Gene Therapy (2000) 7, 481–492 2000 Macmillan Publishers Ltd All rights reserved 0969-7128/00 $15.00 www.nature.com/gt NONVIRAL TRANSFER TECHNOLOGY RESEARCH ARTICLE Regulation of T-helper-1 versus T-helper-2 activity and enhancement of tumor immunity by combined DNA- based vaccination and nonviral cytokine gene transfer K Song, Y Chang, and GJ Prud’homme Department of Pathology, McGill University, 3775 University Street, Room 13, Montreal, Quebec, Canada, H3A 2B4 Intramuscular (i.m.) injections of a plasmid encoding human type 2 (Th2) response. Antitumor immunity was enhanced carcinoembryonic antigen (CEA) elicited both humoral and when the CEA and IL-12 plasmids were coinjected at the cellular immune responses in mice, but only partial inhibition same muscle site, but not at separate sites despite of the growth of transplanted syngeneic CEA-positive P815 increased serum IL-12 levels. Though the tumor cells tumor cells (CEA/P815). Coinjection of the CEA vector with a expressed neomycin phosphotransferase, mice immunized vector encoding either interferon-␥ (IFN␥) or IL-12 promoted with vectors encoding that protein (without CEA) were not IgG2a isotype anti-CEA antibody production, anti-CEA/P815 protected against tumor growth, and produced no CTLs CTL activity and greater resistance to CEA/P815 tumor chal- except for low levels when coinjected with an IL-12 vector. lenge. As well, CEA/P815-stimulated IFN␥ secretion in vitro Thus, we show that immunity elicited by DNA vaccination was increased, but IL-4 diminished, consistent with a T- against CEA can be biased to a protective type (high Th1 helper type 1 (Th1) response. In contrast, coinjection of the and CTL activity) or nonprotective type (high Th2 and low CEA vector with an IL-4 vector increased IgG1 production, CTL activity) by i.m. coinjection of cytokine-expressing plas- but reduced CTL activity and resistance to tumor challenge. mids. IL-12 appears to act locally, but not systemically, The latter treatment inhibited CEA/P815-dependent IFN␥ through an adjuvant effect. Gene Therapy (2000) 7, 481– production but enhanced IL-4 secretion, consistent with a Th 492. Keywords: DNA vaccination; carcinoembryonic antigen; cytokine plasmids; T-helper cells; cytotoxic T lymphocytes; cancer immunotherapy Introduction Immunization usually requires use of adjuvants;15 however, many adjuvants cause undesirable side-effects, It has been shown that DNA vaccination can elicit both such as severe inflammation, which limits or precludes antigen-specific humoral and cell-mediated immune their use in humans.16 We investigated the adjuvant effect responses, including T-helper (Th) cell and cytotoxic T- of plasmids encoding a single cytokine on the immune lymphocyte (CTL) responses. The immune responses responses elicited by a plasmid encoding human CEA. generated by this technique in many experimental animal Thus, we coinjected the CEA plasmid with plasmids models can provide protection against infectious agents encoding mouse IFN␥, IL-12, or IL-4. Though cytokine or malignant tumor cells (reviewed in Ref. 1). A major expression plasmids have been found to enhance or mod- limitation of immunization with conventional vaccines is ify immune responses, most of this work has been perfor- the lack of CTL stimulation. The conventional vaccines med in infectious disease models,17–20 and relatively little stimulate mostly antibody and to varying degrees, Th cell in tumor models. In our study, a transplantable mouse 2–4 responses. The latter responses frequently fail to elim- mastocytoma cell line P815 was stably transfected to inate tumors or virally infected cells, while CTLs are express human CEA (CEA/P815). After five weekly plas- 5–8 more effective. On the other hand, DNA vaccines excel mid i.m. injections, anti-CEA humoral and anti- 9–11 at stimulating CTLs. CEA/P815 cellular immune responses were detected. We used human carcinoembryonic antigen (CEA) as an This immunity, however, only partially inhibited the antigen in mice, where it is not an endogenous protein, growth of CEA/P815 cells. Anti-CEA IgG isotypes, as with the purpose of studying strategies of DNA vacci- well as cytokine secretion by spleen cells were analyzed nation. In humans, CEA is one of the best characterized to reveal the dominant type of the immune response (Th1 tumor-associated antigens in terms of its tissue distri- versus Th2). By this method, we could readily bias the 12,13 bution and biochemistry. It is considered a potential anti-CEA/P815 T cell response to a more protective type 14 target antigen for cancer immunotherapy. (high Th1 and CTL activity) with either IFN␥ or IL-12 vectors, or to a nonprotective type (high Th2 and low CTL activity) with an IL-4 vector. Though circulating lev- Correspondence: GJ Prud’homme els of IL-12 were detectable after IL-12-vector treatment, Received 6 May 1999; accepted 5 November 1999 coinjection of that vector