Gene Therapy (2002) 9, 879–888  2002 Nature Publishing Group All rights reserved 0969-7128/02 $25.00 www.nature.com/gt RESEARCH ARTICLE HER2 peptide-specific CD8+ T cells are proportionally detectable long after multiple DNA vaccinations

K Mukai1, Y Yasutomi2, M Watanabe1, A Kenjo3, T Aota1, L Wang1, H Nishikawa1, M Ishihara1, T Fujita3, K Kuribayashi2 and H Shiku1 1Second Department of Internal Medicine, Mie University School of Medicine, Mie, Japan; 2Department of Bioregulation, Mie University School of Medicine, Mie, Japan; and 3Department of Biochemistry, Fukushima Medical University, Fukushima, Japan

We prepared a plasmid encoding 147 amino acid residues In hosts receiving more than three immunizations, surpris- from the N terminus of c-erbB-2/HER2/neu (HER2), which ingly high numbers of specific CD8+ T cells were persistently included both a cytotoxic T lymphocyte (CTL) epitope detectable. HER2 protein-specific antibodies of IgG class (HER2p63) and a helper epitope (HER2p1), using the mam- with dominance of IgG2a remain detectable 6 months after malian expression vector pCAGGS-New (pCAGGS147 single or multiple immunizations. The antibodies however, HER2). In a parallel analysis with a Tetramer assay and CTL were not reactive with cell surface HER2 antigens. Total assay, good specificity and sensitivity of a quantitative suppression of tumor growth was observed when syngeneic enzyme-linked immunospot (ELISPOT) assay to detect func- HER2+ tumor cells (2 × 106) were injected subcutaneously tional HER2p63-specific CD8+ T cells were demonstrated 14 days after a single immunization with pCAGGS147HER2. after intramuscular immunization of pCAGGS147HER2. In Furthermore, the number of pulmonary metastases an ELISPOT assay for HER2p63, spots of IFN␥-producing decreased significantly when DNA vaccination was initiated cells were first detected 10 days after the first immunization, on the day of, or 3 days after, intravenous injection (1 × and additional immunizations increased the number of 106 cells). spots. HER2p63-specific CD8+ T cells were detected over a Gene Therapy (2002) 9, 879–888. doi:10.1038/sj.gt.3301707 period of more than 10 months after the last immunization.

Keywords: DNA vaccine; c-erbB-2/HER2/neu; CTL; CD8+ T cells

Introduction for in vivo tumor-rejection immune responses.9–11 HLA- A2402, which is expressed by more than 60% of the DNA vaccines are capable of inducing potent cellular and Japanese population and also by a significant proportion humoral immunity in a variety of experimental systems of individuals in other countries, shares a similar peptide- (reviewed in Ref. 1). One of the characteristic features of binding motif with murine MHC class I, Kd. Indeed, we DNA vaccines is their ability to induce lasting immunity. have recently demonstrated that the human homolog of In many systems in mice, antibodies against molecules HER2p63 (TYLPTNASL; one amino acid difference to the encoded by cDNA in plasmids remain detectable for murine peptide) generated CTLs that could lyse HER2- 2–4 more than 1 year after the last immunization. This can expressing tumors in the context of HLA-A2402.12 be explained by persistent antigen expression by the HER2p63 peptides presented by two different MHC class DNA-transduced cells, including professional antigen- I molecules, murine Kd and HLA-A2402, provide us with presenting cells such as dendritic cells, and by persistent a unique system for developing experimental cancer vac- 5,6 memory cells. This is an extremely important feature cine models that could precede their clinical application. beneficial for constructing vaccines against a variety of + + It is becoming clear that CD4 T cells play essential infectious agents. While DNA vaccines induce CD8 cyto- roles in the efficient generation of CD8+ CTL.13–16 We toxic T lymphocytes (CTL) specific for peptides derived therefore prepared a plasmid construct encoding the N- from plasmid-encoded molecules, detailed analysis of the + terminal 147 amino acids of human HER2, which kinetics of CD8 T cells induced by vaccinations is still encompass a HER2p63 CTL epitope and a HER2 p1 7,8 limited. helper epitope (Wang et al, manuscript in preparation). We have recently demonstrated in a murine syngeneic We analyzed detailed kinetics of the induction and main- tumor system that 9mer peptide, murine HER2p63 + + tenance of HER2p63-specific CD8 T cells as a conse- (TYLPANASL), can induce CD8 CTLs with MHC class quence of single or multiple immunizations. I, Kd restriction and more importantly, can be a target Results Dose dependence of immune responses induced by Correspondence: H Shiku, Second Department of Internal Medicine, Mie plasmids encoding 147 amino acid residues from the N University School of Medicine, 2-174 Edobashi, Tsu, Mie, Japan 514-8507 terminus of HER2 (pCAGGS147HER2) Received 18 October 2001; accepted 22 February 2002 To determine the appropriate dose that could elicit the immune response to HER2p63 peptide by intramuscular Kinetics of CD8+ T cells induced by DNA vaccination K Mukai et al 880

