Guanylyl Cyclase C Research Articles

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.

Linaclotide for the treatment of refractory lower bowel manifestations of systemic sclerosis

BackgroundLower gastrointestinal (GI) tract involvement can affect up to 50% of systemic sclerosis (SSc) patients, and may result in malabsorption, pseudo-obstruction, hospitalization, and death. We report our experience with linaclotide, a selective agonist of guanylate cyclase C (GC-C), for SSc patients with refractory lower GI disease.MethodsWe performed an analysis of patients seen at the Johns Hopkins Scleroderma Center and identified patients prescribed linaclotide for refractory lower GI manifestations. Patients had clinical data collected in our longitudinal database. Linaclotide responders were on medication for at least 12 months with documented effectiveness by the treating physician.ResultsThirty-one patients with SSc were treated with linaclotide. At the time of linaclotide initiation, 23 of these patients (74%) were classified as having severe GI disease, as defined by recurrent pseudo-obstruction, malabsorption, and/or need for artificial nutrition (Medsger GI severity score ≥ 3). The majority of patients (90.3%; 28/31) had a treatment response, while only three patients (9.7%) reported ineffectiveness or intolerable side effects. Low-dose linaclotide (≤ 145 mcg daily) was used in 18 patients and was effective in 94%. High-dose therapy (> 145 mcg daily) was effective in 11 of 13 patients (85%). Common side effects were diarrhea, cramping, or bloating (11/31, 35%). Ineffectiveness, cost, and abdominal pain were complaints cited among those who discontinued therapy.ConclusionLinaclotide is a well-tolerated and efficacious pro-secretory and pro-motility agent that can be used to manage refractory lower GI manifestations in SSc. We found that low-dose linaclotide is an effective option and may be better tolerated, though a subset of patients may require high dose regimens.

Changes of guanylate cyclase C in colon tissues of rats with intestinal injury associated with severe acute pancreatitis

To explore the dynamic changes of guanylate cyclase C (GC-C) in the colon tissues of rats with intestinal injury associated with severe acute pancreatitis (SAP). Thirty-six SD rats were randomized equally into two groups to receive either sham operation or retrograde pumping of 5% sodium taurocholate (0.1 mL/100 g) into the pancreaticobiliary duct following laparotomy to induce SAP. At 12, 24, and 48 h after modeling, 6 rats from each group were euthanized and the colon tissues were collected for Western blotting, immunohistochemistry and RT-PCR to determine the changes in GC-C expression, and the lowest GC-C expression was deemed to indicate the most serious intestinal injury and the time window for intervention. Another 18 SD rats were randomized into 3 groups for sham operation, SAP modeling or intragastric administration of linaclotide (a GC-C agonist) solution once daily at the dose of 10 μg/kg. At 12 h after modeling, the pathological changes in the pancreas and colon were observed with HE staining; the serum level of AMY, DAO, D-Lac and TNF-α were measured with ELISA, and the expressions of GC-C and claudin-1 were detected using Western blotting, immunohistochemical and transmission electron microscopy. The expression of GC-C was significantly reduced in the colon of rats in SAP group, and its lowest expression occurred at 12 h after modeling (P < 0.05) followed by gradual increase over time. Claudin-1 showed a similar trend in the colon. Compared with the sham-operated rats, the rats in SAP and Linaclotide groups showed significantly increased pathological scores of the colon tissues (P < 0.05) and serum levels of AMY, DAO, D-Lac and TNF-α and decreased expressions of GC-C and claudin-1 in the colon (P < 0.05). Compared with those in SAP group, the rats in linaclotide group had significantly lower colonic histopathological scores, lower serum levels of AMY, DAO, D-Lac and TNF-α, and higher expression levels of GC-C and claudin-1 in the colon tissue. In rats with SAP-related intestinal injury, the expression of GC-C in the colon tissue decreases to the lowest level at 12 h after SAP onset followed by gradual increase. activating GC-C can increase the expression levels of GC-C and claudin-1 and alleviate intestinal injury, suggesting the role of GC-C in maintaining intestinal barrier integrity by regulating the expression of tight junction proteins.

