Anti-GUCY2C vaccine and vaccination

Abstract

A composition comprising a modified adenovirus AD5.F35 containing a coding sequence for a soluble GUCY2C extracellular domain sequence is disclosed. A composition comprising a Listeria monocytogenes vector containing a coding sequence for a soluble GUCY2C extracellular domain sequence is disclosed. Methods for treating individuals diagnosed with cancer / tumors that express GUCY2C and for preventing micrometastases are disclosed.

Description

[Technical Field]

[0001] Government Rights Statement This invention was made with government support under W81XWH-17-1-0299 awarded by the Department of Defense. The government has certain rights in this invention.

[0002] This invention was made with government support under R01 CA170533 awarded by the National Institutes of Health. The government has certain rights in this invention. [Background technology]

[0003] Background of the Invention Despite improvements and successes in treatment, cancer continues to claim many lives worldwide. Improved screening provides the opportunity to identify many individuals with early-stage cancer, as well as many individuals who do not have cancer but who are genetically predisposed to developing cancer and therefore at high risk of developing cancer. Furthermore, improved treatments have resulted in many individuals whose cancers have been removed or who have gone into remission. These individuals are at risk for relapse or recurrence and are therefore at high risk of developing cancer. Summary of the Invention

[0004] There is a need for improved methods for treating individuals suffering from GUCY2C-expressing tumors / cancers. There is a need for compositions useful for treating individuals suffering from GUCY2C-expressing cancers. There is a need for improved methods for preventing the recurrence of GUCY2C-expressing cancers in individuals who have been treated for such cancers. There is a need for compositions useful for preventing the recurrence of GUCY2C-expressing cancers in individuals who have been treated for such cancers. There is a need for improved methods for preventing GUCY2C-expressing cancers in individuals, particularly those identified as having a genetic predisposition to such cancers. There is a need for compositions useful for preventing GUCY2C-expressing cancers in individuals. There is a need for improved methods for identifying compositions useful for treating and preventing GUCY2C-expressing cancers in individuals. [Brief explanation of the drawings]

[0005] [Figure 1] Figure 1 shows an alignment of Ad5.F35-hGCC-PADRE with wild-type Ad5. The genome of Ad5.F35-hGCC-PADRE differs from that of Ad5 by 1) replacement of the E1 region (E1A and E1B) with the hGCC-PADRE expression cassette, 2) deletion of most of the E3 region, and 3) replacement of the Ad5 fiber with the Ad35 fiber. [Figure 2] FIG. 2 shows the Ad5F35-hGUCY2C-PADRE map. [Figure 3] Figure 3, panels A-E, show Ad5-neutralizing Abs that limit GUCY2C responses in humans. Patient serum samples collected before vaccination (day 0) were analyzed for Ad5 neutralizing antibodies (NAb) using an in vitro Ad5-GFP reporter virus inhibition assay (A). Ad5 NAb titers were calculated as the dilution of serum that resulted in 50% inhibition of GFP reporter expression. (B) Patients were ranked by Ad5 NAb titer; patients with titers <200 were designated Ad5 NAb low and patients with titers >200 were designated Ad5 NAb high (the 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. [Figure 4]Figure 4, panels A–D, show Ad5 neutralizing antibodies that limit GUCY2C responses in mice. BALB / c mice were either naive or preconditioned by immunization twice with 108 IFU of control Ad5 to induce high Ad5 neutralizing antibody (NAb) titers (n = 10 mice per group). (A and B) Two weeks after preconditioning, serum was collected and Ad5 NAb titers (B) were determined using an in vitro assay, and inhibition of A549 cell infection by Ad5-GFP reporter virus in the presence of serum titers (A) was quantified. Ad5 NAb titers were calculated as the dilution of serum that resulted in 50% inhibition of GFP reporter expression. (C and D) Mice were then immunized with 108 IFU of Ad5-mGUCY2C-S1-expressing murine GUCY2C, a CD4+ T helper cell epitope S1-fused antibody. GUCY2C-specific antibody (C) and CD8+ T cell responses (D) were quantified 2 weeks later by ELISA and IFNγ-ELISpot, respectively. NS=not significant, *P<0.05, ***P<0.001, ****P<0.0001 by two-way ANOVA. [Figure 5] Figure 5 shows Ad5 and Ad5.F35 NAb titers in Phase 1 subjects. Pre-vaccination serum samples from Phase 1 study subjects were tested for Ad5 or Ad5.F35 neutralizing antibody (NAb) titers using the Ad5-GFP or Ad5.F35-GFP reporter virus inhibition bioassay. Titers were calculated as the dilution of serum resulting in 50% inhibition by nonlinear regression. Values ​​show the calculated titer and standard deviation for each subject. The dashed line indicates a titer of 200, the standard threshold for a "high" neutralizing titer. All subjects had lower neutralizing immunity to Ad5.F35 than to Ad5. Importantly, while 5 / 10 subjects had "high" Ad5 NAb titers, only one subject had a "high" Ad5.F35 NAb titer. [Figure 6]Figure 6, Panels A–D. Construction and antigen expression of Ad5.F35-GUCY2C-S1. (A) Report of international seroprevalence of Ad5 and Ad35.12. (B) The L5 gene encoding the fiber protein from Ad5 was replaced with the L5 gene from Ad35 to generate the chimeric adenovirus vector Ad5.F35. Recombinant Ad5.F35-GUCY2C-S1 was generated by inserting murine GUCY2C-S1 into the E1 region of E1 / E3-deleted Ad5.F35. (C and D) The human alveolar basal epithelial cell line A549 was transduced with Ad5.F35-GUCY2C-S1 in duplicate at a multiplicity of infection (MOI) of 0–10,000 for 48 hours (C) or at an MOI of 10,000 for 0, 24, 48, and 72 hours (D). Supernatants from infected cells were analyzed for GUCY2C-S1 protein expression by immunoblot. Protein expression was quantified by densitometry and plotted relative to uninfected cells. Error bars indicate mean ± SEM. Ad5, adenovirus serotype 5. [Figure 7] Figure 7, panels A-D. Construction and antigen expression of Ad5.F35-GUCY2C-S1. (A) Report of international seroprevalence of Ad5 and Ad35.12. (B) The L5 gene encoding the fiber protein from Ad5 was replaced with the L5 gene from Ad35 to generate the chimeric adenovirus vector Ad5.F35. Recombinant Ad5.F35-GUCY2C-S1 was generated by inserting murine GUCY2C-S1 into the E1 region of E1 / E3-deleted Ad5.F35. (C and D) The human alveolar basal epithelial cell line A549 was transduced with Ad5.F35-GUCY2C-S1 in duplicate at a multiplicity of infection (MOI) of 0 to 10,000 for 48 hours (C) or at an MOI of 10,000 for 0, 24, 48, and 72 hours (D). Supernatants from infected cells were analyzed for GUCY2C-S1 protein expression by immunoblot. Protein expression was quantified by densitometry and plotted relative to uninfected cells. Error bars indicate mean ± SEM. Ad5, adenovirus serotype 5. [Figure 8]Figure 8, Panels A–D. Antitumor effects of Ad5-GUCY2C-S1 and Ad5.F35-GUCY2C-S1. (A–D) BALB / c mice (n = 10 per group) were immunized intramuscularly with control or 10 vp of Ad5-GUCY2C-S1 or Ad5.F35-GUCY2C-S1 and challenged 7 days later with the murine colorectal cancer cell line CT26 expressing GUCY2C and luciferase. On days 7 and 14 postchallenge, mice were injected with D-luciferin and imaged to quantify tumor burden (A) (day 14; B). Mice were weighed twice weekly (C) and monitored for survival (D). Tumor burden (B) was analyzed by one-way ANOVA, and survival comparisons (D) were analyzed by the Mantel-Cox log-rank test. In (B) and (D), an asterisk (*) indicates a comparison of the GUCY2C vaccine to the control, and brackets ([ ]) indicate a comparison between the Ad5 and Ad5.F35 vaccines. ns, not significant. Ad5, adenovirus serotype 5. [Figure 9]Figure 9, panels A–E. Ad5.F35 resists neutralization associated with preexisting anti-Ad5 immunity in mice and humans. (A–C) To generate preexisting immunity to Ad5, BALB / c mice (n = 10 mice / group) were challenged intranasally with 10 vp of Ad5-GFP once or twice at 4-week intervals. Four weeks after the final Ad5-GFP challenge, Ad5-challenged and naive mice were immunized intramuscularly with 10 vp of Ad5-GUCY2C-S1 or Ad5.F35-GUCY2C-S1. (B) Two weeks after immunization, GUCY2C-specific CD8+ T cell responses in each group were quantified by interferon gamma (IFN-γ) ELISpot and calculated as a percentage of the mean response in naive mice. Values ​​represent individual animals, and bars represent means. Ad5 and Ad5.F35 were compared by two-way analysis of variance. (C) The fraction of animals developing detectable GUCY2C-specific CD8+ T cell responses (filled area) in naive, one-, and two-Ad5-challenged mice was determined from (B). (D and E) Sera from 10 colorectal cancer patients collected before Ad5.GUCY2C-PADRE vaccination were examined for their ability to neutralize Ad5 and Ad5.F35 vectors, and titers were quantified (D; analyzed by paired t-test). The dotted line indicates a titer of 200, with a threshold of 21 for high neutralizing antibody (NAb) titers. (E) Five of 10 subjects had high NAb titers (>200) to Ad5, whereas only one of 10 had high titers to the Ad5.F35 vector (filled area; binomial test). Ad5, adenovirus serotype 5. [Figure 10]Figure 10, Panels A–G. Safety and immunogenicity of multiple doses of Ad5.F35-GUCY2C-S1. (A–G) BALB / c mice (n = 10 per group) were immunized intramuscularly with one or three doses of 10 vp of Ad5.F35-GUCY2C-S1 or control at 4-week intervals. After immunization, body weights ((B) females and (C) males) were recorded weekly, and mice were monitored for survival (D). At 14 and 90 days after the first immunization, mice were euthanized and quantified for organ pathology by weight, biodistribution by quantitative PCR, and GUCY2C-specific CD8+ T cell responses by interferon gamma (IFN-γ) ELISpot (E–G). (G) Pie chart shows the percentage of responding animals. Ad5, adenovirus serotype 5. [Figure 11] Figure 11. Recombinant Lm-GUCY2C secretes GUCY2C into infected J774.A1 macrophages. J774A.1 cells were seeded in 6-well plates and allowed to form monolayers. Macrophages were infected with 4 x 10 CFU of control Lm or Lm-GUCY2C and incubated at 37°C for 1 hour. At 1 hour postinfection, the medium was aspirated, cells were washed once with PBS, and fresh medium containing 10 μg / mL gentamicin was added to remove extracellular Lm. Infected macrophages were incubated for an additional 7 hours at 37°C. Lysates from infected cells were then prepared and stained with antibodies against GUCY2C or the Lm antigen p60. Lane 1: Uninfected J774A.1 cells. Lane 2: J774A.1 infected with control Lm. Lane 3: J774A.1 infected with Lm-ActA-GUCY2C. Lane 4: J774A.1 infected with Lm-ActA-Syn18×5-GUCY2C. [Figure 12]Figure 12. Recombinant Lm-GUCY2C enhances GUCY2C-specific CD8+ T cell responses after Ad5-GUCY2C-S1 vaccination. BALB / c mice were immunized intramuscularly with 1 x 10 plaque-forming units (PFU) of Ad5-GUCY2C-S1 on day 0. On day 21, mice were immunized with 1 x 10 colony-forming units (CFU) of control Lm expressing GUCY2C or recombinant Lm [Lm-ActA-Syn18x5-GUCY2C ("Lm-GUCY2C")]. On day 27, GUCY2C-specific T cells were quantified by IFNγ-ELISpot; DMSO served as a negative control. [Figure 13] Figure 13. Recombinant Lm-GUCY2C boosts GUCY2C-specific CD8+ T cell responses after DNA-GUCY2C vaccination. BALB / c mice were primed with DNA or Lm-GUCY2C (Lm-ActA-Syn18x5-GUCY2C) vaccination on day 0 and boosted with DNA or Lm-GUCY2C (Lm-ActA-Syn18x5-GUCY2C) vaccination on day 21. For DNA vaccination, 50 μg of DNA plasmid encoding GUCY2C protein was injected intramuscularly into each leg and electroporated with 10 pulses (field strength = 100 v / cm, pulse length = 20 ms, pulse interval = 1 s). For Lm vaccination, 1 x 10 CFU of Lm-ActA-Syn18x5-GUCY2C was administered intraperitoneally. On day 27, GUCY2C-specific T cells were quantified by IFNγ-ELISpot, with DMSO serving as a negative control. [Figure 14] Figure 14. First generation Lm-GCC vaccines. Three vaccines were generated: a control Lm containing only the transfer plasmid without GCC; an LLO-GCC fusion protein; and an LLO-GCC-S1 fusion protein. [Figure 15]Figure 15. Quality control of first-generation Lm vaccine. Listeria cultures were grown at 37°C. At OD600 > 0.5, the cultures were centrifuged at 4,000 × G for 10 minutes, and the supernatant was collected. Proteins were precipitated from the supernatant using trichloroacetic acid. Protein (30 μg) was loaded into each lane of a 4-12% Bis-Tris gel. The gel was stained with GCC monoclonal antibody (clone MS20) or polyclonal anti-LLO. The predicted weight of both the LLO-GCC and LLOGCC-S1 fusion proteins is estimated to be approximately 89 kDa. [Figure 16] Figure 16. First-generation Lm vaccine immunogenicity. Control (Lm-LLO), Lm-LLO-GCC, and Lm-LLO-GCC-S1 vaccines were administered to wild-type BALB / c mice at 10 CFU / mouse. Ad5-GCC-S1 was administered at 10 IFU as a positive control. GCC-specific CD8+ T cell responses were measured by IFNγ-ELISpot. No responses to GCC were detected in control, Lm-LLO-GCC, or Lm-LLO-GCC-S1-immunized mice, but were readily detected in Ad5-GCC-S1-immunized mice (left). Lm and Ad5 vaccines induced strong vector-specific T cell responses directed against LLO or DBP, respectively (right), confirming appropriate Lm-LLO-GCC vaccine exposure and immunogenicity. [Figure 17] Figure 17. Second-generation Lm-GCC vaccine designs. While the first-generation vaccine employed the entire extracellular domain of GCC (residues 23-429), the second-generation design employed shorter fragments of GCC. Each fragment contained approximately one-third of GCC with distinct CD4+ and CD8+ epitope profiles, as shown below each construct. [Figure 18] Figure 18. Quality control of second-generation Lm vaccine. Listeria cultures were grown at 37°C. When OD600 was >0.5, the cultures were centrifuged at 4,000 × G for 10 minutes, and the supernatant was collected. Proteins were precipitated from the supernatant using trichloroacetic acid. Protein (30 μg) was loaded into each lane of a 4-12% Bis-Tris gel. The gel was stained with GCC monoclonal antibodies MS7, MS20, and MS24 and detected with an HRP-conjugated goat anti-mouse secondary antibody and a luminescent substrate. [Figure 19] Figure 19, panels A and B. Second-generation vaccine immunogenicity. A second-generation Lm-LLO-GCC vaccine containing GCC fragments was administered to GCC- / - (A) or GCC+ / + (B) mice. GCC- / - mice generated robust CD4+ T cell responses to GCC when immunized with CD4+ T cell epitope-containing fragments 1 and 2 (A). However, GCC+ / + mice failed to generate GCC-specific CD8+ T cell responses when immunized with CD8+ T cell epitope-containing fragments 2 and 3 (B). [Figure 20] Figure 20, panels A, B, and C. Lm-LLO-GCC antitumor immunization. A-C) Wild-type BALB / c mice were immunized with a mixture of LmLLO-GCC fragments 1, 2, and 3 (Figures 9-11). Mice were administered Lm-LLO, lacking GCC, as a negative control, or Ad5-GCCS1 as a positive control. Mice (n=8 per group) were then challenged with CT26 colorectal cancer cells expressing GCC and luciferase. Tumor burden was imaged and quantified by whole-body bioluminescence imaging (BLI). B-C) ​​Lm-LLO-GCC immunization showed no antitumor activity, whereas Ad5-GCC-S1 showed complete protection. C) Representative images at day 14. [Figure 21]Figure 21, Panels A, B, C, and D. Lm-ActA-GCC Vaccines. A) Two ActA-based GCC vaccines, designated Lm-ActA-GCC-A and Lm-ActA-GCC-B, were generated. These contain ActA fused to the full-length GCC extracellular domain (residues 23-429). In addition, vaccine "B" contains a synthetic sequence (Syn18) to enhance Acta-GCC fusion protein production. B) Indeed, the presence of Syn18 significantly improves Acta-GCC production in macrophages infected with various Lm vaccines in vitro. Anti-P60 detects Lm proteins, allowing for normalization of GCC expression levels in Lm-infected macrophages. Wild-type BALB / c mice were immunized with vaccine "A" ((C) or "B" (D) or the positive control Ad5-GCC-S1, and GCC-specific CD8+ T cell responses were measured by IFNγ-ELISpot. No responses were detected in the Lm-immunized group. Individual mice for vaccines "A" and "B" are shown in C and D. [Figure 22] Figure 22 Lm-ActA-GCC multiepitope vaccine. A recombinant Lm containing ActA tandemly fused with five copies of the dominant GCC CD8+ T cell epitope was generated (vaccine "C"), but immunization of mice with this vaccine did not generate a GCC-specific CD8+ T cell response. Individual mice for vaccine "C" are shown. [Figure 23] Figure 23. Lm-ActA-multiepitope vaccine. Recombinant Lm was generated containing ActA fused to four tandem epitopes corresponding to dominant CD8+ T cell epitopes from E. coli β-galactosidase (LacZ), GCC, the murine homolog of Her2, and Ad5 (vaccine "D"). Wild-type BALB / c mice were immunized, and responses were quantified by IFNγ-ELISpot. [Figure 24]Figure 24, panels A-C: Construction of Lm-GUCY2C. A) Lm-GUCY2C secretes a fusion protein composed of ActAN100, an enhancer sequence, and mouse GUCY2C23-429 under the control of the acta promoter. B-C) ​​The J774A.1 macrophage cell line was infected with Lm-GUCY2C or Lm-LacZ at an MOI of 10:1 for 6 hours at 37°C. The GUCY2C fusion protein was detected by (B) Western blot and (C) immunofluorescence. [Figure 25] Figure 25, panels A-D: Heterologous Ad5.F35-GUCY2C-S1 + Lm-GUCY2C immunization enhances GUCY2C-specific CD8+ T cell responses and antitumor immunity. (A-D) BALB / cJ mice (n = 3-9 / group) received a "prime" immunization on day 0 and a "boost" immunization on day 21 with a homologous or heterologous combination of GUCY2C-expressing vaccines and a control vaccine. Adenovirus vaccines were administered intramuscularly (im) at 10 vp of Ad5.F35-GUCY2C-S1 or control Ad5.F35, and Lm vaccines were administered intravenously (iv) at 5 x 10 CFU of Lm-GUCY2C or control Lm. Six days after the final immunization, mice were harvested and GUCY2C-specific CD8+ T cells were quantified by IFNγ-ELISpot (A) or challenged intravenously with 5 x 10 CT26 colorectal cancer cells expressing GUCY2C and firefly luciferase. On days 7 and 14 postchallenge, mice were injected with D-luciferin substrate and imaged (B). Tumor burden, quantified by luminescence (photons / second) upon imaging by ELISpot on day 7, was recorded (C). Survival was monitored throughout the experiment (D). GUCY2C-specific CD8+ T cell responses and tumor burden were analyzed by one-way ANOVA compared with control immunization, and survival comparisons were analyzed by the Mantel-Cox log-rank test. [Figure 26]Figure 26, panels A–C: Pre-existing Ad5 immunity does not affect Ad5.F35-GUCY2C-S1 + Lm-GUCY2C immunity. (A) BALB / cJ (n=4 / group) mice were immunized with decreasing doses (10 vp to 10 vp) of Ad5.F35-GUCY2C-S1 vaccine on day 0 and boosted with 5 × 10 CFU of Lm-GUCY2C on day 21. GUCY2C-specific CD8+ T cell responses were quantified by IFNγ-ELISpot 6 days after the final vaccination. (B–E) BALB / cJ mice (n=15 / group) were intranasally infected with 10 vp of Ad5-GFP or PBS as a control on day 0. On day 28 post-vaccination, mice were bled to confirm the presence of Ad5-specific NAbs before vaccination with 10 vp of Ad5.F35-GUCY2C-S1 (B). Twenty-one days after vaccination with Ad5.F35-GUCY2C-S1, mice were boosted with 5 × 10 CFU of Lm-GUCY2C or control Lm. Six days after the final vaccination, five mice from each group were sacrificed to assess GUCY2C-specific CD8+ T cell responses (C), and the remaining 10 mice were challenged with 5 × 10 GUCY2C and CT26 colorectal carcinoma cells expressing firefly luciferase. [Figure 27]Figure 27, panels A-E: Prime-boost immunization enhances the avidity and polyfunctionality of the GUCY2C-specific CD8+ T cell pool. BALB / cJ mice (n = 5-8 / group) were immunized with Ad5.F35-GUCY2C-S1 (prime) or Ad5.F35-GUCY2C-S1 + Lm-GUCY2C (prime-boost) vaccination regimens. At peak effector responses, 14 days after prime and 6 days after prime-boost vaccination, splenocytes were harvested, and GUCY2C-specific CD8+ T cell avidity was quantified by IFNγ-ELISpot (A), and polyfunctionality was quantified by flow cytometry (B-E). (A) Nonlinear regression of GUCY2C-specific CD8+ T cell avidity (solid line) is shown with 95% confidence intervals (dashed lines). (B-E) Effector function of surviving GUCY2C-specific CD8+ T cells with IFNγ, MIP1α, and CD107a in (B) single-positive and (C) double-positive events, shown as a percentage of all CD8+ T cells. (D-E) Overall polyfunctionality of GUCY2C-specific CD8+ T cells, shown as a percentage of cytokine+ CD8+ T cells. [Figure 28] Figure 28, panels A-D: Heterologous prime-boost does not induce toxicity. (A) BALB / cJ mice (n=10 / group) were immunized with Ad5.F35-GUCY2C-S1 on day 0, Lm-GUCY2C on days 21 and 42, or PBS as a control on all days. (B-D) Survival and body weight were monitored throughout the experiment. DETAILED DESCRIPTION OF THE INVENTION

