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RESEARCH REPORT |
1 Department of Oral and Maxillofacial Surgery, Yokohama City University School of Medicine, 3-9 Fukuura, Kanazawa-ku, Yokohama 236-0004, Japan; and
2 Division of Oral Surgery, Chiba University Hospital, 1-8-1 Inohana, Chuo-ku, Chiba 260-8677, Japan;
* corresponding author, bukawah-cuh{at}umin.ac.jp
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: OK-432 • tumor vaccine • glutaraldehyde • conjugation • tongue cancer
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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The most important strategy for the induction of tumor-specific immunity seems to be the priming of tumor antigens. It is widely accepted that a low level of tumor antigenicity is one of the mechanisms by which a tumor escapes from the host immune system. Cell-based tumor vaccines seem to be more effective on some tumor antigens than single-peptide-based tumor vaccines; however, there are only a few papers describing cell-based tumor vaccine development. Recently, we developed a novel method to increase tumor-specific antigenicity for effective priming. Complexes of tumor cells and other foreign bodies having strong antigenicity for the host immune system may have the potential to increase immunogenicity of the tumor-specific antigen. OK-432, a streptococcal preparation (Okamoto et al., 1966; Saito et al., 1988), was used as the foreign antigen for coupling to the tumor. To evaluate the effects of the newly developed OK-432-conjugated tumor vaccines, we used an experimental murine tongue cancer model (Li et al., 2002). The objective of this study was to examine the feasibility of this vaccine candidate against tongue cancer.
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MATERIALS & METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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The Institutional Animal Care and Use Committee of Yokohama City University School of Medicine approved all procedures for use and care of the mice.
Preparation of the OK-432-conjugated Tumor Vaccines
OK-432, kindly provided by Chugai Pharmaceutical Co., Ltd. (Tokyo, Japan), was used as a binding antigen. The conjugation method with glutaraldehyde (GA) was performed as previously described (Bukawa et al., 1995a,b). Briefly, GA (20%) was diluted to 2.0% with PBS at pH 7.4. KLN-205 cell lines (5 x 105/350 µL) were mixed to OK-432 (0.7 KE/100 µL), followed by addition to 50 µL of 2% GA. The final concentration of GA was adjusted to 0.2% in 500 µL. Conjugation between tumor cells and OK-432 was slowly performed in small rotating columns. Two hrs after conjugation, the OK-432-conjugated tumor cells were centrifuged (at 4°C) at 800 rpm for 5 min 3 times to eliminate unreacted GA and OK-432. OK-432-conjugated KLN-205 cells (5 x 105) were added to 100 µL of PBS as the KLN-205 vaccines. Both B16 and Sq1979 cell lines were conjugated to OK-432 by the same means, and called B16 vaccine and Sq1979 vaccine, respectively. Coupling formation of each tumor vaccine was morphologically observed and confirmed through a microscope in all experiments.
Cytolytic Assay
DBA/2 mice (H-2d) received subcutaneous immunizations of 5 x 105/µL KLN-205 vaccine, KLN-205 treated with 0.2% GA, OK-432 treated with 0.2% GA, or OK-432 alone at seven-day intervals over a three-week period. Control groups of the other DBA/2 mice received no treatment (saline alone). Cytolytic assay was performed 7 days after the final immunization. The spleen cells from immunized mice (3 x 106 cells/well) were co-cultured with MMC-treated KLN-205 cells (3:1 responder:tumor cell ratio) in 2 mL of complete T-cell medium (RPMI 1640 containing Con A) at 37°C in a humidified atmosphere containing 5% CO2. Cytotoxic effector lymphocytes were harvested after 5 days of incubation and subjected to cytolytic assay. Target cells (KLN-205 cells) were labeled with 51Cr and incubated with effector cells for 4 hrs at various E:T ratios. The maximum or spontaneous release was defined as counts from samples incubated with 5% Triton X-100 or medium alone, respectively. Cytolytic activity was calculated according to the following formula:
Percentage of specific 51Cr release = (experimental release - spontaneous release) / (maximum release - spontaneous release) x 100.
