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Strategies to augment immunity to self/neoantigens expressed by cancers are urgently needed to expand the proportion of patients benefiting from immunotherapy, particularly for GI cancers where only a fraction of patients respond to immunotherapies. However, current vaccine strategies are limited by poor immunogenicity, pre-existing vector-specific immunity, and vaccine-induced vector-specific immunity. Here, we examined a prime-boost strategy using a chimeric adenoviral vector (Ad5.F35) that resists pre-existing immunity followed by recombinant Listeria monocytogenes (Lm) to amplify immunity to the GI cancer antigen GUCY2C. This previously unexplored combination enhanced the quantity, avidity, polyfunctionality, and antitumor efficacy of GUCY2C-specific effector CD8 + T cells, without toxicity in any tissue, including GUCY2C-expressing intestines and brain. Importantly, this combination was partially resistant to pre-existing immunity to Ad5 which is endemic in human populations and vector-specific immunity did not limit the ability of multiple Lm administrations to repeatedly enhance GUCY2C-specific responses. Broadly, these findings suggest that cancer patient immunizations targeting self/neoantigens, as well as immunizations for difficult infectious diseases (HIV, malaria, etc), may be most successful using a combination of Ad5.F35-based priming, followed by Lm-based boosting. More specifically, Lm-GUCY2C may be utilized to amplify GUCY2C-specific immunity in patients receiving adenovirus-based GUCY2C vaccines currently in clinical trials to prevent or treat recurrent GI cancer., (© 2022. The Author(s).)

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-2K d -restricted epitope, GUCY2C 254-262 . However, Lm-GUCY2C produced robust CD8 + T-cell responses towards Lm-derived peptides suggesting that GUCY2C 254-262 peptide may be subdominant to Lm-derived peptides. Indeed, incorporating immunogenic Lm peptides into an adenovirus-based GUCY2C vaccine previously shown to induce robust GUCY2C 254-262 immunity completely suppressed GUCY2C 254-262 responses. Comparison of immunogenic Lm-derived peptides to GUCY2C 254-262 revealed that Lm-derived peptides form highly stable peptide-MHC complexes with H-2K d compared to GUCY2C 254-262 peptide. Moreover, amino acid substitution at a critical anchoring residue for H-2K d binding, producing GUCY2C F255Y , significantly improved stability with H-2K d and rescued GUCY2C 254-262 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., Competing Interests: SW is a member of the Board and Chair of the Scientific Advisory Board of, and AS is a consultant for, Targeted Diagnostics & Therapeutics, Inc., which provided research funding that, in part, supported this work and has a license to commercialize inventions related to this work. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Flickinger, Singh, Yarman, Carlson, Barton, Waldman and Snook.)

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.

Introduction: Colorectal cancer (CRC) is one of the most-deadly malignancies worldwide. Current therapeutic regimens for CRC patients are relatively generic, based primarily on disease type and stage, with little variation. As the field of molecular oncology advances, so too must therapeutic management of CRC. Understanding molecular heterogeneity has led to a new-found promotion for precision therapy in CRC; underlining the diversity of molecularly targeted therapies based on individual tumor characteristics., Areas Covered: We review current approaches for the treatment of CRC and discuss the potential of precision therapy in advanced CRC. We highlight the utility of the intestinal protein guanylyl cyclase C (GUCY2C), as a multi-purpose biomarker and unique therapeutic target in CRC. Here, we summarize current GUCY2C-targeted approaches for treatment of CRC., Expert Opinion: The GUCY2C biomarker has multi-faceted utility in medicine. Developmental investment of GUCY2C as a diagnostic and therapeutic biomarker offers a variety of options taking the molecular characteristics of cancer into account. From GUCY2C-targeted therapies, namely cancer vaccines, CAR-T cells, and monoclonal antibodies, to GUCY2C agonists for chemoprevention in those who are at high risk for developing colorectal cancer, the utility of this protein provides many avenues for exploration with significance in the field of precision medicine., Competing Interests: Declaration of interests SAW is the Chair of the Scientific Advisory Board and member of the Board of Directors of, and AES is a consultant for, Targeted Diagnostics & Therapeutics, Inc. which provided research funding that, in part, supported this work and has a license to commercialize inventions related to this work. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