A236 LINACLOTIDE IS NOT DETECTABLE IN BREAST MILK OF LACTATING WOMEN: AN OPEN-LABEL, PHASE 1 STUDY

Abstract Background Linaclotide (LIN) is a guanylate cyclase-C agonist approved to treat adults with irritable bowel syndrome with constipation (IBS-C) and chronic idiopathic constipation (CIC). As LIN is a 14-amino acid peptide, no absorption from the gastrointestinal tract is expected following oral administration. However, LIN levels in human breast milk have not been determined. Aims To determine the levels of LIN and its active metabolite, MM-419447, excreted in breast milk after multiple once-daily doses of LIN (72 µg, 145 μg, or 290 μg) in lactating women. Methods This was a multicenter, open-label, multidose, Phase 1, milk-only lactation study (NCT02220348) in lactating women aged 18–45 years who were actively breastfeeding or pumping for ≥4 weeks and were already taking LIN 72 μg, 145 μg, or 290 μg therapeutically for IBS-C or CIC. Participants continued their LIN dose once daily for the 3 study days, with breast milk extractions at −1 to 0 hours (pre-dose) on Day 1 and −1 to 0 hours and 0–2, 2–4, 4–8, 8–12, 12–16, and 16–24 hours after dosing on Day 3. The pharmacokinetic endpoints were the cumulative amount of LIN and MM-419447 and the percentage dose of LIN excreted into the breast milk over the dosing interval. Adverse events (AEs) were monitored. The study has institutional review board approval. Results Seven women were enrolled in the study (IBS-C, n=1; CIC, n=6) and received LIN (72 μg, n=5; 145 μg, n=1; and 290 μg, n=1). Mean age was 28 (range: 19–35), mean weight was 70 kg (standard deviation [SD]: 12 kg), and mean body mass index was 26 kg/m2 (SD: 4 kg/m2); the majority were white (n=6). Concentrations of LIN and MM-419447 in breast milk samples were below the quantitation limits (&amp;lt;0.25 ng/mL and &amp;lt;1.00 ng/mL, respectively) at all time points for all participants. Cumulative exposure for each dose was 216 μg (72 μg dose), 435 μg (145 μg dose), and 870 μg (290 μg dose). One treatment-emergent AE was reported (mild rash). Conclusions The data collected in this study provide no evidence that breastfeeding infants receive LIN or MM-419447 through breast milk as their concentrations were below the quantitation limit following multiple once-daily doses of LIN 72 μg, 145 μg, or 290 μg. Funding Agencies This study was sponsored by Allergan plc, Dublin, Ireland (prior to acquisition by AbbVie Inc.). Writing and editorial assistance were provided to the authors by Stephanie J. Rippon, MBio, Jane Beck, MA, and Rebecca Fletcher, BA(Hons), of Complete HealthVizion, Inc., Chicago, IL, USA and funded by Allergan plc (prior to acquisition by AbbVie Inc.).

A237 IMPROVEMENT IN ABDOMINAL SYMPTOMS WITH LINACLOTIDE IN PATIENTS WITH IRRITABLE BOWEL SYNDROME WITH CONSTIPATION: RESULTS FROM A PHASE 3B TRIAL

Abstract Background Linaclotide (LIN) is a guanylate cyclase-C agonist approved to treat irritable bowel syndrome with constipation (IBS-C) in adults. Abdominal symptoms are important to patients with IBS-C. In a recent Phase 3b study, LIN significantly improved a composite score of abdominal bloating, pain, and discomfort (Abdominal Score), which was used as the primary endpoint in the study. Aims To evaluate the efficacy of LIN for improving additional efficacy abdominal symptom endpoints in a randomized, double-blind, placebo (PBO)-controlled Phase 3 study of LIN in patients with IBS-C. Methods Adults with IBS-C were randomized to PBO (N=308) or LIN 290 μg (N=306) once daily for 12 weeks. Patients recorded their daily abdominal symptoms, including the individual items of bloating, pain, and discomfort, using an 11-point scale (0–10; 0=none, 10=worst possible). The primary endpoint was the Abdominal Score. Additional efficacy endpoints included 6/12-week abdominal pain and constipation (APC)+1 responder, 6/12-week abdominal bloating responder, 6/12-week abdominal pain responder, and 6/12-week abdominal discomfort responder. For individual symptoms, a responder was a patient who had an improvement from baseline of ≥2 points in the respective endpoint for ≥6 of the 12 weeks. Changes from baseline (CFB) over 12 weeks in abdominal bloating, pain, and discomfort were evaluated using a mixed model with repeated measures framework. Proportions of responders were compared between groups for each responder endpoint using a Cochran-Mantel-Haenszel test. Results 614 patients (mean age, 46.7 years; 81% female; similar baseline abdominal symptoms) were randomized. LIN-treated patients had greater least-squares mean (LSM) CFB in abdominal bloating (LSM difference [95% CI]: –0.889 [–1.249, –0.530], p&amp;lt;0.001), pain (–0.881 [–1.238, –0.524], p&amp;lt;0.001), and discomfort (–0.837 [–1.196, –0.478], p&amp;lt;0.001) compared to PBO-treated patients. There was a greater proportion of LIN-treated vs. PBO-treated patients who were 6/12-week APC+1 (29% vs. 17%; p=0.0003), bloating (40% vs. 24%; p&amp;lt;0.001), pain (42% vs. 25%; p&amp;lt;0.001), and discomfort (42% vs. 26%; p&amp;lt;0.001) responders (Figure). Diarrhea was the most common treatment-emergent adverse event (LIN: 4.6%; PBO: 1.6%). Conclusions LIN significantly improved multiple abdominal symptom and secondary responder endpoints in patients with IBS-C. These results support the effectiveness of LIN for improving a spectrum of abdominal symptoms in IBS-C. Funding Agencies This study was sponsored by Allergan plc, Dublin, Ireland (prior to acquisition by AbbVie Inc.). Writing and editorial assistance were provided to the authors by Brittany Y. Jarrett, PhD, Jane Beck, MA, and Rebecca Fletcher, BA(Hons) of Complete HealthVizion, Inc., Chicago, IL, USA and funded by Allergan plc (prior to acquisition by AbbVie Inc.).