[0006] Brief description of the sequence listing SEQ ID NO: 1: DNA sequence of human GUCY2C (Genbank accession number BC136544), incorporated herein by reference. SEQ ID NO: 2: Predicted amino acid sequence of human GUCY2C. SEQ ID NO: 3: Codon-optimized DNA sequence of human GUCY2C. SEQ ID NO: 4: Predicted amino acid sequence of human GUCY2C. SEQ ID NO: 5: Codon-optimized DNA sequence of soluble human GUCY2C. SEQ ID NO: 6: Predicted amino acid sequence of soluble human GUCY2C. SEQ ID NO: 7: DNA sequence encoding PADRE. SEQ ID NO: 8: Predicted amino acid sequence of PADRE of SEQ ID NO: 6. SEQ ID NO: 9: Codon-optimized DNA sequence of soluble human GUCY2C fused in frame with the DNA sequence encoding PADRE (C-terminal fusion). SEQ ID NO: 10: Predicted amino acid sequence of the codon-optimized DNA sequence of soluble human GUCY2C fused in frame with the DNA sequence encoding PADRE (N-terminal fusion). SEQ ID NO: 11: Ad5.F35-hGUCY2C-PADRE vector sequence. SEQ ID NO: 12: T cell epitope present in the 33 kDa C-terminal region of Plasmodium vivax MSP1. SEQ ID NO: 13: T cell epitope present in the circumsporozoite protein of Plasmodium falciparum. SEQ ID NO: 14: T cell epitope present in the circumsporozoite protein of Plasmodium falciparum. SEQ ID NO: 15: T cell epitope present in the circumsporozoite protein of Plasmodium falciparum. SEQ ID NO: 16: T cell epitope present in the circumsporozoite protein of Plasmodium falciparum. SEQ ID NO: 17: T cell epitope present in the circumsporozoite protein of Plasmodium falciparum. SEQ ID NO: 18: T cell epitope present in the circumsporozoite protein of Plasmodium falciparum. SEQ ID NO: 19: T cell epitope tetanus toxoid TT 830~844 . SEQ ID NO: 20: T cell epitope tetanus toxoid TT 947~967 . SEQ ID NO: 21: T cell epitope tetanus toxoid TT 590~603 . SEQ ID NO: 22: T cell epitope tetanus toxoid TT 615~629 . SEQ ID NO: 23: T cell epitope tetanus toxoid TT 639~652 . SEQ ID NO: 24: T cell epitope tetanus toxoid TT 830~843 . SEQ ID NO: 25: T cell epitope tetanus toxoid TT 947~967 . SEQ ID NO: 26: T cell epitope influenza hemagglutinin residues 306-318. SEQ ID NO: 27: T cell epitope Enterovirus 71 VP1 capsid protein residues 66-77. SEQ ID NO: 28: T cell epitope Enterovirus 71 VP1 capsid protein residues 145-159. SEQ ID NO: 29: T cell epitope Enterovirus 71 VP1 capsid protein residues 247-261. SEQ ID NO: 30: T cell epitope EBV LMP 1159~175 . SEQ ID NO: 31: T cell epitope HIV Gag 131~15 . SEQ ID NO: 32: T cell epitope HIV Gag 211~230 . SEQ ID NO: 33: T cell epitope HIV Gag 241~260 . SEQ ID NO: 34: T cell epitope HIV Gag 263~277 . SEQ ID NO: 35: T cell epitope HIV Gag 271~290 . SEQ ID NO: 36: T cell epitope HIV Gag 291~310 . SEQ ID NO: 37: T cell epitope HIV Gag 301~320 . SEQ ID NO: 38: T cell epitope HIV Gag 321~340 . SEQ ID NO: 39: T cell epitope HIV Gag 331~350 . SEQ ID NO: 40: T cell epitope Ad5 hexon protein residues 556-580. SEQ ID NO: 41: T cell epitope Ad5 hexon protein residues 56-80. SEQ ID NO: 42: T cell epitope Ad5 hexon protein residues 316-335. SEQ ID NO: 43: T cell epitope Ad5 hexon protein residues 906-930.

[0007] Detailed Description of the Invention 1.0 Definition As used herein, "GUCYC2," "GCC," "human GUCYC2," "human GCC," "GUCYC2 protein," "GCC protein," "human GUCYC2 protein," and "human GCC protein" are used interchangeably herein to refer to human guanylyl cyclase C protein. The nucleic acid sequence encoding human GUCYC2 is set forth in SEQ ID NO: 1 (Genbank accession number BC136544). The human GUCYC2 protein encoded by the longest open reading frame of SEQ ID NO: 1 has the amino acid sequence set forth in SEQ ID NO: 2 (Genbank accession number AAB19934). The codon-optimized sequence encoding human GUCYC2 is described by Magee et al. (Magee MS et al. (2018) Cancer Immunol Res 6:509-516, incorporated herein by reference) and is set forth in SEQ ID NO: 3.

[0008] The GUCYC2 protein is a cell surface or membrane-bound receptor protein. GUCYC2 proteins have several generally accepted domains, each of which contributes a separable function to the GUCYC2 molecule. These domains include a signal sequence (also interchangeably referred to as a signal peptide), an extracellular domain, a transmembrane domain, a kinase homology domain, and a guanylyl cyclase catalytic domain (the kinase homology domain and the guanylyl cyclase catalytic domain together may be interchangeably referred to as the intracellular domain or cytoplasmic domain). These domains are listed herein from N-terminus to C-terminus as they occur when the protein is produced intracellularly.

[0009] The GUCYC2 signal sequence is present at the N-terminus of newly synthesized GUCYC2 proteins. The GUCYC2 signal sequence functions in the translocation of proteins from their original site of production within the cell. The GUCYC2 signal peptide is typically excised during maturation to yield a functional mature protein that is part of or involved in protein transport.

[0010] The GUCYC2 extracellular domain is the portion of the GUCYC2 protein that is exposed to the outside of the cell. The GUCYC2 extracellular domain includes the portion of the GUCYC2 protein that binds to naturally occurring agonist and antagonist ligands of the GUCYC2 protein, guanylin and uroguanylin, as well as naturally occurring and synthetic agonist and antagonist ligands, such as the E. coli heat-stable enterotoxin ST. The GUCYC2 extracellular domain includes all amino acid residues between the GUCYC2 signal sequence and the GUCYC2 transmembrane domain. That is, the GUCYC2 extracellular domain includes all amino acid residues from the first residue after the GUCYC2 signal sequence to the last residue before the GUCYC2 transmembrane domain.

[0011] These domains include the signal sequence, the extracellular domain, the transmembrane domain (as bitopic or single-pass transmembrane proteins, only a single transmembrane domain is present), the kinase homology domain, and the guanylyl cyclase catalytic domain (the kinase homology domain and the guanylyl cyclase catalytic domain together may be referred to interchangeably as the intracellular domain or the cytoplasmic domain). These domains are listed herein from N- to C-terminus as they occur when the protein is made inside the cell.

[0012] The transmembrane domain is located within and spans the plasma membrane, anchoring the GCC protein within the plasma membrane.

[0013] The kinase homology domain and the guanylyl cyclase catalytic domain are located inside the cell and function to relay signals when the receptor is activated by binding to an agonist ligand. The kinase homology domain is predicted to have tyrosine kinase activity; the guanylyl cyclase catalytic domain is predicted to have guanylyl cyclase activity.

[0014] As used herein, the terms "Ad5.Fib35 adenoviral vector" and "Ad5.F35 adenoviral vector" are used interchangeably and refer to an adenoviral vector in which the DNA encoding the native Ad5 fiber or the shaft and knob portions of the fiber has been replaced with that derived from the serotype B adenovirus Ad35.

[0015] As used herein, the term "replication-deficient adenoviral vector" refers to an adenoviral vector that lacks one or more regions of the adenoviral genome essential for viral replication (e.g., E1, E2, or E4, or a combination thereof) and therefore cannot propagate in the absence of trans-complementation (e.g., provided by either a complementing cell or a helper virus). Minimal (i.e., gutless) adenoviral vectors that lack all functional genes, including early (E1, E2, E3, and E4) and late (L1, L2, L3, L4, and L5) genes, except for cis-acting sequences, are also included in the definition of "replication-deficient adenoviral vector" (see, e.g., Kovesdi et al., Current Opinion in Biotechnology 8 (1997), 583-589; Yeh and Perricaudet, FASEB 11 (1997), 615-623; WO94 / 12649; WO94 / 28152). Replication-deficient adenoviral vectors can be easily engineered by those skilled in the art taking into account the minimum required sequences and are not limited to these exemplary embodiments. Adenoviral vectors lacking E1, or E1 and E2, or E1 and E3, or E1 and E4, or E1, E2 and E3, or E1, E2 and E4, or E1, E3 and E4, or E1, E2, E3 and E4 are all contemplated by this definition.

[0016] As used herein, the term "gene expression cassette" refers to a DNA construct that is or can be inserted into an adenoviral backbone capable of promoting the expression of soluble GUCYC2 or soluble GUCYC2 fused in-frame to a universal CD4+ epitope. Gene expression cassettes are generally constructed to express one or more foreign antigens of interest under the control of a given promoter and may include other elements, such as a polyadenylation signal, such as the BGH polyadenylation signal or one derived from SV40, and / or a Kozak region and / or additional termination signals not already provided by the host vector or coding region to be expressed. Different promoters can be utilized depending on numerous factors, such as the size, composition, or complexity of the selected promoter and its expression pattern. The CMV IE promoter is commonly used in gene expression cassettes because it is efficiently expressed in many tissue types. For example, other promoters can be used in the gene expression cassette, including the simian virus 40 (SV40) early promoter, the Epstein-Barr virus immediate early promoter, the Rous sarcoma virus promoter, the human hemoglobin promoter, dendritic cell-specific promoters such as the CD11c promoter (Masood, R, et al., 2001 Int J MoI Med 8:335-343; Somia, NV, et al., 1995 Proc Acad Sci USA 92:7570-7574, which are incorporated herein by reference), and muscle-specific promoters including the myosin promoter, the muscle creatine kinase (MCK) promoter, the desmin promoter, and the mammalian troponin 1 promoter.

[0017] As used herein, the terms "soluble GUCYC2," "soluble GCC," "soluble human GUCYC2," "soluble human GCC," "soluble GUCYC2 domain," "soluble GCC domain," "soluble human GUCYC2 domain," and "soluble human GCC domain" are used interchangeably and refer to a fragment of human GUCY2C containing the GUCY2C extracellular domain, or a fragment thereof containing at least 330-423 amino acid residues, including the sequence from amino acid residue 54 to amino acid residue 384 of the functional mature GCC protein, and optionally containing sequences from human GUCY2C fused to a portion of the GUCYC2 transmembrane domain, while still allowing extracellular secretion of soluble GUCYC2 that is not anchored to the cell membrane. That is, to the extent that any sequence from the GUCYC2 transmembrane domain is included, it is insufficient to function as an anchor. Thus, upon intracellular expression, soluble GUCYC2 can be secreted by the cell. Secretion of soluble GCC is facilitated by a signal sequence, and it is understood that the nucleic acid encoding soluble GCC encodes either an endogenous or exogenous signal sequence. Soluble GUCYC2 may be linked to a signal peptide, such as the GUCYC2 signal peptide, and may include additional amino acids.

[0018] As used herein, the terms "secretory signal," "secretory peptide," "signal peptide," and "signal sequence" are used interchangeably and are meant to refer to an amino acid sequence of a protein that, when present, results in the transport and secretion of the protein outside of a cell. Secretory signals are typically cleavable hydrophobic segments of precursor proteins at or near the N-terminus of the precursor protein. During the secretion process, such secretory signals are enzymatically removed, resulting in the secretion of the mature form of the protein, i.e., the form of the protein lacking the secretory signal. In some embodiments, the secretory signal is a GCC secretory signal derived from a GCC protein. The GCC secretory signal is an N-terminal signal peptide comprising residues 1 to about residues 21, 22, or 23 of GUCYC2. In some embodiments, the secretory signal is derived from a source other than GUCYC2 (an "exogenous signal sequence") and replaces the endogenous signal sequence. In the former case, the coding sequence for the GCC antigen, including the signal sequence, is used as is. In the latter case, a nucleotide sequence encoding a signal sequence from another source is linked to the coding sequence GCC in frame with the remainder of the protein, in place of the naturally occurring signal sequence. In such cases, the signal sequence may be any such sequence that is functional in the cells of the individual to whom the genetic construct is administered.

[0019] As used herein, a "universal CD4+ helper epitope" is a peptide sequence that forms a complex with some major histocompatibility complex (MHC) class 2 human leukocyte antigen (HLA), which then binds to CD4 + It is recognized by T cell receptors on T cells, and is discussed in detail below.

[0020] As used herein, a "GUCY2C-expressing tumor" refers to a tumor that expresses guanylyl cyclase C or "GCC." Such tumors / cancer cells generally arise within the gastrointestinal tract and include, but are not limited to, tumors / cancer cells of esophageal, gastric, pancreatic, or colorectal origin. Such tumors may be primary or metastatic.

[0021] As used herein, the term "colorectal tumor" or "cancer arising in the colorectal tract" is intended to include the well-accepted medical definition that defines colorectal cancer as a condition characterized by cancer of cells in the lower intestinal tract of the small intestine (i.e., the large intestine (large intestine) including the cecum, ascending colon, transverse colon, descending colon, and sigmoid colon, and the rectum). Furthermore, as used herein, the term "colorectal cancer" or "cancer arising in the colorectal tract" is intended to further include conditions characterized by cancer of cells in the duodenum and small intestine (jejunum and ileum). The definition of colorectal cancer used herein is broader than the common medical definition, but is provided as such because it also includes cells of the duodenum and small intestine.

[0022] As used herein, the term "stomach cancer" or "gastric cancer" is meant to include the widely accepted medical definition that defines gastric cancer as a medical condition characterized by cancer of the cells of the stomach.

[0023] As used herein, the term "esophageal cancer" is meant to include the widely accepted medical definition that defines esophageal cancer as a medical condition characterized by cancer of the cells of the esophagus.

[0024] As used herein, the term "pancreatic cancer" is meant to include the widely accepted medical definition that defines pancreatic cancer as a medical condition characterized by cancer of the cells of the pancreas.

[0025] As used herein, a "primary tumor" is one that is confined to its original tissue of origin, as opposed to a metastatic tumor that has disseminated from its original primary tissue to other organs.

[0026] As used herein, "outside the colorectal tract" refers to organs that are not within the colorectal tract.

[0027] Defective adenoviral genes are replaced by gene expression cassettes that drive expression of foreign transgenes. These defective vectors are typically constructed from plasmids or Ad DNA containing the genetically modified Ad genome, and the vectors are propagated in complementation cell lines such as HEK293, PER.C6, or N52.E6 that harbor and express the necessary essential genes.

[0028] A functional mature GCC protein is produced upon cleavage of the signal sequence. A functional mature GCC protein typically comprises amino acid residues 22 to 1073 of SEQ ID NO: 2, amino acid residues 23 to 1073 of SEQ ID NO: 2, or amino acid residues 24 to 1073 of SEQ ID NO: 2. The mature GCC protein comprises an extracellular domain, a transmembrane domain, a kinase homology domain, and a guanylyl cyclase catalytic domain.

[0029] The extracellular domain of a functional mature GCC protein typically contains approximately 330-423 amino acid residues, including the sequence from amino acid residue 54 to amino acid residue 384 of the functional mature GCC protein. The extracellular domain may contain the sequence from amino acid residue 22, 23, or 24 to amino acid residue 420-435 of the functional mature GCC protein. The sequence of the extracellular domain of a functional mature GCC protein may contain the sequence from amino acid residue 22 to amino acid residue 435 of the functional mature GCC protein, or a 330-422 amino acid residue fragment of the sequence from amino acid residue 22 to amino acid residue 435 of the functional mature GCC protein, including the sequence from amino acid residue 54 to amino acid residue 384 of the functional mature GCC protein, such as the sequence from amino acid residue 22, 23, or 24 to amino acid residue 420-435 of the functional mature GCC protein. The extracellular domain of GCC does not share homology with guanylyl cyclases A, B, and G, which have wide tissue distribution and different ligand specificities.

[0030] The transmembrane domain of a functional mature GCC protein typically comprises about 16-23 amino acid residues, including the sequence of about amino acid residue 436 to about amino acid residue 452 of the functional mature GCC protein. The transmembrane domain can comprise the sequence of about amino acid residue 431 to about amino acid residue 454 of the functional mature GCC protein, or a 16-22 amino acid fragment of the sequence of amino acid residue 431 to amino acid residue 454 of the functional mature GCC protein, such as, for example, the sequence of amino acid residue 436 to amino acid residue 452 of the functional mature GCC protein or the sequence of amino acid residue 431 to amino acid residue 454 of the functional mature GCC protein.

[0031] The kinase homology domain of a functional mature GCC protein typically comprises about 237-260 amino acid residues, including the sequence of the functional mature GCC protein from about amino acid residue 508 to about amino acid residue 745. The kinase homology domain can comprise the sequence of about amino acid residue 489 to about amino acid residue 749 of the functional mature GCC protein, or a 237-259 amino acid fragment of the sequence of amino acid residue 489 to amino acid residue 749 of the functional mature GCC protein, such as, for example, the sequence of amino acid residue 508 to amino acid residue 745 of the functional mature GCC protein or the sequence of amino acid residue 489 to amino acid residue 749 of the functional mature GCC protein.

[0032] The guanylyl cyclase catalytic domain of a functional mature GCC protein typically comprises about 186-257 amino acid residues, including the sequence from about amino acid residue 816 to about amino acid residue 1002 of the functional mature GCC protein. The guanylyl cyclase catalytic domain can comprise the sequence from about amino acid residue 750 to about amino acid residue 1007 of the functional mature GCC protein, or a 186-256 amino acid fragment of the sequence from amino acid residue 750 to amino acid residue 1007 of the functional mature GCC protein, such as, for example, the sequence from amino acid residue 816 to amino acid residue 1002 of the functional mature GCC protein or the sequence from amino acid residue 750 to amino acid residue 1007 of the functional mature GCC protein.

[0033] The soluble GCC of the present invention preferably comprises soluble GCC and universal CD4 + The soluble GCC coding sequence is preferably expressed as a universal CD4 T epitope fusion protein, resulting in an in-frame translational fusion of the two regions. + The universal CD4+ helper epitope is linked to a sequence encoding a T epitope. The universal CD4+ helper epitope may be attached to either the N-terminus or the C-terminus of the soluble GCC. If attached to the N-terminus, it is inserted downstream of an endogenous or exogenous signal sequence.

[0034] Exemplified herein is a truncated GUCYC2 comprising the GCC signal sequence and extracellular domain of a molecule consisting of approximately the first 429 amino acid residues of GCC fused at the C-terminus to a universal CD4+ helper epitope (PADRE) in an aggregate.

[0035] Methods for detecting tumor markers such as GCC are well known in the art. For example, expression can be detected at the protein level or at the mRNA level. Techniques such as qRT-PCR branched oligonucleotide technology, Panomics QuantiGene® 2.0 (Affymetrix, Inc. Santa Clara, Calif.), quantitative gene expression reagents and assays, MassARRAY® (Sequenom, Inc. San Diego, Calif.), quantitative gene expression systems using detectable probes (such as FISH), in situ hybridization, dot blot assays, and other RNA quantitative amplification techniques and Northern blots are useful for measuring mRNA levels, while protein mass spectrometry, including protein and peptide fractionation combined with mass spectrometry, immunoassays such as immunohistochemistry, ELISA, or Western blot using detectable binding agents, and QProteome FFPE reverse-phase protein microarrays (Qiagen, Valencia, Calif.), are useful for detecting the presence and levels of protein markers. US Patent Publication No. 20170049869A1 describes an RT-PCT detection method applicable to GCC. US Patent Publication No. 20180355062 describes anti-GCC antibody molecules.

[0036] 2.0 Overview Prophylactic and therapeutic vaccines for protecting or treating individuals against primary and / or metastatic GUCY2C-expressing tumors are provided. Compositions useful for producing such vaccines, as well as methods for producing and using the vaccines, are provided.

[0037] One of the greatest obstacles to cancer immunotherapy is the lack of tumor-specific, sufficiently immunogenic, and shared antigens among patients. Instead of ideal targets, antitumor immune responses are generally directed against tissue-specific proteins rather than tumor-specific proteins. Barriers to using self-antigens include the concomitant autoimmunity and potential development of tolerance, which limits the effectiveness of immunotherapy. Attempts to circumvent these limitations include the use of self-proteins expressed in immune-privileged compartments. Their ectopic expression in tumors outside of these compartments provides an opportunity to target guanylyl cyclase C (GCC), a receptor for the heat-stable enterotoxins of diarrheagenic bacteria and the endogenous paracrine hormones guanylin and uroguanylin, expressed on the apical membrane of intestinal epithelial cells and restricted to the mucosal immune compartment. The vaccines of the present invention are useful for prophylactic or therapeutic immunization of human patients with GUCY2C-expressing cancers, including esophageal, gastric, pancreatic, and colorectal cancers, without inducing autoimmunity.

[0038] Some embodiments provide a modified adenoviral vector-based vaccine for cancers affecting the general population, particularly colorectal cancer, and for immunotherapy and cancer prevention, screening, early detection, diagnosis, treatment, and / or survivorship. The vaccine uses a modified version of a recombinant adenoviral vector containing the viral protein F35 to replace the corresponding adenovirus 5 adenoviral protein. The modified adenoviral vector, sometimes referred to as Ad5.F35, contains a coding sequence for a fusion protein containing a cancer-associated protein sequence, such as a portion of the extracellular domain sequence of the cancer-associated protein GUCY2C, combined with a T-cell epitope. The modified adenoviral vector-based anti-GUCY2C vaccine is used to deliver the colorectal cancer protein to immune cells in the body and educate them against colorectal cancer. Some of these educated immune cells may then be able to seek out and kill hidden colorectal cancer cells in the body. Some of these educated immune cells may then be able to produce or induce the production of antibodies that target cells expressing the colorectal cancer protein. Some of these educated immune cells may then be able to induce the production of antibodies and immune cells that target cells expressing the colorectal cancer protein.

[0039] Some embodiments provide a Listeria vaccine for cancers affecting the general population, particularly colorectal cancer, as well as immunotherapy and cancer prevention, screening, early detection, diagnosis, treatment, and / or survivorship. This vaccine uses a "disabled" version of Listeria monocytogenes to deliver colorectal cancer proteins to the body's immune cells, educating them against cells that express colorectal cancer-associated proteins. These educated immune cells may then express the proteins and be able to seek out and kill cancers hiding in the body.

[0040] Some embodiments provide a prime-boost method for vaccinating against cancer, particularly colorectal cancer cells that express a cancer-associated protein, such as the colorectal cancer-associated protein GUCY2C. The combination of an adenovirus-based GUCY2C vaccine with a modified Listeria monocytogenes virus results in an immune response that is superior to that of either vaccine alone.

