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Antitumor Effects of Systemic and Local Immunization with a CTL-Directed Peptide in Combination with a Local Injection of OK-432

Authors' Affiliations: ¹ Cancer Vaccine Development Division, Kurume University Research Center for Innovative Cancer Therapy, Departments of ² Immunology and ³ Otorhinolaryngology and Head and Neck Surgery, Kurume University School of Medicine, ⁴ Center of the 21st Century COE Program for Medical Science, Kurume University, Kurume, Japan

Requests for reprints: Mamoru Harada, Department of Immunology, Kurume University School of Medicine, 67 Asahi-machi, Kurume, Fukuoka 830-0011, Japan. Phone: 81-942-31-7744; Fax: 81-942-31-7745; E-mail: haramamo{at}med.kurume-u.ac.jp.

	Abstract

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Purpose: The accumulation of T cells into the tumor site is crucial for the elicitation of in vivo antitumor effects after cancer vaccination. In this study, we investigated the antitumor effects and associated mechanisms of action that were induced by systemic and local immunization with a CTL-directed peptide in combination with a peritumoral injection of a streptococcal preparation, OK-432.

Experimental Design and Results: The human SART3_315-323 peptide, which has the potential to induce human leukocyte antigen-A24-restricted CTLs, not only has the same amino acid sequence as the mouse SART3, but also has the capacity for binding to H-2K^d molecules. Therefore, the SART3_315-323 peptide could be used as a tumor antigen–derived peptide in H-2^d mice. Systemic immunization with the SART3_315-323 peptide and the subsequent peritumoral injection of both the SART3_315-323 peptide and OK-432 effectively induced peptide-specific and colon26 carcinoma–reactive CTLs in BALB/c mice. The combination therapy suppressed the growth of s.c. established colon26 carcinoma. The accumulation of both CD8⁺ and CD4⁺ T cells into the tumor site was more apparent in mice treated with the combination therapy than in those treated with other protocols. In addition, the level of IgG reactive to the administered SART3_315-323 peptide increased in mice that were treated with the combination therapy.

Conclusion: These results indicate that antitumor effects could be efficiently induced by a combination therapy that included systemic and local immunization with a CTL-directed peptide together with a local injection of OK-432.

OK-432 is a penicillin-killed and lyophilized preparation of a low-virulence strain (Su) of Streptococcus pyogenes (14). When OK-432 is given in vivo, various cytokines, including tumor necrosis factor-, IFN, interleukin (IL)-2, and IL-6, can be induced (15). Therefore, OK-432 has been used for nonspecific immunotherapy (16), and has been suggested to be useful in dendritic cell–based immunotherapy as well (17). In addition, several reports suggest that this reagent is useful to evoke local inflammation and promote accumulation of lymphocytes into tumor sites (18–20).

SART3 protein is a tumor-associated antigen that was identified using human squamous cell carcinoma–reactive CTLs (21). We have identified a 9-mer peptide, SART3_315-323, that has the potential to induce human leukocyte antigen (HLA)-A24-restricted and tumor-reactive CTLs in HLA-A24⁺ cancer patients (21). In addition, we identified a mouse homologue, mouse SART3, which has 80% and 86% homology to the human SART3 at the nucleotide and protein levels, respectively (22). Interestingly, human HLA-A2404 and mouse H-2K^d molecules have similar anchor motifs (23, 24), and the human SART3_315-323 peptide has the same amino acid sequence as the "mouse" SART3_316-324 peptide (22). These lines of evidence indicate that the human SART3_315-323 peptide could be used in order to examine the in vivo antitumor effects of peptide vaccine and the associated mechanisms of action in H-2^d mice. In this study, by using BALB/c mice and a syngeneic colon26 carcinoma, we investigated both antitumor effects and the associated mechanisms of action that were induced by systemic and local immunization with a CTL-directed peptide, human SART3_315-323, in combination with a peritumoral injection of a streptococcal preparation, OK-432.

	Materials and Methods

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Mice. BALB/c female mice (H-2^d, 6-7 weeks old) were purchased from Charles River Japan, Co. Ltd., Japan, and kept under a specific pathogen–free condition. Experiments were carried out in accordance with the guidelines for experimental animal care of our institution.

Peptides and OK432. The SART3_315-323 peptide (AYIDFEMKI) was identified as a peptide with the potential to induce cancer-reactive CTLs in patients with HLA-A24⁺ cancer (21). HIV (RYLRQQLLGI) and Lck_486-494 (TFDYLRSVL) peptides were used as controls. OK-432, which was kindly provided by Chugai Pharmaceutical Co. Ltd. Japan, is a penicillin-killed and lyophilized preparation of a low-virulence strain (Su) of S. pyogenes. OK-432 was prepared at a dose of 1 KE/mL before use.

Tumor cell lines. Three murine tumor cell lines (colon26, P815, and B16) were used in this experiment. Colon26 is a colon carcinoma cell line of BALB/c (H-2^d) mouse origin. P815 is a mastocytoma cell line of DBA/2 (H-2^d) mouse origin. B16 is a melanoma line of C57BL/6 (H-2^b) mouse origin. Both colon26 and P815 were maintained in RPMI 1640 supplemented with 10% fetal bovine serum (FBS), and B16 was maintained in DMEM supplemented with 10% FBS.

