This was a phase I study (ClinicalTrials.gov identifier NCT01972737) of stage I or II (pN0) colon cancer within 3 years of surgery and no clinical or laboratory evidence of local or systemic recurrence. The study protocol and all amendments were approved by the Thomas Jefferson University Institutional Review Board (IRB) and Institutional Biosafety Committee (IBC). The study was conducted in accordance with the protocol, Good Clinical Practice guidelines, the ethical principles outlined in the Declaration of Helsinki, and the NIH Guidelines for Research Involving Recombinant or Synthetic Nucleic Acid Molecules. All patients provided written informed consent to participate.

Ad5 vectors have well-established potency to induce antigen-specific immune responses in animal models and humans, as well as a long and impressive safety record. PADRE is a CD4⁺ T-cell epitope that is active in the context of most human HLA molecules [28] and is required to optimally induce GUCY2C-specific immune responses and efficacy in animal models [17, 18]. Codon-optimized cDNA encoding human GUCY2C residues 1–429 with a C-terminal PADRE epitope (Fig. 1a) was cloned into the E1 region of pAd/CMV/V5 (Life Technologies, Carlsbad, CA) containing E1- and E3-deleted human serotype 5 adenovirus (Ad5; Fig. 1b). Ad5-GUCY2C-PADRE vector used for these studies was produced under GMP conditions at the Baylor College of Medicine in the Cell and Gene Therapy Vector Development Laboratory.Fig1Fig. 1

Ad5-GUCY2C-PADRE design and antigen expression. a GUCY2C is a membrane-spanning enzyme possessing an extracellular ligand-binding domain and intracellular cGMP-producing catalytic domain. The extracellular domain (ECD) of GUCY2C was employed in the vaccine design and included the PADRE epitope on its COOH-terminus. b GUCY2C_ECD-PADRE was inserted into the E1 region of E1/E3-deleted Ad5. c A549 cells were transduced in duplicate with Ad5-GUCY2C-PADRE at an MOI of 10 to 10,000 for 48 h. GUCY2C_ECD-PADRE expression was quantified in supernatants by immunoblot analysis. Densitometry was employed to quantify expression. d A549 cells were transduced in duplicate with Ad5-GUCY2C-PADRE at an MOI of 10,000 for 24–96 h and GUCY2C_ECD-PADRE expression was quantified in supernatants by immunoblot analysis. Densitometry (arbitrary units) was employed to quantify expression. Blots in c and d are representative of two experiments and graphs indicate the mean ± SD from 2 experiments

Venous blood was collected into BD Vacutainer® Glass Serum Tubes for serum collection and BD Vacutainer® CPT™ Mononuclear Cell Preparation Tubes with Sodium Citrate for peripheral blood mononuclear cell (PBMC) isolation. For serum collection, blood samples were incubated 30 min at 37 °C, centrifuged, and supernatants were transferred to cryovials and stored at − 20 °C. PBMCs were collected by centrifugation according to manufacturer’s instructions. PBMCs were then washed with 1x CTL-WASH™ buffer (Cellular Technology Limited, Cleveland, OH), counted using a Muse Cell Analyzer (Millipore, Darmstadt, Germany), and cryopreserved in CTL-Cryo™ ABC freezing medium (Cellular Technology Limited) according to the manufacturer’s instructions. Cryovials were frozen at − 80 °C overnight in a CoolCell® LX Alcohol-free Cryopreservation Container (Biocision, Mill Valley, CA) before long-term storage in LN₂.

Animal studies were approved by the Thomas Jefferson University Institutional Animal Care and Use Committee (IACUC). All mouse studies employed ~ 10 week old female BALB/c mice (Jackson, Bar Harbor, ME). Females were nulliparous and not pregnant. To establish Ad5 NAb titers, vehicle control (naïve) or control Ad5 (Ad5 NAb High) were administered intramuscularly as two 50 μL injections, one in each of the two hind limbs. Ad5 exposure was repeated 21 days later to establish high Ad5 NAb titers. Two weeks after completing the above Ad5 exposures, serum was collected for Ad5 NAb titer determinations and animals were immunized with 10⁸ IFU Ad5-GUCY2C-S1 [18]. Two weeks later, serum and splenocytes were collected and GUCY2C-specific antibody and GUCY2C- and Ad5- specific CD8⁺ T-cell responses were quantified as previously described [16–20].

