Clinical course and therapeutic trial for a case of congenital secretory diarrhea due to novel GUCY2C variant.

Chronic diarrhea presents a significant challenge for managing nutritional and electrolyte deficiencies, especially in children, given the higher stakes of impacting growth and developmental consequence. Congenital secretory diarrhea (CSD) compounds this further, particularly in the case of the activating variants of the guanylate-cyclase 2C (GUCY2C) gene. GUCY2C encodes for the guanylate-cyclase 2C (GC-C) receptor that activates the downstream cystic fibrosis transmembrane receptor (CFTR) that primarily drives the severity of diarrhea with an unclear extent of influence on other intestinal channels. Thus far, management for CSD primarily consists of mitigating nutritional, electrolyte, and volume deficiencies with no known pathophysiology-driven treatments. For activating variants of GUCY2C, experimental compounds have shown efficacy in vitro for direct inhibition of GC-C but are not currently available for clinical use. However, Crofelemer, a CFTR inhibitory modulator with negligible systemic absorption, can theoretically help to treat this type of CSD. Herein, we describe and characterize the clinical course of a premature male infant with a de novo missense variant of GUCY2C not previously reported and highly consistent with CSD. With multi-disciplinary family-directed decision-making, a treatment for CSD was evaluated for the first time to our knowledge with Crofelemer.

Molecular insights into recognition of GUCY2C by T-cell engaging bispecific antibody anti-GUCY2CxCD3.

The intestinal epithelial receptor Guanylyl Cyclase C (GUCY2C) is a tumor-associated cell surface antigen expressed across gastrointestinal malignancies that can serve as an efficacious target for colorectal cancer immunotherapy. Here, we describe a yeast surface-display approach combined with an orthogonal peptide-based mapping strategy to identify the GUCY2C binding epitope of a novel anti-GUCY2CxCD3 bispecific antibody (BsAb) that recently advanced into the clinic for the treatment of cancer. The target epitope was localized to the N-terminal helix H2 of human GUCY2C, which enabled the determination of the crystal structure of the minimal GUCY2C epitope in complex with the anti-GUCY2C antibody domain. To understand if this minimal epitope covers the entire antibody binding region and to investigate the impact of epitope position on the antibody's activity, we further determined the structure of this interaction in the context of the full-length extracellular domain (ECD) of GUCY2C. We found that this epitope is positioned on the protruding membrane-distal helical region of GUCY2C and that its specific location on the surface of GUCY2C dictates the close spatial proximity of the two antigen arms in a diabody arrangement essential to the tumor killing activity of GUCY2CxCD3 BsAb.

Updated Clinical Perspectives and Challenges of Chimeric Antigen Receptor-T Cell Therapy in Colorectal Cancer and Invasive Breast Cancer.

In recent years, the incidence of colorectal cancer (CRC) and breast cancer (BC) has increased worldwide and caused a higher mortality rate due to the lack of selective anti-tumor therapies. Current chemotherapies and surgical interventions are significantly preferred modalities to treat CRC or BC in advanced stages but the prognosis for patients with advanced CRC and BC remains dismal. The immunotherapy technique of chimeric antigen receptor (CAR)-T cells has resulted in significant clinical outcomes when treating hematologic malignancies. The novel CAR-T therapy target antigens include GUCY2C, CLEC14A, CD26, TEM8/ANTXR1, PDPN, PTK7, PODXL, CD44, CD19, CD20, CD22, BCMA, GD2, Mesothelin, TAG-72, CEA, EGFR, B7H3, HER2, IL13Ra2, MUC1, EpCAM, PSMA, PSCA, NKG2D. The significant aim of this review is to explore the recently updated information pertinent to several novel targets of CAR-T for CRC, and BC. We vividly described the challenges of CAR-T therapies when treating CRC or BC. The immunosuppressive microenvironment of solid tumors, the shortage of tumor-specific antigens, and post-treatment side effects are the major hindrances to promoting the development of CAR-T cells. Several clinical trials related to CAR-T immunotherapy against CRC or BC have already been in progress. This review benefits academicians, clinicians, and clinical oncologists to explore more about the novel CAR-T targets and overcome the challenges during this therapy.

