Development of a CD8 co-receptor independent T-cell receptor specific for tumor-associated antigen MAGE-A4 for next generation T-cell-based immunotherapy.

BACKGROUND: The cancer-testis antigen MAGE-A4 is an attractive target for T-cell-based immunotherapy, especially for indications with unmet clinical need like non-small cell lung or triple-negative breast cancer. METHODS: An unbiased CD137-based sorting approach was first used to identify an immunogenic MAGE-A4-derived epitope (GVYDGREHTV) that was properly processed and presented on human leukocyte antigen (HLA)-A2 molecules encoded by the HLA-A*02:01 allele. To isolate high-avidity T cells via subsequent multimer sorting, an in vitro priming approach using HLA-A2-negative donors was conducted to bypass central tolerance to this self-antigen. Pre-clinical parameters of safety and activity were assessed in a comprehensive set of in vitro and in vivo studies. RESULTS: A MAGE-A4-reactive, HLA-A2-restricted T-cell receptor (TCR) was isolated from primed T cells of an HLA-A2-negative donor. The respective TCR-T-cell (TCR-T) product bbT485 was demonstrated pre-clinically to have a favorable safety profile and superior in vivo potency compared with TCR-Ts expressing a TCR derived from a tolerized T-cell repertoire to self-antigens. This natural high-avidity TCR was found to be CD8 co-receptor independent, allowing effector functions to be elicited in transgenic CD4+ T helper cells. These CD4+ TCR-Ts supported an anti-tumor response by direct killing of MAGE-A4-positive tumor cells and upregulated hallmarks associated with helper function, such as CD154 expression and release of key cytokines on tumor-specific stimulation. CONCLUSION: The extensive pre-clinical assessment of safety and in vivo potency of bbT485 provide the basis for its use in TCR-T immunotherapy studies. The ability of this non-mutated high-avidity, co-receptor-independent TCR to activate CD8+ and CD4+ T cells could potentially provide enhanced cellular responses in the clinical setting through the induction of functionally diverse T-cell subsets that goes beyond what is currently tested in the clinic.

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

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

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

Heterologous prime-boost immunization with plasmid DNA and viral vector vaccines is an emerging approach to elicit CD8+ T cell-mediated immunity targeting pathogens and tumor Ags that is superior to either monotherapy. Yet, the mechanisms underlying the synergy of prime-boost strategies remain incompletely defined. In this study, we examine a DNA and adenovirus (Ad5) combination regimen targeting guanylyl cyclase C (GUCY2C), a receptor expressed by intestinal mucosa and universally expressed by metastatic colorectal cancer. DNA immunization efficacy was optimized by i.m. delivery via electroporation, yet it remained modest compared with Ad5. Sequential immunization with DNA and Ad5 produced superior antitumor efficacy associated with increased TCR avidity, whereas targeted disruption of TCR avidity enhancement eliminated GUCY2C-specific antitumor efficacy, without affecting responding T cell number or cytokine profile. Indeed, functional TCR avidity of responding GUCY2C-specific CD8+ T cells induced by various prime or prime-boost regimens correlated with antitumor efficacy, whereas T cell number and cytokine profile were not. Importantly, although sequential immunization with DNA and Ad5 maximized antitumor efficacy through TCR avidity enhancement, it produced no autoimmunity, reflecting sequestration of GUCY2C to intestinal apical membranes and segregation of mucosal and systemic immunity. Together, TCR avidity enhancement may be leveraged by prime-boost immunization to improve GUCY2C-targeted colorectal cancer immunotherapeutic efficacy and patient outcomes without concomitant autoimmune toxicity.

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

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

DNA-PKcs-Mediated Transcriptional Regulation Drives Prostate Cancer Progression and Metastasis.

Emerging evidence demonstrates that the DNA repair kinase DNA-PKcs exerts divergent roles in transcriptional regulation of unsolved consequence. Here, in vitro and in vivo interrogation demonstrate that DNA-PKcs functions as a selective modulator of transcriptional networks that induce cell migration, invasion, and metastasis. Accordingly, suppression of DNA-PKcs inhibits tumor metastases. Clinical assessment revealed that DNA-PKcs is significantly elevated in advanced disease and independently predicts for metastases, recurrence, and reduced overall survival. Further investigation demonstrated that DNA-PKcs in advanced tumors is highly activated, independent of DNA damage indicators. Combined, these findings reveal unexpected DNA-PKcs functions, identify DNA-PKcs as a potent driver of tumor progression and metastases, and nominate DNA-PKcs as a therapeutic target for advanced malignancies.

Challenges to chimeric antigen receptor (CAR)-T cell therapy for cancer.