Ad5-GUCY2C-PADRE is composed of an E1/E3-deleted recombinant human type 5 adenovirus expressing the human GUCY2C extracellular domain (ECD; GUCY2C_1–429) fused on its C-terminus to the universal CD4⁺ T-helper cell epitope PADRE (Fig. 1a and b). Previous studies demonstrated that only the extracellular domain of GUCY2C is a viable vaccine target reflecting the high sequence conservation of the intracellular domains of guanylyl cyclase family members and broad tissue distribution of guanylyl cyclases A, B, and G [20]. GUCY2C_ECD-PADRE and an upstream CMV promoter were cloned into the E1 region of Ad5 (Fig. 1b). Replication-deficient Ad5-GUCY2C-PADRE vector was produced in HEK293 cells and purified by CsCl ultracentrifugation employing GMP procedures at the Center for Cell and Gene Therapy, Baylor College of Medicine. In vitro studies confirmed dose-dependent (Fig. 1c) and time-dependent (Fig. 1d) expression and secretion of GUCY2C_ECD-PADRE protein by western blot.

Ten colorectal cancer patients were enrolled and treated with 10¹¹ vp Ad5-GUCY2C-PADRE. Additional file 1: Table S1 describes the baseline patient characteristics. The median age was 65 (49–76) years, patients were primarily Caucasian (80%) and patients were distributed equally between male and female. All patients had stage I or II colorectal cancer previously treated with surgery but not chemo/radio/immuno-therapy. Treatment-related acute toxicity was assessed in the clinic every 10 min for 30 min after injection and by telephone on days 3 and 8 following vaccination. Patients also returned to the clinic 30, 90, and 180 days after vaccination for safety assessment. All patients completed the study. Adverse events (Table 1) were graded according to The Common Terminology Criteria for Adverse Events (CTCAE version 4.0). Mild grade 1/2 toxicities included injection site pain and fever which are anticipated following a viral vector immunization. No grade 3/4 toxicities occurred at any time during the 6-month follow-up period after vaccination. Moreover, clinical laboratory assessments performed on days 30, 90, and 180, including CBC with differential, comprehensive chemistry panel, and antinuclear antibody (ANA) titers, revealed no vaccine-related adverse events. Importantly, no adverse events related to toxicity in GUCY2C-expressing tissues were observed. GUCY2C is a self protein expressed on the luminal surface of small and large intestinal epithelia [7, 8], as well as anorexigenic hypothalamic and midbrain dopaminergic neurons [9, 10]. However, consistent with mechanisms controlling immune compartmentalization [30], preclinical studies of GUCY2C vaccination in mice [16–20] confirmed the absence of Ad5-GUCY2C-PADRE-induced autoimmunity in intestine or brain.Tab1

Treatment-related toxicities occurring during the 6-months following Ad5-GUCY2C-PADRE vaccination

In preclinical studies, immunization with Ad5-GUCY2C-PADRE induced time- and dose-dependent GUCY2C-specific T-cell and B-cell responses and antitumor immunity mediated by CD8⁺ T cells [17–20, 26]. Here, GUCY2C-specific immune responses were quantified after Ad5-GUCY2C-PADRE administration by ELISA and IFNγ-ELISpot to quantify antibody and T-cell responses, respectively (Additional file 1: Table S2). T-cell responses to PADRE and Ad5 also were quantified by IFNγ-ELISpot. Patient responses typically followed 1 of 4 patterns and representative responses of each are shown in Fig. 2. All other patient responses are shown in Additional file 1: Figure S1. Patient 1001 had no pre-vaccine antibody responses to GUCY2C or T-cell immunity to GUCY2C, PADRE, or Ad5 and Ad5-GUCY2C-PADRE vaccination did not induce responses to these antigens (Fig. 2a). Similarly, while no pre-vaccination responses were observed in patient 1009, vaccination induced Ad5-specific T-cell responses, but not GUCY2C-specific or PADRE-specific responses (Fig. 2b). In contrast to these patients, patient 1008 possessed Ad5-specific T-cell responses prior to vaccination, and Ad5-GUCY2C-PADRE vaccination increased those responses (Fig. 2c). Similarly, GUCY2C-specific T-cell responses also were induced by Ad5-GUCY2C-PADRE vaccination, initially peaking on day 30, followed by a gradual decline through the final 180-day time-point (Fig. 2c). However, this patient produced no PADRE-specific T-cell response or GUCY2C-specific antibody response, recapitulating preclinical studies in mice in which GUCY2C-specific antibody responses require responses to exogenous CD4⁺ “helper” T-cell epitopes, reflecting GUCY2C-specific CD4⁺ T-cell tolerance [17–20, 26]. Patient 1007 was the only patient that produced a response by all three arms of adaptive immunity (Fig. 2d). That patient produced a PADRE-specific CD4⁺ T-cell response, a GUCY2C-specific antibody response, and a GUCY2C-specific CD8⁺ T-cell response that peaked between days 30 and 90, before declining over the remainder of the study.Fig2Fig. 2