Depletion of T-cell Subsets
Rat monoclonal antibodies against the murine CD4 (L3T4) and CD8 (Ly2) were used for depletion of T-cell subpopulations. Anti-CD4 and anti-CD8 antibodies were purchased from PharMingen (San Diego, CA, USA). Depletion of T-cell subsets was carried out as described elsewhere (Mukai et al., 1996). Briefly, effector lymphocytes were suspended at a concentration of 107/mL in 1 mL of RPMI-1640 containing 25 µg of monoclonal antibody. Effector cells were incubated for 45 min at 4°C, washed, and re-suspended to the original volume with complement (C) (Guinea Pig Complement, Inter-Cell Technologies, Inc., Jupiter, FL, USA) at a 1/10 dilution for an additional 45 min at 37°C. After treatment with C, effector cells were washed twice.
Tumor Incidence and Growth
DBA/2 mice were divided into 7 groups of 18 to 20 animals each. The mice were anesthetized by diethyl ether and given subcutaneous immunizations with KLN-205 vaccine, B16 vaccine, Sq1979 vaccine, KLN-205 treated with GA, OK-432 treated with GA, or OK-432 alone. Immunizations were administered into the left flank of each mouse once each week for 3 wks. Control group mice were injected with an equal volume of saline in the same manner. One wk after injection, all mice were inoculated, in the tongue, with 2 x 105/40 µL of KLN-205 cell lines by means of a 100-µL syringe and a 30-gauge needle. Tumor incidence and growth were observed. We also investigated whether post-immunization with KLN-205 vaccine elicited an antitumor effect.
Histological Examination
DBA/2 mice were divided into 2 groups, a KLN-205-vaccine experimental group and a saline control group. There were 20 mice in each group. The mice were given subcutaneous immunizations with KLN-205 vaccine in the left flank once a wk for 3 wks. For the control group, saline was injected in the same manner. At the fourth wk, all mice were inoculated with 2 x 105/40 µL of KLN-205 in the tongue. Three mice from each group were killed randomly at 24 hrs, 5, and 20 days after tumor inoculation, respectively. The excised tongue tissue was fixed in 10% buffered formalin, routinely processed, and embedded in paraffin.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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shows that the KLN-205 vaccine elicited a greater cytolytic response than the others. Although KLN-205 treated with 0.2% GA was somewhat effective in this respect, KLN-205 vaccines induced an approximately two-fold greater response when the effector:target ratio was 40:1. OK-432 treated with GA and OK-432 alone induced almost the same level of response as the controls (saline). KLN-205 vaccine failed to induce cytolytic effect for another DBA/2-derived cell (P815 mastocytoma) (Table 1).
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). There were statistically significant differences between the KLN-205-vaccine group and the other groups. When tumor size was measured, the KLN-205-vaccine group revealed greater inhibition of tumor growth than the other groups, and there were statistically significant differences among them (Fig. 1B). The suppression of mortality rate was also studied among the 7 groups. In the KLN-205-vaccine group, only 56.6% of the mice died of tumor growth by 40 days after tumor inoculation. In contrast, all mice in the other groups and the control group died before 40 days after tumor inoculation (Fig. 1C). As shown in Fig. 1D, antitumor effect induced by post-immunization with KLN-205 vaccine also revealed a stronger inhibition for tumor growth than did the control.
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, 2F). In contrast, a large number of lymphocytes infiltrated around KLN-205 cells in the KLN-205-vaccine group (Figs. 2A, 2B). Five days after tumor inoculation, there was a greater level of lymphocyte infiltration in the vaccine group than in the control group. The former lymphocytes surrounded the KLN-205 cell mass as a barrier, but the latter did not enclose it (data not shown). Fig. 2 (C,D,G,H) shows histological sections of the KLN-205-vaccine group and the control group at 20 days. There were few tumor cells in the KLN-205-vaccine group, while a massive tumor mass was observed in the control group. Moreover, notable fibrosis was observed around the small insular mass. Mitoses were obvious in the control group, but not in the KLN-205-vaccine group.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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The antitumor effects elicited by the KLN-205 vaccines were analyzed for cytolytic activity, suppression of tumor growth, and histological study. In the cytolytic assay, splenocytes from mice immunized with the KLN-205 vaccines indicated a greater cytolytic response than those from mice immunized with the others. In contrast, the others showed almost the same levels of percent-specific lysis as the controls. Table 1
shows that cell-mediated immunity against not P815 but KLN-205 cells can be induced by immunizations with the KLN-205 vaccine, indicating that KLN-205 vaccine elicited anti-KLN-205-specific immunity. These findings also suggest that the conjugation of both (tumor cells and OK-432) may be important for cancer immunotherapy. The cytolytic activity induced by immunization with KLN-205 vaccine was decreased by treatment with anti-CD8 antibody and C. It was assumed that the activity was mediated by CD8+ cells (Table 2).