[0041] An effective immune response to GUCY2C is useful for treating any cancer that expresses such a protein, such as metastatic colorectal cancer, a typically fatal disease, as well as several other cancers that express GUCY2C protein, such as pancreatic, gastric, and esophageal cancers.

[0042] 3.0 Adenovirus-based anti-GUCY2C vaccine (Ad5-GUCY2C) ECD T cell epitopes) Adenovirus represents a widely used viral vector platform for vaccine design. We previously demonstrated that adenovirus (Ad5) and DNA-based vaccines against GUCY2C generate GUCY2C-specific T cell responses, resulting in antitumor immunity. GCC linked to a CD4+ helper epitope ECD Recombinant adenoviral particles expressing chimeric proteins comprising the GUCY2C vector are disclosed in co-pending application Ser. No. 13 / 120,144, which is incorporated herein by reference. ECD The T cell epitope constructs can be used in conjunction with adenoviral Ad5 vectors to generate anti-GUCY2C vaccines.

[0043] Most adenovirus vectors currently used for gene expression in humans rely on serotype 5 Ad vectors. Adenovirus infection is initiated by Ad5 attachment to the cell surface via the fiber protein (Shenk, T. 1996 Fields Virology, Vol. 2, Fields, BN et al. (eds.), Vol. 2, Lippincott-Raven, Philadelphia, PA, 2111-2148). The distal C-terminal domain of the trimeric fiber molecule terminates in a "knob" that binds to a specific cellular receptor, which in the case of Ad5 is the coxsackie adenovirus receptor (CAR) (Bergelson, JM et al. Science, 275, 1320-1323). After binding, cellular internalization leads to interaction of cellular integrins with the Arg-Gly-Asp (RGD-motif) in the penton base in an event unrelated to viral attachment.

[0044] Ad5 is a natural human pathogen that causes mild infections in nearly all human populations (Yu, B. et al. (2012) J Med Virol 84(9):1408-1414). These natural exposures induce Ad5-specific neutralizing antibodies (NAbs), which limit reinfection or Ad5-based vaccination by preventing infection of host cells, a necessary step for target antigen expression and induction of an immune response (Priddy, FH et al. (2008) Clin Infect Dis 46(11):1769-1781; Schirmbeck, R. et al. (2008) Mol Ther 16(9):1609-1616; Small, JC et al. (2014) J Leukoc Biol 96(5):821-831).

[0045] The present inventors have shown that immunization of mice with a human adenovirus type 5 (Ad5) vaccine (Ad5-GUCY2C) expressing guanylyl cyclase C (GUCY2C) elicits immune responses against GUCY2C and immunity against colorectal cancer (Snook AE, et al. J Natl Cancer Inst. 2008;100(13):950-6). The present inventors have extended these observations to humans in a phase I clinical trial. Example 1 reports a correlation between Ad5 neutralizing antibodies and GUCY2C-specific T cell responses, in which responses were significantly greater in Ad5 NAb-low patients. One-time immunization against GUCY2C can generate limited antitumor immunity in some individuals, and repeated vaccination with Ad5-GUCY2C can be ineffective due to vaccine-induced Ad5 neutralizing antibodies after administration.

[0046] 4.0 Modified Adenovirus-Based Vaccines 4.1 Ad5F35 vector Naturally occurring Ad5 NAbs target the fiber molecules on the surface of Ad5 (Cheng, C. et al. (2010) J Virol 84(1):630-638). This suggests that replacing the fiber molecules of Ad5 with those from group B adenovirus Ad35 (to which few human subjects have natural immunity) could generate a chimeric Ad5 viral vector (known as Ad5.F35) that is not affected by naturally occurring Ad5 immunity. Sumida et al. found that functionally significant Ad5-specific NAbs were primarily directed against the Ad5 hexon protein (Sumida et al. Journal of Immunology (2005) 174(11)7179-7185). Hong et al. reported significant neutralizing effects for antibodies directed against the penton base (Hong SS et al. J. Virol. 2003;77:10366-10375).

[0047] Vectors containing group B Ad fibers, including the Ad5.F35 vector, use CD46 for initial cell attachment (Gaggar, A. et al. (2003). Nat. Med. 9:1408-1412). In humans, CD46 is expressed at low levels on all nucleated cells. Although Ad5.F35 vectors efficiently transduce dendritic cells in vitro and potentially in vivo, several findings may argue against the utility of these vectors for in vivo vaccination. (i) CD46 signaling (upon binding of CD46 monoclonal antibodies, recombinant complement factor C3b, or measles virus hemagglutinin) can induce immunosuppression (Schneider-Schaulies, S. and ter Meulen, V. (2002). Springer Semin. Immunopathol. 24:127-148). (ii) Studies of Ad35 outbreaks leading to pneumonia and sepsis revealed transient neutropenia that may have been directly caused by Ad35 infection (Sanchez, MP et al. (1997). J. Infect. Dis. 176:760-763). (iii) Measles virus and HHV6 also use CD46 as a receptor and can cause transient immunosuppression at the level of dendritic cell precursors, bone marrow stromal cells, or CD34+ myeloid progenitor cells (Manchester, M. et al. (2002). J. Virol. 76:6636-6642). These considerations have led to studies with mixed results. One study focusing on the above factors found that AD5.F35 vaccination against a test antigen (hepatitis B core antigen) did not induce immunosuppression in CD46 transgenic mice, noting that the results may be antigen-specific (DiPaolo N, et al. Mol. Ther. 2006;13(4):756-765). However, a primate study found that Ad5.Fib35, carrying a measles hemagglutinin vaccine insert, induced reduced insert-specific humoral and cellular immune responses compared with animals similarly immunized with Ad5 (Ophorst, OJ et al. (2004). Vaccine 22:3035-3044).

[0048] The present invention includes a) an Ad5.F35 vector; and a chimeric adenovirus vaccine vector Ad5.F35 comprising: i) a nucleic acid encoding a soluble human GUCY2C domain; ii) a heterologous promoter operably linked to the nucleic acid encoding the soluble human GUCY2C domain; and iii) a gene expression cassette comprising a universal CD4+ helper epitope, as well as vaccines and methods relying thereon.

[0049] The adenoviral vectors of the present invention can be replication-competent, but preferably, the adenoviral vectors are replication-deficient in the host cell.

[0050] EP 1 693 459 A1, WO2000 / 073478 A9, and EP 1 322 774 A2, each of which is incorporated herein by reference, describe chimeric Ad5.F35 vectors for altering the tropism of Ad5 (cells / tissues targeted by the virus). None describe insensitivity to Ad5 neutralizing immunity.

[0051] Construction of chimeric Ad5.Fib35 viral vectors was described by Shayakhmetov et al. in which three fragments were amplified via a two-step PCR approach: (i) the Ad5 fiber untranslated region and the first 132 bp of the fiber tail domain; (ii) the Ad35 shaft and knob domains; and (iii) the Ad5 E4 region containing the Ad5 fiber polyadenylation signal. After purifying the resulting fragments, they were combined and subjected to an additional PCR reaction with forward and reverse primers directed against the correct ends of the chimeric fiber. The resulting amplified fragment was then ligated into a donor plasmid, and the chimeric fiber was replaced by recombination with the vector genome contained within the plasmid in the Rec+ E. coli strain BJ5183. To generate the corresponding viruses, clones verified to be correct were digested with the restriction enzyme PacI to release the viral genome and transfected into 293 cells (Shayakhmetov DM et al. J Virol 2000;74:2567-2583). EP1550722B1 describes a similar procedure of direct replacement of Ad5 fiber sequences in a donor vector followed by recombination and amplification.

[0052] Numerous strategies have been developed for constructing Ad vectors carrying foreign gene inserts. Traditionally, Ad vectors have been constructed using two standard methods. The first method involves in vitro ligation, which involves ligating a DNA fragment representing the remainder of the Ad genome with a DNA fragment obtained by restriction digestion of a plasmid carrying the foreign gene insert flanked by Ad sequences (Stow ND. J Virol. 1981;37:171-80). The second method involves homologous recombination in a permissive cell line between two plasmids: a shuttle plasmid carrying the foreign gene insert flanked by Ad sequences for site-specific insertion, and a genome plasmid carrying nearly the entire Ad genome (Bett AJ, et al. Proc Natl Acad Sci USA. 1994;91:8802-6). These classical vector construction methods typically have low efficiency and can sometimes be contaminated with parental virus.

[0053] To circumvent the limitations of conventional methods, alternative approaches have been developed. One such strategy relies on the highly efficient homologous recombination mechanism of Escherichia coli (BJ5183) to generate Ad vectors [16-19]. Homologous recombination between a linearized or intact plasmid containing nearly the entire Ad genome and a shuttle plasmid containing an exogenous expression cassette flanked by homologous sequences from an insertion site in the Ad genome generates infectious clones with modifications and / or insertions in the desired region. A similar strategy employing homologous recombination in yeast has been reported

[20] . This strategy involves homologous recombination between Ad DNA containing sequences from the left and right ends of the Ad genome and a yeast artificial chromosome (YAC) vector, resulting in the generation of a yeast artificial chromosome containing an infectious copy of the Ad genome. Transfection of the excised Ad genome into appropriate cells generates infectious virions.

[0054] To overcome the low efficiency of homologous recombination in mammalian cells, a method based on the bacteriophage P1 Cre / LoxP recombination system has been developed (Aoki K, et al. Mol Med. 1999;5:224-31; Hardy S, et al. J Virol. 1997;71:1842-9; Ng et al. Hum Gene Ther. 1999;10:2667-72; Ng et al. Hum Gene Ther. 2000;11:693-9; Ng et al. J Virol. 2002;76:4181-9). Ad vectors are generated as a result of Cre-mediated site-specific recombination between two plasmids after their cotransfection into an appropriate cell line expressing Cre recombinase. The frequency of vector generation using the Cre / LoxP-based system has been found to be 30- to 100-fold higher than that of conventional homologous recombination methods.

[0055] 4.2 GUCY2C ECD A functional mature GCC protein is produced upon cleavage of the signal sequence. A functional mature GCC protein typically comprises amino acid residues 22 to 1073 of SEQ ID NO: 2, amino acid residues 23 to 1073 of SEQ ID NO: 2, or amino acid residues 24 to 1073 of SEQ ID NO: 2. The mature GCC protein comprises an extracellular domain, a transmembrane domain, a kinase homology domain, and a guanylyl cyclase catalytic domain.

[0056] The vaccine includes a soluble truncated form of a GCC protein comprising the extracellular domain of a functional mature GCC protein, which typically comprises approximately 330-423 amino acid residues, including the sequence from amino acid residue 54 to amino acid residue 384 of the functional mature GCC protein. The extracellular domain may comprise the sequence from amino acid residue 22, 23, or 24 to amino acid residue 420-435 of the functional mature GCC protein. The sequence of the extracellular domain of a functional mature GCC protein may comprise the sequence from amino acid residue 22 to amino acid residue 435 of the functional mature GCC protein, or a 330-422 amino acid residue fragment of the sequence from amino acid residue 22 to amino acid residue 435 of the functional mature GCC protein, including the sequence from amino acid residue 54 to approximately amino acid residue 384 of the functional mature GCC protein, such as the sequence from amino acid residue 22, 23, or 24 to amino acid residue 420-435 of the functional mature GCC protein. The construct may also contain sequences from the transmembrane domain, provided that the sequences are sufficiently insufficient to function to prevent the soluble GCC construct from being anchored to the cell membrane. The transmembrane domain of a functional mature GCC protein typically contains about 16-23 amino acid residues, including the sequence from about amino acid residue 436 to about amino acid residue 452 of the functional mature GCC protein. The transmembrane domain may contain the sequence from about amino acid residue 431 to about amino acid residue 454 of the functional mature GCC protein, or a 16-22 amino acid fragment of the sequence from about amino acid residue 431 to about amino acid residue 454 of the functional mature GCC protein, such as, for example, the sequence from about amino acid residue 436 to about amino acid residue 452 of the functional mature GCC or the sequence from about amino acid residue 431 to about amino acid residue 454 of the functional mature GCC. In some preferred embodiments, the transmembrane sequence is absent.

[0057] 4.3 Universal CD4+ helper epitopes To induce an improved anti-GUCY2C immune response, a universal CD4+ helper epitope may be linked to the GUCY2C sequence to generate a fusion protein. A universal CD4+ helper epitope is a peptide sequence that matches multiple HLA types and is therefore recognized by multiple HLA types. An example of a universal CD4+ helper epitope is PADRE (pan-DR epitope peptide).

[0058] The PADRE peptide forms complexes with at least 15 of the 16 most common HLA-DRs. Because humans have at least one DR and PADRE binds to many of those types, PADRE is likely to be effective in most humans. Universal CD4+ helper epitopes such as PADRE and others are disclosed in U.S. Patent No. 5,736,142 to Sette et al., issued April 7, 1998; U.S. Patent No. 6,413,935 to Sette et al., issued July 2, 2002; and U.S. Patent No. 7,202,351 to Sette et al., issued April 10, 2007, and WO1995007707A1.

[0059] The universal HLA-DR epitope PADRE (KXVAAWTLKA) has been described (Alexander, J, et al. J. Immunol, 2000 Feb 1, 164(3):1625-33; Agadjanyan et al. J Immunol 2005;174:1580-6).

[0060] When fusing PADRE to a protein antigen, some researchers have used the peptide sequence AKFVAAWTLKAAA, which is expressed in frame with the antigen of interest. Wei J, et al. Cancer Biother Radiopharm 2008,23:121-8; Bargieri DY et al. Mem Inst Oswaldo Cruz 2007,102:313-7; A T cell epitope present in the 33 kDa C-terminal region of P. vivax MSP1 DYDVVYLKPLAGMYK (SEQ ID NO: 12) has also been found to elicit strong immune responses in non-human primates with different class II HLA haplotypes (Rosa et al. Microbes Infect 2006, 8:2130-7; Sinigaglia et al., Nature 336, 778-780 (1988) described Plasmodium falciparum peptides derived from the circumsporozoite protein that associate with many different MHC class II molecules and are recognized by T cells in mice and humans. Peptides derived from residues 378-398 of DIEKKIAKMEKASSVFNVVNS (SEQ ID NO: 13) and its N and C deletions, e.g., IEKKIAKMEKASSVFNVVNS (SEQ ID NO: 14), EKKIAKMEKASSVFNVVNS (SEQ ID NO: 15), DIEKKIAKMEKASSVFNVVN (SEQ ID NO: 16), DIEKKIAKMEKASSVFNVV (SEQ ID NO: 17), and DIEKKIAKMEKASSVFNV (SEQ ID NO: 18), elicited strong responses.

[0061] Provided herein are such CD4 + These are examples of different proteins and different peptides that are examples of proteins that contain T cell epitopes. These proteins and peptides are + These are intended to be non-limiting examples of T cell epitopes.

[0062] In some embodiments, CD4 + T cell epitopes include tetanus toxin, e.g., TT 830-844 QYIKANSKFIGITEL (SEQ ID NO: 19), and TT 947-967 FNNFTVSFWLRVPKVSASHLE (SEQ ID NO: 20) Panina-Bordignon et al., Eur. J. Immunol. 19, 2237-2242 (1989); Renard V, J Immunol 2003; 171:1588-95; TT 590-603 TKIYSYFPSVISKV (SEQ ID NO: 21), TT 615-629VRDIIDDFTNESSQK (SEQ ID NO: 22), TT 639-652 VSTIVPYIGPALNI (SEQ ID NO: 23), TT 830-843 QYIKANSKFIGITE (SEQ ID NO: 24) and TT 947-967 FNNFTVSFWLRVPKVSASHLE (SEQ ID NO: 25) may be from BenMohamed et al. Hum Immunol 2000;61:764-79.

[0063] In some embodiments, CD4 + The T cell epitope can be derived from influenza hemagglutinin, for example, influenza hemagglutinin residues 306-318 PKYVKQNTLKLAT (SEQ ID NO: 26) (Busch et al., Int. Immunol. 2, 443-451 (1990); Mom et al. BMC Immunol 2005; 6:24).

[0064] In some embodiments, CD4 + The T cell epitope may be derived from the hepatitis B surface antigen (HBsAg) (Litjens et al. J Immunol Methods 2008;330:1-11).

[0065] In some embodiments, CD4 + T cell epitopes can be derived from outer membrane proteins (OMPs) of bacterial pathogens, such as Anaplasma marginale (Macmillan H, Norimine J, Brayton KA, Palmer GH, Brown WC. Physical linkage of naturally complexed bacterial outer membrane proteins enhances immunogenicity. Infect Immun 2008;76:1223-9).

[0066] In some embodiments, CD4 +T cell epitopes can be derived from the VP1 capsid protein from Enterovirus 71 (EV71) strain 41, for example, residues 66-77 IETRCVLNSHSTAET (SEQ ID NO: 27), residues 145-159 EVVPQLLQYMFVPPG (SEQ ID NO: 28), and residues 247-261 LVVRIYMRMKHVRAW (SEQ ID NO: 29) (Wei Foo et al. Viral Immunol 2008).

[0067] In some embodiments, CD4 + T cell epitopes can be derived from EBV BMLF1 (Schlienger K, Craighead N, Lee KP, Levine BL, June C H. Efficient priming of protein antigen-specific human CD4(+) T cells by monocyte-derived dendritic cells. Blood 2000;96:3490-8; Neidhart J, Allen KO, Barlow DL, Carpenter M, Shaw DR, Triozzi PL, Conry RM). Immunization of colorectal cancer patients with recombinant baculovirus-derived KSA (Ep-CAM) formulated with monophosphoryl lipid A in a liposomal emulsion with or without granulocyte-macrophage colony-stimulating factor. Vaccine 2004;22:773-80;Piriou ER, van Dort K, Nanlohy NM, van Oers MH, Miedema F, van Baarle D. Virus-specific CD4 + A novel method for the detection of EBV-specific CD4 T cells in treatment-naive HIV-infected subjects +Novel method for detection of virus-specific CD4+ T cells indicates a decreased EBV-specific CD4+ T cell response in untreated HIV-infected subjects. Eur J Immunol 2005;35:796-805; Heller KN, Upshaw J, Seyoum B, Zebroski H, Munz C. Separate memory CD4 + T cell subsets mediate immune recognition of Epstein-Barr virus nuclear antigen 1 in healthy virus carriers (Distinct memory CD4 + T-cell subsets mediate immune recognition of Epstein Barr virus nuclear antigen 1 in healthy virus carriers) Blood 2007;109:1138-46).

[0068] In some embodiments, CD4 + T cell epitopes are those found in EBV LMPIs, e.g., LMP 1159-175 YLQQNWWTLLVDLLWLL (SEQ ID NO: 30). Kobayashi et al. Cancer Res 2008;68:901-8).

[0069] In some embodiments, CD4 + T cell epitopes include those derived from HIV Gag p24, e.g., Gag 131-15 NYPIVQNIQGQMVHQAISPR (SEQ ID NO: 31), Gag 211-230 EWDRVHPVHAGPIAPGQMRE (SEQ ID NO: 32), Gag 241-260 STLQEQIGWMTNNPPIPVGE (SEQ ID NO: 33), Gag 263-277 KRWIILGLNKIVRMY (SEQ ID NO: 34), Gag 271-290NKIVRMYSPTSILDIRQGPK (SEQ ID NO: 35), Gag 291-310 EPFRDYVDRFYKTLRAEQAS (SEQ ID NO: 36), Gag 301-320 YKTLRAEQASQEVKNWMTET (SEQ ID NO: 37), Gag 321-340 LLVQNANPDCKTILKALGPA (SEQ ID NO: 38), Gag 331-350 KTILKALGPAATLEEMMTAC (SEQ ID NO: 39) (Pajot et al. Eur J Immunol 2007;37:2635-44).

[0070] In some embodiments, CD4 + T cell epitopes can be derived from the adenovirus 5 hexon protein at residues found, for example, at positions 556-580 VPFHIQVPQKFFAIKNLLLLPGSYT (SEQ ID NO: 40), residues 56-80 VTTDRSQRLTLRFIPVDREDTAYSY (SEQ ID NO: 41), residues 316-335 GQQSMPNRPNYIAFRDNFIG (SEQ ID NO: 42), and residues 906-930 EVDPMDEPTLLYVLFEVFDVVRVHRPHR (SEQ ID NO: 43) (Leen et al. J Virol 2008;82:546-544). In total, over 30 CD4 T cell epitopes for multiple MHC-II haplotypes are available. + T cell epitopes have been identified.

[0071] In some embodiments, CD4 + T cell epitopes can be derived from vaccinia virus proteins, for example, F17R protein residues 21-29 (YLVLKAVKV) and A10L protein residues 20-28 (FRIVSTVLP) (Calvo-Calle et al. PLoS Pathog 2007;3:1511-29). Calvo-Calle identified over 25 CD4 T cell epitopes for multiple MHC-II haplotypes from 24 different vaccinia proteins. + T cell epitopes were identified.

[0072] In some embodiments, CD4 + The T cell epitope is derived from Mycobacterium tuberculosis heat shock protein (Liu DW, Tsao YP, Kung JT, Ding YA, Sytwu HK, Xiao X, Chen SL). Recombinant adeno-associated virus expressing human papillomavirus type 16 E7 peptide DNA fused with heat shock protein DNA as a potential vaccine for cervical cancer. J Virol 2000; 74:2888-94.

[0073] In some embodiments, CD4 + T cell epitopes are derived from the Fc portion of IgG (You Z, Huang XF, Hester J, Rollins L, Rooney C, Chen SY). Induction of vigorous helper and cytotoxic T cell as well as B cell responses by dendritic cells expressing a modified antigen targeting receptor-mediated internalization pathway (J Immunol 2000; 165:4581-91).

[0074] In some embodiments, CD4 +The T cell epitope is derived from the lysosomal targeting signal of the human LAMP-1 protein (Su Z, Vieweg J, Weizer AZ, Dahm P, Yancey D, Turaga V, Higgins J, Boczkowski D, Gilboa E, Dannull J). Enhanced induction of telomerase-specific CD4(+) T cells using dendritic cells transfected with RNA encoding a chimeric gene product (Enhanced induction of telomerase-specific CD4(+) T cells using dendritic cells transfected with RNA encoding a chimeric gene product. Cancer Res 2002;62:5041-8).