Ad5-GUCY2C-PADRE-induced immune responses. Patient blood samples were collected before (day 0) and 30, 90 and 180 days after Ad5-GUCY2C-PADRE immunization. GUCY2C-specific antibody titers were quantified by ELISA and GUCY2C, PADRE, and Ad5 -specific T-cell responses were quantified by IFNγ-ELISpot. ELISpot assays employed DMSO as an antigen-negative control. The statistical significance for T-cell responses at each time point (compared to DMSO) was determined by modified DFR(eq) or DFR(2x) after Westfall–Young max-T correction, and p-values < 5% are shown in yellow [mDFR(eq)] or red [mDFR(2x)], respectively. The statistical significance of T-cell responses obtained for each post-vaccination time point (compared to day 0) were determined by a similar modified DFR-like permutation method with Westfall-Young max-T correction. Representative GUCY2C non-responders (a and b) and responders (c and d) are shown. All other patient responses are shown in Additional file 1: Figure S1

Preclinical studies in mice revealed split tolerance to GUCY2C, eliminating CD4⁺ T cells, but not CD8⁺ T or B cells, which could be fully engaged with exogenous CD4⁺ helper T-cell epitopes (S1 or PADRE) to produce antitumor immunity without autoimmunity [18–20]. To extend that observation from mice to humans, CD4⁺ or CD8⁺ T cells were depleted from PBMCs of patients 1007 and 1008 (GUCY2C responders, Fig. 2), prior to quantification of T-cell responses by IFNγ-ELISpot to determine the cell type responsible for GUCY2C-specific responses (Fig. 3). Depletion of CD8⁺, but not CD4⁺, T cells (Fig. 3a, c) eliminated ELISpot responses in both patients (Fig. 3b, d). Thus, vaccine responses in colon cancer patients are mediated exclusively by CD8⁺ T cells, recapitulating GUCY2C immunology in mice [18–20]. Indeed, selective CD4⁺ T-cell tolerance appears to be a universal mechanism regulating GUCY2C-specific immunity, eliminating GUCY2C-specific CD4⁺ T cells, but not B cells or CD8⁺ T cells, in C57BL/6 [17, 19, 20] and BALB/c [18, 20, 26] mice and in humans (Figs. 2 and 3).Fig3Fig. 3

GUCY2C-specific CD8⁺, but not CD4⁺, T-cell responses. PBMCs from GUCY2C-responder patients 1007 (a and b) and 1008 (c and d) collected 30 days after Ad5-GUCY2C-PADRE administration were left unsorted or depleted of CD4⁺ or CD8⁺ T cells by MACS. PBMCs, CD4-depleted PBMCs, and CD8-depleted PBMCs were analyzed by FACS to confirm depletion (a and c) and tested for GUCY2C-specific T-cell responses by IFNγ-ELISpot (b and d). NS = not significant, *** P < 0.001, **** P < 0.0001, Two-way ANOVA. Depletion efficiencies determined by FACS were > 98% for CD4⁺ T cells and ~ 75–95% for CD8⁺ T cells