In the KLN-205-vaccine group, the tumor incidence rates were 25% at 10 days and 56.6% at 30 days after inoculation. The tumor suppression rate was 43.4% (Fig. 1A). In the control (saline-injected) group, tumors were formed at 3 days after inoculation. The tumor incidence rate reached 100% by 10 days. When the mice were immunized with the others (Bib vaccine, Sq1979 vaccine, KLN-205 treated with GA, OK-432 treated with GA, or OK-432 alone), not only the tumor incidence but also the tumor growth and survival period were almost the same as those of the control group (Figs. 1A–1C). Although OK-432 is broadly used for tumor immunotherapy, the results from this study showed that it failed to induce a strong antitumor effect. This may be explained by the use of a low dose of OK-432. Fig. 1D shows that significant antitumor effects were also induced in mice post-immunized with KLN-205 vaccine. This finding suggests that our vaccine method will be effective in planning subsequent treatment in human clinical situations, such as micrometastasis or positive surgical margin after surgery.
To analyze the process of antitumor effects after inoculation of KLN-205 cells, we performed histological examination (Figs. 2A–2H). Infiltrating lymphocytes were markedly observed around the tumor cells at 24 hrs after tumor inoculation in the KLN-205-vaccine group. In contrast, scattered lymphocytes were shown and no infiltrating lymphocytes could be observed in the control group (Figs. 2A, 2B, 2E, 2F). These results indicated that immunized mice acquired an immunological memory for KLN-205 cells. At Day 20 (Figs. 2C, 2D, 2G, 2H), there were few tumor cells in the vaccine group. In contrast, large tumor masses and many mitoses were observed in the control group. All results indicate that OK-432-conjugated tumor vaccines led to acquisition of effective priming, induced tumor-specific cell-mediated immunity, and suppressed tumor activity.
Mukai et al.(1996) reported that in vivo priming of BL6 tumor cells mixed with various doses of OK-432 was effective. They used adoptive immunotherapy of in vitro-sensitized lymphocytes with IL-2 injections. Our conjugation methods induced antitumor effects without adoptive transfer and injections of cytokine such as IL-2. Our results suggest that OK-432-conjugated vaccine may be more effective in priming than a mixture with OK-432. Recently, Vermorken et al.(1999) reported that surgical resection with an autologous tumor-cell-BCG vaccine was more beneficial than resection alone in stage II and stage III colon cancer. Although they used 107 irradiated autologous tumor cells mixed with BCG organisms as a vaccine, the present results suggest that administration of the conjugated tumor cells has the potential to induce stronger antitumor effects than does a simple mixture. Our conjugated tumor vaccines may induce more effective antitumor effects than a mixture of tumor and BCG organisms in clinical trials. The preparation of the OK-432-conjugated tumor vaccines is simple and cost-effective, and many facilities throughout the world might easily be able to construct this type of tumor vaccine candidate. Furthermore, this vaccine candidate seems to be safe for clinical use in humans. It is possible to inject a patient repeatedly from the time of tumor resection to the patient’s death, to prolong his lifetime and to improve quality of life, even if the tumor is not completely cured. This new tumor vaccine method may be a strong candidate for future clinical use.
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ACKNOWLEDGMENTS |
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Received June 27, 2002; Last revision April 21, 2003; Accepted April 29, 2003
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REFERENCES |
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