Guanylyl cyclase C-induced immunotherapeutic responses opposing tumor metastases without autoimmunity

Abstract

Background: One of the greatest impediments to cancer immunotherapy is the paucity of antigens that are tumor specific, sufficiently immunogenic, and shared among patients. Mucosa-restricted antigens that are expressed by tumor cells represent a novel class of vaccine targets that are characterized by immunologic privilege, which limits systemic tolerance to those antigens, and immunologic partitioning, which shields mucosae from systemic autoimmune responses. Here we defined the immunogenicity and antitumor efficacy of guanylyl cyclase C (GCC), a protein that is normally restricted to intestinal mucosa and universally expressed by metastatic colorectal cancer.

Methods: BALB/c mice (n = 197) were immunized with recombinant GCC-expressing viral vectors before (prophylactic) or after (therapeutic) a lethal challenge of GCC-expressing mouse colon cancer cells, and antitumor efficacy was monitored by quantifying metastasis and survival. Induction of autoimmunity was monitored by histopathology. Induction of GCC-specific B-cell and CD4(+) and CD8(+) T-cell responses were determined by enzyme-linked immunosorbent assay and ELISpot, respectively. Tolerance to GCC was quantified by comparing responses in GCC-deficient (n = 45) and wild-type (n = 69) C57BL/6 mice. Statistical tests were two-sided.

Results: Immunization with GCC-expressing viral vectors reduced the formation of metastases to liver (control vs GCC: mean = 30.4 vs 3.55 nodules, difference = 26.9 nodules, 95% confidence interval [CI] = 8.47 to 45.3 nodules; P = .008) and lung (control vs GCC: mean = 263 vs 55.7 nodules, difference = 207, 95% CI = 163 to 251; P < .001) and extended the median survival of mice with established lung metastases following therapeutic immunization (control vs GCC: 29 vs 38 days, P = .024), without autoimmunity. Antitumor efficacy reflected asymmetrical tolerance that was characterized by CD8(+) T-cell, but not CD4(+) T-cell or antibody, responses.

Conclusions: Immunologic partitioning together with immunologic privilege highlight the potential of mucosa-restricted antigens, particularly GCC, as therapeutic targets for metastatic cancer.

Figures

Figure 1

Vaccine design. A) Protein alignment of membrane-bound mouse guanylyl cyclases. Shaded boxes represent amino acid homology. Extracellular ligand-binding domains are nearly devoid of homology across the family. In contrast, there is a high degree of homology within the intracellular domains of the different isoforms. For a full-size version of the alignment, see Supplementary Figure 1 (available online). B) GCC constructs used in these studies: GCCTM lacks cytosolic domains but includes the transmembrane domain and traffics to the cell surface; GCCECD lacks cytosolic and transmembrane domains and is secreted. GCCTM was used in tumor models, and GCCECD was used in viral vaccine vectors. C–D) Cell surface expression of GCCTM in transduced CT26 mouse colon cancer cells, as assessed using anti–pentahistidine antibody and FACS analysis (C) and binding of 125I-ST to membranes prepared from CT26-GCCTM cells (D). Membranes from C57BL/6 mouse small intestine and colon, which endogenously express GCC, and T84 human colon cancer cells were used as positive controls. Binding data indicate means and upper 95% confidence intervals of triplicate measurements, representing two independent experiments.

Figure 2

Prophylactic guanylyl cyclase C (GCC)–specific immunity against subcutaneous colon tumor progression. BALB/c mice were prophylactically immunized with recombinant viruses expressing the extracellular domain of GCC (GCCECD) or control viruses using an escalating heterologous prime-boost strategy and were then injected on day 0 with 1 × 105 CT26-GCCTM cells. A) Adenovirus (AV). B) AV followed by vaccinia virus (VV). C) AV followed sequentially by rabies virus (RV) and VV. Data in (A–C) are means of n = 5–8 mice per immunization, and error bars indicate 95% confidence intervals (***P < .001, two-way analysis of variance of tumor growth in control- vs GCCECD-immunized mice). D) Images of control and GCCECD AV-RV-VV–immunized mice on day 24 with tumors in control mice outlined for clarity. E) Survival analysis of mice from (B) (P = .018, two-sided Mantel–Haenszel log-rank test) in which a tumor volume greater than 1200 mm3 was used as a surrogate endpoint for death. F) Survival analysis of mice from (C) (P < .001, two-sided Mantel–Haenszel log-rank test), in which a tumor volume greater than 1200 mm3 was used as a surrogate endpoint for death. Solid lines in (E) and (F) indicate percent survival; dashed lines indicate 95% confidence intervals.