[0075] Samples of HLA haplotypes of the indicated HLA molecules and representative CD4 + T cell epitopes include: HLADR * 1101 - Tetanus toxoid peptide residues 829-844, hemagglutinin peptide residues 306-318 (Moro M, Cecconi V, Martinoli C, Dallegno E, Giabbai B, Degano M, Glaichenhaus N, Protti MP, Dellabona P, Casorati G. Functional HLA-DR can accept challenge with pathogen- or tumor-derived synthetic peptides. * Generation of functional HLA-DR tetramers * 1101 tetramers receptive for loading with pathogen- or tumor-derived synthetic peptides.BMC Immunol 2005; 6:24. ) HLA-DRB1 *0101(DR1)-Tetanus toxoid peptide residues 639-652, 830-843 or 947-967 and 14 other tetanus toxoid peptides (BenMohamed L, Krishnan R, Longmate J, Auge C, Low L, Primus J, Diamond DJ. HLA A * Induction of CTL response by a minimal epitope vaccine in HLA A*0201 / DR1 transgenic mice: dependence on HLA class II restricted T(H) response. Hum Immunol 2000;61:764-79; and James EA, Bui J, Berger D, Huston L, Roti M, Kwok W, Long mate. Tetramer-derived epitope mapping reveals the role of tetanus toxin-specific CD4 + Tetramer-guided epitope mapping reveals broad, individualized repertoires of tetanus toxin-specific CD4+ T cells and suggests HLA-based differences in epitope recognition (Tetramer-guided epitope mapping reveals broad, individualized repertoires of tetanus toxin-specific CD4+ T cells and suggests HLA-based differences in epitope recognition) Int Immunol 2007; 19:1291-301). HLA-DRB1 *0301-EV71 VP1 residues 145-159 or 247-261 and five different tetanus toxoid peptides (Wei Foo DG, Macary PA, Alonso S, Poh C L. Identification of Human CD4(+) T-Cell Epitopes on the VP1 Capsid Protein of Enterovirus 71. Viral Immunol 2008; and James EA, Bui J, Berger D, Huston L, Roti M, Kwok W, Pooh. Tetramer-induced epitope mapping reveals tetanus toxin-specific CD4 + Tetramer-guided epitope mapping reveals broad, individualized repertoires of tetanus toxin-specific CD4 T cells and suggests HLA-based differences in epitope recognition. + T cells and suggests HLA-based differences in epitope recognition. Int Immunol 2007; 19:1291-301). HLA-DRB1 * 0405 - EV71 VP1 residues 145-159 or 247-261 (Wei Foo DG, Macary PA, Alonso S, Poh C L. Identification of Human CD4(+) T-Cell Epitopes on the VP1 Capsid Protein of Enterovirus 71. Viral Immunol 2008). HLA-DRB1 *1301 - EV71 VP1 residues 145-159 or 247-261 (Wei Foo DG, Macary PA, Alonso S, Poh C L. Identification of Human CD4(+) T-Cell Epitopes on the VP1 Capsid Protein of Enterovirus 71. Viral Immunol 2008). HLA-DR9-Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1) residues 159-175 (Kobayashi H, Nagato T, Takahara M, Sato K, Kimura S, Aoki N, Azumi M, Tateno M, Harabuchi Y, Celis E. Induction of EBV-latent membrane protein 1-specific MHC class II-restricted T-cell responses against natural killer lymphoma cells. Cancer Res 2008;68:901-8). HLA-DR53 EBV LMP1 residues 159-175 (Kobayashi H, Nagato T, Takahara M, Sato K, Kimura S, Aoki N, Azumi M, Tateno M, Harabuchi Y, Celis E. Induction of EBV-latent membrane protein 1-specific MHC class II-restricted T-cell responses against natural killer lymphoma cells. Cancer Res 2008; 68:901-8). HLA-DR15 EBV LMP1 residues 159-175 (Kobayashi H, Nagato T, Takahara M, Sato K, Kimura S, Aoki N, Azumi M, Tateno M, Harabuchi Y, Celis E. Induction of EBV-latent membrane protein 1-specific MHC class II-restricted T-cell responses against natural killer lymphoma cells. Cancer Res 2008; 68:901-8). HLA-DRB1 * 0401-15 tetanus toxoid peptides (James EA, Bui J, Berger D, Huston L, Roti M, Kwok W W. Tetramer-derived epitope mapping of tetanus toxoid-specific CD4 + Tetramer-guided epitope mapping reveals broad, individualized repertoires of tetanus toxin-specific CD4 T cells and suggests HLA-based differences in epitope recognition. + T cells and suggests HLA-based differences in epitope recognition. Int Immunol 2007; 19:1291-301). HLA-DRB1 * 0701-Nine tetanus toxoid peptides (James EA, Bui J, Berger D, Huston L, Roti M, Kwok W W. Tetramer-derived epitope mapping of tetanus toxoid-specific CD4 +Tetramer-guided epitope mapping reveals broad, individualized repertoires of tetanus toxin-specific CD4 T cells and suggests HLA-based differences in epitope recognition. + T cells and suggests HLA-based differences in epitope recognition. Int Immunol 2007; 19:1291-301). HLA-DRB1 * 1501-Seven tetanus toxoid peptides (James EA, Bui J, Berger D, Huston L, Roti M, Kwok W W. Tetramer-derived epitope mapping of tetanus toxoid-specific CD4 + Tetramer-guided epitope mapping reveals broad, individualized repertoires of tetanus toxin-specific CD4 T cells and suggests HLA-based differences in epitope recognition. + T cells and suggests HLA-based differences in epitope recognition. Int Immunol 2007; 19:1291-301). HLA-DRB5 * 0101-Eight tetanus toxoid peptides (James EA, Bui J, Berger D, Huston L, Roti M, Kwok W W. Tetramer-derived epitope mapping of tetanus toxoid-specific CD4 + Tetramer-guided epitope mapping reveals broad, individualized repertoires of tetanus toxin-specific CD4 T cells and suggests HLA-based differences in epitope recognition.+ T cells and suggests HLA-based differences in epitope recognition. Int Immunol 2007; 19:1291-301). These include, but are not limited to:

[0076] 4.4 AD5.F35-GUCY2C vaccine construct As used herein, AD5.F35-GUCY2C, AD5.F35-GUCY2C ECD , AD5.F35-GUCY2C universal T cell epitope, AD5.F35-GUCY2C ECD The universal T cell epitope is GUCY2C bound to a universal T cell epitope fusion protein expressed and secreted by infected cells. ECD GUCY2C bound to a universal T cell epitope, as occurs upon infection with the vaccine. ECD The terms "Ad5.F35" and "Adenoviral vector" are used interchangeably to refer to the Ad5.F35 adenoviral vector having a gene insert encoding the universal T-cell epitope PADRE. In a preferred embodiment, the universal T-cell epitope of the AD5.F35-GUCY2C vaccine is the universal T-cell epitope PADRE. Thus, preferred embodiments are referred to interchangeably as AD5.F35-GUCY2C-PADRE and AD5.F35-GUCY2C. ECD All references to methods using the AD5.F35-GUCY2C vaccine specifically refer to the AD5.F35-GUCY2C vaccine in general and to the AD5.F35-GUCY2C ECD -Intended to describe the PADRE vaccine.

[0077] 4.5 Treatment using the AD5.F35-GUCY2C vaccine Embodiments of the present invention include methods of treating individuals with cancers / tumors that express GUCY2C. Treatment is provided systemically. Treating such individuals with the vaccines described herein can induce an immune response in the peripheral compartment of the individual's immune system that specifically targets cancer cells that express GUCY2C. The vaccines treat primary or metastatic disease, including identified metastatic disease and any undetected metastases, such as micrometastases.

[0078] The vaccine provides an adjuvant therapeutic treatment in conjunction with the usual treatments provided upon diagnosis of cancers involving mucosal tissues. As discussed above in this application, those skilled in the art can diagnose cancers or tumors as expressing GUCY2C. While detection of metastatic disease can be performed using routine methodologies, some microscopic levels of cancer may not be detectable at the time of initial cancer diagnosis. Typical modes of treatment include surgery, chemotherapy, or radiation therapy, or various combinations. A vaccine targeting GUCY2C provides an additional countermeasure with the advantage of selectively detecting and eliminating cancer cells derived from immunologically protected compartments that express GUCY2C, rather than attacking normal tissues.

[0079] Thus, in some embodiments, an individual is diagnosed with cancer and the cancer is identified as expressing GUCY2C, and the AD5.F35-GUCY2C vaccine, which expresses soluble GUCY2C linked to a CD4+ helper epitope, is administered to the patient alone or as part of a treatment regimen that includes surgery, and / or radiation therapy, and / or administration of other anti-cancer agents.

[0080] 4.6 Prophylactic Methods Using AD5.F35-GUCY2C Vaccine The vaccine may be used prophylactically in individuals at risk of developing GUCY2C cancer / tumors. A previous diagnosis of primary disease that has been removed or is in remission places the individual at higher risk.

[0081] Individuals at risk for developing mucosal tissue cancers can be administered a vaccine to induce an immune response that eliminates cancer cells before the individual has detectable disease. In some embodiments, such individuals can also be identified for CD4+ helper epitope type. The vaccine administered to the individual contains a mucosally-restricted antigen recognized by the individual and the protein or genetic code for one or more CD4+ helper epitopes. However, it may be more practical to utilize a universal CD4+ helper epitope, such as PADRE.

[0082] 4.7 Vaccine Composition, Formulation, Dosage and Administration Regimens Vaccines according to some embodiments comprise a pharmaceutically acceptable carrier in combination with an active agent, which may be a nucleic acid molecule, a vector comprising a nucleic acid molecule such as a virus, a protein, or a cell. Pharmaceutical formulations are well known, and pharmaceutical compositions comprising such active agents can be routinely formulated by those skilled in the art. Suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences, a standard reference text in this field. The present invention relates to an injectable pharmaceutical composition comprising a pharmaceutically acceptable carrier and an active agent. The composition is preferably sterile and pyrogen-free.

[0083] In some embodiments, for example, the active agent can be formulated as a solution, suspension, emulsion, or lyophilized powder in association with a pharmaceutically acceptable vehicle. Examples of such vehicles include water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin. Non-aqueous vehicles such as liposomes and fixed oils can also be used. The vehicle or lyophilized powder may contain additives that maintain isotonicity (e.g., sodium chloride, mannitol) and chemical stability (e.g., buffers and preservatives). The formulation is sterilized by commonly used techniques.

[0084] The injectable composition may contain the immunogen in a diluent such as sterile water, electrolytes / dextrose, fatty oils of vegetable origin, fatty acid esters, or polyols such as propylene glycol and polyethylene glycol. The injectable preparation must be sterile and pyrogen-free.

[0085] The vaccine may be administered by any means that allows for the presentation of the immunogenic material to the body's immune system for recognition and induction of an immunogenic response. Pharmaceutical compositions may be administered parenterally, i.e., intravenously, subcutaneously, or intramuscularly.

[0086] The dosage will vary depending on known factors such as the nature of the active agent and the pharmacodynamic properties of the particular agent, as well as its mode and route of administration; the recipient's age, health, and weight; the nature and extent of the condition, the type of concurrent treatment, the frequency of treatment, and the desired effect. A certain amount of immunogen is delivered to induce a protective or therapeutically effective immune response. Those skilled in the art can readily determine ranges and optimal dosages by routine methods.

[0087] The following examples are provided as illustrative embodiments only and are not intended to limit the scope of the present invention.

[0088] 5.0 Listeria monocytogenes vector-based vaccines We have developed a recombinant strain of Listeria monocytogenes (Lm) expressing GUCY2C (Lm-GUCY2C) and demonstrated its ability to secrete GUCY2C within infected macrophages (Figure 11) and enhance GUCY2C-specific T cell responses after Ad5 (Figure 12) or DNA vaccination (Figure 13). These Lm-GUCY2C-based vaccination regimens are expected to induce robust antitumor effects in patients with GUCY2C-expressing cancers, including, but not limited to, colorectal, gastric, esophageal, pancreatic, and salivary cancers. Several different variations of "Lm-GUCY2C" have been disclosed: 1.Lm-LLO-GUCY2C: a. Use a strong Lm promoter such as hly [listeriolysin O; (LLO)]; iap [p60; p60], acta [actin assembly-inducing protein; (actA)]; or other PrfA-dependent promoters. b. Use of the GUCY2C extracellular domain (amino acids about 23 to about 429) fused to the C-terminus of LLO (amino acids about 1 to about 420) to promote cytosolic secretion. 2.Lm-ActA-GUCY2C: a. Use a strong Lm promoter such as hly [listeriolysin O; (LLO)]; iap [p60; p60], acta [actin assembly-inducing protein; (actA)]; or other PrfA-dependent promoters. b.ActAN100 * The GUCY2C extracellular domain (amino acids about 23 to about 429) is used fused to the C-terminus of a modified form of ActA known as (described in U.S. Patent No. 20180030457A1, which is incorporated herein by reference). 3.Lm-ActA-Syn18×5-GUCY2C: a. Use a strong Lm promoter such as hly [listeriolysin O; (LLO)]; iap [p60; p60], acta [actin assembly-inducing protein; (actA)]; or other PrfA-dependent promoters. b. The C-terminal GUCY2C extracellular domain (amino acids about 23 to about 429) (U.S. Patent Publication No. 20180030457A1) linked to a Syn18x5 sequence to enhance expression of the fusion protein, and an N-terminal ActAN100 to promote cytosolic secretion. * A fusion protein consisting of a modified form of ActA known as ActA (described in U.S. Patent Publication No. 20180030457A1) is used. 4. Other variations of Lm-GUCY2C, including promoters that drive expression of GUCY2C proteins / peptides alone or fused to other proteins, are also possible and disclosed.

[0089] In some embodiments, these are: 1. The ΔactA / ΔinLB version of the wild-type 10403S Listeria monocytogenes strain (U.S. Pat. Nos. 7,691,393 and 7,695,725, incorporated herein by reference). 2. A dead but metabolically active (KBMA) version derived from the ΔactA / ΔinLB version described above, which also contains deletions of the uvrA and uvrB genes. 3. dal dat ΔactA Version of the Wild-type 10403S Listeria monocytogenes Strain 4. prfA-deficient Listeria monocytogenes strain XFL-7 Various Lm strain "backbones" may be used, including, but not limited to:

[0090] In some embodiments, the Lm vector contains any one of several different GUCY2C inserts, such as those described in section 4.2 above. In some preferred embodiments, the GUCY2C insert does not contain a universal epitope associated therewith. ECD The coding sequence of is inserted into an Lm vector. In some embodiments, a GUCY2C insert, such as that described in Section 4.2, can be linked to a universal T-cell epitope as described in Section 4.3. As used herein, Lm-GUCY2C, Lm-GUCY2C ECD , Lm-GUCY2C universal T cell epitope and Lm-GUCY2C ECD The universal T cell epitope is GUCY2C bound to a universal T cell epitope fusion protein that is expressed and secreted by infected cells. ECD GUCY2C bound to a universal T cell epitope, as occurs upon infection with the vaccine. ECD In some embodiments containing universal T cell epitopes, the terms "PADRE", "GUCY2C", and "PADRE" are used interchangeably to refer to Lm vectors having a gene insert encoding the universal T cell epitope PADRE. ECD -PADRE is used interchangeably to refer to such embodiments that include PADRE.

[0091] 6.0 Treatment with AD5.F35-GUCY2C vaccine, Lm-GUCY2C vaccine, or a combination of AD5.F35-GUCY2C and Lm-GUCY2C vaccines Embodiments of the present invention include methods of treating individuals with cancers / tumors that express GUCY2C. Treatment is provided systemically. By treating such individuals with the vaccines described herein, an immune response can be induced in the peripheral compartment of the individual's immune system that specifically targets cancer cells that express GUCY2C. The vaccines treat primary or metastatic disease, including identified metastatic disease as well as any undetected metastases, such as micrometastases.

[0092] The vaccine provides an adjuvant therapeutic treatment in addition to the usual treatments provided at the time of diagnosis of cancers involving mucosal tissues. As discussed above in this application, those skilled in the art can diagnose cancers or tumors as expressing GUCY2C. While detection of metastatic disease can be performed using routine methodologies, some microscopic levels of cancer may not be detectable at the time of initial cancer diagnosis. Typical modes of treatment include surgery, chemotherapy, or radiation therapy, or various combinations. A vaccine targeting GUCY2C provides an additional countermeasure with the advantage of selectively detecting and eliminating cancer cells derived from immunologically protected compartments that express GUCY2C, rather than attacking normal tissues.

[0093] In some embodiments, an individual is diagnosed with cancer and the cancer is identified as expressing GUCY2C. The AD5.F35-GUCY2C vaccine, which expresses soluble GUCY2C linked to a CD4+ helper epitope, is administered to the patient alone or as part of a treatment regimen that includes surgery, and / or radiation therapy, and / or administration of other anti-cancer agents.

[0094] In some embodiments, an individual is diagnosed with cancer and the cancer is identified as expressing GUCY2C, and an Lm-GUCY2C vaccine expressing soluble GUCY2C is administered to the patient alone or as part of a treatment regimen that includes surgery, and / or radiation therapy, and / or administration of other anti-cancer agents.

[0095] In some embodiments, an individual is diagnosed with cancer and the cancer is identified as expressing GUCY2C. A prime-boost regimen is performed using both an adenoviral vector-based anti-GUCY2C vaccine and an Lm vector-based anti-GUCY2C vaccine in sequential combination. In some embodiments, the individual is first administered an adenoviral vector expressing a soluble form of GUCY2C, followed by an Lm vector-based anti-GUCY2C vaccine. In some such embodiments, the individual is preferably administered the AD5.F35-GUCY2C vaccine first, followed by the Lm-GUCY2C vaccine, although the vaccines can be delivered in the reverse order. The minimum interval between the first and second vaccinations is about 2-4 weeks. A preferred interval is about 4-6 weeks. The interval can be up to 12 months. The vaccine combination therapy can be administered in combination with surgery, radiation therapy, and / or other anti-cancer agents.

[0096] 7.0 Prophylactic Methods Using AD5.F35-GUCY2C Vaccine, Lm-GUCY2C Vaccine, or a Combination of AD5.F35-GUCY2C Vaccine and Lm-GUCY2C Vaccine The vaccine may be used prophylactically in individuals at risk of developing GUCY2C cancer / tumors. A previous diagnosis of primary disease that has been removed or is in remission places the individual at higher risk.

[0097] Individuals at risk of developing GUCY2C-expressing cancers may be administered the vaccine to induce an immune response that eliminates cancer cells prior to the individual having detectable disease.

[0098] 8.0 Vaccine Composition, Formulation, Dosage and Administration Regimens Vaccines according to some embodiments comprise a pharmaceutically acceptable carrier in combination with an active agent, which may be an adenoviral vector or an Lm vector. Pharmaceutical formulations are well known, and pharmaceutical compositions containing such active agents can be routinely formulated by those skilled in the art. Suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences, a standard reference text in this field. The present invention relates to an injectable pharmaceutical composition comprising a pharmaceutically acceptable carrier and an active agent. The composition is preferably sterile and pyrogen-free.

[0099] In some embodiments, for example, the active agent can be formulated as a solution, suspension, emulsion, or lyophilized powder in association with a pharmaceutically acceptable vehicle. Examples of such vehicles include water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin. Non-aqueous vehicles such as liposomes and fixed oils can also be used. The vehicle or lyophilized powder may contain additives that maintain isotonicity (e.g., sodium chloride, mannitol) and chemical stability (e.g., buffers and preservatives). The formulation is sterilized by commonly used techniques.

[0100] The injectable composition may contain the immunogen in a diluent such as sterile water, electrolytes / dextrose, fatty oils of vegetable origin, fatty acid esters, or polyols such as propylene glycol and polyethylene glycol. The injectable preparation must be sterile and pyrogen-free.

[0101] The vaccine may be administered by any means that allows for the presentation of the immunogenic material to the body's immune system for recognition and induction of an immunogenic response. The pharmaceutical composition may be administered parenterally, i.e., intravenously, subcutaneously, or intramuscularly.

[0102] The dosage will vary depending on known factors such as the nature of the active agent and the pharmacodynamic properties of the particular agent, as well as its mode and route of administration; the age, health, and weight of the recipient; the nature and extent of the condition, the type of concurrent treatment, the frequency of treatment, and the desired effect. A certain amount of immunogen is delivered to induce a protective or therapeutically effective immune response. Those skilled in the art can readily determine ranges and optimal dosages by routine methods.

[0103] The following examples are provided as illustrative embodiments only and are not intended to limit the scope of the present invention.

[0104] The following examples are provided as illustrative embodiments only and are not intended to limit the scope of the present invention.

[0105] Example Example 1 Phase 1 clinical trial patients receiving Ad5-GUCY2C-PADRE had Ad5 neutralizing antibody (NAb) titers ranging from less than 10 to more than 10,000. A clear pattern emerged: half of the patients had titers well below 200 (Ad5 NAb low), and the other half had titers well above 200 (Ad5 NAb high). Dividing patients into Ad5 NAb low and high cohorts revealed a relationship between Ad5 NAb titers and GUCY2C-specific T cell responses, with responses significantly greater in Ad5 NAb low patients (Figure 3). Taken together, these data are consistent with our animal data (Figure 4), which demonstrate that pre-existing Ad5 NAbs eliminate the efficacy of Ad5-GUCY2C-PADRE, despite high dose administration and in contrast to other vaccines (Priddy, FH et al. (2008). Clin Infect Dis 46(11):1769-1781). Additional results are shown in Table 1: Table 1. Ad5-hGCC-PADRE is restricted by Ad5 nAbs. [Table 1]

[0106] Ad5 NAbs are common in most human populations, including approximately 40% in the United States (Nwanegbo, E. et al. (2004) Clin Diagn Lab Immunol 11(2):351-357; Barouch, DH et al. (2011) Vaccine 29(32):5203-5209), and reduce the immunogenicity of Ad5-based vaccines (Priddy, FH et al. (2008). Clin Infect Dis 46(11):1769-1781). Importantly, most naturally occurring Ad5 NAbs target fiber molecules on the surface of Ad5 (Cheng, C. et al. (2010) J Virol 84(1):630-638). In contrast to Ad5, the seroprevalence of Ad35-specific antibodies in most countries is very low (<10%; Nwanegbo, E. et al. (2004) Clin Diagn Lab Immunol 11(2):351-357; Barouch, DH et al. (2011) Vaccine 29(32):5203-5209). Collectively, these observations suggest that replacing the Ad5 fiber molecule with that of Ad35 generates a chimeric Ad5 viral vector (known as Ad5.F35) that is immune to naturally occurring Ad5 immunity. Indeed, 50% of subjects in the Ad5-GUCY2C-PADRE phase 1 trial had high Ad5 NAb (titer >200), whereas only 1 / 10 of subjects had high Ad5.F35 NAb (Figure 5). Therefore, Ad5.F35-GUCY2C-PADRE is expected to overcome pre-existing Ad5-specific immunity that was not achieved by dose escalation, as predicted by published literature (Priddy, FH et al. (2008). Clin Infect Dis 46(11):1769-1781).