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

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. Adult female rats were exposed to water avoidance stress or sham stress for 10days, 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. 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. 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.

Decreased Colonic Guanylin/Uroguanylin Expression and Dried Stool Property in Mice With Social Defeat Stress.

Psychological stress is deeply involved in the pathophysiology of not only mental illness but also functional gastrointestinal disorders. In the present study, we examined the relationship between psychological stress and abnormality of stool properties, focusing on the alteration of plasma glucocorticoid and guanylin (GN)/uroguanylin (UGN) expression in the colon. A murine model of chronic social defeat stress (CSDS) was established by exposing a C57BL/6N intruder mouse to a CD-1 aggressor mouse for 3–5 min. Thereafter the mice were kept in the same cage but separated by a divider for the remainder of the day. This procedure was repeated for 10 consecutive days, and then a social interaction test was performed to evaluate social avoidance. Fresh fecal and blood samples were collected for stool property analysis and measurement of the plasma glucocorticoid level by ELISA. The expression of GN, UGN, and guanylate cyclase 2C in the colonic tissues was examined by real-time RT-PCR and immunohistochemistry. Moreover, Lovo cells were stimulated with dexamethasone, and the expression of GN/UGN mRNA was examined. In the CSDS group, the time spent in the social interaction zone was significantly shorter when the CD-1 aggressor mouse was present than when it was absent. The social interaction ratio was also significantly lower in the CSDS group relative to the controls. The mean Bristol scale score was significantly lower in the CSDS group, but the fecal sodium concentration did not differ between CSDS mice and controls. The level of plasma corticosterone was significantly higher in the CSDS group than in the controls immediately after the 10th day of CSDS. The expression of both GN and UGN was significantly decreased in the CSDS mice. GN was expressed in all colonic epithelial cells, and UGN was expressed in ovoid or pyramidal epithelial cells in the colonic mucosa. The expression of both GN and UGN was significantly decreased in the CSDS mice relative to controls. The expression of both GN and UGN was significantly suppressed in Lovo cells upon stimulation with dexamethasone. Psychological stress-induced glucocorticoid may suppress colonic GN/UGN expression, resulting in a change in stool properties leading to constipation.