Figure 1 Immune response specific for HER2p63 essentially correlates with the injection doses of pCAGGS147HER2 plasmids encoding 147 amino acid residues from the N terminus of HER2. Two BALB/c mice in each group were injected once with varied doses of pCAGGS147HER2. After vaccination, CD8+ T lymphocytes were quantitatively analyzed by ELISPOT assay. Pooled effector cells derived from two spleens of each group (1 × 105) were incubated with same number of peptide-pulsed P1.HTR for 20 h. HER2p63 and a control HER2p780 peptides were used. Data are mean ± s.d. of triplicate wells. Two additional experiments yielded similar results.

injection of pCAGGS147HER2, we titrated the injection dose of pCAGGS147HER2 in mice and measured HER2- specific immune responses by ELISPOT assay to count interferon gamma (IFN␥) producing cells. BALB/c mice were immunized intramuscularly (i.m.) with pCAGGS147HER2 and their spleens were obtained 14days later, and CD8+ T cells were purified as described in Materials and methods. The number of HER2p63-spe- cific CD8+ T cells in these spleen cells increased in a pCAGGS147HER2 dose-dependent manner, and reached a plateau with immunization of 100 ␮g DNA (Figure 1). The IFN␥ spots detected in the assay were antigen-spe- cific, since only a marginal number of spots could be observed when target cells pulsed with a control Kd bind- ing peptide (p780) were used. We therefore used the immunization dose of 100 ␮g in the following experiments.

HER2p63-specific CD8+ T cells are quantitatively measurable with ELISPOT assay and Tetramer assay Mice were immunized i.m. twice at a 3-week interval, with pCAGGS147HER2. Spleen cells were obtained from animals 3 weeks after the second immunization and ana- lyzed for HER2p63-specific CD8+ T cells with ELISPOT and Tetramer assays. A portion of splenocytes was sensit- ized in vitro with mitomycin C (MMC)-treated HER2- expressing CMS17HE cells shown in Figure 2 and they were similarly analyzed with the two assays. In the ELIS- POT assay, about 2 × 102 HER2p63-specific CD8+ T cells were detected in 105 CD8+ T cells (~0.2% of total CD8+ T cells; Figure 3a) before in vitro sensitization, and about 1 × 102 HER2 peptide-specific CD8+ T cells were detected in 103 CD8+ T cells (~10% of total CD8+ T cells; Figure 3d) after in vitro sensitization. A marginal number of spots could be observed when target cells pulsed with a control Kd binding peptide (9wt) were used. In the Tetramer assay, it was difficult to detect HER2p63-specific CD8+ T cells with significance before Figure 2 Three mouse fibrosarcoma cell lines transfected by HER2 cDNA in vitro sensitization (Figure 3b). However, their number express HER2 protein on their surface. Cell lines were described in Materials and methods. Shaded profiles, cells stained with secondary Ab; considerably increased after in vitro sensitization as open profiles, cells stained with anti HER2 monoclonal antibody, Trastu- d shown in Figure 3e. A control Tetramer with a control K zumab. In each panel, the ordinate represents the number of cells and the binding 9wt peptide did not respond to these splenocytes abscissa is the fluorescence intensity in logarithmic arbitrary units.

Gene Therapy Kinetics of CD8+ T cells induced by DNA vaccination K Mukai et al 881

Figure 3 The ELISPOT and Tetramer assays provided comparative enumeration of HER2p63-specific CD8+ T cells induced by DNA vaccination. Mice were immunized i.m. twice at a 3-week interval with 100 ␮g pCAGGS147HER2. Spleen cells were sensitized in vitro with MMC-treated CMS17HE cells as described in Materials and methods. ELISPOT (a, d) and Tetramer (b, c, e, f) assays were performed before (a, b, c) or after (d, e, f) in vitro sensitization. In the ELISPOT assay, effector cells (a, 1 × 105, before sensitization; d, 1 × 103, after sensitization) were incubated with 1 × 105 peptide- pulsed P1.HTR cells for 20 h. HER2p63 and a control 9wt peptides were used. Data are for pooled effector cells of two spleens each and are mean ± s.d. of triplicate wells. In the Tetramer assay, CD8+ cells from the spleen cells were stained with Kd/HER2p63 TetramerPE and anti-CD8FITC (b, e) or Kd/9wt TetramerPE and anti-CD8FITC (c, f). Before in vitro sensitization, to improve the sensitivity of the experiment, CD8+ cells were positively enriched using a miniMACS device. One additional experiment yielded similar results. either before (Figure 3c) or after (Figure 3f) in vitro sensit- these animals were quantitatively analyzed by the ELIS- ization. Following in vitro sensitization, the frequencies POT assay. HER2p63-specific CD8+ T cells became detect- of HER2p63-specific CD8+ T cells detected with the ELIS- able 10 days after the first immunization (data not POT assay were essentially in the same range as those shown), and reached a level of 0.1% of the total CD8+ T determined by the Tetramer assay. These data indicate cells on day 14. For precise evaluation of peptide-specific that the ELISPOT assay could enumerate minute popu- CD8+ T cells in many samples, multiple ELISPOT assays lations of HER2p63-specific CD8+ T cells, and that func- were required as the number of samples in each experi- tional techniques (ELISPOT with IFN␥ production) corre- ment was naturally limited. We therefore included lated well with physical techniques (Tetramer binding). samples of CD8+ T cells obtained from mice 14 days after We therefore used the ELISPOT assay for quantitation priming with pCAGGS147HER2 as a standard in each of HER2p63-specific CD8+ T cells in the following assay. The control samples usually yielded 80 to 120 spots experiments. per 105 CD8+ T cells and these numbers were designated as one relative spot unit. By repeating immunization at Immunization with pCAGGS147HER2 induces persistent 3-week intervals, the number of HER2p63-specific CD8+ HER2p63-specific CD8+ T cells T cells apparently increased, essentially reaching a pla- We chronologically examined the size of the HER2p63- teau after the fourth immunization (Figure 4). Despite the specific CD8+ T cell population maintained in hosts frequency of immunizations, peptide-specific CD8+ T receiving HER2 DNA vaccines. Mice were immunized cells were maintained for a long time. However, the num- i.m. one to four times at 3-week intervals with ber of these cells apparently depended on the frequency pCAGGS147HER2. CD8+ T lymphocytes purified from of immunization, maintaining more than 80% of the