Chimeric antigen receptor (CAR)-expressing T cells have demonstrated potent clinical efficacy in patients with B cell malignancies. However, the use of CAR-T cell therapy targeting other cancers has, in part, been limited by both the induction of antigen-specific toxicities targeting normal tissues expressing the target-antigen, and the extreme potency of CAR-T cell treatments resulting in life-threatening cytokine-release syndromes. Herein, we discuss toxicities associated with CAR-T cell therapy in the clinic. Further, we discuss potential clinical interventions to ameliorate these toxicities and the application of preclinical animal models to predict the clinical utility of CAR-T cell therapy.

GUCY2C lysosomotropic endocytosis delivers immunotoxin therapy to metastatic colorectal cancer.

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

A novel CDX2 isoform regulates alternative splicing.

Gene expression is a dynamic and coordinated process coupling transcription with pre-mRNA processing. This regulation enables tissue-specific transcription factors to induce expression of specific transcripts that are subsequently amplified by alternative splicing allowing for increased proteome complexity and functional diversity. The intestine-specific transcription factor CDX2 regulates development and maintenance of the intestinal epithelium by inducing expression of genes characteristic of the mature enterocyte phenotype. Here, sequence analysis of CDX2 mRNA from colonic mucosa-derived tissues revealed an alternatively spliced transcript (CDX2/AS) that encodes a protein with a truncated homeodomain and a novel carboxy-terminal domain enriched in serine and arginine residues (RS domain). CDX2 and CDX2/AS exhibited distinct nuclear expression patterns with minimal areas of co-localization. CDX2/AS did not activate the CDX2-dependent promoter of guanylyl cyclase C nor inhibit transcriptional activity of CDX2. Unlike CDX2, CDX2/AS co-localized with the putative splicing factors ASF/SF2 and SC35. CDX2/AS altered splicing patterns of CD44v5 and Tra2-ß1 minigenes in Lovo colon cancer cells independent of CDX2 expression. These data demonstrate unique dual functions of the CDX2 gene enabling it to regulate gene expression through both transcription (CDX2) and pre-mRNA processing (CDX2/AS).

Selective antigen-specific CD4(+) T-cell, but not CD8(+) T- or B-cell, tolerance corrupts cancer immunotherapy.

Self-tolerance, presumably through lineage-unbiased elimination of self-antigen-specific lymphocytes (CD4(+) T, CD8(+) T, and B cells), creates a formidable barrier to cancer immunotherapy. In contrast to this prevailing paradigm, we demonstrate that for some antigens, self-tolerance reflects selective elimination of antigen-specific CD4(+) T cells, but preservation of CD8(+) T- and B-cell populations. In mice, antigen-specific CD4(+) T-cell tolerance restricted CD8(+) T- and B-cell responses targeting the endogenous self-antigen guanylyl cyclase c (GUCY2C) in colorectal cancer. Although selective CD4(+) T-cell tolerance blocked GUCY2C-specific antitumor immunity and memory responses, it offered a unique solution to the inefficacy of GUCY2C vaccines through recruitment of self-antigen-independent CD4(+) T-cell help. Incorporating CD4(+) T-cell epitopes from foreign antigens into vaccines against GUCY2C reconstituted CD4(+) T-cell help, revealing the latent functional capacity of GUCY2C-specific CD8(+) T- and B-cell pools, producing durable antitumor immunity without autoimmunity. Incorporating CD4(+) T-cell epitopes from foreign antigens into vaccines targeting self-antigens in melanoma (Trp2) and breast cancer (Her2) produced similar results, suggesting selective CD4(+) T-cell tolerance underlies ineffective vaccination against many cancer antigens. Thus, identification of self-antigens characterized by selective CD4(+) T-cell tolerance and abrogation of such tolerance through self-antigen-independent T-cell help is essential for future immunotherapeutics.

Tumor radiation therapy creates therapeutic vaccine responses to the colorectal cancer antigen GUCY2C.

