Guanylyl Cyclase C–Induced Immunotherapeutic Responses Opposing Tumor Metastases Without Autoimmunity

Abstract

Background One of the greatest impediments to cancer immunotherapy is the paucity of antigens that are tumor-specific, sufficiently immunogenic, and shared among patients. Mucosa-restricted antigens that are expressed by tumor cells represent a novel class of vaccine targets exploiting immunologic privilege, which limits systemic tolerance to those antigens, and immunologic partitioning, which shields mucosae from systemic autoimmune responses. Here we defined the immunogenicity and antitumor efficacy of guanylyl cyclase C (GCC), a protein that is normally restricted to intestinal mucosa and universally expressed by metastatic colorectal cancer. Methods BALB/c mice (n=197) were immunized with recombinant viral vectors before (prophylactic) or after (therapeutic) a lethal challenge of GCC-expressing mouse colon cancer cells, and antitumor efficacy was monitored by quantifying metastasis and survival. Induction of autoimmunity was monitored by histopathology. Induction of GCC-specific B cell and CD4+ and CD8+ T cell responses were determined by enzyme-linked immunosorbent assay (ELISA) and ELISpot, respectively. Tolerance to GCC was quantified by comparing responses in GCC-deficient (n=45) and wild-type (n=69) C57BL/6 mice. Statistical tests were two-sided. Results Immunization with GCC-expressing viral vectors reduced the formation of metastases to liver (control vs GCC: mean = 30.4 vs 3.55 nodules, difference = 26.9 nodules, 95% confidence interval [CI] = 8.47 to 45.3 nodules; P = .008) and lung (control vs GCC: mean =263 vs 55.7 nodules, difference = 207, 95% CI = 163 to 251; P < .001) and extended the median survival of mice with established lung metastases following therapeutic immunization (control vs GCC: 29 vs 38 days, P = .024), without autoimmunity. Antitumor efficacy reflected asymmetric tolerance that was characterized by CD8+ T cell, but not CD4+ T cell or antibody, responses. Conclusions Immunologic partitioning together with immunologic privilege highlight the potential of mucosa-restricted antigens, particularly GCC, as therapeutic targets for metastatic cancer.

A uroguanylin-GUCY2C endocrine axis regulates feeding in mice

“…BALB/c-derived CT26 colorectal cancer cells were obtained from ATCC. This cell line lacks endogenous GUCY2C, established by radiolabeled ligand binding and quantitative RT-PCR (qRT-PCR) analyses (52). For the generation of the stable CT26 cell line expressing GUCY2C, RNA was purified from mucosal scrapings of the jejunum of a 3-month-old C57BL/6 mouse.…”

“…RT-PCR was performed using Ex-Taq (Takara) to amplify cDNA, which was cloned into pENTR/D-TOPO (Invitrogen) and subcloned into pMSCV2.2-Puro to generate Gucy2c-pMSCV2.2-Puro. CT26 cells were transduced with retrovirus produced from 293T cells that were transiently transfected with pCL-Ampho (Imgenex) and Gucy2c-pMSCV2.2-Puro, followed by selection with 50 μg/ml puromycin (EMD Chemicals) (52).…”

“…injection. The truncated GUCY2C1-430-expressing adenovirus (GUCY2CECD-AV) was generated as described previously (52). Three weeks later, mice were boosted i.p.…”

“…in PBS 3 days before spleen collection. GUCY2CECD protein was purified as described previously (52). Spleens were collected 26 days after initial priming and fused with the mouse myeloma cell line Sp2/0-Ag14 (ATCC) to create hybridomas using the ClonaCell-HY Hybridoma Cloning Kit (Stemcell Technologies).…”

“…Spleens were collected 26 days after initial priming and fused with the mouse myeloma cell line Sp2/0-Ag14 (ATCC) to create hybridomas using the ClonaCell-HY Hybridoma Cloning Kit (Stemcell Technologies). Positive clones were screened by detection of purified GUCY2CECD in ELISA (52). After 3 rounds of limiting dilution to ensure stability and clonality, the MS20 hybridoma was grown in the BD CELLine Disposable Bioreactor (BD Biosciences), and antibody was purified from supernatants using a protein G column.…”