A phase I, first-in-human study of TAK-164, an antibody-drug conjugate, in patients with advanced gastrointestinal cancers expressing guanylyl cyclase C.

PURPOSE: Guanylyl cyclase C (GCC) is highly expressed in several gastrointestinal malignancies and preclinical studies suggest that it is a promising target for antibody-based therapeutics. This phase I trial assessed the safety and tolerability of TAK-164, an investigational, anti-GCC antibody-drug conjugate (NCT03449030). METHODS: Thirty-one patients with GCC-positive, advanced gastrointestinal cancers received intravenous TAK-164 on day 1 of 21-day cycles. Dose escalation proceeded based on cycle 1 safety data via a Bayesian model. RESULTS: Median age was 58 years (range 32-72), 25 patients (80.6%) had colorectal carcinoma, and median number of prior therapies was four. No dose-limiting toxicities (DLTs) were reported during cycle 1 DLT evaluation period. After cycle 2 dosing, 3 patients reported dose-limiting treatment-emergent adverse events (TEAEs): grade 3 pyrexia and grade 5 hepatic failure (0.19 mg/kg), grade 4 hepatic failure and platelet count decreased (0.25 mg/kg), grade 3 nausea, grade 4 platelet and neutrophil count decreased (0.25 mg/kg). The recommended phase II dose (RP2D) was 0.064 mg/kg. Common TAK-164-related TEAEs included platelet count decreased (58.1%), fatigue (38.7%), and anemia (32.3%). There was a dose-dependent increase in TAK-164 exposure over the range, 0.032-0.25 mg/kg. TAK-164 half-life ranged from 63.5 to 159 h. One patient (0.008 mg/kg) with high baseline GCC expression had an unconfirmed partial response. CONCLUSIONS: TAK-164 appeared to have a manageable safety profile at 0.064 mg/kg. Hepatic toxicity was identified as a potential risk. The RP2D of 0.064 mg/kg was considered insufficient to derive clinical benefit; there are no plans for further clinical development. CLINICAL TRIAL REGISTRATION: NCT03449030.

Guanylyl cyclase C as a diagnostic and therapeutic target in colorectal cancer.

Colorectal cancer remains a major cause of mortality in the USA, despite advances in prevention and screening. Existing therapies focus primarily on generic treatment such as surgical intervention and chemotherapy, depending on disease severity. As personalized medicine and targeted molecular oncology continue to develop as promising treatment avenues, there has emerged a need for effective targets and biomarkers of colorectal cancer. The transmembrane receptor guanylyl cyclase C (GUCY2C) regulates intestinal homeostasis and has emerged as a tumor suppressor. Further, it is universally expressed in advanced metastatic colorectal tumors, as well as other cancer types that arise through intestinal metaplasia. In this context, GUCY2C satisfies many characteristics of a compelling target and biomarker for gastrointestinal malignancies.

Colorectal cancer is a leading cause of death in the USA. In recent years, there has been a shift in the field of oncology from generic treatments, such as surgery and chemotherapy, to personalized molecular therapies, which focus on targeting specific attributes of each patient's unique cancer. Guanylyl cyclase C is a receptor expressed in the intestinal tract, where it regulates fluid secretion and prevents tumor formation. Beyond its function in the healthy intestine, it is expressed in colorectal tumors, and other types of cancer, where it regulates transformation. Therefore, guanylyl cyclase C can serve as a useful target in cancer for prevention and therapy, as well as a marker for tumor cell detection.

Receptor Guanylyl Cyclase C and Cyclic GMP in Health and Disease: Perspectives and Therapeutic Opportunities.

Receptor Guanylyl Cyclase C (GC-C) was initially characterized as an important regulator of intestinal fluid and ion homeostasis. Recent findings demonstrate that GC-C is also causally linked to intestinal inflammation, dysbiosis, and tumorigenesis. These advances have been fueled in part by identifying mutations or changes in gene expression in GC-C or its ligands, that disrupt the delicate balance of intracellular cGMP levels and are associated with a wide range of clinical phenotypes. In this review, we highlight aspects of the current knowledge of the GC-C signaling pathway in homeostasis and disease, emphasizing recent advances in the field. The review summarizes extra gastrointestinal functions for GC-C signaling, such as appetite control, energy expenditure, visceral nociception, and behavioral processes. Recent research has expanded the homeostatic role of GC-C and implicated it in regulating the ion-microbiome-immune axis, which acts as a mechanistic driver in inflammatory bowel disease. The development of transgenic and knockout mouse models allowed for in-depth studies of GC-C and its relationship to whole-animal physiology. A deeper understanding of the various aspects of GC-C biology and their relationships with pathologies such as inflammatory bowel disease, colorectal cancer, and obesity can be leveraged to devise novel therapeutics.