Novel CoupledCARTM Technology for Treating Colorectal Cancer

Chimeric antigen receptor (CAR) T cell therapy has made significant progress in the treatment of blood cancers such as leukemia, lymphoma, and myeloma. However, the therapy faces many challenges in treating solid tumors. These challenges include physical barriers, tumor microenvironment immunosuppression, tumor heterogeneity, target specificity, and limited reactive cell expansion in vivo. Conventional CAR T cell therapy has thus far shown weak cell expansion in solid tumor patients and achieved little or no therapeutic responses. Here, we developed CAR T cells based on a novel CoupledCAR® technology to treat solid tumors. In contrast to conventional CAR T cells, CoupledCAR T cells significantly improved the expansion of the CAR T cells in vivo and enhanced the CAR T cells' migration ability and resistance to immunosuppression by the tumor microenvironment. The enhanced migration ability and resistance allow the CAR T cells to infiltrate to tumor tissue sites and increase anti-tumor activities. Specifically, we engineered CoupledCAR T cells with lentiviral vectors encoding an anti-GCC (guanylate cyclase 2C) CAR molecule. Furthermore, anti-GCC CAR T cells showed anti-tumor activities in vitro and in vivo experiments. To verify the safety and efficacy of CoupledCAR T cells for treating solid tumors, we conducted several clinical trials for different solid tumors, including seven patients with colorectal cancer. These seven patients failed multiple rounds of chemotherapy and radiotherapy. In the clinical trial, the patients were infused with autologous anti-GCC CoupledCAR T cells range from 4.9×10^5/kg to 2.9×10^6/kg. All patients using anti-GCC CoupledCAR T cells showed rapid expansion of CoupledCAR T cells and killing of tumor cells. Specifically, we observed that CoupledCAR T cells expanded significantly in the patients and infiltrated tumor tissue sites, demonstrating enhanced anti-tumor activities. PET/CT showed significant tumor shrinkage and SUV max declined, and the ongoing responses were monitored. Patient 3 achieved complete response and the best overall response rate (ORR, include complete remission, complete metabolic response, partial response, and partial metabolic response.) was 71.4% (5/7), complete remission (CR) rate was 14.3% (1/7). The clinical data demonstrated that CoupledCAR T cells effectively expanded, infiltrated tumor tissue sites, and kill tumor cells in patients with colorectal cancer. We used immunotherapy to achieve complete remission in patients with advanced colorectal cancer for the first time. We are recruiting more colorectal cancer patients to further test the safety and efficacy of anti-GCC CoupledCAR T cells. Since our CoupledCAR® technology is a platform technology, we are expanding it to treat other solid tumors using different target tumor markers . Disclosures Xiao: Innovative Cellular Therapeutics: Other: stockholder.

S0506 Efficacy and Safety of Plecanatide in Patients With Chronic Idiopathic Constipation: Pooled Analysis of Two Phase 3 Trials Stratified by Concomitant Acid Suppression Use

INTRODUCTION: Plecanatide, an approved treatment for chronic idiopathic constipation (CIC), replicates the pH-sensitive activity of uroguanylin in binding to guanylate cyclase-C receptors on intestinal epithelium. Since many individuals with CIC experience reflux and use concomitant acid suppression therapy, we explored the impact of concomitant acid suppressor use on the efficacy and safety of plecanatide in adults with CIC. METHODS: Data on two 12-week, randomized, double-blind, placebo-controlled phase 3 clinical trials of plecanatide were pooled and results were stratified by concomitant acid suppressor use. For each group, durable overall complete spontaneous bowel movement (CSBM) response rates, changes from baseline in bowel movement frequency, stool consistency (Bristol Stool Form Scale), and daily CIC symptom scores (Likert Scale, 0 = none to 4 = very severe) were assessed. Safety was analyzed in patients who received ≥1 dose. RESULTS: The intention-to-treat population (N = 2639) included 12.6% of patients with concomitant acid suppressor use. Patients using acid suppressors were 54.8 years (mean), vs 43.9 years in those not using acid suppressors. Gastroesophageal reflux disease was more common in patients using acid suppressors (83.1%), compared to those not on acid suppressors (6.1%). Proton pump inhibitors (PPIs) were used by 86.4% of patients using acid suppressors with omeprazole as the most common PPI (45.1%); H2 blockers were used by 17.8% of subjects with ranitidine as the most common H2 blocker (9.8%). Regardless of acid suppressor use, plecanatide-treated patients demonstrated significant improvements in the durable overall CSBM responder rates (Figure 1) and greater efficacy across almost all reported endpoints (Table 1) vs placebo, with benefits observed as early as Week 1. Comparisons between acid suppressor use groups (yes vs no) were not significantly different for plecanatide-treated patients (both doses, Table 1). Of the plecanatide-treated patients on acid suppressors, 6.4% experienced diarrhea, 1.4% discontinued due to diarrhea, and 2.3% had ≥ 1 serious adverse event (SAE), compared to 5.0% (diarrhea), 2.1% (discontinued due to diarrhea), and 1.0% (SAE) of patients not using acid suppressors. CONCLUSION: Plecanatide is a safe and effective treatment for CIC when administered with acid suppression therapy. Regardless of acid suppressor use, efficacy responses in plecanatide groups were significantly greater than placebo.Figure 1.: Percentage of Patients With Durable Overall CSBM Response Stratified by Concomitant Acid Suppressor Use (ITT).Table 1.: Change From Baseline in Bowel Movement Frequency, Stool Consistency, and CIC Symptoms in Patients With and Without Concomitant Acid Suppression Therapy (ITT)

The endogenous ligand for guanylate cyclase-C activation reliefs intestinal inflammation in the DSS colitis model.