Figure 3

Prophylactic guanylyl cyclase C (GCC)–specific immunity against colon cancer metastases in liver. BALB/c mice were immunized with control adenovirus (AV) or GCCECD-AV and challenged 1 week later by intrasplenic injection (on day 0) with 1 × 105 CT26-GCCTM mouse colon cancer cells. Mice were killed on day 21, and livers were collected. A) Livers of control- and GCCECD-immunized mice. B) Liver weights of control- and GCCECD-immunized mice. Data shown are means of n = 5 mice per immunization, and error bars indicate upper 95% confidence intervals (P = .088, two-sided Student t test). C) Numbers of liver nodules (n = 11–12 mice per immunization). Bars indicate means (**P = .008, two-sided Welch t test).

Figure 4

Guanylyl cyclase C (GCC)–specific immunotherapy of colon cancer metastases in lung. BALB/c mice were immunized with control adenovirus (AV) or GCCECD-AV (day 7) and then challenged with 5 × 105 CT26-GCCTM cells by tail vein injection on day 0. A) On day 14, metastases were visualized by positron emission tomography/micro computer tomography (PET/microCT). Lungs are outlined for clarity. B) Lungs surgically removed following PET/microCT analysis were stained with India ink to visualize lung nodules. C) PET/microCT–quantified tumor burden, determined by absolute quantification of the percent of injected dose per gram of tissue (% ID/g) in a defined region of interest. Average lung values were normalized to uptake in liver within each mouse (lung minus liver). Bars indicate means of n = 5–9 mice per immunization (***P < .001, two-sided Welch t test). D) Numbers of lung nodules as determined by visual inspection of lungs in (C). Bar indicates mean of n = 6–10 mice per immunization (***P < .001, two-sided Welch t test). E) Survival analysis of prophylactic GCCECD immunization. F) Survival analysis of therapeutic GCCECD immunization. Mice were challenged with 1 × 105 CT26-GCCTM cells by tail vein injection. Beginning 3 days after tumor challenge, mice were immunized sequentially with control or GCCECD AV, rabies virus (RV), and vaccinia virus (VV), and survival was monitored (P = .024, two-sided Mantel–Haenszel log-rank test). Solid lines in (E) and (F) indicate percent survival, and dashed lines indicate 95% confidence intervals.

Figure 5

Adaptive systemic cellular responses to guanylyl cyclase C (GCC) immunization. BALB/c mice were naive or immunized with LacZ-adenovirus (AV) or GCCECD-AV, and sera and splenocytes were collected 10–14 days later for quantification of antibody and T-cell responses. A) Enzyme-linked immunosorbent assay (ELISA) analysis of AV-specific IgG antibody responses in immunized BALB/c mice. Bars indicate mean absorbance at 405 nm of reciprocal serum dilutions of 200, 400, 800, and 1600 of n = 4 mice per group, and error bars indicate upper 95% confidence intervals (CIs). B) ELISA analysis of GCCECD-specific IgG antibody responses in immunized BALB/c mice. Bars indicate means at reciprocal serum dilutions of 25, 50, 100, and 200 of n = 4 mice per group, and error bars indicate upper 95% CIs. Anti–hexahistidine mouse IgG (αHis) (concentrations of 200, 100, 50, and 25 ng/mL αHis IgG) served as a positive control for immunodetection of the plated hexahistidine-tagged GCCECD. C) AV-specific CD4+ T-cell responses in immunized BALB/c mice as measured by interferon gamma (IFNγ) ELISpot. Data indicate pooled analysis of n = 2 mice per group, and error bars indicate 95% CIs of triplicate measurements. Two independent experiments were performed with similar results. D) GCCECD-specific CD4+ T-cell responses in immunized BALB/c mice as measured by IFNγ ELISpot. Data indicate pooled analysis of n = 2 mice per group, and error bars indicate 95% CIs of triplicate measurements. Two independent experiments were performed with similar results. E) β-galactosidase–specific CD8+ T-cell responses in LacZ-AV–immunized BALB/c mice as measured by IFNγ ELISpot. Data indicate pooled analysis of n = 2 mice per group, and error bars indicate 95% CIs of triplicate measurements. Results are representative of those obtained in four independent experiments (***P < .001, two-way analysis of variance [ANOVA]). F) GCCECD-specific CD8+ T-cell responses in GCCECD-AV–immunized BALB/c mice as measured by IFNγ ELISpot. Data indicate pooled analysis of n = 2 mice per group, and error bars indicate 95% CIs of triplicate measurements and are representative of four independent experiments (***P < .001, two-way ANOVA). Symbols indicate means in (C–F).