Gene Therapy Kinetics of CD8+ T cells induced by DNA vaccination K Mukai et al 882

Figure 4 HER2-specific CTLs were persistently present for 10 months. Four groups of mice that had one to four immunizations with 100 ␮g pCAGGS147HER2 were prepared. After vaccination, CD8+ T lymphocytes were quantitatively analyzed by ELISPOT assay. Effector cells (1 × 105) and P1.HTR cells (1 × 105) pulsed with HER2p63 peptide were used. Data are for pooled effector cells of two spleens each and are mean ±s.d. of triplicate wells. For standardization of different experiments, CD8+ T cells taken 14 days after the first immunization were analyzed simultaneously in all experimental sets as a standard control. The standard control usually yielded 80 to 120 spots per 105 CD8+ T cells and their spot number was designated as one relative spot unit in each experiments. All counts of spots represent the count relative to the standard control. One additional experiment yielded similar results.

maximal level when hosts received more than three immunized i.m. twice and the proliferative response of immunizations. their T cells was examined by stimulating them with a CTL epitope or a helper epitope in 147HER2. T cells pro- CD4+ T lymphocyte dependency in DNA vaccination liferated significantly upon stimulation with the peptides Mice extensively treated by anti-CD4 monoclonal anti- p1-25 (HER2p1, a helper epitope) and p63-71 (HER2p63, body (mAb) (as described in Materials and methods) a CTL epitope), when compared with stimulation with were immunized i.m. once or twice at a 3-week interval the control peptides p45-70 and p780-788 (Figure 6). with pCAGGS147HER2. In both cases, the number of These results indicate that antigen-presenting cells of HER2p63-specific CD8+ T cells decreased significantly in hosts receiving immunization with pCAGGS147HER2 mice treated with anti-CD4 mAb, indicating the contri- display the immunogenic peptides both on MHC class I bution of CD4+ helper T cells (Figure 5). Mice were also and class II molecules.

Figure 6 T cells from immunized mice proliferated in response to class I Figure 5 CD4+ T cells were required for the priming and boosting of and class II binding peptides of HER2. Mice were immunized i.m. twice CD8+ T cells. Anti-CD4 monoclonal antibody (GK1.5) or control mono- with 100 ␮g pCAGGS147HER2. T cells were enriched from immunized clonal antibody (Ly2.1) was injected into the tail vein. CD4+ T cell- spleen cells using Nylon filter. The proliferative response of T cells was depleted mice were immunized i.m. with 100 ␮g pCAGGS147HER2. The examined by incorporation of 3H-thymidine with MHC class I binding number of HER2p63-specific T cells at 2 weeks after one (left panel) or two peptides, HER2p63 and HER2p780, and class II binding peptides, immunizations (right panel) was determined by ELISPOT assay. Data are HER2p1 and HER2p45. Data are for pools of two spleens each and are for pools of two spleens each and are mean ±s.d. of triplicate wells. One mean ±s.d. of triplicate wells. Two additional experiments yielded additional experiment yielded similar results. similar results.

Gene Therapy Kinetics of CD8+ T cells induced by DNA vaccination K Mukai et al 883 Induction of HER2-specific CD8+ CTLs by in vivo pCAGGS-New (data not shown). The killer activity was immunization with pCAGGS147HER2 blocked in vitro with mAb against CD8 or Kd, but not We examined the specific cytolytic activity of CD8+ T with mAbs against CD4, Dd,Ld, or I-Ad (Figure 7B), sug- cells generated by immunization with pCAGGS147HER2. gesting that the induced killer cells were Kd-restricted Mice were i.m. immunized twice at a 3-week interval CD8+ CTLs. with pCAGGS147HER2 or control pCAGGS-New. Spleen cells were prepared 3 weeks after the second immuni- Immunizations with pCAGGS147HER2 can induce long- zation and sensitized in vitro with MMC-treated term persistent IgG class antibodies reactive with CMS17HE cells. Killer cells specific for HER2-expressing recombinant HER2 protein but not with cell surface target cells recognizing the HER2p63 were induced from HER2 antigen the spleen cells of animals immunized with Mice were immunized i.m. one to four times with pCAGGS147HER2 (Figure 7A), but not with control pCAGGS147HER2 at 3-week intervals. Serums were obtained 2 weeks or 6 months after the last immuni- zations, and were examined for the presence of anti- HER2 IgG class antibodies in ELISA with recombinant truncated HER2 protein with N terminal 147 amino acid residues. As shown in Figure 8, antibody titers increased with repeated immunization. IgG2a class antibodies were dominant relative to IgG1 antibodies. Antibody titers were well-maintained 6 months after the last immuni- zation, regardless of immunization frequencies. None of the serum samples showed significant reactivity with car- bonic anhydrase 2 (CAB) protein as a negative control when examined at a dilution of 1:100 (data not shown). None of the serum samples showed positive reactivity with cell surface HER2 antigens of CMS5HE or CMS7HE in FACS analysis (data not shown).