[0107] Example 2 To generate Ad5.F35-hGCC-PADRE, the hGCC-PADRE coding sequence was synthesized, and to increase the efficiency of translation of hGCC-PADRE mRNA into protein, a mammalian codon-optimized sequence was generated, as set forth in SEQ ID NO: 3. This construct was subcloned into the Ad5.F35 vector to generate Ad5.F35-hGCC-PADRE.

[0108] Overall, the structure of Ad5.F35-hGCC-PADRE is identical to that of wild-type Ad5 (Genbank: AY339865.1) with three exceptions. First, the hGCC-PADRE expression cassette replaces the E1A and E1B regions of Ad5 (nucleotides 455-3512), rendering Ad5.F35-hGCC-PADRE replication incompetent. Second, Ad5.F35-hGCC-PADRE has a large deletion of the E3 region (nucleotides 28586-30464). The E3 region contains several proteins with immunosuppressive functions. All of these E3 proteins are deleted in Ad5.F35-hGCC-PADRE.

[0109] Finally, the Ad5 fiber is replaced with the Ad35 fiber to minimize neutralization of the vector by pre-existing antibodies to Ad5 in patients.

[0110] Ad5.F35-hGCC-PADRE DNA construct construction The hGCC-PADRE coding sequence was synthesized to contain a Kozak sequence (GCCGCCACC) immediately 5' to the initiating ATG and two stop codons immediately following the PADRE sequence. The hGCC-PADRE construct was subcloned into the pShuttle vector and then recombined into the Ad5.F35 vector. Ad5.F35 contains human Ad5 sequences encoding all elements necessary to generate replication-incompetent adenovirus, including left and right inverted terminal repeats (ITRs), an encapsidation signal for packaging, the E2 and E4 regions, and late genes. The plasmid lacks the E1 and E3 proteins, and the fiber is replaced with the Ad35 fiber.

[0111] Preparation, structure, and composition of Ad5.F35-hGCC-PADRE A master virus bank of the adenoviral vector Ad5.F35-hGCC-PADRE was generated under GMP conditions at the Vector Production Facility (VPF) of the Center for Cellular and Gene Therapy (CAGT), Baylor College of Medicine, Houston, Texas.

[0112] The recombinant adenoviral vector was produced in HEK293 cells, which carry the E1 gene required for viral replication. Viable Ad5.F35-hGCC-PADRE virus was formed and harvested. The virus was plaque-purified twice in HEK293 cells. Individual plaques were tested for the presence of hGCC-PADRE DNA, sequenced for hGCC-PADRE cassette integrity, and tested for their ability to produce hGCC-PADRE protein when infected with cells in vitro. The plaque-purified vector was then propagated in HEK293 cells in tissue culture flasks and further amplified using 10-layer cell factories. After large-scale amplification, Ad5.F35-hGCC-PADRE was purified by CsCl banding. The Ad5.F35-hGCC-PADRE master virus bank (MVB) was fully sequenced. A portion of the MVB was aliquoted for subject administration as part of this study.

[0113] Example 3 Vaccination Protocol The Ad5.F35-hGCC-PADRE vaccine to be used consists of a recombinant, replication-deficient, E1 / E3-deleted Ad5.F35 virus carrying a GCC-PADRE expression cassette. This vaccine vector uses recombinant human adenovirus type 5 (rAd5), which carries deletions in the early E1 and E3 regions of the vector, allowing for insertion of the GCC-PADRE cassette. The E1 / E3 deletions disable viral replication, enhancing the safety associated with its clinical use.

[0114] Ad5.F35-hGCC-PADRE:10 formulated in TG buffer (20 mM Tris-HCl, pH 8.0, 25 mM NaCl, 2.5% glycerol) 11 , 10 12 , or 5 x 10 12 Viral particles are administered intramuscularly to human patients with selected solid tumors (colorectal, pancreatic, gastric, or esophageal) at risk of recurrence after radical surgery and standard adjuvant therapy. Three doses are administered, with four-week intervals between doses.

[0115] The subjects' cellular (T cell) responses to Ad5.F35-hGCC-PADRE and humoral immune responses to GCC will be assessed at weeks 5, 9, and 14 after the first vaccination. The effect of pre-existing neutralizing antibodies to Ad5 will be reduced compared to what has been observed previously.

[0116] Example 4 A Phase 2A Dose-Finding Study of the Ad5.F35-hGCC-PADRE Vaccine in Adults with Gastrointestinal Adenocarcinoma at Risk of Recurrence After Curative Surgery and Standard Therapy This is an open-label, dose-finding, Phase 2A study of Ad5.F35-hGCC-PADRE as a vaccine against gastrointestinal (GI) malignancies (pancreatic, colorectal, esophageal, and gastric adenocarcinomas) following surgical resection and standard adjuvant therapy. Patients will receive multiple intramuscular doses of Ad5.F35-hGCC-PADRE at one of three dose levels. Treatment-related toxicity and immune response development against GCC will be assessed at weeks 5, 9, and 13 after the first (week 1) vaccination. Primary safety endpoints will examine adverse events (AEs), injection site reactions, and clinically significant changes in safety laboratory tests. The primary efficacy endpoint will include a 10-day follow-up period across different dose levels (10 11 , 10 12 , and 5 × 10 12 This involves the development of a GCC-specific T cell response to viral particles (vp).

[0117] the purpose The main objectives of this study are as follows: 1) In subjects with high-risk colorectal, pancreatic, gastric, or esophageal adenocarcinoma who have no evidence of disease after surgery and standard therapy, 3 dose levels (10 11 , 10 12 , and 5 × 10 12 To evaluate the safety and tolerability of sequential Ad5.F35-hGCC-PADRE vaccine doses delivered intramuscularly (IM) (vp). 2) In subjects with high-risk colorectal, pancreatic, gastric, or esophageal adenocarcinoma who have no evidence of disease after surgery and standard therapy, 3 different dose levels (10 11 , 10 12 , and 5 × 10 12 vp) to evaluate the cellular (T cell) response to Ad5.F35-hGCC-PADRE.

[0118] Observed goals include: 1) Three different dose levels (10 ) administered IM as three consecutive doses at 4-week intervals in subjects with high-risk colorectal, pancreatic, gastric, or esophageal adenocarcinoma with no evidence of disease after surgery and standard therapy. 11 , 10 12 , and 5 × 10 12 To evaluate the humoral (antibody) response to Ad5.F35-hGCC-PADRE (vp). 2) To evaluate the relationship between neutralizing antibodies against Ad5 and immunological responses. 3) To evaluate the correlation of immune response to GCC protein expression in tumors and assess immune tolerance. 4) Evaluate disease-free survival (DFS). 5) If possible, assess overall survival (OS).

[0119] group The target final sample size is 72 subjects with evaluable response data. Up to 81 subjects with GI adenocarcinoma who meet all eligibility criteria will be enrolled. We expect to enroll approximately 3 patients per month. A futility analysis will be conducted in each arm after 15 patients have completed treatment, potentially discontinuing the arm at that time. Therefore, the minimum sample size is 45.

[0120] protocol Ad5.F35-hGCC-PADRE:10 formulated in TG buffer (20 mM Tris-HCl, pH 8.0, 25 mM NaCl, 2.5% glycerol) and administered intramuscularly 11 , 10 12 , or 5 x 10 12 Virus particles.

[0121] The study will enroll a minimum of 45 and a maximum of 81 subjects. Subjects will be randomized to one of three treatment arms stratified according to tumor type and surgical resection margin (R0, i.e., negative resection margin, vs. R1, i.e., positive resection margin). Treatment arm A will consist of three 10-week follow-up injections at 4-week intervals. 11 Treatment arm B will receive three 10-day vp vaccine doses at 4-week intervals. 12 Treatment arm C will receive three 5 × 10 12 Subjects will receive the vp vaccine. Subjects will be randomized to each treatment arm and begin treatment concurrently with ongoing monitoring for adverse events.

[0122] After completion of the 13-week study evaluation for immune response and safety, subjects will be followed by telephone or optional clinic visits for subsequent cancer-related therapy, DFS rate for at least 24 months of follow-up, recurrence, or death, whichever occurs first.

[0123] The duration of a treatment cycle (i.e., three doses) is 9 weeks. Subjects will be followed during the treatment period until 4 weeks after the final study dose, and then until death, early discontinuation, or completion of follow-up. Subjects will be followed throughout the study until all subjects have reached at least 24 months of follow-up or death. At the end of the study, all remaining subjects will be offered enrollment in a long-term follow-up protocol.

[0124] Introduction Background information The study will be conducted in subjects with solid tumors of the gastrointestinal (GI) tract who are at high risk of recurrence after definitive surgery and / or standard therapy. The target population includes individuals with colorectal, pancreatic, gastric, or esophageal adenocarcinoma who meet the study eligibility criteria.

[0125] Epidemiology and current treatment of pancreatic, colorectal, esophageal, and gastric cancer Pancreatic, colorectal, gastric, and esophageal cancers each continue to represent a major cancer burden in the United States, despite improvements in detection and treatment. In each case, a significant number of patients undergo surgical resection and standard therapy in an attempt to achieve a cure, but a small proportion (varies depending on disease histology, stage, and other factors) remain in remission. Adjuvant therapy in these cases in the form of chemotherapy and / or radiation therapy improves overall clinical outcomes. However, there is significant room for further improvement, especially for patients with high-risk features.

[0126] GI malignancies were responsible for 26% of cancer-related deaths in the United States in 2016. Colorectal cancer was the fourth most common and second most deadly cancer, while pancreatic, esophageal, and gastric cancers had the third, tenth, and sixteenth highest estimated cancer-related mortality rates in the United States in 2016. Five-year relative survival rates for GI malignancies vary by disease stage. In general, pancreatic adenocarcinoma has the worst 5-year survival rate (7%) across all stages, compared with 2% for stage 4 disease (distant metastasis), 11% for locally widespread disease, and 27% for localized disease. The majority (>80%) of exocrine pancreatic adenocarcinomas are not diagnosed until the development of regional or distant metastases, which are no longer amenable to surgical resection and have an estimated median survival of 3-6 months.2 The estimated median survival for surgically resected pancreatic adenocarcinoma is 18 months, but only 20% of patients survive 5 years.

[0127] For patients with localized pancreatic, colorectal, gastric, and esophageal cancer who are still considered at high risk of recurrence, the mainstay of treatment may involve surgery, chemotherapy, and radiation. Adjuvant therapy with monoclonal antibodies is also used in colorectal and esophageal cancer. Immunotherapy, which has established utility in patients with melanoma, prostate cancer, and non-small cell lung cancer, is increasingly being investigated in different settings as a means of reducing the risk of recurrence.

[0128] This study aims to evaluate a novel vaccine immunotherapy delivered intramuscularly (IM) sequentially at three dose levels to identify the dose that is best tolerated and most effective in inducing a cellular immune response. The ultimate therapeutic goal of this vaccine, currently in late-stage clinical development, is to reduce the risk of recurrence in subjects with GI adenocarcinoma.

[0129] Pancreatic adenocarcinoma and available treatments Of the estimated 46,000 Americans diagnosed with pancreatic adenocarcinoma in 2014, approximately 20% are considered resectable. After recovery from surgery, the primary modalities of adjuvant therapy are chemotherapy regimens such as capecitabine and gemcitabine or irinotecan, oxaliplatin, and 5-fluorouracil (modified FOLFIRNOX). 8 It is well established that chemotherapy is superior to best supportive care, reducing mortality by approximately one-third with a hazard ratio of approximately 0.60. 9 The modified FOLFIRNOX regimen improved 3-year disease-free survival to 39.7% compared with 21.4% in the gemcitabine-alone group, while improving overall survival to 63.4% compared with 48.6% in the gemcitabine-alone group. 8However, regardless of surgery and adjuvant therapy, at least 80% of patients will experience recurrence and ultimately die. Survival is related to pathological features, preoperative functional status, surgical outcome, and adjuvant therapy. According to the American Cancer Society, for all stages of pancreatic cancer combined, the 1-year relative survival rate is 20% and the 5-year survival rate is 6%. If surgical resection can be performed, the average survival rate is 18-20 months. The overall 5-year survival rate is approximately 10%, but can reach 20-25% if the tumor is completely removed and the cancer has not spread to the lymph nodes.

[0130] Colorectal Cancer and Available Treatments Of the estimated 132,000 Americans diagnosed with colorectal cancer (CRC) annually, approximately two-thirds undergo surgical resection. Five-year survival rates range from 25 to 70%, depending on the type and stage of the cancer.

[0131] When CRC is diagnosed at an early stage, it is often curative with surgery alone. In later stages, where cure is still considered possible with surgery, chemotherapy in the form of fluorouracil, fluoropyrimidines, capecitabine, or oxaliplatin is often used as adjuvant therapy. Two studies found a 10-15% increase in 3-year survival rate when patients received adjuvant therapy after surgery, while other studies also found some beneficial benefit from adjuvant treatment.

[0132] Recent improvements in both surgical and adjuvant treatment and in early diagnosis have increased the 3-year DFS for patients who undergo surgical resection. Targeted therapy in the form of monoclonal antibodies against VEGF and EGFR is also available for patients with advanced disease. 4However, cure rates remain low and recurrence rates are high. Two anti-PD-1 checkpoint immunotherapies, nivolumab (Opdivo®) and pembrolizumab (Keytruda®), have also been approved for colorectal cancer patients with advanced recurrent tumors characterized by high microsatellite instability (MSI-hi), but overall response rates with these therapies remain low.

[0133] Gastric Cancer and Available Treatments An estimated 25,000 Americans are diagnosed with gastric cancer each year. The majority of patients with early-stage cancer have surgically resectable disease—in localized distal gastric cancer, more than 50% of patients can be cured. However, early-stage disease accounts for only 10–20% of all cases diagnosed in the United States. Five-year OS rates range from almost zero for patients with disseminated disease to approximately 50% for patients with localized distal gastric cancer and resectable localized disease. While the clinical benefit of adjuvant therapy has been debated, recent studies demonstrate potential benefit. In one study, patients receiving adjuvant treatment with capecitabine and oxaliplatin had an improved 5-year DFS with a hazard ratio of 0.58. Another study using an adjuvant fluoropyrimidine found that the treatment group demonstrated a 10% increase in survival within the first year compared with surgery alone. Current treatments, with or without adjuvant therapy, result in an average 5-year survival rate of 10-15% in patients with proximal gastric cancer and a recurrence rate of over 50% in resected patients, suggesting the ineffectiveness of current treatment regimens. Furthermore, pembrolizumab (Keytruda®), an anti-PD-1 checkpoint immunotherapy, was recently approved for patients with advanced PD-L1-positive gastric cancer, albeit with a low response rate.

[0134] Esophageal Cancer and Available Treatments According to the National Cancer Institute (Cancer.gov), an estimated 16,940 people will be diagnosed with esophageal cancer in 2017, and 15,690 will die. Approximately half will have resectable tumors. On average, 18.8% of patients survive five years after diagnosis, but the 5-year survival rate drops sharply to 4.6% for patients with distal disease. Esophagectomy remains the cornerstone treatment for clinically localized esophageal cancer, but the nature of this disease is driven solely by surgical failure. In squamous cell carcinoma of the esophagus, neoadjuvant cisplatin was not found to confer an improvement in survival, while neoadjuvant cisplatin and fluorouracil increased 5-year DFS by 10%. In contrast, neoadjuvant chemotherapy (carboplatin and paclitaxel) and radiation therapy in esophageal adenocarcinoma resulted in a median OS of 49.4 months compared with 24 months in the surgery-alone group. Other studies have also found beneficial effects of neoadjuvant chemotherapy with epirubicin, 5-fluorouracil, and / or cisplatin in patients with gastric or gastroesophageal junction adenocarcinoma. In addition, antibody therapy targeting HER2 in the small number of esophageal cancers that overexpress HER2 and inhibiting angiogenesis by targeting the VEGF pathway has emerged in the treatment of esophageal cancer. 4 A recent meta-analysis of 13 randomized controlled trials of neoadjuvant chemoradiotherapy compared with surgery alone showed a pooled hazard ratio of 0.78, corresponding to an absolute survival benefit at 2 years of 8.7% and a number needed to treat of 11. Although the efficacy of neoadjuvant therapy in esophageal cancer is generally beneficial compared with best supportive care, recurrence remains high, and many patients ultimately die. As with gastric cancer, anti-PD-1 checkpoint immunotherapy, pembrolizumab (Keytruda®), was recently approved for patients with PD-L1-positive cancer of the gastroesophageal junction, but response rates are low. Given the incidence and prognosis of this disease, effective adjuvants with minimal side effects are needed to improve disease outcomes.

[0135] Rationale for the proposed study Guanylyl cyclase C (GCC) as a tumor-associated antigen in colon cancer Guanylyl cyclase C (GCC or GUCY2C), a member of a family of homologous proteins that synthesize cyclic GMP (cGMP), is specifically expressed by intestinal epithelial cells. GCC is a receptor for the paracrine hormones guanylin and uroguanylin and the diarrheal bacterial heat-stable enterotoxin (ST), whose interaction with its extracellular domain activates the cytoplasmic catalytic domain and induces cGMP accumulation. While the catalytic domains share approximately 50% homology among family members, the extracellular domains of GCC show less than 20% homology, creating an antigenically unique structure. GCC has been detected in over 500 samples of normal intestine but not in over 1,000 extragastrointestinal tissues. Furthermore, in intestinal epithelial cells, GCC specifically localizes to the apical brush border membrane, the "outside" of the mucosal wall. This anatomical and functional compartmentalization suggests that GCC is normally restricted to the mucosa, a finding confirmed by radioligand imaging, biodistribution, and immunological studies. Importantly, GCC protein (>200 samples) and / or mRNA (>900 samples) were detected in nearly all primary and metastatic human colorectal tumors, uniformly expressed by tumor cells regardless of anatomical location or grade, but were not detected in extra-GI tumors (>200). GCC was also overexpressed in >80% of colorectal tumors. The stringency of expression by enterocytes and universal overexpression by metastases highlights the utility of GCC as a marker for staging pN0 patients.

[0136] GCC in pancreatic, gastric, and esophageal cancer GCC is normally expressed in intestinal epithelial cells, and expression is maintained during colorectal neoplastic transformation in both local and metastatic settings. In addition, GCC is found to be expressed in approximately 60% of pancreatic, gastric, and esophageal cancers. GCC's disruption of epithelial tight junctions, cell polarity, and apical localization makes it a desirable target for systemic agents in tumor tissue, while sparing normal tissue.

[0137] Ad5.F35-hGCC-PADRE vaccine Preclinical studies using Ad5-hGCC-PADRE Preclinical studies have demonstrated that an Ad5 vector vaccine incorporating murine GCC generates GCC-specific immune responses (antibodies and cytotoxic T cells) in mice. These responses are durable and persist for several months after vaccination. In addition, GCC-specific cytotoxic T cells induced by the vaccine can kill GCC-expressing CRC cells in vitro. Mice bearing GCC-expressing metastatic colorectal tumors growing in the lung or liver are protected by GCC vaccination: GCC vaccination reduces (or eliminates) the number of detectable metastatic tumors and significantly improves animal survival. Importantly, these immune responses selectively target metastatic CRC but do not target normal intestinal tissue expressing GCC, nor cause intestinal pathology. Therefore, GCC-targeted vaccination induces GCC-specific immune responses capable of preventing / treating metastatic CRC in mouse models, but does so without causing adverse effects. This targeted therapy is expected to be clinically advantageous compared to established cancer therapies, which have low efficacy and cause significant off-target toxicity.

[0138] Clinical data to date on the Ad5-hGCC-PADRE vaccine The Ad5-hGCC-PADRE vaccine was first evaluated in humans in an open-label, single-arm, single-dose feasibility study to determine the safety and immunogenicity of Ad5-hGCC-PADRE in patients with early-stage colorectal cancer (ClinicalTrials.gov NCT01972737). Subjects received a single intramuscular injection of 10 particles of Ad5-hGCC-PADRE.

[0139] Safety Data Subjects in this study were evaluated for acute events in the clinic every 10 minutes for 30 minutes after injection and by telephone on days 3 and 8 after vaccination. Patients also returned to the clinic 30, 90, and 180 days after vaccination for safety assessments. Adverse events were graded according to CTCAE version 5.0. Of the 10 subjects who received Ad5-hGCC-PADRE, 10 (100%) experienced an AE. The most frequently reported vaccine-related AEs were chills / rigidity (20%), injection site pain / swelling (20%), dizziness (10%), sweating (10%), pain (10%), and fever (10%). There were no serious adverse events (SAEs), and no subjects died during the study.

[0140] All AEs were grade 1, and no grade 3 / 4 toxicities occurred during the 6-month follow-up period after vaccination. Furthermore, laboratory evaluations performed on days 30, 90, and 180, including CBC with differential and comprehensive chemistry panels, and antinuclear antibody (ANA) titers, revealed no clinically relevant changes. Based on reported clinical experience with similar treatments, mild grade 1 / 2 toxicities, such as injection site pain and fever, were expected after viral vector immunization and were observed in some patients. Importantly, no adverse events related to toxicity in GCC-expressing tissues were observed. GCC is a self-protein expressed on the luminal surface of the small intestinal and colonic epithelium and in anorexigenic hypothalamic neurons. However, consistent with mechanisms controlling immune compartmentalization and preclinical studies of GCC vaccination, there was no evidence of Ad5-hGCC-PADRE-induced autoimmunity in GCC-expressing intestinal or brain tissues.

[0141] Immunogenicity data In preclinical studies, immunization with Ad5-hGCC-PADRE induced time- and dose-dependent GCC-specific T- and B-cell responses and CD8 +In clinical trials, GCC-specific immune responses after administration of Ad5-hGCC-PADRE were quantified by ELISA and IFNγ-ELISpot to quantify antibody and T cell responses, respectively. T cell responses to PADRE and Ad5 were also quantified by IFNγ-ELISpot. Patient immune responses typically followed one of four patterns: 1) In the absence of a pre-vaccination antibody response to GCC or T cell immunity to GCC, PADRE, or Ad5 and Ad5-hGCC-PADRE vaccinations, no targeted responses were induced to any antigen. 2) There was no pre-vaccination response, and vaccination induced Ad5-specific T cell responses, but not GCC- or PADRE-specific responses. 3) Ad5-hGCC-PADRE vaccination induced GCC-specific T cell responses. GCC-specific antibody responses were induced by exogenous CD4 + Similar to animal studies requiring responses to "helper" T cell epitopes, the absence of PADRE-specific T cell responses or GCC-specific antibody responses was consistent with GCC-specific CD4 + Reflects T-cell tolerance. 4) Vaccine-induced responses through all three arms of adaptive immunity: PADRE-specific CD4 + T cell responses, GCC-specific antibody responses, and GCC-specific CD8 + T cell response.

