Epitope-targeted cytotoxic T cells mediate lineage-specific antitumor efficacy induced by the cancer mucosa antigen GUCY2C

Abstract

Guanylyl cyclase C (GUCY2C) is the index cancer mucosa antigen, an emerging class of immunotherapeutic targets for the prevention of recurrent metastases originating in visceral epithelia. GUCY2C is an autoantigen principally expressed by intestinal epithelium, and universally by primary and metastatic colorectal tumors. Immunization with adenovirus expressing the structurally unique GUCY2C extracellular domain (GUCY2C(ECD); Ad5-GUCY2C) produces prophylactic and therapeutic protection against GUCY2C-expressing colon cancer metastases in mice, without collateral autoimmunity. GUCY2C antitumor efficacy is mediated by a unique immunological mechanism involving lineage-specific induction of antigen-targeted CD8(+) T cells, without CD4(+) T cells or B cells. Here, the unusual lineage specificity of this response was explored by integrating high-throughput peptide screening and bioinformatics, revealing the role for GUCY2C-directed CD8(+) T cells targeting specific epitopes in antitumor efficacy. In BALB/c mice vaccinated with Ad5-GUCY2C, CD8(+) T cells recognize the dominant GUCY2C(254-262) epitope in the context of H-2K(d), driving critical effector functions including interferon gamma secretion, cytolysis ex vivo and in vivo, and antitumor efficacy. The ability of GUCY2C to induce lineage-specific responses targeted to cytotoxic CD8(+) T cells recognizing a single epitope mediating antitumor efficacy without autoimmunity highlights the immediate translational potential of cancer mucosa antigen-based vaccines for preventing metastases of mucosa-derived cancers.

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

“…Immune responses to the dominant GUCY2C-specific CD8 + T cell epitope GUCY2C 254–262 (26) were measured in splenocytes 14 days after the final immunization.…”

“…Intravenous administration of CT26 cells is a well-established model of metastatic colorectal cancer, preferentially forming metastases in the lungs (13–15, 26, 28, 29). …”

“…Further, its expression is maintained throughout colorectal tumorigenesis and GUCY2C is found in >95% of colorectal cancer metastases (24, 25). Previously, we demonstrated that recombinant replication-deficient adenoviral vaccines (Ad5) expressing the extracellular domain of GUCY2C induce protective GUCY2C-specific CD8 + T-cell responses, without toxicity, in a mouse model of metastatic colorectal cancer (13–15, 26). …”

Preclinical Evaluation of a Replication-Deficient Recombinant Adenovirus Serotype 5 Vaccine Expressing Guanylate Cyclase C and the PADRE T-helper Epitope

“…Virus was added to the cultures at the indicated doses, and culture supernatants were collected at the indicated time points. Relative GUCY2C levels were quantified in supernatants by Western blot using MS20 mouse anti-GUCY2C monoclonal antibody 3,10,16 and HRP-conjugated goat anti-mouse secondary antibody ( Jackson Immuno Research Laboratories).…”

“…Mice were immunized as above with 10 11 vp Ad5-GUCY2C-PADRE or Ad5-Control-PADRE, 12 and splenocytes were frozen (CTLCryoABC; Cellular Technology Ltd.), thawed (CTL-Anti-Aggregate; Cellular Technology Ltd.), and stimulated with GUCY2C 217-225 , GUCY2C 377-386 , and GUCY2C [254][255][256][257][258][259][260][261][262] in an IFN-c ELISpot plate, as previously described. [11][12][13]16 To quantify antitumor immunity, immunized mice were challenged with CT26 cells expressing mouse GUCY2C and K b , lungs were collected and stained with India ink 16-17 days later, and tumors were enumerated (tumor burden).…”