First generation, E1-deleted Adenovirus subtype 5 (Ad5)-based vectors, although promising platforms for use as cancer vaccines, are impeded in activity by naturally occurring or induced Ad-specific neutralizing antibodies. Ad5-based vectors with deletions of the E1 and the E2b regions (Ad5 [E1-, E2b-]), the latter encoding the DNA polymerase and the pre-terminal protein, by virtue of diminished late phase viral protein expression, were hypothesized to avoid immunological clearance and induce more potent immune responses against the encoded tumor antigen transgene in Ad-immune hosts. Indeed, multiple homologous immunizations with Ad5 [E1-, E2b-]-CEA(6D), encoding the tumor antigen CEA, induced CEA-specific cell-mediated immune (CMI) responses with antitumor activity in mice despite the presence of pre-existing or induced Ad5-neutralizing antibody. In the present phase I/II study, cohorts of patients with advanced colorectal cancer were immunized with escalating doses of Ad5 [E1-, E2b-]-CEA(6D). CEA-specific CMI responses were observed despite the presence of pre-existing Ad5 immunity in a majority (61.3%) of patients. Importantly, there was minimal toxicity, and overall patient survival (48% at 12 months) was similar regardless of pre-existing Ad5 neutralizing antibody titers. The results demonstrate that, in cancer patients, the novel Ad5 [E1-, E2b-] gene delivery platform generates significant CMI responses to the tumor antigen CEA in the setting of both naturally acquired and immunization-induced Ad5-specific immunity.

Adenovirus serotype 5 (Ad5) has been widely used in clinical trials because it expresses inserted transgenes robustly and augments the innate immune response. Strategies to improve Ad5 vectors that can circumvent Ad5 immunity have become a critical issue, especially for use as a cancer immunotherapeutic in which repeated immunization is required. In this study, we constructed a novel Ad5 vector with unique deletions of the viral DNA polymerase and the pre-terminal protein region (Ad5 [E1-, E2b-]). This vector contains the carcinoembryonic antigen (CEA) gene insert and is designed to induce cell-mediated immunity (CMI) against the tumor-associated target. The CEA immunogenicity and in vivo anti-tumor effects of repeated immunizations with Ad5 [E1-, E2b-]-CEA compared with those observed with current generation Ad5 [E1-]-CEA were tested in Ad5 pre-immunized mice. We report that Ad5-immune mice immunized multiple times with Ad5 [E1-, E2b-]-CEA induced CEA-specific CMI responses that were significantly increased over those detected in Ad5-immune mice immunized multiple times with a current generation Ad5 [E1-]-CEA. Ad5 immune mice bearing CEA-expressing tumors that were treated with Ad5 [E1-, E2b-]-CEA had increased anti-tumor response as compared with Ad5 [E1-]-CEA treated mice. These results demonstrate that Ad5 [E1-, E2b-]-CEA can induce CMI immune responses which result in tumor growth inhibition despite the presence of pre-existing Ad5 immunity. Multiple re-immunizations using the same vector platform are now possible with the novel Ad5 [E1-, E2b-] platform.

SummaryAdenovirus vectors have been shown to be highly effective as vaccine platforms capable of inducing both humoral and cell mediated immune responses. An Ad serotype 5 vector containing unique deletions in the E2b region (Ad5 [E1-, E2b-]) has been reported to have several advantages over conventional Ad5 vectors deleted in only the E1 region (Ad5 [E1-]), including increased carrying capacity and diminished viral late gene expression. Here, we evaluated a novel Ad5 [E1-, E2b-] vector utilizing the E.C7 cell line for viral packaging. Its' effectiveness as a potential vaccine platform as compared to the currently utilized Ad5 [E1-] based platform was assessed in both Ad5 naïve and Ad5 immune mice. We employed the HIV-1 Gag gene as the antigenic transgene expressed by the novel vector. Cellular expression of the Gag was confirmed by Western Blot analysis. Dose response studies using three intradermal immunizations of 10 7 to 10 10 virus particles (VP) of each construct revealed that immunization with 10 10 VP resulted in the maximum immunological response. Fax: (206) 838-2978, E-mail: beth@etubics.com. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. NIH Public Access Author ManuscriptImmunol Lett. Author manuscript; available in PMC 2010 January 29. Published in final edited form as:Immunol Lett. Materials and Methods AnimalsSpecific pathogen-free, BALB/c mice (Jackson Laboratory, Bar Harbor, Maine) ages 6 to 8 weeks were housed in animal facilities at the Infectious Disease Research Institute (Seattle, Washington) and all procedures were conducted according to IACUC approved protocols. Cynomolgus macaques were housed at the Southern Research Institute (SRI, Frederick, MD) and all protocols were reviewed and approved by appropriate animal care and biosafety committees before initiation of the study. NHP peripheral blood mononuclear cells (PBMC) and serum samples were collected by SRI and shipped overnight to Etubics Corporation (Seattle, WA) for analysis. Vaccine VectorsUsing the HXB Gag gene (Genbank Accession # K03455) derived from pVRC3900 Infectious titers were determined on 293 cell monolayers which had plaque-forming titers of 3.0 × 10 10 and 5.0 × 10 10 PFU/ml for the Ad5 [E1-]-gag and Ad5 [E1-, E2b-]-gag virus preparations, respectively. The manufacturing particle concentrations were determined spectrophotometrically and were 1.1 × 10 12 virus particles/ml for both viral lots. Thus, the ratios of particle number to PFU were similar for both virus lots, 36 versus 22 VP/PFU, respectively. ImmunizationMice were injected with Ad5 [E1-]-gag or Ad5 [E1-, E2b-]-gag ...