[0142] Ad5 nAb limits Ad5-hGCC-PADRE immunogenicity Ad5 is a naturally occurring human pathogen that causes mild infections in nearly the entire human population. These natural exposures induce Ad5-specific neutralizing antibodies (nAbs), which prevent reinfection or Ad5-based vaccination by preventing infection of host cells, a necessary step for target antigen expression and induction of an immune response. Ad5 nAbs were quantified in patient sera collected before Ad5-hGCC-PADRE vaccination (day 0) using an Ad5-GFP reporter virus inhibition bioassay. Titers ranged from less than 10 to more than 10,000, with a clear pattern emerging: half of the patients had titers well below 200 (Ad5 nAb low) and the other half had titers well above 200 (Ad5 nAb high). Dividing patients into Ad5 nAb low and high cohorts revealed a relationship between Ad5 nAb titers and GCC-specific T cell responses, with responses significantly greater in Ad5 nAb low patients. PADRE-specific T cell responses were generally low but did not correlate with pretreatment Ad5 nAb titers. Similar to GCC-specific T cell responses, Ad5-specific T cell responses were also restricted by Ad5 nAb in the high-Ad5 nAb group. Collectively, these data are consistent with preclinical data demonstrating that pre-existing Ad5 nAb immunity eliminates Ad5-hGCC-PADRE viral particles in vivo before they enter host cells, preventing subsequent gene expression and induction of host immune responses.

[0143] Rationale for Ad5.F35-hGCC-PADRE Ad5 nAbs are common in most human populations, including approximately 40% in the United States (Nwanegbo, E. et al. (2004) Clin Diagn Lab Immunol 11(2):351-357.; Barouch, DH et al. (2011) Vaccine 29(32):5203-5209), and reduce the immunogenicity of Ad5-based vaccines (Priddy, FH et al. (2008). Clin Infect Dis 46(11):1769-1781). Importantly, most naturally occurring Ad5 nAbs target the fiber molecules on the surface of Ad5 (Cheng, C. et al. (2010) J Virol 84(1):630-638). In contrast to Ad5, the seroprevalence of Ad35-specific antibodies in most countries is very low (<10%, Nwanegbo, E. et al. (2004) Clin Diagn Lab Immunol 11(2):351-357; Barouch, DH et al. (2011) Vaccine 29(32):5203-5209). Collectively, these observations suggest that replacing the Ad5 fiber molecule with that of Ad35 generates a chimeric Ad5 viral vector (known as Ad5.F35) that is immune to naturally occurring Ad5 immunity. Indeed, in a cohort of 12 patients with preexisting Ad5 nAbs, sera from 8 patients (67%) failed to neutralize chimeric Ad5.F35 in vitro. Furthermore, 50% of subjects in a Phase 1 trial of Ad5-hGCC-PADRE had high Ad5 nAb titers (titers >200), whereas only 1 / 10 of subjects had high Ad5.F35 nAb titers (Figure 5). Therefore, Ad5.F35 offers an advantage over Ad5 by reducing the expected seroprevalence of Ad5.F35 nAb to less than 25% without any additional safety risks. Ad5.F35-GUCY2C-PADRE is expected to overcome pre-existing Ad5-specific immunity, which was not achieved by dose escalation as predicted by published literature (Priddy, FH et al. (2008). CL1n Infect Dis 46(11):1769-1781).

[0144] The similarities between Ad5 and Ad5.F35 vectors in immunogenicity, safety, and biodistribution profiles are supported by published nonclinical studies. Indeed, there are some indications that the safety profile of Ad5.F35 may be better. While Ad5.F35 transduction of muscle (vaccination site) is comparable to Ad5, liver (1000x), spleen (3x), kidney (100x), heart (10x), and lung (100x) demonstrate significantly lower transduction efficiency with Ad5.F35 than Ad5. In the context of Ad5.F35's lower tropism for the liver, Ad5.F35 produced significantly lower increases in serum proinflammatory cytokines and liver enzymes, suggesting that Ad5.F35 virus has a better safety profile than Ad5 vectors. Furthermore, the Ad5.F35 vector is significantly less susceptible to Ad5 nAbs than the Ad5 vector, increasing the magnitude of target antigen-specific immune responses in animals with high Ad5 nAb titers (>200). Therefore, the Ad5.F35 vector exhibits better biodistribution, safety, and efficacy in the context of Ad5 nAbs than the Ad5 vector. Collectively, these data suggest that neutralizing immunity to Ad5.F35-hGCC-PADRE is significantly reduced in patients, increasing the rate and magnitude of GCC-specific T cell responses in human subjects. In this context, we modified the vaccine by replacing the Ad5 fiber molecule with an Ad35 fiber molecule, generating the chimeric viral vector Ad5.F35. This study examines the safety, immunogenicity, and pre-existing nAb resistance of Ad5.F35-hGCC-PADRE in patients with GI malignancies.

[0145] Design of Ad5.F35-hGCC-PADRE The Ad5.F35-hGCC-PADRE vaccine consists of a recombinant, replication-deficient, E1 / E3-deleted Ad5.F35 virus carrying a GCC-PADRE expression cassette. This vaccine vector uses recombinant human adenovirus type 5 (rAd5), a stable, non-enveloped, icosahedral virus with a linear, double-stranded DNA genome of approximately 38 kb encoding over 30 proteins. rAd5 contains deletions of the early E1 and E3 regions of the vector, allowing for insertion of the GCC-PADRE cassette. The E1 / E3 deletion renders the virus replication-incompetent, enhancing safety for clinical use.

[0146] The GCC-PADRE cassette contains the extracellular domain of human GCC (residues 1-430) and CD4 + The Ad5.F35-hGCC-PADRE vector is composed of the human cytomegalovirus (HCMV) immediate-early promoter, which drives expression of a fusion product consisting of the T cell epitope PADRE. In vivo, the Ad5.F35-hGCC-PADRE vector induces expression of the GCC-PADRE immunogen by infected cells, leading to an immune response against GCC and PADRE. PADRE stimulates the expression of GCC-specific B cells and CD8 + CD4 on T cells + It provides T cell "help" and is included in the vaccine, resulting in superior immune and antitumor effects in preclinical models compared to vaccination with GCC without PADRE.

[0147] Rationale for Ad5.F35-hGCC-PADRE administration Our phase 1 study results confirm an immunological response to the Ad5-hGCC-PADRE vaccine that was suppressed by Ad5 neutralizing antibodies. Given the high prevalence of natural Ad5 exposure in the human population and the resulting development of neutralizing antibodies in approximately 40% of patients, this study will determine whether sequential dosing, higher dose levels, and / or modification of the Ad5 backbone (Ad5 to Ad5.F35) can overcome this obstacle. This approach is supported by results with other Ad5 vaccines, where higher dose levels were able to overcome pre-existing high Ad5 nAb titers and generate a cellular immune response.

[0148] The dose selected for this study was based on previous human experience with the Ad5-hGCC-PADRE vaccine and clinical trials of other Ad5-based vaccines. 11 The viral particle (vp) dose was tested as a single dose in a previous Phase 1 clinical trial and was found to be safe and tolerable. In this study, 1 × 10 vp administered intramuscularly (IM) at 4-week intervals for a total of three injections was used. 11 Repeated administration of vp Ad5.F35-hGCC-PADRE will be investigated. Additionally, this study will also investigate two additional dose levels administered three times at four-week intervals. In summary, the doses are 1 x 10 11 vp, 1×10 12 vp, and 5 × 10 12 vp. 5×10 12 The maximum dose of vp is approximately the maximum achievable dose, given the limited solubility of the vaccine.

[0149] The higher dose regimen used in this Phase 2A study (1 × 10 12 and 5 x 10 12 The Ad5.F35-hGCC-PADRE vector has not been previously tested in humans, and other adenovirus-based vaccines have been administered at similar dose levels and have been well tolerated. Similarly, repeated administration of the GCC-PADRE vector has not been clinically evaluated, but other studies have used repeated administration of adenovirus-based vaccines at comparable dose levels and have been well tolerated.

[0150] Research content An investigational product is defined as the pharmaceutical form of the active ingredient being tested or used as the reference in this study. The active investigational product used in this study, Ad5.F35-hGCC-PADRE, is a replication-deficient adenovirus vaccine against the human GCC protein formulated in TG buffer for intramuscular injection. [Table 2]

[0151] Characteristics of Ad5.F35-hGCC-PADRE Virus: Adenoviridae; non-enveloped, icosahedral virion, 70-90 nm diameter, double-stranded, linear DNA genome. Wild-type virus is lytic, but the E1 / E3 deleted version in the vaccine is non-replicative.

[0152] Pathogenicity: Ad5.F35-hGCC-PADRE is a replication-deficient adenovirus, so the risk of productive infection is low. Symptoms of wild-type adenovirus infection include fever, rhinitis, pharyngitis, cough, and conjunctivitis. Adenoviruses can elicit strong immune responses. Adenoviruses do not integrate into the host cell genome, and Ad5.F35-hGCC-PADRE lacks additional features that promote integration. The transgene expressed by Ad5.F35-hGCC-PADRE produces a secreted GCC-PADRE fusion protein, which generates an immune response. These responses are expected to have antitumor activity in patients.

[0153] Ad5.F35-hGCC-PADRE is a milky-white suspension of recombinant adenovirus type 5 particles, which may contain visible proteinaceous particles in a liquid composed of Ad5.F35-hGCC-PADRE viral particles formulated in TG buffer (20 mM Tris-HCl, pH 8.0, 25 mM NaCl, 2.5% glycerol). The vaccine is supplied at the required concentration in vials containing 1.2 mL of clear, colorless, sterile solution. Ad5.F35-hGCC-PADRE must be protected from light and stored at -70°C or below. Vials should not be thawed unless dispensed. Freeze-thaw cycles reduce vaccine potency and should be avoided.

[0154] Example 5 The ability of adenovirus serotype 5 (Ad5) to mediate gene transfer and induce potent immune responses has made it a popular vector for experimental vaccines against cancer and infectious diseases. Indeed, there are over 400 clinical trials using Ad5 vectors, with most trials focused on the development of cancer treatments. However, during natural infection, the host immune system develops neutralizing antibodies (NAb) against the Ad5 capsid, limiting viral spread and preventing reinfection. Because Ad5 infection is endemic to many human populations, pre-existing NAb, present in over 70% of the global population, limits Ad5-based vaccine strategies. These considerations highlight the need for improved vectors for use in vaccines targeting cancer- and pathogen-associated antigens that can generate therapeutic immune responses in the largest number of patients. Importantly, although the adenovirus capsid is composed of hexon, penton, and fiber proteins, NAb elicited by natural Ad5 infection in humans is primarily directed against the Ad5 fiber, suggesting that strategies to circumvent pre-existing immunity to this element could improve Ad5-based vaccines.

[0155] To improve antitumor immunity in a mouse model expressing the gastrointestinal (GI) cancer antigen guanylyl cyclase C (GUCY2C), we aimed to overcome pre-existing Ad5 NAb by replacing the Ad5 fiber with that of the rare adenovirus serotype Ad35 (international seroprevalence approximately 10%). Preclinical models demonstrated that an Ad5-based GUCY2C-directed vaccine (Ad5-GUCY2C-S1) elicited CD8+ T cell and antibody responses without autoimmunity. Furthermore, vaccination of mice with Ad5-GUCY2C-S1 induced long-term T cell-mediated protection against metastatic colorectal cancer in the lung and liver. These results were replicated in a recent first-in-human phase I clinical trial (NCT01972737), demonstrating that a humanized version of the vaccine (Ad5-GUCY2C-PADRE) safely induced GUCY2C-specific CD8+ T cell responses in colorectal cancer patients following conventional therapy. However, patients with high pre-existing NAb titers against Ad5 were unable to develop GUCY2C-specific immunity after vaccination with Ad5-GUCY2C-PADRE. To overcome Ad5 NAb, we constructed a chimeric Ad5 vector (Ad5.F35) carrying the Ad35 fiber, which has comparable safety and antitumor activity to Ad5 in mice and humans and is resistant to Ad5 NAb. This chimeric vaccine can be administered to GI cancer patients, not only in patients with low Ad5 immunity but also in patients with high pre-existing Ad5 NAb, to safely induce GUCY2C-specific immunity.

[0156] Materials and Methods Adenovirus vectors Influenza HA, also known as site 1 (S1) 107-119 CD4 + Mouse extracellular domain (GUCY2C) containing T cell epitopes 1-429The adenovirus containing GUCY2C was previously described (Ad5-GUCY2C-S1). Here, GUCY2C-S1 was cloned into pShuttle and subcloned into the E1 region of a previously generated replication-deficient chimeric adenovirus (Ad5.F35), replacing the Ad5 fiber with the Ad35 fiber to generate Ad5.F35-GUCY2C-S1. All adenovirus vaccines used in this study were produced in HEK293 cells under good laboratory practices at the Cell and Gene Therapy Vector Development Institute at Baylor College of Medicine, purified by cesium chloride ultracentrifugation, and certified negative for replication-competent adenovirus, mycoplasma, and host cell DNA contamination. In vitro GUCY2C expression experiments (dose-response and time-course) were performed in A549 (American Type Culture Collection (ATCC)) cells. Virus was added to cultures at the indicated doses, and culture supernatants were collected at the indicated time points. Relative GUCY2C levels in the supernatants were quantified by Western blot using 2 μg / mL of MS7 mouse anti-GUCY2C monoclonal antibody and 0.1 μg / mL of horseradish peroxidase-conjugated goat anti-mouse secondary antibody (Jackson Immuno).

[0157] Mice and immunization Eight-week-old male and female BALB / cJ mice were purchased from The Jackson Laboratory for this experiment. The animal protocol was approved by the Thomas Jefferson University Institutional Animal Care and Use Committee (Protocol 02092). For immunization, 10 mL of insulin was administered as two intramuscular injections of 50 μL, one into each hind leg, using a 0.5 mL insulin syringe. 10 or 10 11 Mice were administered vp Ad5-GUCY2C-S1, Ad5.F35-GUCY2C-S1, or Ad5.F35-GFP (control).

[0158] Quantification of T cell responses by ELISpot ELISpot assays were performed using a mouse interferon-γ (IFN-γ) single-color ELISpot kit (Cellular Technology) according to the manufacturer's protocol. Briefly, 96-well plates were coated with IFN-γ capture antibody overnight at 4°C. The next day, plates were washed with phosphate-buffered saline (PBS), and splenocytes from immunized mice were incubated with GUCY2C without peptide or at 10 μg / mL in 0.1% dimethyl sulfoxide (DMSO) in CTL-TEST medium (Cellular Technology). 254-262 Peptide was seeded at 500,000 cells / well for 24 hours at 37°C. For T cell avidity studies, 10 6 GUCY2C normalized to cells / well 254-262 Splenocytes were seeded at 600,000–800,000 cells / well with decreasing peptide concentrations (10 μg / mL to 56 pg / mL). After incubation, cells were removed and a colorimetric reagent was added to detect IFNγ-producing, spot-forming cells. The number of spot-forming cells per well was determined using the SmartCount and Autogate functions of an ImmunoSpot S6 Universal Analyzer (Cellular Technology). GUCY2C-specific responses were calculated by subtracting the average number of spots in 0.1% DMSO wells from the peptide-stimulated wells.

[0159] Oncology Research GUCY2C-expressing mouse (BALB / c) CT26 colorectal cancer cells were used for in vivo tumor studies. Luciferase-expressing cells were generated by co-transduction of pLenti4-V5-GW-luciferase with lentiviral supernatant produced by 293FT cells (Invitrogen). For tumor experiments, 5 × 10 5 BALB / cJ mice were transfected with 10 10Mice were immunized with vp Ad5-GUCY2C-S1, Ad5.F35-GUCY2C-S1, or PBS (control). Tumor burden was quantified weekly by subcutaneous injection of 3.75 mg of D-luciferin potassium salt (Gold Biotechnologies) in PBS, followed by 8 minutes of incubation and imaging with a 10-second exposure using a Caliper IVIS Lumina XR imaging station (PerkinElmer). Total radiance (photons / second) was measured using Living Image in vivo imaging software (PerkinElmer).

[0160] Antibody neutralization assay Serum samples were obtained from patients prior to immunization with Ad5-GUCY2C-PADRE (NCT01972737), which was approved by the Thomas Jefferson University Institutional Review Board. Neutralizing antibody titers against Ad5 and Ad5.F35 vectors were quantified. Briefly, dilutions of heat-inactivated serum samples were diluted 10 to 10. 5 Add 10 ml of 10 ... 8 Cells were infected with GFP-expressing Ad5 or Ad5.F35 viruses (Ad5-CMV-eGFP or Ad5.F35-CMV-eGFP, respectively; Baylor Vector Development Lab). After 41 h of incubation at 37°C, eGFP fluorescence (excitation at 490 nm, emission at 510 nm) was quantified using a POLARstar Optimate plate reader (BMG Labtech). Sample fluorescence was normalized to control wells containing cells and virus (0% neutralization) or wells containing cells only (100% neutralization). Titers were determined using nonlinear regression as the serum dilution resulting in 50% neutralization (Prism v8, GraphPad Software).

[0161] Ad5 neutralization immunoassay To induce anti-Ad5 immunity, 10 10 Mice were challenged intranasally with 100 mg of Ad5-GFP once or twice at 4-week intervals. Thirty days after the final challenge, Ad5 NAb was quantified in serum as described above, and mice were challenged with 100 mg of Ad5-GFP.11 Mice were immunized intramuscularly with vp Ad5-GUCY2C-S1 or Ad5.F35-GUCY2C-S1.

[0162] Biodistribution and Toxicity Studies BALB / cJ mice were injected with 10 11 One dose of Ad5.F35-GUCY2C-S1, 10 vp 11 Mice were immunized intramuscularly with three doses of vp Ad5.F35-GUCY2C-S1 or PBS (control). Animals were monitored daily for adverse events, with additional evaluations performed on the day of dosing (5 min, 1 h, and 3 h post-dose). On days 14 and 90, designated animals were sacrificed, and the brain, salivary glands, stomach, small intestine, colon, heart, lungs, kidneys, liver, and injection site were collected and weighed for histopathological analysis by a blinded pathologist (pathological evaluation was performed by IDEXX BioAnalytics). Viral DNA was detected by quantitative PCR (qPCR) using the assay described above for the GUCY2C transgene. Spleens were also collected for histopathological analysis and detection of viral DNA as described above, and for quantification of GUCY2C-specific T cell responses by IFNγ-ELISpot as described above.

[0163] statistical analysis Statistical analysis was performed using GraphPad Prism software v8. Statistical significance was determined as follows: ns = p > 0.05; * p<0.05, ** p<0.01, *** p<0.001, and **** p<0.0001 was considered significant. Cohort size was based on previous studies with β=0.2 and α=0.05. In the case of multiple comparisons of survival outcomes, the significance threshold was corrected using the Bonferroni method. To identify vaccine-induced T cell responders and non-responders, a modified distribution-free resampling approach was used, with DMSO and >20 specific spots / 10 6A positive T cell response was defined as 2-fold compared to 100%. To determine the effect of sex and number of vaccinations on responses, the magnitude of the log-transformed vaccine response was compared for up to three-way interactions among mice of different sexes, cohorts, and treatment regimens using stepwise backward variable selection with the Akaike Information Criterion using the R package MASS.

[0164] result Ad5-GUCY2C-S1 and Ad5.F35-GUCY2C-S1 vectors While Ad5 seroprevalence worldwide exceeds 70% (over 90% in some regions), Ad35 is approximately 10% and is associated with lower titers (Figure 6, panel A). Therefore, we constructed a chimeric adenovirus (Ad5.F35) consisting of Ad5 with its fiber replaced with the Ad35 fiber and evaluated its ability to induce GUCY2C-specific immunity and resist Ad5-specific immunity in humans and mice. Ad5-GUCY2C-S1 is a replication-deficient human Ad5 expressing the mouse GUCY2C extracellular domain fused to the I-Ed-restricted CD4+ epitope known as site 1 at its C-terminus. To generate Ad5.F35-GUCY2C-S1, the Ad5 fiber (L5) was replaced with the Ad35 fiber (Figure 6, panel B). Replication-deficient Ad5-GUCY2C-S1 and Ad5.F35-GUCY2C-S1 produced in HEK293 cells resulted in dose-dependent (Figure 6, panel C) and time-dependent (Figure 6, panel D) expression of GUCY2C-S1 protein in A549 human alveolar basal epithelial cells in vitro.

[0165] Ad5.F35-GUCY2C-S1 induces GUCY2C-specific antitumor immunity Following in vitro validation of GUCY2C expression by Ad5.F35-GUCY2C-S1, its ability to induce GUCY2C-specific immune responses in vivo after vaccination was confirmed. 10 BALB / c mice immunized intramuscularly with vp Ad5.F35-GUCY2C-S1 showed 54% lower GUCY2C-specific CD8 +The Ad5 and Ad5.F35 vaccines generated T cell responses (Figure 7, panel A) and no GUCY2C-specific antibody responses (Figure 7, panel B). Importantly, the Ad5 and Ad5.F35 vaccines generated GUCY2C-specific CD8 T cells of comparable avidity, a key determinant of the antitumor efficacy of GUCY2C-targeted vaccines. + T cells (Figure 7, panel C). In contrast, GUCY2C-specific antibody responses have no detectable antitumor activity. Similarly, Ad5 and Ad5.F35 vaccines generated comparable S1-specific CD4 + This resulted in a T cell response (Figure 7 panel D).

[0166] Previous studies have shown that the Ad5-GUCY2C vaccine induces protective antitumor CD8 responses in a mouse model of metastatic colorectal cancer. + These results demonstrate that Ad5.F35 induces T cell responses. Therefore, BALB / c mice were immunized with Ad5 or Ad5.F35 expressing GUCY2C-S1 and challenged 7 days later with CT26 colorectal cancer cells expressing GUCY2C and firefly luciferase. This model specifically emulates secondary protection against metastatic disease in the clinical setting where GUCY2C vaccines are being developed. As previously demonstrated, Ad5 vaccination nearly eliminated metastatic tumor burden (Figure 8, panels A and B), delayed disease progression (Figure 8, panel C), and improved survival (Figure 8, panel D). Similarly, Ad5.F35 reduced tumor burden (Figure 8, panels A and B), disease progression (Figure 8, panel C), and prolonged survival (Figure 8, panel D). Importantly, the efficacy of Ad5- and Ad5.F35-based GUCY2C vaccines was identical in reducing tumor burden, preventing disease progression, and promoting survival (Figure 8, panels A–D).