Immunization with pCAGGS147HER2 can induce in vivo resistance against HER2-expressing tumor cells Naive BALB/c mice were immunized i.m. once or twice with pCAGGS147HER2 or a mock vector. Two weeks after immunization, the animals were challenged with 2 × 106 CMS7HE cells injected subcutaneously. Complete protection from CMS7HE tumor cells was observed in mice immunized either once or twice with pCAGGS147HER2 (Figure 9a). No reappearance of tumor cells was observed during the subsequent long obser- vation period. When DNA vaccination was initiated on the day of subcutaneous tumor challenge or later, no sup- pression of tumor growth was observed (data not shown). We then analyzed the effect of DNA vaccination on suppression of pulmonary metastasis of HER2- expressing tumors. As shown in Figure 9b, the number of pulmonary metastases decreased significantly when DNA vaccination was initiated on the day of, or 3 days after, intravenous injection of CMS5mHE cells. No sup- pression of pulmonary metastasis, however, was observed when DNA vaccination was initiated 7 days later.

Discussion Figure 7 HER2-specific CD8+ CTLs were induced by immunization with Extensive analyses of DNA vaccination targeting ␮ pCAGGS147HER2. (A) Mice were immunized i.m. twice with 100 g HER2/neu have been reported by the use of transgenic pCAGGS147HER2. Pooled spleen cells from two mice were sensitized in mice with either activated rat neu gene17–21 or over vitro with MMC-treated CMS17HE cells as described in Materials and 22,23 methods. Killer cell activity was measured by 51Cr release assay. E/T ratio expressing wild-type neu gene. Investigators aimed to was adjusted as indicated. (a) Target cells used were CMS7HE expressing examine whether DNA vaccination of plasmids of cog- whole HER2 protein (̆), CMS7neo (̅) and parental CMS7 (̃). (b) nate cDNA may break immunological tolerance against P1.HTR pulsed by two Kd binding peptides from HER2, p63 HER2/neu-derived peptides in those transgenic mice. (TYLPTNASL) (᭿) and p780 (PYVSRLLGI) (ᮀ) and non-pulsed ᭺ They were successful either in inducing preventive P1.HTR ( ) were used as target cells. (B) Cytotoxic activity of killer cells effects against HER2/neu expressing tumors or in delay- was measured by 51Cr release assay in the presence of 50 ␮l of monoclonal antibody as indicated. The E/T ratio of the CTL assay was 5. Data are ing the appearance of HER2 expressing mammary for pools of two spleens each. Two additional experiments yielded tumors in response of single or multiple DNA vacci- similar results. nations. Thus, based on the experimental results available

Gene Therapy Kinetics of CD8+ T cells induced by DNA vaccination K Mukai et al 884

Figure 8 DNA vaccination effectively produced antibody against recombinant HER2 protein. Sera prepared from mice immunized with 100 ␮g pCAGGS147HER2 one to four times were analyzed by ELISA against HER2 protein. Sera were obtained from mice 2 weeks (a) or 6 months (b) after last immunization. Sera of mice from non-immunized mice were used as a negative control. Each serum sample was diluted sequentially as indicated and antibody subtypes were specifically quantitated. Data are for pools of sera from three mice each. Two additional experiments yielded similar results.

Figure 9 Intramuscular DNA vaccination effectively protected against HER2-expressing tumor growth and potentially had a therapeutic effect. (a) Naive BALB/c mice were immunized i.m. once or twice with 100 ␮g pCAGGS147HER2 or control pCAGGS-New. Two weeks after immunization, the animals were challenged subcutaneously with 2 × 106 CMS7HE cells. The size of subcutaneous tumors was measured. Groups of five mice were tested in each case, and data displayed in the panels represent individual mice. (b) Mice were injected intravenously with 1 × 106 CMS5mHE tumor cells to establish pulmonary metastases. On day 0, 3 or 7, mice were immunized with pCAGGS147HER2, pCAGGS control, or none. On day 14, mice were killed and metastatic lung nodules were enumerated. Data are mean ±s.d. of seven mice. Since results in multiple experimental groups had to be compared, mean value of pulmonary metastasis in control animals with no treatment was expressed as 100 in relative metastasis counts. Actual number of pulmonary metastasis in this control group ranged from 62 to 112. One additional experiment yielded similar results.

