1B)

1B). mice. This study characterized the biodistribution, immunogenicity, and safety of a vector expressing GUCY2C fused with the human CD4+T helper cell epitope PADRE (Ad5-GUCY2C-PADRE) to advance this vaccine into clinical trials in colorectal cancer patients. Ad5-GUCY2C-PADRE levels were highest in the injection site and distributedin vivoprimarily to draining lymph nodes, the liver, spleen and, unexpectedly, to the bone marrow. Immune responses following Ad5-GUCY2C-PADRE administration were characterized by PADRE-specific CD4+T-cell and GUCY2C-specific B-cell and CD8+T-cell responses, producing antitumor immunity targeting GUCY2C-expressing colorectal cancer metastases in the lungs, without acute or chronic autoimmune or other toxicities. Collectively, these data support Ad5-GUCY2C-PADRE as a safe and effective vaccination strategy in preclinical models and position Ad5-GUCY2C-PADRE for Phase I clinical testing in colorectal cancer patients. Keywords:: colorectal cancer, GUCY2C, vaccine == Introduction == Guanylate cyclaseC (GUCY2C), one of a family of homologous proteins, is selectively expressed by intestinal epithelial cells, 1, 2where it catalyzes the production of cGMP following binding of its paracrine hormones guanylin and uroguanylin. 2GUCY2C is normally expressed in intestinal epithelium and neurons of the hypothalamus3and substantia nigra, 4but not by other normal tissues. 1, 2, 5, 6GUCY2C is also found in all colorectal cancers, and a subset of gastric, esophageal, Sophoradin and pancreatic cancers. 1, 58Limited expression in normal tissues and universal overexpression by primary and metastatic colorectal cancer (CRC) makes GUCY2C a useful biomarker for lymph node metastases in CRC patients6, 9and potential chemotherapeutic10and immunotherapeutic1113target in humans with GUCY2C-expressing cancers. Development of a specific vaccine has leveraged the structurally distinct GUCY2C extracellular domain (GUCY2CECD), which is absent from other guanylate cyclase isoforms. 13Indeed, while catalytic domains across family members share 50% homology, the extracellular domain of GUCY2C exhibits <20% homology with other proteins and is antigenically unique. 2, 13In mouse models, replication deficient (E1 and E3 deleted) human serotype 5 adenovirus (Ad5) expressing the extracellular domain of GUCY2C produced GUCY2C-specific CD8+T-cell responses, but no CD4+T- or B -cell responses. 11, 13Because GUCY2C-deficient mice produced robust GUCY2C-specific CD4+T- and B -cell responses, 11, 13their absence in wild-type mice reflected self-tolerance, a complex program of immune cell selection mechanisms that eliminate or suppress self-reactive immunity to prevent autoimmunity. 14, 15Importantly, Sophoradin Ad5-GUCY2C vaccination producing CD8+T-cell responses protected mice with metastatic colorectal cancer in the lungs and liver, primary sites of colorectal cancer metastasis in humans, without autoimmune toxicity in GUCY2C-expressing tissues. 11, 13 Subsequent studies of GUCY2C tolerance uncovered a previously unknown mechanism of self-tolerance mediated by selective CD4+T-cell tolerance with preservation of self-antigen-specific CD8+T and B cells. 12Indeed, selective CD4+T-cell tolerance was the primary mechanism limiting vaccine efficacy targeting GUCY2C, tyrosinase-related protein 2 (Trp2) and Her2 in colorectal, melanoma, and breast cancers, respectively. 12Importantly, cancer vaccine efficacy could be restored by adding a foreign ( non-self ) CD4+T-cell epitope to these antigens, optimizing antitumor immunity. 12Initially, a CD4+T-cell epitope restricted to mouse H-2bmolecules was fused to GUCY2C to confirm proof-of-concept. 12To translate those findings into a clinical vaccine approach, this study explored the biodistribution, immunogenicity, and safety of Ad5-GUCY2C-PADRE, containing the human CD4+T-cell epitope PADRE (PAn DR Epitope), which can be advanced into Phase I clinical trials in colorectal cancer patients. == Materials and Methods == == Vector design and manufacturing == Mouse (NP_001120790) and human (NP_004954) GUCY2C sequences were aligned (Supplementary Fig. S1; Supplementary Data are available online atwww.liebertpub.com/hgtb) using Geneious v9 (Biomatters Ltd. ). Codon-optimized cDNA encoding mouse GUCY2C residues 1-429 with a C-terminal PADRE epitope (Fig. 1A) was cloned into the E1 region of pAd/CMV/V5 (Life Technologies) containing E1- and E3-deleted human serotype 5 adenovirus (Ad5; Fig. 1B). Ad5-GUCY2C-PADRE vector used for these studies was produced under Good Laboratory Practice (GLP) conditions in HEK293 cells and purified by CsCl ultracentrifugation at the Baylor College of Medicine in the Cell and Gene Therapy Vector Development Lab. Ad5-GUCY2C-PADRE vector was tested for replication-competent adenovirus (negative), sterility (negative), mycoplasma (negative), and host cell DNA contamination (negative), before employing it in preclinical studies. == Figure 1 . == Ad5-GUCY2C-PADRE design and antigen expression. (A) GUCY2C is a membrane-spanning enzyme possessing an extracellular ligand-binding domain, intracellular cGMP-producing catalytic domain, and intervening regulatory domains. The extracellular domain (ECD) of GUCY2C was employed in the vaccine design and Sophoradin included the PADRE epitope on its COOH-terminus. (B) GUCY2CECD-PADRE was inserted into the E1 region of E1/E3-deleted Ad5. T (C) HEK293 cells were transduced with Ad5-GUCY2C-PADRE at a multiplicity of infection (MOI) of 11, 000 for 48 h. Supernatants were analyzed for GUCY2C-PADRE expression by Western blot. Densitometry analysis was employed to quantify relative expression to dose 0 vp. (D) A549 cells were transduced with Ad5-GUCY2C-PADRE at a.