Ulcerative colitis (UC) is a major type of inflammatory bowel disease (IBD) and significantly impacts patient quality of life. Previous research revealed that the guanylate cyclase-C (GC-C) signaling pathway is associated with the severity of UC. We aimed to investigate the effect of the GC-C agonist, guanylin (Gn), on inflammatory injury in mice with colitis. An experimental UC model was established in Balb/c mice. Mesalamine served as a positive control. The Gn overexpression vector was administered once per day for 1 week. Intestinal permeability of the mice was measured using fluorescein isothiocyanate-dextran after the treatment. Histopathologic grading was estimated to assess the inflammatory injury of the colon. The expression level of crucial mediators of the GC-C signaling pathway (Gn, Ugn and GC-C) and tight junction proteins (occludin, claudin-1 and ZO-1) was measured in the colon. Additionally, the level of pro-inflammatory cytokines (IL-8 and TNF-α) in serum was measured. After injecting the UC mice with the Gn overexpression vector, the body weight increased, and the frequency of loose stools and bloody stools was decreased. Intestinal permeability and histopathologic score were significantly reduced (P<0.05). The expression level of GC-C, Gn, Ugn, claudin-1 and ZO-1 was significantly increased (P<0.05). The level of IL-8 and TNF-α in the serum was significantly decreased (P<0.01). Therefore, the application of Gn overexpression vector can ameliorate the intestinal inflammatory injury and repair the mucosal barrier in colitis mice, which further suggests the clinical therapeutic potential of GC-C agonists in IBD.

Pharmacokinetics and Catabolism of [3H]TAK-164, a Guanylyl Cyclase C Targeted Antibody-Drug Conjugate.

TAK-164 is an antibody-drug conjugate (ADC) comprising human anti-guanylyl cyclase C (GCC) monoclonal antibody conjugated to indolinobenzodiazepine DNA alkylator IGN-P1 through a cleavable alanine-alanine dipeptide linker. TAK-164 is currently being evaluated for the treatment of gastrointestinal cancers expressing GCC. The catabolism of TAK-164 was studied using 3H-labeled ADC using GCC-expressing HEK-293 (GCC-HEK-293) cells, rat tritosomes, cathepsin B, and tumor-bearing mice. Time- and target-dependent uptake of [3H]TAK-164 was observed in GCC-HEK-293 cells with approximately 12% of radioactivity associated with DNA after 24 hours of incubation. Rat liver tritosomes and cathepsin B yielded IGN-P1 aniline, sulfonated IGN-P1 (s-IGN-P1) aniline, and a lysine conjugate of IGN-P1 (IGN-P1-Lys) aniline as catabolites. In tumor-bearing mice, [3H]TAK-164 exhibited a terminal half-life of approximately 41 and 51 hours in plasma and blood, respectively, with low plasma clearance (0.75 ml/h per kilogram). The extractable radioactivity in plasma and tumor samples revealed the presence of s-IGN-P1 aniline and IGN-P1 aniline as payload-related components. The use of a radiolabeled payload in the ADC in tumor uptake investigations provided direct and quantitative evidence for tumor uptake, DNA binding, and proof of mechanism of action of the payload. SIGNIFICANCE STATEMENT: Since payload-related species are potent cytotoxins, a thorough characterization of released products of ADCs, metabolites, and their drug interaction potential is necessary prior to clinical investigations. This study characterized in vitro and in vivo DNA binding mechanisms and released products of TAK-164. The methodologies described here will be highly useful for characterization of payload-related products of ADCs in general.

Abstract 974: Immune modulation following combination of TAK-164 with checkpoint inhibitors

Abstract TAK-164 is an antibody drug conjugate comprised of a full length, fully human IgG1 monoclonal antibody (mAb) directed towards the human extra-cellular domain of guanylyl cyclase C (GCC). The mAb is conjugated using ImmunoGen's peptide-linked indolinobenzodiazepine DNA alkylator DGN549. Previously presented preclinical studies using TAK-164 have demonstrated single agent activity of TAK-164 in a panel of patient derived xenograft models of colorectal cancer grown in immunodeficient mice. With emerging evidence demonstrating the immunostimulatory activity of cytotoxic antibody drug conjugates we sought to explore the potential of TAK-164 in immune competent mice. A CT26 model was engineered to express human GCC, allowing us to test the ability of TAK-164 to modulate the tumor microenvironment, thereby potentiating the activity of checkpoint inhibitors. huGCC-CT26 tumor cells were implanted in the right flank of 6-8 week old BALB/c female mice and randomized when tumors were between 50-150mm3 and treated with a single intravenous administration of TAK-164 alone or in combination with checkpoint inhibition administered Intraperitoneally biweekly for 2 weeks and monitored for tumor growth. To understand if responses observed were driven by a response capable of inducing immunological memory we separated mice with complete responses and rechallenged with CT26 tumor cells on the opposite flank. While memory was observed in the mice that achieved complete response in the single agent groups, both frequency of complete response and tumor rejection was increased in groups treated with a combination of TAK-164 and immune checkpoint inhibition. We performed immunophenotyping studies which revealed broad immunomodulation of lymphoid and myeloid cells by TAK-164 and combination regimens. Collectively these data support further interrogation of immune checkpoint inhibition in combination with TAK-164. Citation Format: Victoria A. Appleman, Michael D. Smith, Michelle Ganno, Tiquella Hatten, Maria Borland, Adnan O. Abu-Yousif. Immune modulation following combination of TAK-164 with checkpoint inhibitors [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 974.