so far, it is justifiable to use DNA vaccinations for preven- while others stress the role of antibodies.19,20 Since tion of HER2/neu expressing tumors. The proposed HER2/neu is expressed in a variety of normal murine underlying mechanisms involved in the eradication of tissues and also in some limited normal adult human HER2/neu expressing tumors in those DNA vaccinated tissues, difficulty inducing effective in vivo anti-HER2 hosts are controversial. Some investigators claim the immune responses leading to tumor destruction is still possible involvement of T cell-mediated immunity,17,21 conceivable. Our previous reports on preventive, as well

Gene Therapy Kinetics of CD8+ T cells induced by DNA vaccination K Mukai et al 885 as therapeutic in vivo tumor suppression by immuni- at present. A recent view on the significance of CD4+ zation with defined peptides derived from murine HER2 helper T cells in maintaining the activity of peptide-spe- gene9,11 are in agreement with multiple analyses of cific CD8+ T cells may well fit with our obser- HER2/neu transgenic mice and may provide a strong vation,24,25since immunization with pCAGGS147HER2 rationale setting HER2/neu molecules as targets of active can present a MHC class I binding peptide, as well as a immunotherapy. Based on these experimental evidences, MHC class II binding peptide (Figure 6). we intended to examine detailed kinetics of CD8+ T cells DNA vaccination with pCAGGS147HER2 resulted in recognizing defined HER2-derived peptide following induction of strong resistance against HER2-expressing DNA vaccinations. tumors. When the immunization was administered We applied a sensitive ELISPOT assay for detection of before challenge with the tumor, even after a single single cell secreting IFN␥ to detect frequencies of immunization, the immunized host became completely + HER2p63-specific CD8 T lymphocytes freshly isolated resistant to syngeneic tumors. Tumors did not reappear from immunized hosts. Specificity of the ELISPOT assay in these animals during the extended observation. How- + was confirmed by parallel analyses of the same CD8 T ever, no measurable resistance against subcutaneous cell population with a CTL assay and also a Tetramer challenge tumors was observed, when the immunization assay using HER2p63 peptide-Kd complex molecules was initiated on the day of tumor challenge or later. This + (Figure 3). Peptide-specific CD8 T cells became detect- is somewhat surprising since our experimental model is able 10 days after the priming immunization with plas- targeted against human HER2, which is expected to be mid DNA. They remained detectable for at least 10 immunogenic with xenogeneic epitopes. Studies of vari- months after immunization. The peak activity, as determ- + ous animal models reported that DNA vaccination with ined by the number of specific CD8 T cells, apparently a variety of antigen systems was successful in preventing became higher following repetitive immunization at 3- 26–29 + subsequent growth of challenged tumors. However, week intervals. A surprisingly high level of specific CD8 it appears to be extremely difficult to demonstrate that T cells has been maintained in these animals immunized DNA vaccination is effective in suppressing tumor more than three times (Figure 4), which indicates the use- + growth if the immunization is given after the tumor chal- fulness of DNA vaccination in maintaining CD8 T cell- lenge. One possible interpretation for this is a delay of the dependent immunity in hosts. appearance of anti-tumor immune responses after DNA Several points about our analysis require emphasis. vaccination. In our system, after a single immunization Firstly, we used the ELISPOT assay for quantitative with pCAGGS147HER2, CD8+ T cells specific for ␥ + measurement of the number of IFN -producing CD8 T HER2p63 became detectable in spleen 10 days after the cells reactive with HER2p63 peptide. The assay enabled immunization. Relatively slow induction of anti-HER2 us to measure the number of positive cells in the order 4 immunoresponses after DNA vaccination may make the of 1 to 10 cells (0.01%), which fitted extremely well with suppression of continuously growing tumors extremely our goal of analyzing the kinetics of peptide-specificT difficult in the setting of these animal models.30 cells. Parallel analysis with CTL, Tetramer, and ELISPOT Since one of the attractive characteristics of DNA vacci- assays confirmed the specificity as well as the biological nation is the long-lasting specific immune response to significance of our measurement. Since the lymphocytes cognate antigens, DNA vaccination could be an attractive were maintained in culture for only 20 h for the purpose approach for preventing recurrence and metastasis of of analysis, they may accurately reflect the estimation of cancer, both of which often occur even after long latent the number of peptide-specific T cells in vivo. periods. When the effect of DNA vaccination was investi- One of the surprising findings in our analysis was, in addition to the persistence of peptide-specific CD8+ T gated in pulmonary metastasis models of HER2-express- cells, the number of peptide-specific T cells in the host ing tumors, we noted significant suppression of metasta- after DNA vaccination. Detailed analysis clearly showed sis when immunization was initiated on the day of that a surprisingly high number of peptide-specific CD8+ intravenous tumor challenge or 3 days after the tumor T cells were maintained in the host receiving more than challenge. A similar experiment was reported using the CT26 tumor line transfected with the ␤-galactosidase three immunizations, ranging over 80% of the highest 31 detectable number of T cells attainable after immuni- gene as a model tumor antigen system. In that system, zation. The number of peptide-specific T cells was well DNA administered by ‘gene gun’ could suppress pul- maintained for long periods with only limited variation monary metastasis of CT26 tumors injected intravenously between hosts. In a number of experimental systems, 2 days before immunization. One possible interpretation long-term persistence of humoral immunity has been of this finding could be the presence of a much smaller reported,2–4 in accordance with our results in measure- number of tumors present in situ after intravenous injec- ment of antibodies reactive with truncated HER2 protein tion of tumors when anti-HER2 immune responses encoded by plasmids for vaccines. The persistent immun- were elicited. ity has been accounted for by either long-term mainte- Alternatively, there could be different mechanisms of nance of genes encoded by plasmids in myocytes or of cellular anti-HER2 tumor immune responses involved in synthesized antigen molecules5 rather than cDNA, or by the suppression of metastatic tumor cells. In either case, maintenance of memory lymphocytes specific for the cog- we are rather reluctant in our experimental condition to nate target antigens.6 Much less information is available speculate that antibodies reactive with cell surface HER2 on cellular immunity. Hassett et al7 reported that antigen- antigens play a major role in in vivo tumor rejection, since specific CD8+ T cells remained detectable at 1 year after we repeatedly fail to detect them. The present data sug- vaccination by directly measuring the presence of pep- gest that DNA vaccinations might be more effective in tide-specific CD8+ T cells, also similar to the present suppressing micrometastasis rather than in eradication of results. The mechanism of their persistence is unknown established massive tumors and therefore provide an