Background: Adenovirus serotype 5 (Ad5) is a commonly used viral vector for transient delivery of transgenes, primarily for vaccination against pathogen and tumor antigens. However, endemic infections with Ad5 produce virus-specific neutralizing antibodies (NAbs) that limit transgene delivery and constrain target-directed immunity following exposure to Ad5-based vaccines. Indeed, clinical trials have revealed the limitations that virus-specific NAbs impose on the efficacy of Ad5-based vaccines. In that context, the emerging focus on immunological approaches targeting cancer self-antigens or neoepitopes underscores the unmet therapeutic need for more efficacious vaccine vectors., Methods: Here, we evaluated the ability of a chimeric adenoviral vector (Ad5.F35) derived from the capsid of Ad5 and fiber of the rare adenovirus serotype 35 (Ad35) to induce immune responses to the tumor-associated antigen guanylyl cyclase C (GUCY2C)., Results: In the absence of pre-existing immunity to Ad5, GUCY2C-specific T-cell responses and antitumor efficacy induced by Ad5.F35 were comparable to Ad5 in a mouse model of metastatic colorectal cancer. Furthermore, like Ad5, Ad5.F35 vector expressing GUCY2C was safe and produced no toxicity in tissues with, or without, GUCY2C expression. Importantly, this chimeric vector resisted neutralization in Ad5-immunized mice and by sera collected from patients with colorectal cancer naturally exposed to Ad5., Conclusions: These data suggest that Ad5.F35-based vaccines targeting GUCY2C, or other tumor or pathogen antigens, may produce clinically relevant immune responses in more (≥90%) patients compared with Ad5-based vaccines (~50%)., Competing Interests: Competing interests: SAW is the Chair of the Scientific Advisory Board and member of the Board of Directors of, and AES is a consultant for, Targeted Diagnostics and Therapeutics, which provided research funding that, in part, supported this work and has a license to commercialize inventions related to this work., (© Author(s) (or their employer(s)) 2020. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

The ability of the immune system to precisely target and eliminate aberrant or infected cells has long been studied in the field of infectious diseases. Attempts to define and exploit these potent immunological processes in the fight against cancer has been a longstanding effort dating back over 100 years to when Dr. William Coley purposefully infected cancer patients with a cocktail of heat-killed bacteria to stimulate anti-cancer immune processes. Although the field of cancer immunotherapy has been dotted with skepticism at times, the success of immune checkpoint inhibitors and recent FDA approvals of autologous cell therapies have pivoted immunotherapy to center stage as one of the most promising strategies to treat cancer. This review aims to summarize historic milestones throughout the field of cancer immunotherapy as well as highlight current and promising immunotherapies in development.

Gastrointestinal cancers remain a tremendous burden on society. Despite advances in therapy options, including chemotherapy and radiation, cancer mortality from recurrences and metastases occur frequently. Cancer stem cells (CSCs) drive disease recurrence and metastasis, as these cells are uniquely equipped to self-renew and evade therapy. Therefore, cancer eradication requires treatment strategies that target CSCs in addition to differentiated cancer cells. This review highlights current literature on therapies targeting CSCs in gastrointestinal cancer.

Background: The colorectal cancer antigen GUCY2C exhibits unique split tolerance, evoking antigen-specific CD8 + , but not CD4 + , T-cell responses that deliver anti-tumor immunity without autoimmunity in mice. Here, the cancer vaccine Ad5-GUCY2C-PADRE was evaluated in a first-in-man phase I clinical study of patients with early-stage colorectal cancer to assess its safety and immunological efficacy., Methods: Ten patients with surgically-resected stage I or stage II (pN0) colon cancer received a single intramuscular injection of 10 11 viral particles (vp) of Ad5-GUCY2C-PADRE. Safety assessment and immunomonitoring were carried out for 6 months following immunization. This trial employed continual monitoring of both efficacy and toxicity of subjects as joint primary outcomes., Results: All patients receiving Ad5-GUCY2C-PADRE completed the study and none developed adverse events greater than grade 1. Antibody responses to GUCY2C were detected in 10% of patients, while 40% exhibited GUCY2C-specific T-cell responses. GUCY2C-specific responses were exclusively CD8 + cytotoxic T cells, mimicking pre-clinical studies in mice in which GUCY2C-specific CD4 + T cells are eliminated by self-tolerance, while CD8 + T cells escape tolerance and mediate antitumor immunity. Moreover, pre-existing neutralizing antibodies (NAbs) to the Ad5 vector were associated with poor vaccine-induced responses, suggesting that Ad5 NAbs oppose GUCY2C immune responses to the vaccine in patients and supported by mouse studies., Conclusions: Split tolerance to GUCY2C in cancer patients can be exploited to safely generate antigen-specific cytotoxic CD8 + , but not autoimmune CD4 + , T cells by Ad5-GUCY2C-PADRE in the absence of pre-existing NAbs to the viral vector., Trial Registration: This trial (NCT01972737) was registered at ClinicalTrials.gov on October 30th, 2013. https://clinicaltrials.gov/ct2/show/NCT01972737.

