by T-cell number (ELISpot; Fig. 3B) and polyfunctional cytokine production (Fig. 3C), revealed modest increases by DNA+Ad5 and no increase by Ad5+DNA immunization. However, the functional TCR avidity of GUCY2C-specifc CD8⁺ T-cell responses (Fig. 3D) induced by DNA+Ad5 (0.30 ug/mL) and Ad5+DNA (0.82 ug/mL) were substantially increased compared to Ad5 immunization alone (1.92 ug/mL). Importantly, GUCY2C-specific CD8⁺ T-cell numbers (Fig. 3B), polyfunctional cytokine responses (Fig. 3C), TCR avidity (Fig. 3D) and antitumor efficacy (Fig. 3A) were not improved by homologous Ad5 prime-boost (Ad5+Ad5) compared to Ad5 alone, confirming the role of heterologous prime-boosting in GUCY2C-specific CD8⁺ T-cell enhancement.

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.

Functional TCR avidity enhancement is required for GUCY2C-targeted antitumor efficacy

In the context of little or no enhancement of GUCY2C-specific CD8⁺ T-cell number and effector cytokine responses by heterologous prime-boost, yet substantial increases in TCR avidity and antitumor immunity, we hypothesized that TCR avidity enhancement underlies the improved antitumor efficacy of GUCY2C-specific prime-boost regimens. To test this hypothesis by manipulating TCR avidity, while preserving the magnitude and cytokine profile of GUCY2C-specific CD8⁺ T cells, we created an adenovirus vaccine expressing only the dominant, pre-processed GUCY2C-specific CD8⁺ T-cell epitope (Ad5-GUCY2C_254–262). We have previously shown that viral vectors expressing processed MHC class I epitopes, result in high surface peptide-MHC density, producing low avidity T-cell responses (32). Indeed, the quantity (Fig. 4A) and effector cytokine profiles (Fig. 4B) of Ad5-GUCY2C_254–262-induced CD8⁺ T-cell responses were comparable to DNA+Ad5 immunization, while the TCR avidity of Ad5-GUCY2C_254–262-induced T cells was ~20-fold lower than DNA+Ad5 (5.88 vs. 0.30 ug/mL, P<0.0001). In turn, Ad5-GUCY2C_254–262 produced only an 11-day improvement in median survival compared to control immunization, while the majority of DNA+Ad5-immunized mice survived >200 days (Fig. 4D). When comparing antitumor efficacy to T-cell quantity (Fig. 5A), T-cell cytokine polyfunctionality (Fig. 5B) and T-cell avidity (Fig. 5C) across all tested vaccination combinations, only T-cell avidity predicted tumor outcomes, revealing the previously unrecognized mechanism of functional T-cell avidity enhancement mediating synergy of DNA+Ad5 prime-boost vaccinations.

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.

TCR avidity and antitumor efficacy enhancement by DNA+Ad5 immunization does not produce autoimmune toxicity

Enhancing TCR avidity during adoptive T cell therapy for melanoma enhances antitumor immunity and autoimmunity, suggesting a strong relationship between efficacy and toxicity in T-cell immunotherapy (33). However, immunologic compartmentalization between systemic and mucosal immune systems prevents autoimmunity in mice receiving Ad5-based GUCY2C vaccines, suggesting that the increased TCR avidity and antitumor immunity observed with DNA+Ad5 prime-boost vaccination would not come at the expense of increased autoimmunity (13–15). Indeed, mice immunized against GUCY2C with DNA or the DNA+Ad5 prime-boost strategy were free of acute (2 weeks after final immunization) or chronic (6 months after final immunization) autoimmune toxicity (Fig. 6A–B) in tissues associated with GUCY2C expression (small and large intestines, cecum, salivary gland and hypothalamus), as well as tissues devoid of GUCY2C (heart, lung, liver, kidneys and stomach). Moreover, oral administration of DSS, an established model of experimental colitis, resulted in equivalent clinical and histopathologic colitis and recovery in control and GUCY2C DNA+Ad5-immunized mice (Fig. 6C–E). Thus, GUCY2C DNA+Ad5 immunization did not exacerbate experimentally-induced colitis, allowing clinical recovery. Together, DNA+Ad5 prime-boost immunization enhances TCR avidity, maximizing antitumor immunity, without concomitant autoimmunity.

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).

Discussion

Despite emerging oncoimmunotherapeutics (34), cancer causes about 8 million deaths worldwide each year and the total economic burden of cancer, exceeding 1 trillion dollars annually, is higher than that of any other disease (35, 36), highlighting the need for effective, safe and inexpensive strategies to treat cancer in developed and developing regions around the world. In that context, vaccines have had one of the largest impacts on public health in human history, eradicating several infectious diseases and nearly eliminating morbidity and mortality associated with many others (37). However, cancer vaccines have been largely unsuccessful reflecting, in part, their poor immunogenicity and a lack of biomarkers predictive of patient outcomes. Here, we show that a strategy employing heterologous prime-boost with GUCY2C-targeted DNA and Ad5 vaccines cures ~50% of mice with metastatic colorectal cancer. More importantly, the efficacy of this strategy reflected increased TCR functional avidity compared to Ad5 vaccination alone. Indeed, TCR functional avidity, but not CD8⁺ T-cell number, cytokine or polyfunctional cytokine responses, were highly correlated (P < 0.0001) with outcomes, and experimental reduction of TCR functional avidity, while maintaining effector T-cell numbers and cytokine profiles, eliminated vaccine efficacy.