Gene Therapy Kinetics of CD8+ T cells induced by DNA vaccination K Mukai et al 886 important approach for the prevention of micrometasta- (TYLPTNASL), HER2p780 (PYVSRLLGI),9 HER2p1 sis or recurrence of tumors. (MELAALCRWGLLLALLPPGAASTQV), HER2p45 (LRH LYQGCQVVQGNLELTYLPTNAS) and mouse wild-type ERK2-derived peptide 9wt (KYIHSANVL)36were synthe- Materials and methods sized by the Mitsubishi Chemical Corporation at the Yokohama Research Center (Yokohama, Japan). Mice Six- to 8-week-old female BALB/c mice purchased from Immunization the Shizuoka Animal Laboratory Center (Shizuoka, Six- to 8-week-old BALB/c mice were intramuscularly Japan) were used for all experiments and were main- (i.m.) injected in tibialis anterior muscles bilaterally with tained at the Animal Center of Mie University School of 5 to 100 ␮g of plasmids in 50 ␮l of phosphate buffered Medicine, Tsu, Japan. The experimental protocols were saline (PBS). Immunization was repeated every 3 weeks approved in advance by the Animal Experimentation if necessary. Ethics Committee of our institution. Generation of CTL effector cells and target cells Tumors and cell lines Spleen cells from mice at 3 weeks after the final immuni- CMS5, CMS7 and CMS17 are 3-methylcholanthrene- zation were treated with ACK lysis buffer (0.15 M NH4Cl, 32 induced fibrosarcoma cell lines of BALB/c mice origin. 1 mM KHCO3, and 0.1 mM Na2EDTA, pH 7.2) to remove These cell lines were transfected with full-length human erythrocytes. Spleen cells (5 × 106) were co-cultured with HER2 cDNA and designated CMS5HE, CMS7HE and 5 × 105 mitomycin C (MMC)-treated CMS17HE cells in CMS17HE, respectively. Transformants derived by trans- RPMI 1640 (GIBCO BRL, Grand Island, NY, USA) with fection with mock vectors were designated CMS5neo, 10% fetal calf serum (FCS) (Intergen, Purchase, NY, CMS7neo and CMS17neo, as described previously.9 USA), 2-mercaptoethanol (50 ␮M), penicillin (100 CMS5mHE was cloned from a metastatic lung lesion of units/ml), streptomycin (100 ␮g/ml), and 20 IU/ml CMS5HE.33 P1.HTR is a mastocytoma cell line of DBA/2 recombinant human IL-2 (rIL-2, Takeda Pharmaceutical, 34 ° mice origin. The expression of recombinant-HER2 was Tokyo, Japan) in 24-well plates at 37 Cin5%CO2. The evaluated by using anti-HER2 monoclonal antibody effector cells generated were harvested 6 days later. (mAb), Trastuzumab (Genentech, South San Francisco, P1.HTR cells (1 × 106) were washed with plain RPMI and CA, USA). Cells were stained using a standard indirect then resuspended in 500 ␮l of plain RPMI. Ten ␮l of pep- immunofluorescence procedure with the primary Ab fol- tide solution (1 mg/ml in PBS) were added and incu- lowed by a fluorescein-conjugated anti-human IgG. bated for 15 min at room temperature. RPMI (500 ␮l) Stained cells were analyzed by FACScalibur (Becton with 20% FCS was added and incubated for 45 min at Dickinson, San Jose, CA, USA) using CellQuest software room temperature, followed by another round of incu- (Figure 