T-Cell Responses to Immunodominant Listeria Epitopes Limit Vaccine-Directed Responses to the Colorectal Cancer Antigen, Guanylyl Cyclase C.

The Gram-positive bacterium Listeria monocytogenes (Lm) is an emerging platform for cancer immunotherapy. To date, over 30 clinical trials have been initiated testing Lm cancer vaccines across a wide variety of cancers, including lung, cervical, colorectal, and pancreatic. Here, we assessed the immunogenicity of an Lm vaccine against the colorectal tumor antigen GUCY2C (Lm-GUCY2C). Surprisingly, Lm-GUCY2C vaccination did not prime naïve GUCY2C-specific CD8+ T-cell responses towards the dominant H-2Kd-restricted epitope, GUCY2C254-262. However, Lm-GUCY2C produced robust CD8+ T-cell responses towards Lm-derived peptides suggesting that GUCY2C254-262 peptide may be subdominant to Lm-derived peptides. Indeed, incorporating immunogenic Lm peptides into an adenovirus-based GUCY2C vaccine previously shown to induce robust GUCY2C254-262 immunity completely suppressed GUCY2C254-262 responses. Comparison of immunogenic Lm-derived peptides to GUCY2C254-262 revealed that Lm-derived peptides form highly stable peptide-MHC complexes with H-2Kd compared to GUCY2C254-262 peptide. Moreover, amino acid substitution at a critical anchoring residue for H-2Kd binding, producing GUCY2CF255Y, significantly improved stability with H-2Kd and rescued GUCY2C254-262 immunogenicity in the context of Lm vaccination. Collectively, these studies suggest that Lm antigens may compete with and suppress the immunogenicity of target vaccine antigens and that use of altered peptide ligands with enhanced peptide-MHC stability may be necessary to elicit robust immune responses. These studies suggest that optimizing target antigen competitiveness with Lm antigens or alternative immunization regimen strategies, such as prime-boost, may be required to maximize the clinical utility of Lm-based vaccines.

High plasma and lingual uroguanylin as potential contributors to changes in food preference after sleeve gastrectomy.

BACKGROUND: The biological mediators supporting long-term weight loss and changes in dietary choice behaviour after sleeve gastrectomy remain unclear. Guanylin and uroguanylin are gut hormones involved in the regulation of satiety, food preference and adiposity. Thus, we sought to analyze whether the guanylin system is involved in changes in food preference after sleeve gastrectomy in obesity. METHODS: Proguanylin (GUCA2A) and prouroguanylin (GUCA2B) were determined in patients with severe obesity (n = 41) as well as in rats with diet-induced obesity (n = 48), monogenic obesity (Zucker fa/fa) (n = 18) or in a food choice paradigm (normal diet vs high-fat diet) (n = 16) submitted to sleeve gastrectomy. Lingual distribution and expression of guanylins (GUCA2A and GUCA2B) and their receptor GUCY2C as well as the fatty acid receptor CD36 were evaluated in the preclinical models. RESULTS: Circulating concentrations of GUCA2A and GUCA2B were increased after sleeve gastrectomy in patients with severe obesity as well as in rats with diet-induced and monogenic (fa/fa) obesity. Interestingly, the lower dietary fat preference observed in obese rats under the food choice paradigm as well as in patients with obesity after sleeve gastrectomy were negatively associated with post-surgical GUCA2B levels. Moreover, sleeve gastrectomy upregulated the low expression of GUCA2A and GUCA2B in taste bud cells of tongues from rats with diet-induced and monogenic (fa/fa) obesity in parallel to a downregulation of the lingual lipid sensor CD36. CONCLUSIONS: The increased circulating and lingual GUCA2B after sleeve gastrectomy suggest an association between the uroguanylin-GUCY2C endocrine axis and food preference through the regulation of gustatory responses.