Abstract LB-380: Novel coupledCARTMtechnology for treating colorectal cancer

Abstract Chimeric antigen receptor (CAR) T cell therapy has made significant progress in the treatment of blood cancers such as leukemia, lymphoma, and myeloma. However, the therapy faces many challenges in treating solid tumors. These challenges include physical barriers, tumor microenvironment immunosuppression, tumor heterogeneity, target specificity, and limited reactive cell expansion in vivo. Here, we developed CAR -T cells based on a novel CoupledCARTM technology to treat solid tumors. CoupledCAR T cells significantly improved the expansion of the CAR-T cells in vivo and enhanced the CAR-T cells' migration ability and resistance to immunosuppression by the tumor microenvironment, allowing the CAR-T cells to infiltrate to tumor tissue sites and increase anti-tumor activities.Specifically, we engineered CoupledCAR-T cells with lentiviral vectors encoding an anti-GUCY2C (guanylate cyclase 2C) CAR molecule. To verify the safety and efficacy of CoupledCAR T cells for treating solid tumors, we conducted several clinical trials for different solid tumors, including two patients with colorectal cancer. After the infusion of anti-GUCY2C CoupledCAR T cells, these two patients showed rapid expansion of CoupledCAR T cells and the killing of tumor cells. Spherically, we observed that CoupledCAR T cells expanded significantly in the patients and infiltrated tumor tissue sites, demonstrating enhanced anti-tumor activities. Patient Profile: Patient 1: Male, 55Y, Colon Adenocarcinoma. In May 2016, 8 cycles of XELOX chemotherapy and 1 dose of radiotherapy were performed. In Step 2016, “radical rectal resection and terminal ileum double ileostomy” was performed. After surgery, gemcitabine chemotherapy was performed for 2 cycles. In January 2018, relapse and metastasis of prostate and left lung were observed. Starting from March 2018, seven cycles of "Iritican + Retitrazepam + Epitope Chemotherapy," one cycle of "Iritican + Retitrazepam," implantation of radioactive particles, and three cycles of "Oxaliplatin + Capecitabine" were performed. In April 2019, relapse and metastasis were observed. Patient 2: Female, 57Y, Colon Adenocarcinoma. In December 2014, DT46Gy/2Gy/23 radiotherapy was performed. In December 2014 and January 2015, the single drug chemotherapy of Xeloda was taken orally. In February 2015, laparoscopic radical resection of rectal cancer was performed. In April, May, June, and July 2015, mFOLFOX6 chemotherapy was performed. In June 2019, CT showed tumor metastasis. In June, July, August 2019, irinotecan + fluorouracil regimen chemotherapy was performed. Observations and Results: Patient 1: One month after infusion (M1), the patient was evaluated as PR (Partial Response); most of the target lesions were significantly reduced by more than 50%, and the primary tumor volume was reduced by ~45%. Patient 2: M1, the patient was also evaluated as PR; the tumor in the left upper lobe tip posterior segment was reduced by approximately 75%. The clinical data demonstrated that CoupledCAR-T cells effectively expanded, infiltrated tumor tissue sites, and killed tumor cells in patients with colorectal cancer. We are recruiting more colorectal cancer patients to further test the safety and efficacy of anti-GUCY2C CoupledCAR T cells. Further, since our CoupledCARTM technology is a platform technology, we are developing it to treat other solid tumors using different target markers. Citation Format: Song Li, Chengfei Pu, Zhiyuan Cao, Cheng Lu, Hang Yang, Xi Huang, Xiaogang Shen, Xiuwen Wang, Zhao Wu, Lei Xiao. Novel coupledCARTMtechnology for treating colorectal cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr LB-380.