2). bation at 37°C for 1 h. Finally, cells were washed twice with plain RPMI and used as target cells for CTLs. Plasmids The truncated HER2 mutant encompassing the N-ter- Purification of CD8+ T cells minus 147 amino acids was first cloned into the BglII and CD8+ T cells were prepared by positive enrichment using BamHI sites of the pET-15b vector (Novagen, Madison, the MACS system (Miltenyi Biotec, Bergisch Gladbach, WI, USA). Cloned cDNA was digested with BglII and Germany). In brief, spleen cells were labeled with CD8a BamHI and subcloned into the BglII site of pCAGGS- (Lyt2) microbeads (Miltenyi Biotec; 10 ␮l/107 cells) in New35 (kindly provided by Dr J Miyazaki, Osaka Univer- incubation buffer (PBS/0.5% BSA; 90 ␮l/107 cells) for 15 sity, Graduate School of Medicine, Japan) min at 4°C. After washing, cells were resuspended in (pCAGGS147HER2). The plasmid was maintained in incubation buffer (0.5 ml buffer/108 cells), and were Escherichia coli DH5␣ (Toyobo, Osaka, Japan) and purified applied on to MS columns equipped with a MiniMACS through large-scale plasmid preparations using the magnet according to the instructions provided by the Qiagen Endofree Giga kit (Qiagen, Hilden, Germany) as manufacturer. The resulting CD8+ T cell population was directed by the manufacturer. more than 95% pure as determined by flow cytometric analysis for the surface marker CD8 (Lyt2.2). Antibodies and peptides Anti-L3T4/CD4 (GK1.5), anti-Lyt2.2/CD8 (19/178), anti- ELISPOT assay Lyt2.1 (49-31.1), anti-H-2Kd (20-8-4), anti-H-2Dd (34-5-8S), The ELISPOT assay described previously37 was modified anti-H-2Ld (30-5-7), and anti-I-Ad (MKD-6) monoclonal to detect HER2-specific CD8+ T cells. Briefly, 96-well antibodies (mAbs) were prepared as described pre- nitrocellulose MAHA S4510 Millipore plates (Millipore, viously.36 Anti-HER2 mAb Trastuzumab were purchased Bedford, MA, USA) were incubated overnight with 100 from Genentech. Anti-mouse IFN␥ (R4-6A2), biotinylated ␮l/well of rat anti-mouse IFN␥ antibody (R4-6A2) at a anti-mouse IFN␥(XMG1.2) antibodies and fluorescein- concentration of 2 ␮g/ml in PBS at 4°C. Plates were conjugated anti-human IgG antibodies were purchased washed with PBS and blocked with RPMI supplemented from BD PharMingen (San Diego, CA, USA). Anti-mouse IgG with 10% FCS for 1 to 2 h at 37°C. Effector cells (1 × 105) conjugated with horseradish peroxidase was purchased were incubated with 1 × 105 MMC-treated P1.HTR cells ° from Jackson Immuno Research Laboratories (West Balti- pulsed with peptide for 20 h at 37 Cin5%CO2. After more Pike, MD, USA), and anti-mouse IgG1 and IgG2a incubation, the wells were washed thoroughly with PBS conjugated with horseradish peroxidase were purchased containing 0.05% Tween 20 (PBST). One hundred ␮lof from Nordic Immunological Laboratories (Tilburg, The biotinylated anti-mouse IFN␥ antibody (XMG 1.2), at a Netherlands). Human HER2-derived peptides HER2p63 concentration of 1.25 ␮g/ml in PBS, was added to wells

