The generation and analyses of a novel combination of recombinant adenovirus vaccines targeting three tumor antigens as an immunotherapeutic

Abstract

Phenotypic heterogeneity of human carcinoma lesions, including heterogeneity in expression of tumor-associated antigens (TAAs), is a well-established phenomenon. Carcinoembryonic antigen (CEA), MUC1, and brachyury are diverse TAAs, each of which is expressed on a wide range of human tumors. We have previously reported on a novel adenovirus serotype 5 (Ad5) vector gene delivery platform (Ad5 [E1-, E2b-]) in which regions of the early 1 (E1), early 2 (E2b), and early 3 (E3) genes have been deleted. The unique deletions in this platform result in a dramatic decrease in late gene expression, leading to a marked reduction in host immune response to the vector. Ad5 [E1-, E2b-]-CEA vaccine (ETBX-011) has been employed in clinical studies as an active vaccine to induce immune responses to CEA in metastatic colorectal cancer patients. We report here the development of novel recombinant Ad5 [E1-, E2b-]-brachyury and-MUC1 vaccine constructs, each capable of activating antigen-specific human T cells in vitro and inducing antigen-specific CD4+ and CD8+ T cells in vaccinated mice. We also describe the use of a combination of the three vaccines (designated Tri-Ad5) of Ad5 [E1-, E2b-]-CEA, Ad5 [E1-, E2b-]-brachyury and Ad5 [E1-, E2b-]-MUC1, and demonstrate that there is minimal to no "antigenic competition" in in vitro studies of human dendritic cells, or in murine vaccination studies. The studies reported herein support the rationale for the application of Tri-Ad5 as a therapeutic modality to induce immune responses to a diverse range of human TAAs for potential clinical studies.

Keywords: adenovirus vaccines; brachyury; cancer vaccines; immunotherapy; tumor antigens.

Conflict of interest statement

CONFLICTS OF INTEREST

Laboratory of Tumor Immunology and Biology, NCI:

No potential conflicts of interest were disclosed.

Etubics Corporation:

Elizabeth S. Gabitzsch is a shareholder and employee of Etubics and has stock options in the Company. Adrian Rice is an employee of Etubics and has stock options in the Company. Yvette Latchman is an employee of Etubics and has stock options in the Company. Joseph P. Balint is a shareholder and employee of Etubics and has stock options in the Company. Frank R. Jones is a shareholder and employee of Etubics and has stock options in the Company.

Figures

Figure 1. Expression of brachyury and MUC1 protein in human dendritic cells (DCs) infected with Ad5 [E1-, E2b-]- brachyury and Ad5 [E1-, E2b-]-MUC1

SW620 tumor cells were used as positive control. Actin was used as a loading control. A. Expression of brachyury was robust in DCs infected with Ad5 [E1-, E2b-]-brachyury. B. MUC1 expression was observed in human DCs infected with Ad5 [E1-, E2b-]-MUC1 vector as compared to DCs infected with Ad5 [E1-, E2b-]-null (no transgene).

Figure 2. Analysis of IFN-γ− and IL-2−expressing splenocytes following vaccination of mice with Ad5 [E1-, E2b-]- brachyury, Ad5 [E1-, E2b-]-CEA, Ad5 [E1-, E2b-]-MUC1, Tri-Ad5, or Ad5 [E1-, E2b-]-null

C57Bl/6 mice (n = 5/group) were vaccinated three times at 2-week intervals with 1010 VP (viral particle) of Ad5 [E1-, E2b-]-brachyury (white bar), Ad5 [E1-, E2b-]-CEA (grey bar), Ad5 [E1-, E2b-]-MUC1 (black bar) or Tri-Ad5 (1:1:1 mixture of 1010 VP each of Ad5 [E1-, E2b-]-brachyury, Ad5 [E1-, E2b-]-CEA, Ad5 [E1-, E2b-]-MUC1) (diagonal hatched bar). Controls received 3 × 1010 VP of Ad5 [E1-, E2b-]-null (horizontal striped bar). Splenocytes were collected 14 days after the final vaccination and assessed for IFN-γ−secreting cells A. or IL-2-secreting cells B. by ELISPOT assay. For positive controls, splenocytes were exposed to Concanavalin A (Con A) (data not shown). Data reported as the number of spot forming cells (SFCs) per 106 splenocytes. The error bars depict the SEM. Significant differences (p < 0.05) between columns are reported in p-values, not significant = ns.

