[CANCER RESEARCH 61, 4497– 4505, June 1, 2001] Synergy of Vaccine Strategies to Amplify Antigen-specific Immune Responses and Antitumor Effects Douglas W. Grosenbach, Jacqueline C. Barrientos, Jeffrey Schlom, 1 and James W. Hodge Laboratory of Tumor Immunology and Biology, National Cancer Institute [D. W. G., J. S., J. W. H.] and Howard Hughes Medical Institute, Research Scholars at the NIH [J. C. B.], NIH, Bethesda, Maryland 20892-1750 ABSTRACT Several different vaccine strategies have been evaluated and combined in an attempt to amplify T-cell responses toward induction of antitumor immunity. The model tumor antigen used was carcinoembryonic antigen (CEA). While initial T-cell activation studies were conducted in conven- tional mice, combined vaccine strategy studies and antitumor studies were conducted in transgenic mice in which CEA is expressed in normal gastrointestinal tissue and CEA protein is found in sera. The studies reported here demonstrate: (a) A recombinant avipox (fowlpox, rF) vector expressing the signal 1 (CEA) and the B7-1 costimulatory molecule trans- genes (designated rF-CEA/B7-1) is more potent in inducing CEA-specific T-cell responses than rF-CEA; one administration of recombinant fowl- pox vector expressing CEA and three different costimulatory molecule transgenes (B7-1, ICAM-1, LFA-3, designated rF-CEA/TRICOM) was more potent in inducing CEA-specific T-cell responses than four vaccina- tions with rF-CEA or two vaccinations with rF-CEA/B7-1. Moreover, up to four vaccinations with rF-CEA/TRICOM induced greater CEA-specific T-cell responses with each vaccination. (b) A diversified prime and boost strategy using a prime with a recombinant vaccinia vector expressing CEA and the triad of costimulatory molecules (designated rV-CEA/TRI- COM) and a boost with rF-CEA/TRICOM was more potent in inducing CEA-specific T-cell responses than the repeated use of rF-CEA/TRICOM alone. (c) The addition of granulocyte macrophage colony-stimulating factor (GM-CSF) to the rF-CEA or rF-CEA/TRICOM vaccinations via the simultaneous administration of a rF-GM-CSF vector enhanced CEA- specific T-cell responses. These strategies (TRICOM/diversified prime and boost/GM-CSF) were combined to treat CEA-expressing carcinoma liver metastases in CEA-transgenic mice; vaccination was initiated 14 days posttumor transplant. Antitumor effects in terms of survival and CD8  and CD4  responses specific for CEA were also observed in this CEA-transgenic mouse model. These studies demonstrate that the use of cytokines and diversified prime and boost regimens can be combined with the use of recombinant vectors expressing signal 1 and multiple costimu- latory molecules to further amplify T-cell responses toward more effective vaccine strategies. INTRODUCTION The general hypothesis involving the induction of immune re- sponses to TAAs 2 is that the antigens are extremely weak immuno- gens or functionally nonimmunogenic in the tumor-bearing host (1– 5). Antitumor effects in many experimental vaccine studies have been correlated with T-cell responses to TAAs. However, this does not rule out the possibility that other immune mechanisms involving antibod- ies or other effector cells are also involved in antitumor effects. Nonetheless, the vast majority of experimental vaccine studies have demonstrated the role of both CD8 + and CD4 + T cells in antitumor responses (6 – 8). Most experimental and some clinical vaccine studies to date have used various strategies to enhance T-cell responses to specific TAAs. Among these are: (a) the use of vectors for more efficient delivery of the TAA to the APC (9 –12) and for more efficient processing with MHC molecules (13, 14); (b) the use of T-cell costimulation either by antibody-mediated blockade mechanisms (15, 16) or via inserting costimulatory molecules into vectors (17–19); (c) the use of cytokines to enhance either APC function or T-cell function (20 –25); and (d) the use of diversified prime and boost vaccine strategies (26 –32). Al- though several studies have used two or more of the above vaccine strategies, few studies, if any, have analyzed multiple strategies to determine maximum ability to activate T cells. In previous experimental studies, we and others have demonstrated the following: (a) The use of recombinant orthopox vectors such as vaccinia or avipox [fowlpox and/or canarypox (ALVAC)] to enhance T-cell responses to TAAs and to induce antitumor immunity as compared to the use of the TAA protein in adjuvant (5, 27). (b) Diversified vaccine prime and boost regimens were advantageous in enhancing T-cell responses. A rV vector cannot be used multiple times because of the induction of host immunity to the virus (33–36). However, it has been shown that a primary vaccination with a rV vector followed by booster vaccinations with recombinant avipox viruses leads to optimal induction of T-cell responses (32). In these studies and others, it has been shown that the use of two different recombinant vectors, in prime and boost strategies, can be more potent in inducing T-cell responses than the continued use of one single vector. (c) The insertion of genes for a costimulatory molecule in vectors also containing the gene for a TAA enhances T-cell responses to the TAA. Moreover, the use of rV or rF vectors containing a triad of costimulatory molecules (B7-1, ICAM-1, LFA-3; designated TRI- COM) has been shown to activate T cells to greater levels than the use of any one or two of these costimulatory molecules in recombinant vectors (18). (d) The use of cytokines such as GM-CSF has been shown to enhance the infiltration of APC, including dendritic cells, to regional nodes and consequently enhance T-cell responses when given for 4 consecutive days following vaccine (5, 37). More recently, it has been shown that a single administration of rF-GM-CSF given with vaccine is as potent as four daily administrations of recombinant GM-CSF in enhancing T-cell responses (38). In addition, it has been previously shown that when IL-2 has been used in low doses several days following vaccination, antigen-specific T-cell responses can be further enhanced (25). Although the vast majority of experimental vaccine studies in mouse models have used conventional mice, and the target antigen is not “self,” most human TAAs identified to date have been shown to be “self-antigens,” with some expression on normal adult tissues. CEA is a TAA, which is overexpressed in most carcinomas, including gastrointestinal carcinomas, and is also expressed at lower levels in normal colonic mucosa (39). The most common site of metastases of CEA-positive malignancies in patients is the liver; CEA protein is also shed into the serum of many patients with metastatic CEA-positive malignancies (39). There are several clinical studies that have now Received 11/27/00; accepted 4/3/01. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 To whom requests for reprints should be addressed, at Laboratory of Tumor Immu- nology and Biology, National Cancer Institute, NIH, 10 Center Drive, Room 8B09, Bethesda, MD 20892. Phone: (301) 496-4343; Fax: (301) 496-2756; E-mail address: js141c@nih.gov. 2 The abbreviations used are: TAA, tumor-associated antigen; APC, antigen-presenting cell; TRICOM, triad of costimulatory molecules; rV, recombinant vaccinia; rF, recombi- nant fowlpox; GM-CSF, granulocyte macrophage colony-stimulating factor; IL-2, inter- leukin 2; CEA, carcinoembryonic antigen; FP-WT, fowlpox wild type; V-WT, vaccinia WT; pfu, plaque-forming unit; ConA, concanavalin A. 4497 Research. on February 25, 2016. © 2001 American Association for Cancer cancerres.aacrjournals.org Downloaded from