Human GUCY2C-Targeted Chimeric Antigen Receptor (CAR)-Expressing T Cells Eliminate Colorectal Cancer Metastases.

One major hurdle to the success of adoptive T-cell therapy is the identification of antigens that permit effective targeting of tumors in the absence of toxicities to essential organs. Previous work has demonstrated that T cells engineered to express chimeric antigen receptors (CAR-T cells) targeting the murine homolog of the colorectal cancer antigen GUCY2C treat established colorectal cancer metastases, without toxicity to the normal GUCY2C-expressing intestinal epithelium, reflecting structural compartmentalization of endogenous GUCY2C to apical membranes comprising the intestinal lumen. Here, we examined the utility of a human-specific, GUCY2C-directed single-chain variable fragment as the basis for a CAR construct targeting human GUCY2C-expressing metastases. Human GUCY2C-targeted murine CAR-T cells promoted antigen-dependent T-cell activation quantified by activation marker upregulation, cytokine production, and killing of GUCY2C-expressing, but not GUCY2C-deficient, cancer cells in vitro GUCY2C CAR-T cells provided long-term protection against lung metastases of murine colorectal cancer cells engineered to express human GUCY2C in a syngeneic mouse model. GUCY2C murine CAR-T cells recognized and killed human colorectal cancer cells endogenously expressing GUCY2C, providing durable survival in a human xenograft model in immunodeficient mice. Thus, we have identified a human GUCY2C-specific CAR-T cell therapy approach that may be developed for the treatment of GUCY2C-expressing metastatic colorectal cancer. Cancer Immunol Res; 6(5); 509-16. ©2018 AACR.

GUCY2C Signaling Opposes the Acute Radiation-Induced GI Syndrome.

High doses of ionizing radiation induce acute damage to epithelial cells of the gastrointestinal (GI) tract, mediating toxicities restricting the therapeutic efficacy of radiation in cancer and morbidity and mortality in nuclear disasters. No approved prophylaxis or therapy exists for these toxicities, in part reflecting an incomplete understanding of mechanisms contributing to the acute radiation-induced GI syndrome (RIGS). Guanylate cyclase C (GUCY2C) and its hormones guanylin and uroguanylin have recently emerged as one paracrine axis defending intestinal mucosal integrity against mutational, chemical, and inflammatory injury. Here, we reveal a role for the GUCY2C paracrine axis in compensatory mechanisms opposing RIGS. Eliminating GUCY2C signaling exacerbated RIGS, amplifying radiation-induced mortality, weight loss, mucosal bleeding, debilitation, and intestinal dysfunction. Durable expression of GUCY2C, guanylin, and uroguanylin mRNA and protein by intestinal epithelial cells was preserved following lethal irradiation inducing RIGS. Oral delivery of the heat-stable enterotoxin (ST), an exogenous GUCY2C ligand, opposed RIGS, a process requiring p53 activation mediated by dissociation from MDM2. In turn, p53 activation prevented cell death by selectively limiting mitotic catastrophe, but not apoptosis. These studies reveal a role for the GUCY2C paracrine hormone axis as a novel compensatory mechanism opposing RIGS, and they highlight the potential of oral GUCY2C agonists (Linzess; Trulance) to prevent and treat RIGS in cancer therapy and nuclear disasters. Cancer Res; 77(18); 5095-106. ©2017 AACR.

GUCY2C-directed CAR-T cells oppose colorectal cancer metastases without autoimmunity.

Adoptive T-cell therapy (ACT) is an emerging paradigm in which T cells are genetically modified to target cancer-associated antigens and eradicate tumors. However, challenges treating epithelial cancers with ACT reflect antigen targets that are not tumor-specific, permitting immune damage to normal tissues, and preclinical testing in artificial xenogeneic models, preventing prediction of toxicities in patients. In that context, mucosa-restricted antigens expressed by cancers exploit anatomical compartmentalization which shields mucosae from systemic antitumor immunity. This shielding may be amplified with ACT platforms employing antibody-based chimeric antigen receptors (CARs), which mediate MHC-independent recog-nition of antigens. GUCY2C is a cancer mucosa antigen expressed on the luminal surfaces of the intestinal mucosa in mice and humans, and universally overexpressed by colorectal tumors, suggesting its unique utility as an ACT target. T cells expressing CARs directed by a GUCY2C-specific antibody fragment recognized GUCY2C, quantified by expression of activation markers and cytokines. Further, GUCY2C CAR-T cells lysed GUCY2C-expressing, but not GUCY2C-deficient, mouse colorectal cancer cells. Moreover, GUCY2C CAR-T cells reduced tumor number and morbidity and improved survival in mice harboring GUCY2C-expressing colorectal cancer metastases. GUCY2C-directed T cell efficacy reflected CAR affinity and surface expression and was achieved without immune-mediated damage to normal tissues in syngeneic mice. These observations highlight the potential for therapeutic translation of GUCY2C-directed CAR-T cells to treat metastatic tumors, without collateral autoimmunity, in patients with metastatic colorectal cancer.

GUCY2C lysosomotropic endocytosis delivers immunotoxin therapy to metastatic colorectal cancer.