Figure 3. Analysis of CD8+ and CD4+ and multifunctional cellular populations following vaccination with Ad5 [E1-, E2b-]-brachyury, Ad5 [E1-, E2b-]-CEA, Ad5 [E1-, E2b-]-MUC1, Tri-Ad5, or Ad5 [E1-, E2b-]-null

C57Bl/6 mice (n = 5/group) were vaccinated three times at 2-week intervals with 1010 VP (viral particle) of Ad5 [E1-, E2b-]-brachyury (white bar), Ad5 [E1-, E2b-]-CEA (grey bar), Ad5 [E1-, E2b-]-MUC1 (black bar) or Tri-Ad5 (1:1:1 mixture of 1010 VP (viral particle) each of Ad5 [E1-, E2b-]-brachyury, Ad5 [E1-, E2b-]-CEA, Ad5 [E1-, E2b-]-MUC1) (diagonal hatched bar). Controls received 3 × 1010 VP of Ad5 [E1-, E2b-]-null (horizontal striped bar). Splenocytes were collected 14 days after the final vaccination and were assessed by FACS for CD8α+A. and CD4+B. IFN-γ–secreting cells, or for CD8α+C. and CD4+D. cells secreting IFN-γ and TNF-α. For positive controls, splenocytes were exposed to Concanavalin A (Con A) (data not shown). The error bars depict the SEM. Significant differences (p < 0.05) between columns are reported in p-values, not significant = ns.

Figure 4. CEA antibody activity from sera from mice vaccinated with Ad5 [E1-, E2b-]-CEA or Tri-Ad5

CEA IgG levels in mice vaccinated three times with 1010 VP (viral particle) of Ad5 [E1-, E2b-]-CEA (grey bar), Tri-Ad5 (diagonal hatched bar) or 3 × 1010 VP of Ad5 [E1-, E2b-]-null (horizontal striped bar) were determined by ELISA A. Complement-dependent cytotoxicity (CDC) against MC38-CEA2 cells was performed B. The error bars depict the SEM. Significant differences (p < 0.05) between columns are reported in p-values, not significant = ns.

Figure 5. Comparison of immunotherapy of MUC1-expressing tumors using Ad5 [E1-, E2b-]-MUC1 vs. Tri-Ad5

C57Bl/6 mice (n = 7/group) were inoculated with 106 MC-38-MUC1 cells subcutaneously in the left flank. Mice were administered 1010 VP (viral particle) of Ad5 [E1-, E2b-]-MUC1 or Tri-Ad5 (1:1:1 mixture of 1010 VP each of Ad5 [E1-, E2b-]-CEA, Ad5 [E1-, E2b-]-MUC1, and Ad5 [E1-, E2b-]-brachyury, 3 × 1010 VP total). A control group of mice received 3 × 1010 VP of Ad5 [E1-, E2b-]-null (no transgene). Tumor growth was monitored and volumes calculated. (*) indicates days when Ad5 [E1-, E2b-]-MUC1 treated mice had significantly smaller (p < 0.05) tumors than control mice and (^) indicates days when Tri-Ad5–treated mice had significantly smaller (p < 0.05) tumors than control mice. There was no significant difference (p > 0.1) between Ad5 [E1-, E2b-]-MUC1 vs. Tri-Ad5–treated mice at any time point. Error bars represent the SEM.