A ß-Catenin-TCF-Sensitive Locus Control Region Mediates GUCY2C Ligand Loss in Colorectal Cancer.

BACKGROUND & AIMS: Sporadic colorectal cancers arise from initiating mutations in APC, producing oncogenic ß-catenin/TCF-dependent transcriptional reprogramming. Similarly, the tumor suppressor axis regulated by the intestinal epithelial receptor GUCY2C is among the earliest pathways silenced in tumorigenesis. Retention of the receptor, but loss of its paracrine ligands, guanylin and uroguanylin, is an evolutionarily conserved feature of colorectal tumors, arising in the earliest dysplastic lesions. Here, we examined a mechanism of GUCY2C ligand transcriptional silencing by ß-catenin/TCF signaling. METHODS: We performed RNA sequencing analysis of 4 unique conditional human colon cancer cell models of ß-catenin/TCF signaling to map the core Wnt-transcriptional program. We then performed a comparative analysis of orthogonal approaches, including luciferase reporters, chromatin immunoprecipitation sequencing, CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats) knockout, and CRISPR epigenome editing, which were cross-validated with human tissue chromatin immunoprecipitation sequencing datasets, to identify functional gene enhancers mediating GUCY2C ligand loss. RESULTS: RNA sequencing analyses reveal the GUCY2C hormones as 2 of the most sensitive targets of ß-catenin/TCF signaling, reflecting transcriptional repression. The GUCY2C hormones share an insulated genomic locus containing a novel locus control region upstream of the guanylin promoter that mediates the coordinated silencing of both genes. Targeting this region with CRISPR epigenome editing reconstituted GUCY2C ligand expression, overcoming gene inactivation by mutant ß-catenin/TCF signaling. CONCLUSIONS: These studies reveal DNA elements regulating corepression of GUCY2C ligand transcription by ß-catenin/TCF signaling, reflecting a novel pathophysiological step in tumorigenesis. They offer unique genomic strategies that could reestablish hormone expression in the context of canonical oncogenic mutations to reconstitute the GUCY2C axis and oppose transformation.

A Potential Treatment of Congenital Sodium Diarrhea in Patients With Activating GUCY2C Mutations.

INTRODUCTION: Gain-of-function mutations in guanylyl cyclase C (GCC) result in persistent diarrhea with perinatal onset. We investigated a specific GCC inhibitor, SSP2518, for its potential to treat this disorder. METHODS: We investigated the effect of SSP2518 on GCC-mediated intracellular cyclic guanosine monophosphate (cGMP) levels and on GCC-mediated chloride secretion in intestinal organoids from 3 patients with distinct activating GCC mutations and from controls, with and without stimulation of GCC with heat-stable enterotoxin. RESULTS: Patient-derived organoids had significantly higher basal cGMP levels than control organoids, which were lowered by SSP2518 to levels found in control organoids. In addition, SSP2518 significantly reduced cGMP levels and chloride secretion in patient-derived and control organoids (P < 0.05 for all comparisons) after heat-stable enterotoxin stimulation. DISCUSSION: We reported in this study that the GCC inhibitor SSP2518 normalizes cGMP levels in intestinal organoids derived from patients with GCC gain-of-function mutations and markedly reduces cystic fibrosis transmembrane conductance regulator-dependent chloride secretion, the driver of persistent diarrhea.

Phase I double-blind, placebo-controlled trial of dolcanatide (SP-333) 27 mg to explore colorectal bioactivity in healthy volunteers.

Guanylyl cyclase C (GUCY2C) is a tumor-suppressing receptor silenced by loss of expression of the luminocrine hormones guanylin and uroguanylin early in colorectal carcinogenesis. This observation suggests oral replacement with a GUCY2C agonist may be an effective targeted chemoprevention agent. Previous studies revealed that linaclotide, an oral GUCY2C agonist formulated for gastric release, did not persist to activate guanylyl cyclase signaling in the distal rectum. Dolcanatide is an investigational oral uroguanylin analog, substituted with select D amino acids, for enhanced stability and extended persistence to activate GUCY2C in small and large intestine. However, the ability of oral dolcanatide to induce a pharmacodynamic (PD) response by activating GUCY2C in epithelial cells of the colorectum in humans remains undefined. Here, we demonstrate that administration of oral dolcanatide 27 mg daily for 7 d to healthy volunteers did not activate GUCY2C, quantified as accumulation of its product cyclic GMP, in epithelial cells of the distal rectum. These data reveal that the enhanced stability of dolcanatide, with persistence along the rostral-caudal axis of the small and large intestine, is inadequate to regulate GUCY2C across the colorectum to prevent tumorigenesis. These results highlight the importance of developing a GUCY2C agonist for cancer prevention formulated for release and activity targeted to the colorectum.