These results contrast with previous studies, primarily in infectious disease, which suggest that polyfunctional cytokine responses are the key determinant in outcomes (38). Indeed, analysis of untreated patients with poor HIV control (progressors) and those with HIV control (nonprogressors) demonstrated that nonprogressors possessed higher functionality than progressors (39). More recently, suppression of HIV replication, through killing of HIV-infected targets by high avidity CD8⁺ T cells (40) was found to be the exclusive determinant of HIV viremic control in progressors (41), suggesting that CD8⁺ T cell avidity, rather than polyfunctionality, may also underlie HIV control. Similarly, experimental manipulation of TCR avidity employing adoptive transfer of T cells genetically engineered to express TCRs of varying affinities to the melanoma antigen gp100 demonstrated that antitumor efficacy was determined by TCR affinity (33). Of note, anti-melanoma efficacy and melanocyte-targeted autoimmunity were tightly coupled, suggesting that there may not be a window of TCR affinity in which selective tumor, but not self, tissues may be targeted (33). In that context, anatomical localization of GUCY2C to intestinal apical membranes (18–20) and restricted immune trafficking to intestinal mucosa by T cells induced by systemic vaccination (42–44) decouples antitumor activity and autoimmunity. Immunization with Ad5 alone produces antitumor immunity targeting metastatic colorectal cancer in lungs and liver, without concomitant autoimmunity (Fig. 6 and Ref. 13–15). Similarly, TCR avidity and antitumor efficacy were improved ~10-fold following DNA+Ad5 prime-boost, without acute (2 weeks) or chronic autoimmunity (~6 months; Fig. 6). Thus, while TCR avidity may not be exploited to decouple antitumor immunity and autoimmunity (33), antigen and immune compartmentalization may be exploited to produce safe and effective immunotherapeutics targeting GUCY2C, as well as other antigens selectively expressed in mucosa and by metastatic cancer (cancer mucosa antigens Ref. 12–15).

While DNA+Ad5 produced superior antitumor efficacy through TCR avidity enhancement, the mechanism(s) mediating TCR avidity enhancement are not yet defined and likely reflect selective enrichment of individual T-cell clones possessing high TCR avidity and/or T-cell intrinsic TCR avidity enhancement. DNA vaccination may preferentially prime T-cell clones of high TCR avidity by eliciting both direct presentation and indirect (cross) presentation of encoded antigens (45, 46), which can be expanded by subsequent Ad5 boosts. The relative contribution of direct and indirect presentation of GUCY2C to induction of CD8⁺ T-cell responses following DNA or Ad5 immunization has not yet been explored, preventing predictions of their contributions to TCR avidity enhancement. Studies employing cell/tissue-specific promotors, such as the CD11c promoter to limit expression to professional APCs (direct presentation) or the human desmin gene 5′ regulatory region (DES) to limit expression to myocytes (indirect presentation) may be employed to determine the role of direct and indirect antigen presentation in TCR avidity enhancement (47). Moreover, spectratype analysis (48) or next-generation sequencing (49) may reveal shifts in antigen-specific T-cell populations following DNA+Ad5 immunizations, while TCR transgenic models possessing a fixed TCR repertoire may prevent TCR avidity enhancement by DNA+Ad5 immunization. In that context, a recent study employing ovalbumin-specific TCR transgenic mice, suggests that TCR avidity enhancement following DNA+vaccinia prime-boost is independent of clonal selection or proximal TCR signaling, but instead depends on decreased TCR activation threshold via a T-cell intrinsic MyD88 pathway (50). However, while TCR avidity enhancement was dependent on MyD88 signaling, those studies employed Rag^+/+ TCR transgenic mice permitting thymic recombination of endogenous TCR alleles, expanding the TCR repertoire and permitting a contribution by T-cell clone selection to TCR avidity enhancement. Defining the mechanism(s) mediating TCR-avidity enhancement by DNA+Ad5 immunization may lead to the development of targeted adjuvants that enhance TCR avidity without requiring heterologous prime-boost strategies, reducing vaccination complexity and potentially decreasing the time required to generate high-avidity responses, a critical consideration in the development of therapeutic vaccines in cancer and infectious diseases.

TCR avidity enhancement following prime-boost is not limited to immunization combinations composed of DNA and Ad5. DNA+vaccinia (50), as well as fowlpox+vaccinia (51) prime-boost regimens, similarly enhance TCR avidity. Moreover, we have shown that heterologous viral vector immunization regimens targeting GUCY2C produce increasing antitumor efficacy with each successive boost (15). Together, they suggest that TCR avidity enhancement may be a generalizable outcome of heterologous prime-boost immunizations, independent of antigen target and vaccine design. In the context of ongoing clinical studies of Ad5-GUCY2C (52), GUCY2C-specific TCR avidity and clinical efficacy could be enhanced, without autoimmunity, employing heterologous prime-boost strategies in patients with GUCY2C-expressing cancers, including colorectal and a subset of gastric, esophageal and pancreatic cancers.

Abbreviations

Ad5

adenovirus serotype 5

GUCY2C

guanylyl cyclase c