PURPOSE: Radiation therapy (RT) is thought to produce clinical responses in cancer patients, not only through direct toxicity to cancer cells and supporting tumor stroma cells, but also through activation of immunologic effectors. More recently, RT has potentiated the local and systemic effects of cancer immunotherapy (IT). However, combination regimens that maximize immunologic and clinical efficacy remain undefined. METHODS AND MATERIALS: We evaluated the impact of local RT on adenoviral-mediated vaccination against the colorectal cancer antigen GUCY2C (Ad5-GUCY2C) in a murine subcutaneous tumor model using mouse CT26 colon cancer cells (CT26-GUCY2C). Immune responses were assessed by ELISpot, and clinical responses were assessed by tumor size and incidence. RESULTS: The specific sequence of tumor-directed RT preceding Ad5-GUCY2C IT transformed inactive therapeutic Ad5-GUCY2C vaccination into a curative vaccine. GUCY2C-specific T cell responses were amplified (P<.05), tumor eradication was maximized (P<.01), and tumor volumes were minimized (P<.001) in mice whose tumors were irradiated before, compared with after, Ad5-GUCY2C vaccination. The immunologic and antitumor efficacy of Ad5-GUCY2C was amplified comparably by unfractionated (8 Gy × 1), or biologically equivalent doses of fractionated (3.5 Gy × 3), RT. The antitumor effects of sequential RT and IT (RT-IT) depended on expression of GUCY2C by tumor cells and the adenoviral vaccine vector, and tumor volumes were inversely related to the magnitude of GUCY2C-specific T cell responses. Moreover, mice cured of CT26-GUCY2C tumors by RT-IT showed long-lasting antigen-dependent protection, resisting tumors formed by GUCY2C-expressing 4T1 breast cancer cells inoculated 50 days after CT26 cells. CONCLUSIONS: Optimal sequencing of RT and IT amplifies antigen-specific local and systemic immune responses, revealing novel acute and long-term therapeutic antitumor protection. These observations underscore the importance of modality sequence optimization before the initiation of clinical trials of RT and IT to maximize immune and antitumor responses.

Colorectal cancer immunotherapy.

Antitumor immunotherapy for colorectal cancer has been studied at the bench and bedside for decades. Some clinical trials of cancer immunotherapy have demonstrated a potential benefit for patients with colorectal cancer, yet immunotherapy remains only an experimental option for this disease. Here, we review the major immunotherapeutic approaches currently under investigation for colorectal cancer, including cancer vaccines and adoptive cell therapy. Weakness and advantages of each strategy and progress in clinical trials will be described. Examination of previous and ongoing research in colorectal cancer therapy should define a path towards identification, approval, and mainstream adoption of colorectal cancer immunotherapeutics.

Immunotherapeutic strategies to target prognostic and predictive markers of cancer.

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

Convergence of oncogenic and hormone receptor pathways promotes metastatic phenotypes.

Cyclin D1b is a splice variant of the cell cycle regulator cyclin D1 and is known to harbor divergent and highly oncogenic functions in human cancer. While cyclin D1b is induced during disease progression in many cancer types, the mechanisms underlying cyclin D1b function remain poorly understood. Herein, cell and human tumor xenograft models of prostate cancer were utilized to resolve the downstream pathways that are required for the protumorigenic functions of cyclin D1b. Specifically, cyclin D1b was found to modulate the expression of a large transcriptional network that cooperates with androgen receptor (AR) signaling to enhance tumor cell growth and invasive potential. Notably, cyclin D1b promoted AR-dependent activation of genes associated with metastatic phenotypes. Further exploration determined that transcriptional induction of SNAI2 (Slug) was essential for cyclin D1b-mediated proliferative and invasive properties, implicating Slug as a critical driver of disease progression. Importantly, cyclin D1b expression highly correlated with that of Slug in clinical samples of advanced disease. In vivo analyses provided strong evidence that Slug enhances both tumor growth and metastatic phenotypes. Collectively, these findings reveal the underpinning mechanisms behind the protumorigenic functions of cyclin D1b and demonstrate that the convergence of the cyclin D1b/AR and Slug pathways results in the activation of processes critical for the promotion of lethal tumor phenotypes.

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

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

GUCY2C-targeted cancer immunotherapy: past, present and future.

For the last decade, we have focused on guanylyl cyclase C (GUCY2C) as a potentially ideal target antigen for colorectal cancer immunotherapy. GUCY2C is expressed only in intestinal epithelial cells and by nearly 100% of colorectal cancers. We have developed and tested a recombinant adenoviral vector possessing GUCY2C (Ad5-GUCY2C) as a candidate vaccine for colorectal cancer patients. Murine studies have revealed that this vaccine is safe and effective against GUCY2C-expressing targets, and Ad5-GUCY2C is poised for phase I clinical testing in colorectal cancer patients with minimal residual disease. Moreover, we are developing second-generation GUCY2C-targeted therapeutics, including the use of chimeric antigen receptor (CAR)-expressing T cells, for treatment of patients with advanced colorectal cancer for whom Ad5-GUCY2C immunization is not appropriate. Thus, a family of GUCY2C-targeted immunotherapeutics may bridge the gap in effective treatments for the 500,000 patients worldwide who die annually from colorectal cancer.

A uroguanylin-GUCY2C endocrine axis regulates feeding in mice.

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

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

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