Diarrheal pathogens trigger rapid evolution of the guanylate cyclase-C signaling axis in bats.

The pathogenesis of infectious diarrheal diseases is largely attributed to enterotoxins that cause dehydration by disrupting intestinal water absorption. We investigated patterns of genetic variation in mammalian guanylate cyclase-C (GC-C), an intestinal receptor targeted by bacterially encoded heat-stable enterotoxins (STa), to determine how host species adapt in response to diarrheal infections. Our phylogenetic and functional analysis of GC-C supports long-standing evolutionary conflict with diarrheal bacteria in primates and bats, with highly variable susceptibility to STa across species. In bats, we further show that GC-C diversification has sparked compensatory mutations in the endogenous uroguanylin ligand, suggesting an unusual scenario of pathogen-driven evolution of an entire signaling axis. Together, these findings suggest that conflicts with diarrheal pathogens have had far-reaching impacts on the evolution of mammalian gut physiology.

Preclinical Evaluation of 89Zr-Df-IAB22M2C PET as an Imaging Biomarker for the Development of the GUCY2C-CD3 Bispecific PF-07062119 as a T Cell Engaging Therapy.

PURPOSE: A sensitive and specific imaging biomarker to monitor immune activation and quantify pharmacodynamic responses would be useful for development of immunomodulating anti-cancer agents. PF-07062119 is a T cell engaging bispecific antibody that binds to CD3 and guanylyl cyclase C, a protein that is over-expressed by colorectal cancers. Here, we used 89Zr-Df-IAB22M2C (89Zr-Df-Crefmirlimab), a human CD8-specific minibody to monitor CD8+ T cell infiltration into tumors by positron emission tomography. We investigated the ability of 89Zr-Df-IAB22M2C to track anti-tumor activity induced by PF-07062119 in a human CRC adoptive transfer mouse model (with injected activated/expanded human T cells), as well as the correlation of tumor radiotracer uptake with CD8+ immunohistochemical staining. PROCEDURES: NOD SCID gamma mice bearing human CRC LS1034 tumors were treated with four different doses of PF-07062119, or a non-targeted CD3 BsAb control, and imaged with 89Zr-Df-IAB22M2C PET at days 4 and 9. Following PET/CT imaging, mice were euthanized and dissected for ex vivo distribution analysis of 89Zr-Df-IAB22M2C in tissues on days 4 and 9, with additional data collected on day 6 (supplementary). Data were analyzed and reported as standard uptake value and %ID/g for in vivo imaging and ex vivo tissue distribution. In addition, tumor tissues were evaluated by immunohistochemistry for CD8+ T cells. RESULTS: The results demonstrated substantial mean uptake of 89Zr-Df-IAB22M2C (%ID/g) in PF-07062119-treated tumors, with significant increases in comparison to non-targeted BsAb-treated controls, as well as PF-07062119 dose-dependent responses over time of treatment. A moderate correlation was observed between tumor tissue radioactivity uptake and CD8+ cell density, demonstrating the value of the imaging agent for non-invasive assessment of intra-tumoral CD8+ T cells and the mechanism of action for PF-07062119. CONCLUSION: Immune-imaging technologies for quantitative cellular measures would be a valuable biomarker in immunotherapeutic clinical development. We demonstrated a qualification of 89Zr-IAB22M2C PET to evaluate PD responses (mice) to a novel immunotherapeutic.

Novel GUCY2C variant causing familial diarrhea in a Mennonite kindred and a potential therapeutic approach.