Characterizing self-tolerance mechanisms and their failure is critical to understand immune homeostasis, cancer immunity, and autoimmunity. However, examination of self-tolerance mechanisms has relied primarily on transgenic mice expressing TCRs targeting well-characterized, but nonphysiologic, model Ags, such as OVA and hemagglutinin. Identifying TCRs directed against bona fide self-antigens is made difficult by the extraordinary diversity of TCRs and the low prevalence of Ag-specific clones (<10-100 naive cells per organism), limiting dissection of tolerance mechanisms restricting immunity to self-proteins. In this study, we isolated and characterized TCRs recognizing the intestinal epithelial cell receptor and colorectal cancer Ag GUCY2C to establish a model to study self-antigen-specific tolerance mechanisms. GUCY2C-specific CD4 + effector T cells were isolated from immunized, nontolerant Gucy2c -/- mice. Next-generation sequencing identified GUCY2C-specific TCRs, which were engineered into CD4 + T cells in vitro to confirm TCR recognition of GUCY2C. Further, the generation of "retrogenic" mice by reconstitution with TCR-transduced hematopoietic stem cells resulted in normal CD4 + T cell development, responsiveness to immunization, and GUCY2C-induced tolerance in recipient mice, recapitulating observations in conventional models. This retrogenic model can be employed to define self-tolerance mechanisms restricting T and B cell responses to GUCY2C to optimize colorectal cancer immunotherapy without autoimmunity., (Copyright © 2019 by The American Association of Immunologists, Inc.)

In 2017, the US Food and Drug Administration approved the first two novel cellular immunotherapies using synthetic, engineered receptors known as chimeric antigen receptors (CARs), tisagenlecleucel (Kymriah) and axicabtagene ciloleucel (Yescarta), expressed by patient-derived T cells for the treatment of hematological malignancies expressing the B-cell surface antigen CD19 in both pediatric and adult patients. This approval marked a major milestone in the use of antigen-directed "living drugs" for the treatment of relapsed or refractory blood cancers, and with these two approvals, there is increased impetus to expand not only the target antigens but also the tumor types that can be targeted. This state-of-the-art review will focus on the challenges, advances, and novel approaches being used to implement CAR T-cell immunotherapy for the treatment of solid tumors., (© 2018 American Society for Clinical Pharmacology and Therapeutics.)

Listeria monocytogenes , a Gram-positive facultative anaerobic bacterium, is becoming a popular vector for cancer immunotherapy. Indeed, multiple vaccines have been developed utilizing modified Listeria as a tool for generating immune responses against a variety of cancers. Moreover, over a dozen clinical trials testing Listeria cancer vaccines are currently underway, which will help to understand the utility of Listeria vaccines in cancer immunotherapy. This review aims to summarize current views on how Listeria -based vaccines induce potent antitumor immunity and the current state of Listeria -based cancer vaccines in clinical trials.

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 ., (©2018 American Association for Cancer Research.)

Purpose: Laboratory studies have documented radioprotective effects with carbamazepine. We sought to determine whether carbamazepine or other anticonvulsant/neuroleptic drugs would show significant radioprotective effects in patients undergoing high-dose small-volume radiosurgery for trigeminal neuralgia., Methods and Materials: We conducted a retrospective review of 200 patients undergoing Gamma Knife (Elekta Instrument AB, Stockholm, Sweden) stereotactic radiosurgery for trigeminal neuralgia between February 1995 and May 2008. We selected patients treated with a maximum dose of 80 Gy with 4-mm diameter collimators, with no previous microvascular decompression, and follow-up ≥6 months (median, 24 months; range, 6-153 months). At the time of radiosurgery, 28 patients were taking no anticonvulsants, 62 only carbamazepine, 35 only gabapentin, 21 carbamazepine plus gabapentin, 17 carbamazepine plus other anticonvulsants, and 9 gabapentin plus other anticonvulsants, and 28 were taking other anticonvulsants or combinations., Results: Pain improvement developed post-radiosurgery in 187 of 200 patients (93.5%). Initial complete pain relief developed in 84 of 200 patients (42%). Post-radiosurgery trigeminal neuropathy developed in 27 of 200 patients (13.5%). We could not significantly correlate pain improvement or initial complete pain relief with use of carbamazepine, gabapentin, or use of any anticonvulsants/neuroleptic drugs or other factors in univariate or multivariate analysis. Post-radiosurgery numbness/paresthesias correlated with the use of gabapentin (1 of 36 patients with gabapentin vs. 7 of 28 without, p = 0.017). In multivariate analysis, decreasing age, purely typical pain, and use of gabapentin correlated (p = 0.008, p = 0.005, and p = 0.021) with lower risks of developing post-radiosurgery trigeminal neuropathy. New post-radiosurgery numbness/paresthesias developed in 3% (1 of 36), 5% (4 of 81), and 13% (23 of 187) of patients on gabapentin alone, with age ≤70 years, and Type 1 typical trigeminal neuralgia pain compared with 25% (7 of 28), 20% (23 of 114), and 33% (4 of 12) of patients taking no anticonvulsants, age >70 years, and partly atypical Type 2 trigeminal neuralgia, respectively., Conclusions: The use of carbamazepine or gabapentin at the time of radiosurgery does not decrease the rates of obtaining partial or complete pain relief after radiosurgery, but gabapentin may reduce the risks of developing post-radiosurgery trigeminal neuropathy., (Copyright © 2012 Elsevier Inc. All rights reserved.)