at the same site as the CEA vec- Regulation of anti-tumor response by cytokine vectors K Song et al 482 tor was required for optimal immunization. The use of preliminary experiments, we found that two DNA injec- cytokine vectors to regulate immunity is discussed. tions were not sufficient to generate CTL responses, while anti-CEA antibodies were detectable but could be further Results enhanced by additional injections. Four or five DNA injections were required consistently to generate CTLs and optimal antibody titers. For this reason, all experi- Construction of expression vectors ments in this study were performed with five DNA injec- Human CEA cDNA (full length) was inserted into the tions at weekly intervals. One week after the last injec- vector pCI-neo, and the resulting plasmid was desig- tion, serum samples were obtained for quantitation of nated pCI-CEA (Figure 1). This vector has the neo gene, IgG anti-CEA antibody. It has been shown that the Th1 encoding neomycin phosphotransferase (NPT). In some + and Th2 subsets of CD4 T cells promote different pro- experiments we also used pCMV and pCMV-CEA, that files of IgG isotype production. Strong IgG1 production do not encode NPT, but are otherwise identical to pCI- is an important indication of a Th2-dominant immune neo and pCI-CEA, respectively. Cell surface expression of response, whereas strong IgG2a production reflects a CEA directed by pCI-CEA or pCMV-CEA was confirmed Th1-dominant immune response.22 initially by transient transfection of COS-7 cells with As shown in Figure 2a, production of IgG anti-CEA these vectors, and analyzed by flow cytometry with anti- antibodies was elicited by i.m. injection of the CEA vector CEA mAb (data not shown). Mouse IFN␥ cDNA and into mice. Coinjection of pCI-CEA and VR-IL12 increased mouse IL-4 cDNA were also cloned into the pCI-neo vec- the production of IgG anti-CEA antibody compared with tor, and the resulting vectors were designated pCI-IFN␥ the group injected with pCI-CEA and control vector. and pCI-IL4, respectively. The plasmid encoding mouse Injection of a mixture of pCI-CEA and pCI-IL4, but not IL-12, designated VR-IL12, was constructed by cloning a pCI-IFN␥, also induced an increase in the total IgG anti- segment consisting of p35 cDNA, a synthetic intron (IVS), CEA antibody production. an internal ribosome entry site (IRES) sequence and p40 In all cases, the profiles of IgG1 and IgG2a isotypes in cDNA into the VR1255 plasmid (Figure 1). the serum of mice were influenced by injection of a mix- The in vitro production and secretion of IFN␥, IL-4, and ture of pCI-CEA and a cytokine vector as compared with IL-12 were confirmed by transient transfection of COS-7 a control vector (Figure 2b). A mixture of pCI-CEA with cells with the respective vectors. In all cases the respect- either pCI-IFN␥ or VR-IL12 induced higher IgG2a levels ive cytokines, as detected by ELISA, were secreted at lev- and lower IgG1 levels, while coinjection of pCI-CEA and els ranging from 100 to 200 ng/ml. The biological activity pCI-IL4 had the opposite effect (higher IgG1, lower of these cytokines was confirmed with appropriate bio- IgG2a), and these changes were statistically significant assays (data not shown). (Figure 2b). These results are consistent with Th1 (IgG2a) and Th2 (IgG1) responses, respectively. Detection of anti-CEA IgG antibody and isotyping To study the effects of the plasmid pCI-CEA and the cytokine-encoding vectors on production of anti-CEA Detection of CTL response against CEA-expressing antibodies, groups of DBA/2 mice were injected i.m. with tumor cells in vitro a mixture of pCI-CEA and either a control vector or a CTLs play a critical role in cell-mediated immune cytokine-encoding vector. To induce an optimal immune responses against virally infected cells and tumor response to CEA, we adapted a protocol previously dem- cells.7,8,11 The generation of CTLs is highly influenced by onstrated to be effective by Conry and colleagues.21 In the cytokines produced during an immune response. Figure 1 Schematic diagram of the expression vectors. CEA was cloned into pCI-Neo or pCMV (lacking the neo gene), to produce pCI-CEA and pCMV-CEA (not shown), respectively. The major transcriptional control elements are shown: in pCI-CEA, cytomegalovirus (CMV) immediate–early enhancer/promoter (CMV-I-E/E-P); a chimeric intron; the human CEA cDNA sequence; the SV40 late polyadenylation and transcriptional termination sequence (SV40 poly A). Mouse IFN␥ and IL4 cDNAs were cloned into pCI-neo vector respectively to generate cytokine-expressing vectors pCI-IFN␥, and pCI-IL4. Mouse IL-12 p35 cDNA, a synthetic intron (IVS), an internal ribosome entry site sequence (IRES) and p40 cDNA were cloned into the VR1255 vector, which also has the CMV-I-E/E-P, CMV intron A, and the minimal rabbit beta globin terminator sequence (RBG poly A), to generate a IL-12-expressing vector, VR-IL12.