Guanylate cyclase 2C (GC-C), encoded by the GUCY2C gene, is implicated in hereditary early onset chronic diarrhea. Several families with chronic diarrhea symptoms have been identified with autosomal dominant, gain-of-function mutations in GUCY2C. We have identified a Mennonite patient with a novel GUCY2C variant (c.2381A > T; p.Asp794Val) with chronic diarrhea and an extensive maternal family history of chronic diarrhea and bowel dilatation. Functional studies including co-segregation analysis showed that all family members who were heterozygous for this variant had GI-related symptoms. HEK-293 T cells expressing the Asp794Val GC-C variant showed increased cGMP production when stimulated with Escherichia coli heat-stable enterotoxin STp (HST), which was reversed when 5-(3-Bromophenyl)-5,11-dihydro-1,3-dimethyl-1H-indeno[2',1':5,6]pyrido[2,3-d]pyrimidine-2,4,6(3H)-trione (BPIPP; a GC-C inhibitor) was used. In addition, cystic fibrosis transmembrane conductance regulator (CFTR) activity measured with SPQ fluorescence assay was increased in these cells after treatment with HST, indicating a crucial role for CFTR activity in the pathogenesis of this disorder. These results support pathogenicity of the GC-C Asp794Val variant as a cause of chronic diarrhea in this family. Furthermore, this work identifies potential candidate drug, GC-C inhibitor BPIPP, to treat diarrhea caused by this syndrome.

The shifting paradigm of colorectal cancer treatment: a look into emerging cancer stem cell-directed therapeutics to lead the charge toward complete remission.

Introduction: Colorectal cancer (CRC) is one of the most common forms of cancer worldwide and is the second leading cause of cancer-related death in the United States. Despite advances in early detection, ~25% of patients are late stage, and treated patients have <12% chance of survival after five years. Tumor relapse and metastasis are the main causes of patient death. Cancer stem cells (CSCs) are a rare population of cancer cells characterized by properties of self-renewal, chemo- and radio-resistance, tumorigenicity, and high plasticity. These qualities make CSCs particularly important for metastasic seeding, DNA-damage resistance, and tumor repopulating.Areas Covered: The following review article focuses on the role of CRC-SCs in tumor initiation, metastasis, drug resistance, and tumor relapse, as well as on potential therapeutic options for targeting CSCs.Expert Opinion: Current studies are underway to better isolate and discriminate CSCs from normal stem cells and to produce CSC-targeted therapeutics. The intestinal receptor, guanylate cyclase C (GUCY2C) could potentially provide a unique therapeutic target for both non-stem cells and CSCs alike in colorectal cancer through immunotherapies. Indeed, immunotherapies targeting CSCs have the potential to break the treatment-recurrence cycle in the management of advanced malignancies.

Guanylyl cyclase 2C (GUCY2C) in gastrointestinal cancers: recent innovations and therapeutic potential.

INTRODUCTION: Gastrointestinal (GI) cancers account for the second leading cause of cancer-related deaths in the United States. Guanylyl cyclase C (GUCY2C) is an intestinal signaling system that regulates intestinal fluid and electrolyte secretion as well as intestinal homeostasis. In recent years, it has emerged as a promising target for chemoprevention and therapy for GI malignancies. AREAS COVERED: The loss of GUCY2C signaling early in colorectal tumorigenesis suggests it could have a significant impact on tumor initiation. Recent studies highlight the importance of GUCY2C signaling in preventing colorectal tumorigenesis using agents such as linaclotide, plecanatide, and sildenafil. Furthermore, GUCY2C is a novel target for immunotherapy and a diagnostic marker for primary and metastatic diseases. EXPERT OPINION: There is an unmet need for prevention and therapy in GI cancers. In that context, GUCY2C is a promising target for prevention, although the precise mechanisms by which GUCY2C signaling affects tumorigenesis remain to be defined. Furthermore, clinical trials are exploring its role as an immunotherapeutic target for vaccines to prevent metastatic disease. Indeed, GUCY2C is an emerging target across the disease continuum from chemoprevention, to diagnostic management, through the treatment and prevention of metastatic diseases.

Guanylyl cyclase C as a biomarker for immunotherapies for the treatment of gastrointestinal malignancies.