The emergence of targeted cancer therapy has been limited by the paucity of determinants which are tumor-specific and generally associated with disease, and have cell dynamics which effectively deploy cytotoxic payloads. Guanylyl cyclase C (GUCY2C) may be ideal for targeting because it is normally expressed only in insulated barrier compartments, including intestine and brain, but over-expressed by systemic metastatic colorectal tumors. Here, we reveal that GUCY2C rapidly internalizes from the cell surface to lysosomes in intestinal and colorectal cancer cells. Endocytosis is independent of ligand binding and receptor activation, and is mediated by clathrin. This mechanism suggests a design for immunotoxins comprising a GUCY2C-directed monoclonal antibody conjugated through a reducible disulfide linkage to ricin A chain, which is activated to a potent cytotoxin in lysosomes. Indeed, this immunotoxin specifically killed GUCY2C-expressing colorectal cancer cells in a lysosomal- and clathrin-dependent fashion. Moreover, this immunotoxin reduced pulmonary tumors>80% (p<0.001), and improved survival 25% (p<0.001), in mice with established colorectal cancer metastases. Further, therapeutic efficacy was achieved without histologic evidence of toxicity in normal tissues. These observations support GUCY2C-targeted immunotoxins as novel therapeutics for metastatic tumors originating in the GI tract, including colorectum, stomach, esophagus, and pancreas.

Intestinal GUCY2C prevents TGF-ß secretion coordinating desmoplasia and hyperproliferation in colorectal cancer.

Tumorigenesis is a multistep process that reflects intimate reciprocal interactions between epithelia and underlying stroma. However, tumor-initiating mechanisms coordinating transformation of both epithelial and stromal components are not defined. In humans and mice, initiation of colorectal cancer is universally associated with loss of guanylin and uroguanylin, the endogenous ligands for the tumor suppressor guanylyl cyclase C (GUCY2C), disrupting a network of homeostatic mechanisms along the crypt-surface axis. Here, we reveal that silencing GUCY2C in human colon cancer cells increases Akt-dependent TGF-ß secretion, activating fibroblasts through TGF-ß type I receptors and Smad3 phosphorylation. In turn, activating TGF-ß signaling induces fibroblasts to secrete hepatocyte growth factor (HGF), reciprocally driving colon cancer cell proliferation through cMET-dependent signaling. Elimination of GUCY2C signaling in mice (Gucy2c(-/-)) produces intestinal desmoplasia, with increased reactive myofibroblasts, which is suppressed by anti-TGF-ß antibodies or genetic silencing of Akt. Thus, GUCY2C coordinates intestinal epithelial-mesenchymal homeostasis through reciprocal paracrine circuits mediated by TGF-ß and HGF. In that context, GUCY2C signaling constitutes a direct link between the initiation of colorectal cancer and the induction of its associated desmoplastic stromal niche. The recent regulatory approval of oral GUCY2C ligands to treat chronic gastrointestinal disorders underscores the potential therapeutic opportunity for oral GUCY2C hormone replacement to prevent remodeling of the microenvironment essential for colorectal tumorigenesis.

Immunotherapeutic strategies to target prognostic and predictive markers of cancer.

Throughout the last century medical advances in cancer treatment in the fields of surgery, radiation therapy and chemotherapy have greatly impacted patients' survival rates. Nevertheless, cancer remains a significant cause of mortality, with an estimated 7.6 million deaths worldwide in 2008, reflecting the inability of existing therapies to effectively cure disease. The emergence of vaccines and their successes in preventing the spread of infectious diseases has demonstrated the unique specificity and therapeutic potential of the immune system. This potential has driven the development of novel cancer immunotherapeutics. This review focuses on the current status of the use of immunologic effectors to target known biomarkers in cancer.

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

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.

A uroguanylin-GUCY2C endocrine axis regulates feeding in mice.

Intestinal enteroendocrine cells are critical to central regulation of caloric consumption, since they activate hypothalamic circuits that decrease appetite and thereby restrict meal size by secreting hormones in response to nutrients in the gut. Although guanylyl cyclase and downstream cGMP are essential regulators of centrally regulated feeding behavior in invertebrates, the role of this primordial signaling mechanism in mammalian appetite regulation has eluded definition. In intestinal epithelial cells, guanylyl cyclase 2C (GUCY2C) is a transmembrane receptor that makes cGMP in response to the paracrine hormones guanylin and uroguanylin, which regulate epithelial cell dynamics along the crypt-villus axis. Here, we show that silencing of GUCY2C in mice disrupts satiation, resulting in hyperphagia and subsequent obesity and metabolic syndrome. This defined an appetite-regulating uroguanylin-GUCY2C endocrine axis, which we confirmed by showing that nutrient intake induces intestinal prouroguanylin secretion into the circulation. The prohormone signal is selectively decoded in the hypothalamus by proteolytic liberation of uroguanylin, inducing GUCY2C signaling and consequent activation of downstream anorexigenic pathways. Thus, evolutionary diversification of primitive guanylyl cyclase signaling pathways allows GUCY2C to coordinate endocrine regulation of central food acquisition pathways with paracrine control of intestinal homeostasis. Moreover, the uroguanylin-GUCY2C endocrine axis may provide a therapeutic target to control appetite, obesity, and metabolic syndrome.

Bacterial heat-stable enterotoxins: translation of pathogenic peptides into novel targeted diagnostics and therapeutics.

Heat-stable toxins (STs) produced by enterotoxigenic bacteria cause endemic and traveler's diarrhea by binding to and activating the intestinal receptor guanylyl cyclase C (GC-C). Advances in understanding the biology of GC-C have extended ST from a diarrheagenic peptide to a novel therapeutic agent. Here, we summarize the physiological and pathophysiological role of GC-C in fluid-electrolyte regulation and intestinal crypt-villus homeostasis, as well as describe translational opportunities offered by STs, reflecting the unique characteristics of GC-C, in treating irritable bowel syndrome and chronic constipation, and in preventing and treating colorectal cancer.