[0167] Ad5.F35 resists Ad5-directed immunity in mice and humans NAb to Ad5 correlated with poor GUCY2C-specific immune responses in patients vaccinated with Ad5-GUCY2C-PADRE, and pre-exposure of mice to Ad5 similarly blunted vaccine-induced immunity. Resistance to pre-existing Ad5 immunity was quantified using an Ad5.F35-based vaccine in a model of respiratory pre-exposure to Ad5, a natural route of infection in patients, followed by vaccination and quantification of GUCY2C-specific T cell responses. Control mice (naive) and mice pre-exposed intranasally to Ad5 once (1x) or twice (2x) were vaccinated intramuscularly with Ad5 or Ad5.F35 expressing GUCY2C-S1 four weeks later, and immune responses were quantified two weeks later (Figure 9, panel A). As expected, one pre-exposure to Ad5 induced moderate (<1:200) Ad5 NAb and reduced GUCY2C-specific T cell responses by approximately 75%, whereas two pre-exposures induced high (>1:200) Ad5 NAb and reduced GUCY2C-specific T cell responses by more than 90% after Ad5 vaccination (Figure 9, panel B). In contrast, GUCY2C-specific T cell responses were reduced by only 60% (one pre-exposure) and 80% (two pre-exposures) after Ad5.F35 vaccination (Figure 9, panel B). Importantly, after sequential pre-exposures to Ad5, Ad5.F35 generated T cell responses in a significantly larger proportion of the population (80% cohort response) compared with Ad5 (30% cohort response) (Figure 9, panel C).

[0168] These observations in mice were replicated using sera from colorectal cancer patients in the Ad5-GUCY2C-PADRE Phase I trial (NCT01972737). NAb titers to Ad5 and Ad5.F35 were quantified using an established Ad5 / Ad5.F35 reporter virus inhibition bioassay in serum samples collected before vaccination with Ad5-GUCY2C-PADRE. In these patients, Ad5.F35-specific NAb titers were substantially lower than Ad5-specific titers (Figure 9, panel D). Most importantly, 50% of patients possessed low (<1:200) Ad5 NAb titers (Figure 9, panels D and E), which closely correlated with the 40% GUCY2C-specific response rate. In stark contrast, 90% had low Ad5.F35 NAb titers, suggesting that the majority of patients immunized with an Ad5.F35-based vaccine could generate GUCY2C-specific responses (Figure 9, panel E). Overall, these observations suggest that pre-existing viral immunity induced by repeated environmental exposure that neutralizes the Ad5 delivery platform can be overcome by the chimeric Ad5.F35 vector, enhancing a modest herd vaccine response.

[0169] Safety, biodistribution, and toxicity of Ad5.F35-GUCY2C-S1 A Food and Drug Administration IND (Investigational New Drug)-enabled study used three schemes to quantify the toxicity, biodistribution, and immunogenicity of Ad5.F35-GUCY2C-S1 in BALB / c mice and to examine acute and chronic effects (Figure 10, panel A). As shown, a gender-balanced cohort received 10 doses of Ad5.F35-GUCY2C-S1 as either a single intramuscular injection or three intramuscular injections spaced 4 weeks apart. 11Mice were administered Ad5.F35-GUCY2C-S1, monitored daily, and sacrificed for analysis on days 14 or 90 (Figure 10, panel A). There were no signs of acute or chronic toxicity during lifespan by observation, weight change, or survival (Figure 10, panels A-D). Similarly, there were no clinically significant differences in organ weight or histopathology at necropsy (not shown). Small statistical differences in organ weight were considered clinically insignificant and were unrelated to vaccine exposure (dose, time). Biodistribution quantified by qPCR detected Ad5.F35-GUCY2C-S1 at the injection site and in the spleen after acute and chronic exposure, but not in other organs. Furthermore, robust CD8 + T cell responses were quantified on day 14 and persisted for 90 days in 70% of mice after a single dose (Figure 10, panels E-G). As expected, CD8 + T cell responses were greater and sustained in more mice (100%) at day 90 after three vaccinations (Figure 10 panels E-G).

[0170] Consideration Although Ad5 has remained a popular vector throughout decades of gene therapy trials, high Ad5 seroprevalence remains an obstacle to universal vaccination. Natural respiratory infections can generate long-lived antibodies that neutralize Ad5-based vaccines, precluding transgene delivery and potential therapeutic benefit. In this context, Ad5 seroprevalence is greater than 70% in several countries, highlighting the unmet need for alternative vectors. Herein, we demonstrate that chimeric Ad5.F35 resists pre-existing Ad5 immunity and induces transgene-specific antitumor immunity. Indeed, Ad5.F35 is less susceptible to neutralization associated with Ad5 exposure in mice and humans, resulting in significantly higher rates of vaccine responders in mice pre-exposed to Ad5. These observations support the suggestion that Ad5.F35 results in a higher rate of vaccine responders in patient populations.

[0171] The extent to which NAbs against Ad5 fibers limit reinfection remains controversial. Some studies have shown that replacing the Ad5 fiber with that of a different serotype circumvents pre-existing Ad5 immunity. In contrast, other studies have suggested that these chimeric adenoviruses do not circumvent pre-existing Ad5 NAbs, suggesting that the hexon is the primary target of antibody neutralization. In contrast to previous studies that generated pre-existing Ad5 immunity via intramuscular or intravenous administration, here Ad5 immunity was induced by intranasal challenge in mice, recapitulating natural human respiratory infection. Furthermore, naturally pre-existing Ad5 NAbs in colorectal cancer patients were uniformly generated by repeated respiratory infections and similarly overcome by the Ad5.F35 vector. Importantly, the quality of the antibody response after adenovirus infection depends on the route of exposure. Indeed, respiratory infection induces fiber-specific NAbs, while intramuscular challenge induces capsid-specific NAbs. These qualitative differences in NAb responses reflect different routes of immunity and may contribute to observed discrepancies between laboratories. This study, confirmed and validated in patient samples using relevant animal models, supports the suggestion that an Ad5.F35-based vaccine should result in a clinically relevant immune response in a substantial (approximately 90%) proportion of patients.

[0172] Recognizing the widespread limitations imposed by inherent Ad5 immunity in global populations, renewed interest has emerged in alternative serotypes and chimeric constructs as tractable strategies for vaccine development. Ad26, Ad35, and Ad48 vectors have advanced into Phase I clinical trials. In that regard, comparison of Ad5, Ad26, Ad35, and Ad48 immunity among healthy patients revealed that inherent Ad35 seropositivity rates were lowest across the global population, strengthening the chimeric strategy used herein. Similarly, the first hexon-chimeric adenovirus containing Ad5 and Ad48 components was safe and immunogenic in patients. Interestingly, Ad5-Ad35 chimeric vectors transduce a variety of human cell types more efficiently in vitro than either parental vector.

[0173] Although the antitumor effects were comparable, Ad5.F35-GUCY2C-S1 significantly increased CD8 + T cell responses were lower and there was no antibody response. However, the antitumor effect of GUCY2C-directed immunotherapy is primarily driven by T cell avidity, not by the amount of effector T cells. In this context, GUCY2C-specific CD8 + The functional avidity of T cells was comparable, consistent with their comparable antitumor efficacy. The quantitative differences in transgene-specific immunity between vectors may reflect a variety of factors. Thus, the amount and persistence of the GUCY2C-S1 transgene after Ad5.F35 immunization was lower compared to Ad5, consistent with previous observations that Ad5 transduction efficiency in vivo may be several-fold higher than Ad5.F35. Furthermore, the Ad5 fiber binds to CXADR (the coxsackievirus and adenovirus receptor), whereas the Ad35 fiber binds to CD46, suggesting that the two viruses may infect distinct cell types.

[0174] While checkpoint inhibitors have resulted in a practice shift in clinics and defined immunotherapy as an effective strategy for the treatment of several malignancies, they have not been universally successful. In this context, the lack of neoepitopes in many cancer types, including microsatellite-stable colorectal and pancreatic cancers (the second and third leading causes of cancer mortality, respectively), renders them unresponsive to checkpoint blockade. Indeed, examination of neoepitopes displayed on the surface of five colorectal cancer specimens revealed a total of three neoepitopes. Therefore, vaccines targeting cancer-associated autoantigens, alone and in combination with checkpoint inhibitors, have reemerged as strategies for preventing and treating metastasis from these cold tumors.

[0175] While checkpoint inhibitors have become first-line therapy in the metastatic setting for some cancers, chimeric antigen receptor expressing T cells (CAR-T cells) have been deployed in patients with metastatic and refractory disease. In contrast, few cancer immunotherapies have been developed for early-stage cancer patients who have "no evidence of disease" (NED) after conventional surgery, radiation, and chemotherapy, and who are at very high risk for disease recurrence. Indeed, approximately 25% of stage II and 50% of stage III colorectal cancer patients relapse after surgery and chemotherapy, while 70% of resectable pancreatic cancer patients experience recurrence. Vaccines targeting tumor-associated antigens, such as Ad5.F35-GUCY2C-PADRE, may offer a safe and effective immunotherapy for secondary prevention of metastatic disease, particularly in NED patients who are ineligible to receive checkpoint inhibitors or CAR-T cells.

[0176] This study suggests that the chimeric adenovirus vector Ad5.F35 may be preferable to the widely used Ad5 vector, and further investigation is warranted. Indeed, ongoing clinical studies of GUCY2C-directed immunotherapy in patients with GUCY2C-expressing cancers, including colorectal, pancreatic, gastric, and esophageal cancers, suggest that they may benefit from the use of Ad5.F35 rather than Ad5 vectors. In this context, upcoming clinical trials will examine the safety, immunogenicity, and tolerance to pre-existing immunity of Ad5.F35-GUCY2C-PADRE in patients with GI cancers (NCT04111172). The safe generation of GUCY2C-targeted immunity in a high proportion of patients may be beneficial for the treatment of cancers that account for 25% of all cancer deaths. 51 This will lead to an efficacy trial to establish the ability of Ad5.F35-GUCY2C-PADRE to prevent recurrence after standard therapy in patients with GI cancers for whom established immunotherapies are ineffective.

[0177] Example 6 BACKGROUND: Recombinant attenuated Listeria monocytogenes (Lm) is an emerging platform for cancer immunotherapy, partly reflecting its insusceptibility to vector-specific neutralizing immunity, a limitation of many viral vectors. Furthermore, Lm is an intracellular bacterium with tropism for antigen-presenting cells (APCs) and CD4 + T helper cells (Th cells) and CD8 + The vaccine targets immune cells involved in initiating immune responses by activating cytotoxic T cells (CTLs). These advantages are consistent with the limitations of the Ad5-GUCY2C-PADRE vaccine, which was recently tested in a phase I clinical trial in colorectal cancer (CRC) patients. Ad5-GUCY2C-PADRE immunogenicity is limited in the human population by pre-existing neutralizing antibodies (NAbs) that target the Ad5 viral vector. Furthermore, GUCY2C-specific immune responses are limited by CD8 + CD4 is essential for the induction of T and B cell responses + In contrast, Lm vaccines incorporating GUCY2C (Lm-LLO-GUCY2C) should be resistant to vector-specific immunity and should not induce GUCY2C-specific CD8 T cells, reflecting its tropism for APCs. + CD4 on T and B cells + Lm-specific CD4 provides T cell help + It should induce a T cell response, leading to CRC elimination.

[0178] Objectives / Hypothesis: Lm-LLO-GUCY2C is superior to the current GUCY2C vaccine and will increase neutralizing antibodies and GUCY2C-specific CD4 + Overcoming T-cell tolerance and generating safe and effective CRC immunotherapies.

[0179] Specific Objectives: This proposal defines the (1) immunogenicity, (2) antitumor activity in metastatic CRC models, and (3) safety of Lm-LLO-GUCY2C.

[0180] Study design: Lm-LLO-GUCY2C was generated and its immunogenicity was assessed by assessing the GUCY2C-specific immune response (CD4+ This is tested in mice by quantifying Lm-specific CD4 T cells and CD8+ T cells and B cells. + T cell responses will be quantified and their role in inducing GUCY2C-specific responses will be determined by CD4 depletion studies. The antitumor efficacy of Lm-LLO-GUCY2C will be tested in a mouse model of metastatic CRC and compared with a benchmark GUCY2C vaccine (Ad5-GUCY2C-S1). Finally, Lm-LLO-GUCY2C toxicity will be examined specifically in normal GUCY2C-expressing tissues. Positive and negative controls for the proposed study include Ad5-GUCY2C-S1 and Lm-LLO (lacking GUCY2C), respectively.

[0181] Innovation: As GUCY2C vaccine development progressed from inception to clinical trials, our understanding of GUCY2C immunobiology and vaccine platform has advanced sufficiently to allow for more informed GUCY2C vaccine design. In that context, we have combined the properties of Lm (resistance to vector immunity; APC tropism) and GUCY2C (gut-specific; ubiquitous expression in CRC; selective CD4 + "These findings, combined with the potential for T-cell tolerance, may be mutually beneficial, resulting in effective immunotherapy for CRC. With the establishment of Lm vaccine safety and efficacy in patients and GUCY2C immunogenicity, our findings could be readily translated into phase I clinical trials, potentially extending this hypothesis from animals to humans and ultimately leading to effective immunotherapy in civilian and military CRC populations."

[0182] Immunogenicity, efficacy, and safety of a Listeria-based cancer vaccine (Lm-LLO-GUCY2C) targeting GUCY2C as a potential CRC immunotherapy. CRC is the fourth most common neoplasm, with approximately 150,000 new cases per year, and the second leading cause of cancer mortality in civilians and military personnel, with a mortality rate of approximately 50%.

[0183] Example 7 A vaccine for treating GUCY2C-expressing cancers. The vaccine uses attenuated Listeria monocytogenes to deliver the colorectal cancer antigen GUCY2C to "antigen-presenting cells" (APCs) and induces GUCY2C-specific T cell responses against GUCY2C that can find and eliminate GUCY2C-expressing cancers. A novel recombinant Listeria monocytogenes vaccine will be initially tested in a mouse model of colorectal cancer to determine its activity, efficacy, and safety.

[0184] Key Project 1: Development of Lm-LLO-GCC and control Lm vaccines Key Question 2: Determine the ability of Lm-LLO-GCC to overcome tolerance in WT mice. Quantify Lm-LLO-GCC-induced immune responses in WT and KO mice. Key issue 3: GCC-specific responses are CD4 + Determining T cell dependency Quantification of the antitumor activity of LmLLO-GCC in metastatic CRC models Comparison of antitumor effects between LmLLO-GCC and Ad5-GCC-S1 Key Question 5: Comparison of the impact of vector-specific immunization on immune responses to Lm-LLO-GCC and Ad5-GCC-S1. Quantification of immune responses in Lm-LLO-GCC or Ad5-GCC-S1 immunized mice with pre-existing vector-specific immunity. Key Question 6: Comparison of the influence of vector-specific immunity on the antitumor efficacy of Lm-LLO-GCC and Ad5-GCC-S1. Quantification of the antitumor efficacy of Lm-LLO-GCC or Ad5-GCC-S1 in mice with pre-existing vector-specific immunity. Demonstration of the safety of Lm-LLO-GCC Key Question 7: Examination of Lm-LLO-GCC immunized mice for signs of toxicity. Establishment of Lm-LLO-GCC symptoms in GCC-expressing and non-expressing tissues.

[0185] First-generation recombinant Lm-LLO-GCC vaccine Using the integrative plasmid pPL2, we induced integration of recombinant GCC or GCC-S1 into the genome of attenuated (ΔactA / ΔinLB) Listeria monocytogenes (Lm) strains containing a truncated listeriolysin O (LLO) expression cassette and downstream of the LLO promoter, generating LLO-GCC fusion proteins secreted into the cytosol of Lm-infected cells (Figure 14). These constructs contain the complete extracellular domain of GCC (residues 23–429), which has been used in previous viral vector-based vaccines.

[0186] The quality of the first-generation Lm-GCC and control vaccines was controlled by confirming GCC expression in the supernatants of Lm cultures (Fig. 15). The LLO-GCC or LLO-GCC-S1 fusion proteins were detected in the supernatants using anti-GCC or anti-LLO antibodies.

[0187] The immunogenicity of the first-generation Lm-GCC vaccine was tested in BALB / c mice (Figure 16). A well-established adenovirus-based vaccine (Ad5-GCC-S1) was used as a positive control. Following immunization, CD8 + T cell responses were quantified by IFNγ-ELISpot. As expected, the control Lm-LLO did not induce GCC-specific responses. However, Lm-LLO-GCC and Lm-LLO-GCC-S1 also failed to induce responses. Responses were induced by the positive control Ad5-GCC-S1 vaccine, which generated responses against the antigens LLO and DBP in the Lm and Ad5 vectors, respectively. Collectively, these data suggest that the Lm-LLOGCC vaccine is a poor inducer of GCC-specific responses.

[0188] Second-generation recombinant Lm-LLO-GCC vaccine In the context of the low expression of the LLO-GCC fusion protein (Figure 15), we hypothesized that the large size of the LLO-GCC fusion protein may hinder its expression and immune induction. Indeed, similar studies using large tumor antigens fused to LLO show similar difficulties in generating the fusion protein. Therefore, three different second-generation vaccines were created, each containing approximately one-third of GCC (Figure 17). These designs included different GCC CD4 + T cell epitopes and CD8 + T cell epitopes are included, allowing responses to each epitope to be tested in two different fragments.

[0189] Expression of the LLO-GCC fusion protein was confirmed in the second-generation Lm-LLO-GCC vaccines by Western blot. Because each second-generation vaccine contains a different GCC fragment, multiple GCC-specific monoclonal antibodies were used to detect the GCC-LLO fusion protein in Lm supernatants (Figure 18). These data confirm LLO-GCC fusion protein expression by each recombinant Lm vaccine. As expected, the shorter fusion proteins were expressed at higher levels than the full-length LLO-GCC product produced in the first-generation vaccines.

[0190] The second-generation Lm-LLO-GCC vaccine containing the GCC fragment was administered to - / - (Figure 19 Panel A) or GCC + / + (Figure 19 Panel B) Mice were administered GCC - / - Mice are CD4 + Generates only T cell responses and GCC + / + is CD8 + To generate only T cell responses, mice were administered only the vaccine fragments containing these epitopes. - / - Mice were immunized with CD4 containing fragments 1 and 2. + Robust CD4 against GCC when immunized with T cell epitopes + induced a T cell response (Figure 19 Panel A). + / + Mice are CD8 +GCC-specific CD8 + failed to generate a T cell response (Figure 19 Panel B). + / + CD8 against GCC in mice + To confirm the lack of T cell responses, we performed antitumor immunity experiments (Figure 20, panels A-C). Consistent with the IFNγ-ELISpot data (Figure 19, panels A and B), GCC + / + Immunization of mice with the Lm-LLO-GCC fragment did not show antitumor effects compared to Ad5-GCC-S1, which was 100% effective (FIG. 20 panels A, B, and C).

[0191] Second-generation recombinant Lm-LLO-GCC vaccine In light of the poor immunogenicity of the Lm-LLO-GCC vaccine in mice vaccinated with vaccines containing full-length GCC or fragments of GCC (Figures 14-20), we hypothesized that GCC may not be properly processed and presented in the context of the LLO fusion protein. Therefore, we generated recombinant Lm vaccines containing GCC fused to ActA, another Lm protein highly expressed in the cytosol of Lm-infected cells (Figure 21, panel A). These vaccines induced GCC fusion protein production in Lm-infected macrophages, and inclusion of the Syn18 enhancer sequence significantly enhanced expression (Figure 21, panel B). However, the LmActA-GCC vaccine did not induce GCC fusion protein production. + / + GCC-specific CD8 in mice + failed to induce a T cell response (Figure 21, panels C-D).

[0192] Next, GCC CD8 + By generating a novel construct containing five tandem repeats of the T cell epitope but lacking other domains of GCC, we were able to generate GCC-specific CD8 + We attempted to enhance T cell responses by fusing these epitopes to ActA and Syn18 (Figure 22). +We generated what was predicted to be a highly immunogenic construct capable of inducing a T cell response, but immunization of wild-type BALB / c mice with this vaccine failed to produce any response (Figure 22).

[0193] Finally, there may be a fundamental flaw in our construct design, which is that it is not possible to induce CD8 + We hypothesized that these antibodies would be unable to generate a CD8 T cell response. To test this hypothesis, we used antibodies against Escherichia coli β-galactosidase (LacZ), mouse Her2, and Ad5 DNA-binding protein (DBP). + Similar to T cell epitopes, the predominant CD8 + We generated a novel construct containing ActA-Syn18 fused to a T cell epitope (Figure 23). The vaccine generated robust responses against LacZ and Ad5, but failed to generate meaningful responses against GCC and Her2 (Figure 23). We now report that the GCC CD8 + Novel Lm constructs designed to enhance antigen processing and presentation of T cell epitopes are being generated.

[0194] We demonstrate increased GCC epitope presentation and GCC-specific CD8 + We have designed and are currently producing several novel Lm constructs that we expect will generate T cell responses. Over the remaining project period, we will complete the production of these vaccines and test their immunogenicity. Collectively, these studies and the completed work will establish the potential utility of Listeria-based vaccines targeting GCC for colorectal cancer immunotherapy.

[0195] Example 8 Colorectal cancer (CRC) is the second leading cause of cancer deaths in the United States and the fourth most commonly diagnosed cancer. Furthermore, the 5-year survival rate for patients with metastatic CRC is currently 14% and has remained relatively stagnant for decades, highlighting the lack of effective therapies for preventing and treating metastatic CRC. In this context, the intestinal receptor and tumor-associated antigen guanylyl cyclase C (GUCY2C) has emerged as an immunotherapy option under development. A recent phase I trial in CRC patients demonstrated the immunogenicity and safety of an adenovirus-based vaccine against GUCY2C (Ad-GUCY2C). However, optimal immunity to vaccine antigens is routinely achieved using a combination approach with heterologous vaccine vectors, which helps circumvent limitations associated with vector-specific immunity and is therefore often superior to homologous vaccination using the same vector. Herein, we constructed a recombinant strain of Listeria monocytogenes secreting GUCY2C (Lm-GUCY2C) and examined its immunogenicity in combination with Ad-GUCY2C to define an optimal GUCY2C vaccination regimen. We demonstrate that optimal GUCY2C and anti-tumor immunity are achieved using a heterologous prime-boost regimen that uses Ad-GUCY2C to "prime" the GUCY2C immune response and Lm-GUCY2C to "boost" the memory GUCY2C immune response. We demonstrate that this combination stimulates GUCY2C-specific CD8 + Quantitative T cell count and GUCY2C-specific CD8 + We report that this immunization leads to a significant enhancement of the avidity and polyfunctionality of the T cell pool. Furthermore, we demonstrate that this immunization is safe, and histopathological evaluation of vaccinated mice demonstrates no signs of toxicity. Collectively, these findings suggest that Lm-GUCY2C can be utilized to enhance GUCY2C immunity in patients with pre-existing GUCY2C immune responses and may inform future GUCY2C-targeted vaccine clinical trials.