Gastrointestinal cancers encompass a diverse class of tumors arising in the GI tract, including esophagus, stomach, pancreas and colorectum. Collectively, gastrointestinal cancers compose a high fraction of all cancer deaths, highlighting an unmet need for novel and effective therapies. In this context, the transmembrane receptor guanylyl cyclase C (GUCY2C) has emerged as an attractive target for the prevention, detection and treatment of many gastrointestinal tumors. GUCY2C is an intestinally-restricted protein implicated in tumorigenesis that is universally expressed by primary and metastatic colorectal tumors as well as ectopically expressed by esophageal, gastric and pancreatic cancers. This review summarizes the current state of GUCY2C-targeted modalities in the management of gastrointestinal malignancies, with special focus on colorectal cancer, the most incident gastrointestinal malignancy.

Discovery and optimization of a novel anti-GUCY2c x CD3 bispecific antibody for the treatment of solid tumors.

We report here the discovery and optimization of a novel T cell retargeting anti-GUCY2C x anti-CD3ε bispecific antibody for the treatment of solid tumors. Using a combination of hybridoma, phage display and rational design protein engineering, we have developed a fully humanized and manufacturable CD3 bispecific antibody that demonstrates favorable pharmacokinetic properties and potent in vivo efficacy. Anti-GUCY2C and anti-CD3ε antibodies derived from mouse hybridomas were first humanized into well-behaved human variable region frameworks with full retention of binding and T-cell mediated cytotoxic activity. To address potential manufacturability concerns, multiple approaches were taken in parallel to optimize and de-risk the two antibody variable regions. These approaches included structure-guided rational mutagenesis and phage display-based optimization, focusing on improving stability, reducing polyreactivity and self-association potential, removing chemical liabilities and proteolytic cleavage sites, and de-risking immunogenicity. Employing rapid library construction methods as well as automated phage display and high-throughput protein production workflows enabled efficient generation of an optimized bispecific antibody with desirable manufacturability properties, high stability, and low nonspecific binding. Proteolytic cleavage and deamidation in complementarity-determining regions were also successfully addressed. Collectively, these improvements translated to a molecule with potent single-agent in vivo efficacy in a tumor cell line adoptive transfer model and a cynomolgus monkey pharmacokinetic profile (half-life>4.5 days) suitable for clinical development. Clinical evaluation of PF-07062119 is ongoing.

Peripheral Guanylate Cyclase-C modulation of corticolimbic activation and corticotropin-releasing factor signaling in a rat model of stress-induced colonic hypersensitivity.

BACKGROUND: Psychological stress is a risk factor for irritable bowel syndrome, a functional gastrointestinal pain disorder featuring abnormal brain-gut connectivity. The guanylate cyclase-C (GC-C) agonist linaclotide has been shown to relieve abdominal pain in IBS-C and exhibits antinociceptive effects in rodent models of post-inflammatory visceral hypersensitivity. However, the role GC-C signaling plays in psychological stress-induced visceral hypersensitivity is unknown. Here, we test the hypothesis that GC-C agonism reverses stress-induced colonic hypersensitivity via inhibition of nociceptive afferent signaling resulting in normalization of stress-altered corticotropin-releasing factor (CRF) expression in brain regions involved in pain perception and modulation. METHODS: Adult female rats were exposed to water avoidance stress or sham stress for 10 days, and the effects of linaclotide on stress-induced changes in colonic sensitivity, corticolimbic phospho-extracellular signal-regulated kinase (pERK), and CRF expression were measured using a combination of behavioral assessments, immunohistochemistry, and qRT-PCR. KEY RESULTS: Stressed rats exhibited colonic hypersensitivity and elevated corticolimbic pERK on day 11, which was inhibited by linaclotide. qRT-PCR analysis revealed dysregulated CRF expression in the medial prefrontal cortex, paraventricular nucleus of the hypothalamus, and central nucleus of the amygdala on day 28. Dysregulated CRF expression was not affected by linaclotide treatment. CONCLUSIONS AND INFERENCES: Our results demonstrate that exposure to repeated stress induces chronic colonic hypersensitivity in conjunction with altered corticolimbic activation and CRF expression. GC-C agonism attenuated stress-induced colonic hypersensitivity and ERK phosphorylation, but had no effect on CRF expression, suggesting the analgesic effects of linaclotide occur independent of stress-driven CRF gene expression in corticolimbic circuitry.