Figure 6

Differential systemic immune cell tolerance to guanylyl cyclase C (GCC). A) Enzyme-linked immunosorbent assay (ELISA) analysis of adenovirus (AV)-specific IgG antibody responses in GCC+/+ (+/+) or GCC–/– (−/−) C57BL/6 mice 10–14 days after immunization with AV or a GST-fusion protein (negative control). Data represent means (symbols) of n = 8 AV-immunized mice per genotype or pooled samples of n = 3 control-immunized mice per genotype at reciprocal serum dilutions from 100 to 800. Error bars indicate 95% confidence intervals (CIs). B) ELISA analysis of GCCECD-specific antibody responses in +/+ or −/− C57BL/6 mice 14 days after immunization with GCCECD-AV or LacZ-AV. Responses from individual GCCECD-immunized mice are shown, and those from controls are presented as means (n = 3 per group, P < .001, two-way analysis of variance [ANOVA] for GCCECD-AV−/− vs all other groups). Data are representative of three independent experiments. C) AV-specific CD4+ T-cell responses in GCCECD-AV–immunized +/+ or −/− C57BL/6 mice as measured by interferon gamma (IFNγ) ELISpot. D) GCCECD-specific CD4+ T-cell responses in LacZ-AV– or GCCECD-AV–immunized +/+ and −/− C57BL/6 mice as measured by IFNγ ELISpot. Data in (C) and (D) indicate pooled analysis of n = 2 mice per group, and error bars indicate 95% CIs of triplicate measurements and are representative of four independent experiments (***P < .001, two-way ANOVA for GCCECD-AV−/− vs all other groups). E) β-galactosidase–specific CD8+ T-cell responses in GCC+/+ C57BL/6 mice following LacZ-AV immunization as measured by IFNγ ELISpot. F) GCCECD-specific CD8+ T-cell responses in GCC+/+ C57BL/6 mice following GCCECD-AV immunization and measured by IFNγ ELISpot. Data in (E) and (F) indicate pooled analysis of n = 2 mice per group, and error bars indicate 95% CIs of triplicate measurements and are representative of six independent experiments (***P < .001, two-way ANOVA). G) β-galactosidase–specific CD8+ T-cell responses in +/+ and −/− C57BL/6 mice following LacZ-AV immunization measured by IFNγ ELISpot. Data indicate pooled analysis of n = 2 mice per group, and error bars indicate 95% CIs of two independent experiments. H) GCCECD-specific CD8+ T-cell responses in +/+ and −/− C57BL/6 mice following GCCECD-AV immunization measured by IFNγ ELISpot. Data indicate pooled analysis of n = 2 mice per group, and error bars indicate 95% CIs of two independent experiments (*P = .019, two-way ANOVA).

Figure 7

Absence of autoimmunity upon systemic guanylyl cyclase C (GCCECD) immunization. A) Serum antinuclear antibody (ANA) quantification from naive, LacZ-adenovirus (AV)–, or GCCECD-AV–immunized C57BL/6 or naive MRL/MpJ-Faslpr/J mice (positive control). Data represent mean serum ANA concentrations and upper 95% confidence intervals. B) Histopathologic analysis of tissues collected from BALB/c mice immunized with control or GCCECD-AV or control or GCCECD-ARV (sequential AV, rabies, and vaccinia virus from Figure 2, C–F). Images are representative sections from n = 3 mice per immunization group. Bar = 200 μm.