Gene Therapy Kinetics of CD8+ T cells induced by DNA vaccination K Mukai et al 887 and incubated overnight at 4°C. After another wash with 51Cr release cytotoxicity assay PBST, 100 ␮l of alkaline phosphatase streptavidin Cytotoxicity assays were performed as described pre- (Mabtech AB, Nacka, Sweden) at a concentration of 0.2 viously.43 Briefly, target cells labeled with 51Cr (1 × 104 ␮g/ml in PBS was added to each well and incubated at cells in 100 ␮l) were added to varying numbers of effector room temperature for 1.5 h. Finally, spots were cells (100 ␮l) in V-bottom 96-well plates. After a 4-h (10- developed by the addition of 100 ␮l alkaline phosphatase h for CMS7HE, CMS7neo and CMS7 target cells) incu- ° ␮ conjugate substrate kit (BioRad Laboratories, Hercules, bation at 37 Cin5%CO2,20 l supernatant were col- CA, USA). After spot development, the plate was rinsed lected and the percent specific lysis was calculated as thoroughly with ddH2O and allowed to dry. Spots were (c.p.m. released from the test sample – spontaneous counted with the aid of a dissecting microscope, and ana- release)/(c.p.m. released by detergent – spontaneous lyzed by the Axioplan 2 imaging system (Carl Zeiss release) × 100%. Vision, Hallbergmoos, Germany). Enzyme-linked immunosorbent assay Tetrameric MHC class I/peptide complexes and FACS Sera from immunized mice were serially diluted and the analysis titer for recombinant truncated HER2 protein with N ter- Tetrameric MHC class I/peptide complexes were pre- minal 147 amino acid residues was measured by ELISA pared as described by Altman et al.38 Briefly, recombinant as described previously.10 Flat-bottomed 96-well human ␤2-microglobulin and the extracellular portion of microtiter plates (Nunc-Immuno, Roskilde, Denmark) H-2Kd containing the BirA recognition sequence in frame were coated with 10 ng/50 ␮l of truncated HER2 or car- at its C terminus were expressed in E. coli as insoluble bonic anhydrase 2 (CAB) protein (Sigma, St Louis, MO, aggregates. Purified inclusion bodies were solubilized in USA) as a control at 4°C overnight and stored at 4°C until 6M-guanidine hydrochloride. Six mg of monomeric HLA just before use. Serum (75 ␮l) was added into pre-coated class I molecule, 5 mg of ␤2-microglobulin and 5 mg of 96-well plates and detected by anti-mouse IgG, IgG1, or peptide were refolded by dilution of denaturing con- IgG2a conjugated with horseradish peroxidase (1:5000). ditions. Refolded complex solution was concentrated and Finally, absorbance was measured at 450 nm. purified with monoQ (Amersham Pharmacia Biotech, Buckinghamshire, UK) anion exchange column chroma- tography. After buffer exchange, a specific lysine residue Acknowledgements in the BirA recognition sequence tag was biotinylated by We thank Ms Megumi Goto and Mr Shoichi Kita who BirA enzyme (Avidity LLC, Denver, CO, USA). The provided excellent assistance and useful information. monomeric complex was purified by gel filtration column This work was supported in part by grants from the (TSK3000SW, Tosoh, Tokyo, Japan) connected to FPLC Scientific Research on Priority Areas (C) from the Minis- (AKTA 10S, Amersham). Tetrameric arrays of MHC class try of Education, Culture, Sports Science and Technology I peptide complexes were formed by the addition of PE- of Japan. labeled avidin (Biomeda, Foster City, CA, USA). Effector T cells (3 × 105) were washed in tissue culture medium and incubated with 1 ␮g of PE-labeled tetramers at 37°C References for 30 min,39 along with FITC-labeled anti-mouse CD8 antibody (BD PharMingen) as a counterstain. Stained 1 Gurunathan S, Klinman DM, Seder RA. DNA vaccines: immu- cells were analyzed by FACScalibur using the CellQu- nology, application, and optimization. Annu Rev Immunol 2000; est software. 18: 927–974. 2 Raz E et al. Intradermal gene immunization: the possible role of DNA uptake in the induction of cellular immunity to viruses. Depletion of CD4+ T cells in vivo + Proc Natl Acad Sci USA 1994; 91: 9519–9523. Depletion of CD4 T cells in vivo was performed as 3 Davis HL, Mancini M, Michel ML, Whalen RG. DNA-mediated described previously40 with modifications. Ascites fluid immunization to hepatitis B surface antigen: longevity of pri- containing anti-CD4 antibody (G.K.1.5, 25 ␮l) or control mary response and effect of boost. Vaccine 1996; 14: 910–915. antibody (Lyt2.1, 25 ␮l) was diluted 1:4 with PBS and 4 Deck RR et al. Characterization of humoral immune responses injected into the tail vein of mice 1 day before immuni- induced by an influenza hemagglutinin DNA vaccine. Vaccine zation, followed by injection every 5 days. Disappearance 1997; 15:71–78. of splenic CD4+ T cells was confirmed by FACS analysis. 5 Wolff JA et al. Long-term persistence of plasmid DNA and foreign gene expression in mouse muscle. Hum Mol Genet 1992; 1: 363–369. T cell proliferation assay 6 Akbari O et al. DNA vaccination: transfection and activation of The T cell proliferation assay was performed as described dendritic cells as key events for immunity. J Exp Med 1999; 189: 41 previously with modifications. Spleen cells were 169–177. obtained 2 weeks after the final immunization. Nylon 7 Hassett DE, Zhang J, Slifka MK, Whitton JL. Immune responses fiber-purified suspensions of T cells (5 × 105 cells)42 from following neonatal DNA vaccination are long-lived, abundant, spleen cells of immunized mice were plated into 96-well and qualitatively similar to those induced by conventional flat-bottom microtiter plates as responder cells. Irradiated immunization. J Virol 2000; 74: 2620–2627. × 8 Hassett DE, Slifka MK, Zhang J, Whitton JL. Direct ex vivo kin- spleen cells (46 Gy) from SCID mice were plated at 5 + 105 for antigen presenting cells. Peptide solutions were etic and phenotypic analyses of CD8 T-cell responses induced ␮ by DNA immunization. J Virol 2000; 74: 8286–8291. added to the plates (1 g/well). After 3 days incubation 9 Nagata Y et al. Peptides derived from a wild-type murine proto- ␮ 3 in RPMI 1640 supplemented with 1 Ci/well H-thymid- oncogene c-erbB-2/HER2/neu can induce CTL and tumor sup- ine during the last 5 h of culture, the incorporated radio- pression in syngeneic hosts. J Immunol 1997; 159: 1336–1343. activity was determined by microplate scintillation 10 Gu XG et al. A novel hydrophobized polysaccharide/oncopro- counter. tein complex vaccine induces in vitro and in vivo cellular and

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