CANCER MUCOSA ANTIGENS A NOVEL PARADIGM IN CANCER IMMUNOTHERAPEUTICS

Abstract

Employing antigens with expression restricted to normal intestinal mucosa and derivative colorectal tumors – cancer mucosa antigens (CMAs) – represents a novel paradigm in anti-tumor immunotherapy. Immune compartmentalization limits tolerance to CMAs and restricts mucosa-targeted autoimmunity, allowing safe and effective immunotherapy for metastatic colorectal cancer. Guanylyl cyclase C (GCC), an intestine/colorectal cancer-restricted protein, is poised for clinical evaluation as the index CMA.

Colorectal Cancer Immunotherapy

Colorectal cancer is a leading cause of cancer-related deaths in the world, producing ~1 million cases and ~500,000 deaths worldwide each year [1]. Surgery and adjuvant fluoropyrimidine therapy for advanced disease, remains the standard of care, yet the overall 5-year mortality of this disease remains ~60% [1]. This underscores the unmet need for effective colorectal cancer therapeutics. Several observations suggest that immunological targeting may offe r a therapeutically advantageous approach to the management of patients with colorectal cancer, including the positive prognosis of patients with infiltrating immune cells [2] suggesting that immune responses favorably impact clinical outcome [3]. Yet, clinical evaluation of immunotherapy for colorectal cancer has been disappointing, resulting in a clinical response rate of <1% from >30 studies examining >500 patients [4]. However, examination of tested antigens suggests a paucity of suitable targets for this disease, and a conspicuous absence of tissue-specific differentiation antigens targeted in colorectal cancer [5,6]. Their dearth may reflect a presumed lack of efficacy targeting self-antigens or risk of collateral autoimmune disease. However, exploitation of immune compartmentalization may offer a unique immunotherapeutic opportunity in colorectal cancer targeting mucosa-restricted differentiation antigens.

Immune Compartmentalization

An established paradigm in immunity suggests the segregation of immune compartments including systemic and mucosal compartments, as well as those of the central nervous system, testes, eye, etc. Immune compartmentalization contributes significantly to immunity against invading microorganisms and prevention of autoimmune disease by increasing the efficiency of regional immune responses, decreasing tissue antigen cross-reactivity and establishing appropriate tissue-compatible effector mechanisms (e.g., IgA in mucosa) [7]. Structural and functional compartmentalization is reflected by the tissue-restricted recirculation of effector lymphocytes such that central compartment immune responses rarely extend to mucosal surfaces [5,6]. Independence of immune responses in mucosae is reminiscent of immunologically privileged tissues, wherein central immune responses rarely extend to privileged compartments. This immunological independence may be exploited to generate therapeutic responses to cancers employing antigens that are normally restricted to mucosa, but expressed during carcinogenesis – cancer mucosa antigens. These antigens are advantageous due to potentially limited central compartment tolerance, enhancing antitumor immunity and the absence of compartment cross-talk, limiting autoimmune disease. Employing mucosal self-antigens expressed by normal intestinal epithelium and derivative tumors for targeted immunotherapy of colorectal cancer represents a significant paradigm shift, since these antigens have remained unexplored.

Guanylyl Cyclase C

GCC is a member of the guanylyl cyclase family of receptors, converting cytosolic GTP to the second messenger cyclic GMP upon engagement with its endogenous hormones, guanylin and uroguanylin, and the diarrheagenic bacterial heat-stable enterotoxins. GCC is expressed selectively by intestinal epithelial cells from the duodenum to the rectum and expression persists through all stages of colorectal tumorigenesis, including primary and metastatic tumors [8]. GCC expression has been identified in 100% of intestinal specimens including normal intestine and primary and metastatic colorectal tumors (>100 specimens) but not in normal extra-intestinal tissues or tumors (>100 specimens). This persistent expression of GCC by metastatic tumors of the intestine but not by normal extra-intestinal tissues makes GCC a sensitive and specific molecular marker for staging patients with colorectal tumors, a critical prognostic determinant of patient management and survival [9]. The uniform expression by colorectal tumors also underscores the potential utility of GCC as a therapeutic target in managing patients with colorectal cancer.

Immunological compartmentalization suggests that CMAs may be ideal immunotherapeutic targets. Reflecting its mucosa- and colorectal cancer- restricted expression, GCC-targeted immunotherapy has been examined in pre-clinical models [10]. Immunization with recombinant viral vectors expressing GCC protected mice against challenge with GCC-expressing colorectal cancer cells, reducing tumor burden ~80–90% in mice with lung or liver metastases (Fig. 1). Interestingly, GCC-targeted anti-tumor immunity is mediated by unique lineage-specific immune cell responses following immunization, which included CD8⁺ T cells, but not CD4⁺ T or B cells (Fig. 2). Importantly, GCC-immunization did not induce intestinal autoimmune disease, suggesting remarkable safety despite generation of self antigen-targeted immunity.

Figure 1.

GCC-targeted immunity against colorectal cancer metastatic to lung. Mice immunized with recombinant adenovirus (AV) expressing GCC, but not a control AV, were protected against GCC-expressing colorectal cancer metastatic to the lungs. Tumor burden was reduced 80–90% in metastatic models to lung or liver. Reproduced from reference 10.

Figure 2.

Lineage-specific immune responses to GCC. Mice immunized with AV expressing GCC generated CD8+ T cell, but not CD4+ T or B cell, responses to GCC. A) Antibody responses were measured by ELISA. CD4+ T cell (B) and CD8+ T cell (C) responses were measured by IFNγ-ELISpot. Reproduced from reference 10.

Conclusion

These results establish proof-of-principle of CMA-targeted anti-tumor immunity confirming that immune compartmentalization provides a previously unexplored opportunity for immunization against antigens selectively expressed in mucosae, providing therapeutic immunity against CMA-expressing tumors. While molecular mechanisms underlying immune compartmentalization beyond the GI tract remain poorly understood, CMA-directed immunotherapy may be applicable to malignancies derived from other mucosae, such as oral, respiratory, mammary and urogenital tissues, to treat head and neck, lung, breast and bladder cancers, respectively.