Adenovirus, including serotype 5 (Ad5) used in Ad5-GUCY2C-PADRE, is a natural pathogen producing mild infections in humans. Natural exposures induce Ad5-neutralizing antibodies (NAbs) that inhibit future infections or gene delivery by recombinant adenoviruses, including Ad5-based vaccines, by preventing infection of host cells required for antigen expression and induction of immune responses [31–34]. To determine the impact of Ad5 NAbs on Ad5-GUCY2C-PADRE immunogenicity, Ad5 NAbs were quantified in patient serum collected prior to Ad5-GUCY2C-PADRE vaccination (day 0) using an Ad5-GFP reporter virus inhibition bioassay (Fig. 4a). Titers ranged from < 10 to > 10,000 and an obvious pattern emerged in which 50% of the patients had titers below 200 (Ad5 NAb Low) and the other 50% were characterized by titers above 200 (Ad5 NAb High; Fig. 4b). Separating patients into Ad5 NAb Low and High cohorts revealed a relationship between Ad5 NAb titer and GUCY2C-specific T-cell responses in which responses were significantly greater in Ad5 NAb Low patients (Fig. 4c). PADRE-specific T-cell responses, which were generally low, showed no relationship to Ad5 NAb titer (Fig. 4d). Similar to GUCY2C-specific T-cell responses (Fig. 4c), Ad5-specific T-cell responses also were limited in the Ad5 NAb High group (Fig. 4e).Fig4Fig. 4

Ad5 neutralizing Abs limit GUCY2C responses in humans. a-e) patient serum samples collected prior to vaccination (day 0) were analyzed for Ad5 neutralizing antibodies (NAbs) employing an in vitro Ad5-GFP reporter virus inhibition assay (a). The Ad5 NAb titer was calculated as the dilution of serum that produced 50% inhibition of GFP reporter expression. b patients were rank-ordered by Ad5 NAb titers and patients with titers < 200 were designated as Ad5 NAb Low, while those with titers > 200 where designated Ab5 NAb High (dotted line indicates a titer of 200). c-e antigen-specific responses to GUCY2C (c), PADRE (d), and Ad5 (e) were compared in Ad5 NAb Low and High patient populations by IFNγ-ELISpot (mixed effect model). # P = 0.052, *** P < 0.001

To confirm the impact of Ad5 NAbs on GUCY2C vaccination with Ad5 vectors in a mouse model, mice were exposed to control Ad5 vector by two intramuscular immunizations, producing animals with high Ad5 NAb titers (~ 3000; Fig. 5a and b). Ad5-naïve mice or mice with high Ad5 NAb titers were then immunized with Ad5-GUCY2C-S1 (a mouse GUCY2C vaccine analogous to Ad5-GUCY2C-PADRE [18]), and GUCY2C-specific antibody (Fig. 5c) and CD8⁺ T-cell responses (Fig. 5d) were quantified. Consistent with previous mouse studies [31] and human responses to Ad5-GUCY2C-PADRE (Fig. 4), pre-existing Ad5 NAbs eliminated GUCY2C-specific antibody (Fig. 5c) and CD8⁺ T-cell responses (Fig. 5d) in mice. Together, these data suggest that pre-existing Ad5 NAb immunity eliminates Ad5-GUCY2C-PADRE viral particles in vivo prior to entry into host cells, preventing subsequent gene expression and induction of host immune responses, establishing pre-existing Ad5 immunity as a potential barrier to Ad5-GUCY2C-PADRE vaccination in human populations.Fig5Fig. 5

Ad5 neutralizing antibodies limit GUCY2C responses in mice. BALB/c mice were naïve or preconditioned by immunizing 2 times with 10⁸ IFU control Ad5 to induce High Ad5 neutralizing antibody (NAb) titers (n = 10 mice/group). a, b two weeks after preconditioning, serum was collected and an in vitro assay quantifying inhibition of A549 cell infection by an Ad5-GFP reporter virus in the presence of serum titrations (a) was used to calculate Ad5 NAb titers (b). The Ad5 NAb titer was calculated as the dilution of serum that produced 50% inhibition of GFP reporter expression. c, d mice were then immunized with 10⁸ IFU of Ad5-mGUCY2C-S1 expressing mouse GUCY2C fused to the CD4⁺ T-helper cell epitope S1. GUCY2C-specific antibody (c) and CD8⁺ T-cell responses (d) were quantified two weeks later by ELISA and IFNγ-ELISpot, respectively. Bars in (c) indicate serum dilutions of 1/20, 1/40, 1/80, and 1/160 (left to right). NS = not significant, ** P < 0.01, **** P < 0.0001, T-test (b) or Two-way ANOVA (c and d)