Deciphering ion transporters, kinases and PDZ-adaptor molecules that mediate guanylate cyclase C agonist-dependent intestinal fluid loss in vivo

BackgroundThe molecular basis for heat-stable Escherichia coli enterotoxin (STa) action and its synthetic analogue linaclotide is well understood at the enterocyte level. Pharmacologic strategies to prevent STa-induced intestinal fluid loss by inhibiting its effector molecules, however, have achieved insufficient inhibition in vivo. Aims and experimental approachTo investigate whether the currently discussed effector molecules and signaling mechanisms of STa/linaclotide-induced diarrhea have similar relevance in vivo than at the enterocyte level, we studied the effect of 10−7M of the STa analogue linaclotide on short circuit current (Isc) of chambered isolated jejunal mucosa, and on the in vivo action on fluid transport in a perfused segment of proximal jejunum of anesthetized mice. The selected mice were deficient of transport (NHE3, CFTR, Slc26a3/a6), adaptor (NHERF1-3), or signal transduction molecules [cGMP-dependent kinase II (GKII)] considered to be downstream effectors after STa/linaclotide binding to guanylate cyclase C (GCC). Selective NHE3 inhibition by tenapanor was also employed. Key results, conclusions and implicationsThe comparison allowed the separation of effectors for stimulation of electrogenic anion secretion and for inhibition of electrolyte/fluid absorption in response to STa/linaclotide. The cGKII-NHERF1-CFTR and cGKII-NHERF2-NHE3 interactions are indeed major effectors of small intestinal fluid loss downstream of GCC activation in vitro and in vivo, but 50% of the linaclotide-induced fluid loss in vivo, while dependent on CFTR activation and NHE3 inhibition, does not involve cGKII, and 30% does not depend on NHERF1 or NHERF2. A combined NHERF1 and NHERF2 inhibition appears nevertheless a good pharmacological strategy against STa-mediated fluid loss.

Mutational landscape of receptor guanylyl cyclase C: Functional analysis and disease-related mutations.

Guanylyl cyclase C (GC-C) is the receptor for the heat-stable enterotoxin, which causes diarrhea, and the endogenous ligands, guanylin and uroguanylin. GC-C is predominantly expressed in the intestinal epithelium and regulates fluid and ion secretion in the gut. The receptor has a complex domain organization, and in the absence of structural information, mutational analysis provides clues to mechanisms of regulation of this protein. Here, we review the mutational landscape of this receptor that reveals regulatory features critical for its activity. We also summarize the available information on mutations in GC-C that have been reported in humans and contribute to severe gastrointestinal abnormalities. Since GC-C is also expressed in extra-intestinal tissues, it is likely that mutations thus far reported in humans may also affect other organ systems, warranting a close observation of these patients in future.

Liquid-Phase Total Synthesis of Plecanatide Aided by Diphenylphosphinyloxyl Diphenyl Ketone (DDK) Derivatives.

Plecanatide is an oral guanylate cyclase-C agonist for the treatment of gastrointestinal disorders. The large-scale supply of plecanatide is restrained primarily by its industrial manufacture. Herein we developed diphenylphosphinyloxyl diphenyl ketone (DDK) derivatives as greener supports with unique precipitation-inducing properties to aid the liquid-phase total synthesis of plecanatide without the use of chromatography. Plecanatide could be obtained in high yield, and the ultimately sheared DDK derivative residue could be directly recycled or regenerated for reuse.

Plasma levels of guanylins are reduced in patients with Crohn’s disease

Background: Guanylin (GN) and uroguanylin (UGN) are endogenous ligands for the intestinal receptor guanylate cyclase C (GC-C), an important regulator of intestinal fluid homeostasis. Gene expression and protein levels of GN are suppressed in inflamed intestinal tissue from patients with inflammatory bowel disease (IBD), but knowledge about plasma levels of guanylins in these conditions is sparse. We aimed to investigate the fasting plasma levels of the prohormones proGN and proUGN in patients with Crohn’s Disease (CD) and relate these to levels found in persons with other diarrheal conditions, as well as persons with normal bowel habits.Methods: Plasma from patients with CD, patients with Familial GUCY2C Diarrheal Disease (FGDS), diarrhea-predominant irritable bowel syndrome (IBS-D) and healthy controls (HC) was analyzed using ELISA assays.Results: Significantly lower fasting plasma levels of proguanylins were found in CD and FGDS patients, compared to HC. In CD patients, plasma proGN levels correlated negatively with Harvey Bradshaw Index and with number of stools/24 h.Conclusion: Our data indicate that diarrhea may be a determinant for levels of proGN in plasma, and should be further explored in studies of different diarrheal disorders.

Circulating guanylyl cyclase C (GCC) mRNA is a reliable metastatic predictor and prognostic index of colorectal cancer.