[0196] Materials and Methods vaccine Influenza HA, also known as site 1 (S1)107-119 CD4 + Mouse GUCY2C fused to a T cell epitope 1-429 The chimeric replication-deficient adenovirus expressing Ad5.F35-GUCY2C-S1 was previously described (Ad5.F35-GUCY2C-S1). The Ad5.F35-GUCY2C-S1 and Ad5.F35-GFP vaccines used in this study were produced by the Cell and Gene Therapy Vector Development Laboratory at Baylor College of Medicine and were certified negative for replication-competent adenovirus, mycoplasma, and host cell DNA contamination.

[0197] An attenuated Lm strain, LmΔactA / ΔinlB, containing deletions of the virulence factors internalin B and actA, was ordered from ATCC and serves as the parent strain for all Lm vaccines utilized in this study. Recombinant Lm-GUCY2C and Lm-LacZ each contain the mouse GUCY2C extracellular domain in frame with the first 100 amino acids of the ActA protein under the control of the actA promoter. 23-429 or β-galactosidase 618-1024 The resulting sequence was cloned into the pPL2 integration vector and integrated into the Lm chromosome. 600 The viruses were grown in Brain Heart Infusion Broth (Fisher Scientific) until the Mn was approximately 1, aliquoted, and stored at −80°C. On the day of the experiment, aliquots were thawed, incubated at 37°C for 60 min, washed twice with PBS, and diluted to the desired concentration in PBS for vaccination.

[0198] In vitro infection The murine macrophage cell line J774A.1 was cultured in DMEM supplemented with 10% fetal bovine serum. J774A.1 cells were infected with Lm-GUCY2C or control Lm at a multiplicity of infection of 10:1. After 1 h of incubation, cells were washed twice with PBS, resuspended in medium containing 10 μg / mL gentamicin to eliminate free extracellular bacteria, and incubated for an additional 5 h. For immunofluorescence studies, Lm was labeled by incubation with 2 mM CellTracker Red CMPTPX dye at 37°C for 10 min before infection. For Western blot studies, proteins were extracted from cells using M-PER Reagent (Pierce) supplemented with protease inhibitors. GUCY2C protein was stained with anti-GUCY2C monoclonal antibody MS20, and p60 was stained with anti-p60 monoclonal antibody p6017 (AdipoGen).

[0199] Mice and immunization Eight-week-old male and female BALB / cJ mice were purchased from The Jackson Laboratory for this experiment. The animal protocol was approved by the Thomas Jefferson University Institutional Animal Care and Use Committee (Protocol 01956). For adenoviral immunization, mice were administered 10 mg of adenovirus as two 50 μL injections, one into each hind leg. 10 vp of Ad5.F35-GUCY2C-S1 or Ad5.F35-GFP as a control was administered intramuscularly (im). For Lm immunization, 5 × 10 6 Colony-forming units (CFU) of Lm-GUCY2C, empty Lm, or Lm-LacZ were administered intravenously (iv) as a 100 μL suspension in PBS. In ELISpot experiments, empty Lm was used as a control, and in tumor experiments, Lm-LacZ was used as a control due to the ability of the ActA protein to function as an adjuvant and enhance antitumor responses. For prime-boost immunizations, vaccines were delivered 21 days apart.

[0200] Ad5 neutralization immune research 10 10Ad5 immunity was induced in BALB / cJ mice by intranasal exposure to Ad5-GFP. At day 28 post-infection, mice were bled, serum was collected, and 10 11 vp Ad5.F35-GUCY2C-S1, followed by 5 × 10 6 Mice were immunized with CFU of Lm-GUCY2C or control Lm. Ad5 neutralizing antibody titers in the mice were quantified as previously described.

[0201] IFNγ-ELISpot assay ELISpot assays were performed using a mouse interferon-γ (IFN-γ) single-color ELISpot kit (Cellular Technology Limited) according to the manufacturer's protocol. Briefly, 96-well plates were coated with IFN-γ capture antibody at 4°C. After overnight incubation, the plates were washed with PBS, and splenocytes from immunized mice were incubated with 10 μg / mL of GUCY2C. 254-262 The cells were seeded in 0.1% DMSO in CTL-TEST medium (Cellular Technology Limited) with or without peptide and incubated for 24 hours at 37°C. For TCR avidity studies, splenocytes were incubated with various concentrations of GUCY2C. 254-262 The cells were seeded with peptide (10 μg / mL to 3 pg / mL). The next day, cells were removed and a colorimetric reagent was added to detect IFN-γ-producing, spot-forming cells. The number of spot-forming cells per well was calculated using the SmartCount and Autogate functions of an ImmunoSpot S6 Universal Analyzer (Cellular Technology Limited). GUCY2C-specific responses were calculated by subtracting the average number of spots in 0.1% DMSO wells from that in peptide-pulsed wells.

[0202] Intracellular cytokine staining 10 of immunized mice 6 Splenocytes were incubated with DMSO or 10 μg / mL GUCY2C 254-262Peptides were seeded into 96-well plates in the presence of peptide and anti-CD107-FITC (clone 1D4B). Cells were incubated at 37°C for 1 hour, and then a protein transport inhibitor cocktail (eBioscience) was added. Splenocytes were incubated for an additional 5 hours at 37°C. Cells were stained using the LIVE / DEAD Fixable Aqua Dead Cell Staining Kit (Invitrogen), anti-CD8 PerCP-Cy5.5 (53-6.7; BD Biosciences), and anti-CD19 BV510 (6D5; Biolegend). After permeabilization using the BD Cytofix / Cytoperm Kit (BD Biosciences), intracellular cytokines were stained using anti-IFN-γ (XMG1.2; BD Biosciences) and anti-MIP1α-APC (39624; R&D Systems). Cells were fixed with 4% paraformaldehyde and analyzed using a BD LSR II flow cytometer. Analysis was performed using FlowJo software (TreeStar).

[0203] Oncology Research GUCY2C and luciferase-expressing mouse (BALB / c) CT26 colorectal cancer cell lines were generated as described previously and used for in vivo tumor studies. Six days after the final immunization, mice were injected with 5 × 10 cells into the tail vein. 5 CT26 cells were administered intravenously. Tumor burden was quantified weekly by subcutaneous injection of 3.75 mg of D-luciferin potassium salt (Gold Biotechnologies) in PBS, followed by 8 min of incubation and imaging after a 10 s exposure using a Caliper IVIS Lumina XR imaging station (PerkinElmer). Total radiance (photons / sec) was measured using Living Image In Vivo imaging software (PerkinElmer).

[0204] result Lm-GUCY2C vaccine design Mouse GUCY2C 23-429The extracellular domain of ActA was codon-optimized for L. monocytogenes using the Java Codon Adaptation Tool and synthesized downstream of the actA promoter, ActAN100, and the enhancer sequence shown in Figure 24, panel A. The resulting sequence was cloned into the pPL2 integration vector and stably integrated into the genome of the live-attenuated double-deficient strain Lm ΔactAΔinlB. To confirm secretion of the ActA-GUCY2C fusion protein, the J774A.1 macrophage cell line was infected with Lm-GUCY2C or a control Lm strain. After 6 hours of incubation, macrophages were stained for GUCY2C protein expression by Western blot (Figure 24, panel B) and immunofluorescence (Figure 24, panel C).

[0205] The heterologous Ad5.F35-GUCY2C-S1+Lm-GUCY2C immunization regimen stimulated GUCY2C-specific CD8 + Enhances T cell responses and anti-tumor immunity After in vitro confirmation of GUCY2C fusion protein expression by Lm-GUCY2C, we next sought to identify the optimal GUCY2C immunization regimen. Therefore, the Lm-GUCY2C vaccine was tested in combination with a chimeric adenovirus-based vaccine against GUCY2C (Ad5.F35-GUCY2C-S1) in a current Phase II trial (NCT04111172). GUCY2C immunogenicity and antitumor immunity were evaluated using a prime-boost regimen in which Lm-GUCY2C and Ad5.F35-GUCY2C-S1 vaccines were administered 21 days apart as homologous or heterologous vaccinations. Notably, the heterologous regimen using Ad5.F35-GUCY2C-S1 to "prime" the GUCY2C immune response, followed by Lm-GUCY2C to "boost," resulted in significantly higher GUCY2C-specific CD8+ cells as determined by IFNγ-ELISpot compared with all other regimens. +T cell responses were generated (Figure 25, panel A). Similarly, in the context of colorectal tumor burden, Ad5.F35-GUCY2C-S1 + Lm-GUCY2C immunization significantly reduced metastatic tumor burden (Figure 25, panels B and C) and increased median survival (Figure 25, panel D) compared with other vaccination strategies. Importantly, the order of immunization was essential for optimal GUCY2C immunization, with Ad5.F35-GUCY2C-S1 + Lm-GUCY2C generating significantly more GUCY2C-specific CD8 T cells compared with Lm-GUCY2C + Ad5.F35-GUCY2C-S1 vaccination. + It induced a >15-fold increase in T cells (p<0.0001) and significantly improved median survival (71 d vs. 38 d, p<0.05). Furthermore, we found that Lm-GUCY2C enhanced GUCY2C immune responses 100 days after Ad5.F35-GUCY2C-S1 priming, suggesting that Lm-GUCY2C may enhance GUCY2C memory responses long after the first priming vaccination.

[0206] Effect of vector-specific immunity on Ad5.F35-GUCY2C-S1+Lm-GUCY2C immunity A known limitation of virus-based vaccines is the ability of vector-specific immunity to interfere with the immunogenicity of target vaccine antigens. Specifically, neutralizing antibodies (NAb) against the common adenovirus serotype 5 (Ad5) are detectable in over 70% of healthy donors and have been demonstrated to blunt the efficacy of adenovirus-based vaccines. Chimeric adenoviruses such as Ad5.F35 are less susceptible to the neutralization associated with Ad5 NAb, but the effect is modest, such that the GUCY2C-specific immune response of Ad5.F35-GUCY2C-S1 is partially diminished in the presence of prior Ad5 exposure. In contrast, Lm infection in humans does not result in NAb against Lm, and prior exposure does not limit the immunogenicity of target vaccine antigens. Consistent with these observations, we found that prior Lm exposure did not limit the GUCY2C immunogenicity of Lm-GUCY2C. Therefore, we focused on the impact that prior Ad5 exposure may have on limiting the Ad5.F35-GUCY2C-S1+Lm-GUCY2C vaccination approach. First, we wanted to define the ability of Lm-GUCY2C to boost with a low priming dose of Ad5.F35-GUCY2C-S1 vaccine, which may be associated with antibody neutralization. Therefore, we immunized mice with reduced doses of Ad5.F35-GUCY2C-S1 and compared the efficacy of Lm-GUCY2C with a similar 10 11 vp to 10 9 GUCY2C-specific CD8 at doses ranging from vp + We found that the Ad5.F35-GUCY2C-S1+Lm-GUCY2C vaccination regimen enhanced T cell proliferation and proliferation of Ad5-specific T cells (Figure 26 Panel A). Next, we wanted to mimic pre-existing Ad5 immunity in vivo. 28 days before the start of the Ad5.F35-GUCY2C-S1+Lm-GUCY2C vaccination regimen, mice were vaccinated with 10 10 Mice were intranasally challenged with VP of Ad5-GFP or PBS. As expected, Ad5-GFP challenge induced NAbs in the mice upon vaccination (Figure 26, panel B). However, Lm-GUCY2C did not induce GUCY2C-specific CD8+ antibodies in naive mice or mice pre-challenged with Ad5-GFP. +Lm-GUCY2C enhanced T cell responses (Figure 26, panel C). Similarly, Lm-GUCY2C vaccination of naive and Ad5-GFP-challenged mice similarly enhanced antitumor immunity as quantified by reduced metastatic tumor burden (Figure 26, panel D) and prolonged survival (Figure 26, panel E). Thus, Lm-GUCY2C may be effective in enhancing GUCY2C-specific immunity in situations associated with inadequate GUCY2C prime vaccination.

[0207] GUCY2C-specific CD8 after prime-boost vaccination + Qualitative changes in the T cell pool In addition to increasing the quantity of vaccine-specific T cells, previous studies have demonstrated that prime-boost immunization affects the quality of vaccine-specific T cells. Therefore, we investigated the effect of GUCY2C-specific CD8+ cells in peak effector responses after Ad5.F35-GUCY2C-S1 prime and Ad5.F35-GUCY2C-S1+Lm-GUCY2C prime-boost vaccinations. + By comparing the avidity and polyfunctionality of T cells, GUCY2C-specific CD8 + We wanted to determine the effect on the T cell pool. + To assess the avidity of the T cell pool, we used decreasing concentrations of GUCY2C 254-262 Splenocytes from mice immunized with the peptide were pulsed and IFN-γ responses were quantified. Compared to mice immunized with prime alone, the EC50 for mice immunized with prime-boost was shifted by approximately 2.5-fold (0.0046 μg / mL vs. 0.0018 μg / mL, p<0.0001), suggesting enrichment of high avidity GUCY2C-specific T cells in mice after prime-boost immunization (Figure 27, panel A). Next, flow cytometry was used to quantify GUCY2C-specific CD8 T cells after prime and prime-boost vaccination. +T cell polyfunctionality was assessed. Consistent with the ELISpot experiments, prime-boost significantly enhanced IFN-γ responses, as well as the effector cytokines MIP1α and surface markers of degranulation CD107a, compared with prime immunization alone (Figure 27, panel B). Furthermore, double-positive (Figure 27, panels C and E) and triple-positive (Figure 27, panels D and E) CD8 + The proportion of GUCY2C-specific CD8 T cells significantly increased after prime-boost vaccination, and prime-boost immunization resulted in multiple effector functions. + Therefore, it was suggested that GUCY2C-specific CD8 + In addition to significantly amplifying T cell numbers, the enhanced antitumor immunity after Lm-GUCY2C booster immunization was due to the GUCY2C-specific CD8 + It is likely to be conferred through qualitative changes in the avidity and polyfunctionality of the T cell pool.

[0208] Heterologous prime-boost does not induce toxicity We previously demonstrated the ability of GUCY2C vaccine to induce systemic antitumor immunity without eliciting autoimmunity against endogenous GUCY2C-expressing tissues. GUCY2C-specific CD8 + Given the changes in the quantity, avidity, and polyfunctionality of the T cell pool, we wished to characterize the safety of this immunization regimen. To this end, we used two vaccination and evaluation schedules to elucidate potential toxicity of Ad5.F35-GUCY2C-S1+Lm-GUCY2C immunization. As shown in Figure 28, panel A, mice were vaccinated with 10 10 vpA d5.F35-GUCY2C-S1, followed by 5 × 10 6Two booster immunizations of CFU of Lm-GUCY2C were administered. Seven and 30 days after the final immunization, mice were sacrificed, and organs were harvested for acute and / or chronic toxicity assessments, respectively. Additional cohorts were administered PBS on all immunization days. No signs of toxicity were observed in the acute or chronic cohorts during life observation or survival (Figure 28, panel B). Notably, male mice, but not female mice, in the acute and chronic cohorts lost weight throughout the study compared with the control cohort (Figure 28, panels C and D). Similarly, statistically significant decreases in brain and small intestinal organ weights were observed only in male mice in the acute and / or chronic cohorts, likely due to differences in general body size. Interestingly, female mice in the acute and chronic cohorts exhibited decreased stomach weights. Furthermore, mice in the acute cohort recently immunized with Lm-GUCY2C exhibited splenomegaly, consistent with previous studies and likely due to Lm tropism for the spleen. Overall, no differences in organ weights were maintained across sex groups, with the exception of splenomegaly. Despite slight differences in organ size, histopathological scoring by blinded pathologists revealed no differences in inflammation between known GUCY2C-expressing tissues (small intestine, colon, brain) and GUCY2C-deficient tissues (salivary gland, stomach, heart, lung, kidney, and liver). Increased splenic inflammation was observed in the acute cohort, consistent with the findings of splenomegaly. Collectively, these data suggest, consistent with previous studies, that Ad5.F35-GUCY2C-S1+Lm-GUCY2C immunization enhances GUCY2C immunity without generating autoimmunity against endogenous GUCY2C-expressing tissues.

[0209] Consideration The main limitations in CRC management, particularly for patients with microsatellite-stable, mismatch repair-competent tumors, remain the potential for disease recurrence after surgical resection and the ineffectiveness of current therapies for treating metastatic disease. In these situations, vaccination may be an ideal treatment by eliminating CRC cells that escape conventional treatment and conferring long-term immunity, thereby protecting against future recurrence. Herein, we define an optimal GUCY2C immunization regimen utilizing a heterologous combination of vaccine vectors that is immunogenic and induces potent antitumor immunity without causing autoimmunity.

[0210] Consistent with previous studies, we found that prime-boost vaccination not only increased the amount of vaccine-specific T cells but also significantly affected the quality of the vaccine-specific T cell pool. We found that GUCY2C-specific CD8 + We report that the T cell pool has higher avidity and exhibits increased effector function. In particular, multiple studies have demonstrated that higher avidity TCR and polyfunctional T cells can be more effective in eliminating cancer cells and potentially eliminating viral infections. Therefore, the additional antitumor immunity conferred by Lm-GUCY2C boosting may be mediated through a combination of all three factors.

[0211] Adenovirus-based cancer vaccines have been hampered in part by the high proportion of individuals with Ad5-specific NAb, which prevents reinfection and therefore limits vaccine efficacy upon immunization. While the use of rare serotypes and chimeric adenovirus vectors, such as Ad5.F35, are less susceptible to the neutralization associated with preexisting Ad5 immunity, the induction of vector-specific NAb upon vaccination limits the usefulness of booster immunization. Therefore, heterologous immunization utilizing two different vectors may be preferable to homologous immunization in this setting. Furthermore, in contrast to viral vectors, bacterial vectors, such as Lm, do not induce NAb upon vaccination. Therefore, as GUCY2C immunity declines over time, repeated vaccinations with Lm-GUCY2C are tolerated and may be necessary to consistently raise GUCY2C immunity to therapeutic levels in patients.

[0212] Lm-GUCY2C expresses GUCY2C-specific memory CD8 + Although Lm-GUCY2C induced a strong proliferation of T cells, Lm-GUCY2C alone was ineffective as monotherapy. Indeed, vaccination with the homologous Lm-GUCY2C induced GUCY2C-specific CD8 + T cells were apparently absent, providing no protection against CRC challenge. Interestingly, studies examining Lm vaccines as monotherapy have reported mixed success. While Lm vaccines against the tumor antigens HER2 and PSA generate potent antitumor immunity as monotherapy, Lm vaccines against other tumor antigens, including PAP and mesothelin, have similarly demonstrated only limited immunogenicity and were utilized as heterologous prime-boost immunizations. Furthermore, enhanced antitumor immunity has been reported when Lm vaccines are combined with immunomodulators, such as anti-GITR and anti-PD-1 antibodies. Therefore, understanding why Lm-GUCY2C alone does not stimulate detectable GUCY2C immune responses and further investigating its combination with immune modulators may yield important insights into Lm-GUCY2C biology and lead to further enhancement of GUCY2C immunity.

[0213] In the context of an ongoing clinical trial (NCT04111172) testing the Ad5.F35-GUCY2C-PADRE vaccine in gastrointestinal cancer, these studies suggest that patients enrolled in this trial may benefit from booster vaccination with Lm-GUCY2C. Our study demonstrated that Ad5.F35-GUCY2C-S1+Lm-GUCY2C induced superior GUCY2C antitumor immunity and CD8 upregulation compared to homologous immunization with either vector. + Furthermore, our study suggests that Lm-GUCY2C boosting can be effective long after the initial priming with Ad5.F35-GUCY2C-S1, suggesting that patients may benefit from Lm-GUCY2C boosting despite a long interval since the initial priming immunization.

Claims

1. a) Adenovirus Ad5.F35 vector: b) i) an operably linked heterologous promoter; ii) a nucleic acid encoding a soluble human GUCY2C domain fused in frame; iii) a nucleic acid encoding a universal CD4+ helper epitope A gene expression cassette comprising: A recombinant Ad5.F35 adenovirus comprising:

2. 2. The recombinant Ad5.F35 adenovirus of claim 1, wherein the gene expression cassette comprises a nucleic acid encoding a soluble human GUCY2C domain set forth in SEQ ID NO:

5.

3. The gene expression cassette is a universal CD4 ＋ 3. The recombinant Ad5.F35 adenovirus of claim 2, comprising a nucleic acid encoding a soluble human GUCY2C domain according to SEQ ID NO: 5 fused in frame to a nucleic acid encoding a helper epitope.

4. 4. The recombinant Ad5.F35 adenovirus of claim 3, wherein the heterologous promoter is CMV IE.

5. 4. The recombinant Ad5.F35 adenovirus of claim 3, wherein the fusion sequence encodes PADRE fused to the C-terminus of a soluble human GUCY2C domain and is the sequence set forth in SEQ ID NO:

9.

6. 6. An injectable pharmaceutical composition comprising the adenovirus of claim 5 and a pharmaceutically acceptable carrier or diluent.

7. An injectable pharmaceutical for use in treating cancer, comprising the injectable pharmaceutical composition of claim 6, wherein cells of the cancer express GUCY2C.

8. An injectable pharmaceutical composition comprising recombinant Listeria monocytogenes for use in combination with the injectable pharmaceutical composition of claim 6 to treat cancer, wherein the recombinant Listeria monocytogenes comprises a gene expression cassette comprising a heterologous promoter operably linked to a nucleic acid encoding a soluble human GUCY2C domain fused in-frame to a nucleic acid encoding a universal CD4+ helper epitope, and wherein cells of the cancer express GUCY2C.

9. The injectable pharmaceutical composition described in Claim 8, wherein the gene expression cassette of the recombinant Listeria monocytogenes comprises a nucleic acid encoding a soluble human GUCY2C domain described in SEQ ID NO:

5.

10. The injectable pharmaceutical composition of claim 8, wherein the gene expression cassette of the recombinant Listeria monocytogenes comprises a nucleic acid encoding a soluble human GUCY2C domain described in sequence 5 fused in frame to a nucleic acid encoding a universal CD4+ helper epitope described in sequence number 7 to form a fusion sequence.

11. 11. The injectable pharmaceutical composition of claim 10, wherein the fusion sequence encodes PADRE fused to the C-terminus of a soluble human GUCY2C domain and is the sequence set forth in SEQ ID NO: 9.

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