BackgroundCurrently, few specific biomarkers or standard cutoff values are available for circulating tumor cells (CTCs) detection and survival prediction in patients with early stage colorectal cancer (CRC). Guanylyl cyclase C (GCC) presents as a specific expression in intestinal tumor cells and during their metastases, indicating its potential application as a metastatic predictor of CRC.MethodsThe circulating GCC mRNA of 160 colorectal cancer patients at stage I–III was detected via quantitative real-time (qRT)-PCR in our study, and the correlation of GCC mRNA level with tumor metastasis and long-term survival was explored.ResultsGCC mRNA was found to be positive in 43 out of 160 CRC patients and negative in ten healthy controls. It was found that GCC mRNA over the baseline (>100 copies/µL and 200 copies/µL) showed a significant correlation with disease-free survival (DFS) and overall survival (OS) in the stage II subgroup. It was further revealed that GCC mRNA over 300 copies/µL or higher than the median value of copy numbers was significantly correlated with reduced OS and DFS in CRC patients. A nomogram model based on variables including GCC mRNA copy number was established for predicting the OS of CRC patients (AUC =0.98).ConclusionsCirculating GCC mRNA over baseline is a reliable predictor for tumor metastasis and can be a prognostic index in CRC patients.

Abnormal Uroguanylin Immunoreactive Cells Density in the Duodenum of Patients with Diarrhea-Predominant Irritable Bowel Syndrome Changes following Fecal Microbiota Transplantation.

Altered densities of enteroendocrine cells play an important role in patients with irritable bowel syndrome (IBS). Uroguanylin activates guanylate cyclase-C to regulate intestinal electrolyte and water transport. Aim. To quantify uroguanylin immunoreactive cells density in the duodenum of diarrhea-predominant IBS (IBS-D) patients compared to controls and to investigate the effect of fecal microbiota transplantation (FMT) on these cell densities. Method. Twelve patients with IBS-D according to Rome III criteria were included. The cause was identified as post infectious (PI, n = 6) or idiopathic (n = 6). They completed the IBS-symptom questionnaire before and 3 weeks after FMT. Thirty grams of fresh feces donated from healthy relatives were diluted with 60 ml normal saline and instilled via endoscope into the duodenum. Biopsies were taken from the patients' duodenum before and 3 weeks after FMT. Duodenal biopsies taken from eight healthy controls were also included. The biopsies were immunostained for uroguanylin and quantified using computerized image analysis. Results. Uroguanylin immunoreactive cells were found both in duodenal villi and crypts in both controls and IBS-D patients. The densities of uroguanylin immunoreactive cells were significantly lower in the villi (P < 0.0001) and higher in the crypts (P < 0.0001) for the patients than the controls. Following FMT, the densities of uroguanylin immunoreactive cells for the total group and idiopathic subgroup decreased significantly in the duodenal crypts (P = 0.049 and 0.04, respectively) but not in the villi. No significant changes were shown in the PI-IBS subgroups. The cells density in only the crypts correlated with diarrhea (r = 0.97, P = 0.001) and bloating (r = –0.91, P = 0.01) in the PI-IBS subgroup before FMT and with abdominal pain (r = 0.63, P = 0.03) in the total group of IBS-D patients after FMT. Conclusion. Altered uroguanylin immunoreactive cells density was found in IBS-D patients compared to controls. Changes in these cells density following FMT correlated with IBS symptoms (diarrhea, bloating, and abdominal pain).

Oral Pharmacological Activation of Hypothalamic Guanylate Cyclase 2C Receptor Stimulates Brown Fat Thermogenesis to Reduce Body Weight

Linaclotide is a synthetic peptide approved by the FDA for the treatment of constipation-predominant irritable bowel syndrome and chronic constipation. Linaclotide binds and activates the transmembrane receptor guanylate cyclase 2C (Gucy2c). Uroguanylin (UGN) is a 16 amino acid peptide that is mainly secreted by enterochromaffin cells in the duodenum and proximal small intestine. UGN is the endogenous ligand of Gucy2c and decreases body weight in diet-induced obese (DIO) mice via the activation of the thermogenic program in brown adipose tissue. Therefore, we wanted to evaluate whether oral linaclotide could also improve DIO mice metabolic phenotype. In this study, we have demonstrated that DIO mice orally treated with linaclotide exhibited a significant reduction of body weight without modifying food intake. Linaclotide exerts its actions through the central nervous system, and more specifically, via Gucy2c receptors located in the mediobasal hypothalamus, leading to the activation of the sympathetic nervous system to trigger the thermogenic activity of brown fat stimulating energy expenditure. These findings indicate for first time that, in addition to its effects at intestinal level to treat irritable bowel syndrome with constipation and chronic constipation, linaclotide also exerts a beneficial effect in whole body metabolism.