RT-PCR. Total RNA was isolated according to the manufacturer's instructions. First-strand cDNA synthesis was done from 2 µg of total RNA in a 20 µL volume with oligo(dT) priming using the Superscript First-Strand Synthesis System (Invitrogen, Carlsbad, CA). PCR was done with 1 µL of the generated cDNA using 1 unit of Ex Tag polymerase (Takara, Tokyo, Japan). The following mouse SART3 primer pairs were used: sense 5'-AGAGGCTTCGGCTATGAAGG-3', and antisense 5'-ATACTCCACAT ACGCCAGGC-3'. The following ß-actin primer pairs were used: sense 5'-GTGGGCC GCTCTAGGCACCA-3', and antisense 5'-TGGCCTTAGGGTTCAGGGG G-3'. Denaturation, annealing, and extension were carried out at 95°C for 1 minute, 60°C for 1 minute, and 72°C for 1 minute. Twenty-five cycles were carried out for both SART3 (588 bp) and ß-actin (242 bp).

Immunotherapy protocol. The immunotherapy protocols are shown in Table 1. S.c. immunization into the abdomen of BALB/c mice was done with complete Freund's adjuvant (CFA) mixed with saline or the SART3_315-323 peptide (100 µg). Two weeks after the first immunization, a boosting immunization with incomplete Freund's adjuvant (IFA) mixed with saline or the SART3_315-323 peptide (100 µg) was done thrice weekly. Each group consisted of five mice. Two weeks after the boosting immunization, 2 x 10⁵ colon26 cells were injected s.c. into the lateral flank of the mice. On day 9 after the tumor cell inoculation, the mice were injected peritumorally with saline (100 µL), either, or both of the SART3_315-323 peptide (100 µg) and OK-432 (0.1 KE) every 3 days, for a total of six times. Tumor size (mm²) was measured every 3 days.

Assay of cytotoxicity. A standard ⁵¹Cr-release assay was done. Effector cells were incubated with target cells (1.0 x 10⁴) at the indicated E:T ratios for 6 hours. After incubation, ⁵¹Cr-release in the supernatant was calculated according to the following formula: [(experimental release – spontaneous release) / (maximum release – spontaneous release)] x 100.

Preparation of tumor-infiltrating lymphocytes. Tumors were removed from the lateral flank of each mouse, and the tissues were fragmented with scissors and incubated in 0.2% collagenase/0.04% DNase1 (Sigma, Steinheim, Germany)/10% FBS/RPMI 1640. Suspended tumor cells and tumor-infiltrating lymphocytes (TIL) were placed on lymphocyte separation medium (Wako, Osaka, Japan) and centrifuged at 2,500 rpm. Thereafter, TILs were analyzed by flow cytometry.

Flow cytometry. One million cells were prepared and suspended in 50 µL of PBS/0.1% NaN/1.0% FBS. To prevent nonspecific binding to the Fc receptor, the cell suspensions were incubated with 1 µg of Mouse BD Fc Block at 4°C for 5 minutes, and were then further incubated with 20 µL of Mouse 2 Color FCM Reagent at 4°C for 40 minutes. Cell suspensions were washed twice with PBS/0.1% NaN/1.0% FBS and fixed with 1% formalin/PBS. Analysis was done using Coulter Epics XL (Beckman Coulter, Fullerton, CA).

Measurement of peptide-specific IgG. On day 29 after the tumor cell inoculation, blood samples were collected individually by retro-orbital plexus puncture after anesthesia with ether. To prepare the serum, the collected blood was left to stand for 30 minutes, and was then centrifuged at 10,000 x g. Peptide-specific IgG levels in the sera were measured by ELISA as reported previously (26). In brief, the peptides were immobilized on the surface of 96-well plates using a chemical cross-linker, disuccinimidyl suberate (Pierce). Serum samples were diluted at 1:100. The peptide-coated plates were blocked with 100 µL/well of Block Ace (milk proteins; Yukigirushi, Tokyo, Japan) for 2 hours at 4°C. After the plates were blocked, 100 µL/well of serum sample diluted with 0.05% Tween 20-Block Ace was added to the wells. After a 2-hour incubation at 37°C, the plates were washed thrice with 0.05% Tween 20-PBS and were incubated at 37°C for 2 hours with 100 µL/well of 1:1,000-diluted rabbit anti-mouse IgG (-chain specific).

Statistics. The statistical significance of the data was determined using Student's t test. P < 0.05 was considered to be statistically significant.

	Results

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Induction of peptide-specific and colon26-reactive CTLs in BALB/c mice by vaccination with the SART3_315-323 peptide. First, we examined the expression of mouse SART3 mRNA in three murine tumor cell lines, colon26, P815, and B16. Normal spleen cells from BALB/c mice were used as a control. As shown in Fig. 1, the expression of mouse SART3 mRNA was detected in these three tumor cell lines, but not in the spleen cells.

View larger version (15K): [in this window] [in a new window]   Fig. 1. Expression of mouse SART3 mRNA in murine tumor cell lines. RT-PCR analysis was done to examine SART3 mRNA expression. ß-Actin mRNA expression was examined as a control.

View larger version (24K): [in this window] [in a new window]   Fig. 2. Induction of SART3 peptide–specific and colon26-reactive CTLs. BALB/c mice were treated as shown in Table 1. Ten days after the last peritumoral injection of either or both of the SART3315-323 peptide and OK-432, the spleen cells and lymph nodes were harvested and stimulated in vitro with the SART3315-323 peptide, as described in Materials and Methods. After 5 days, the cultured cells were harvested and purified for CD8+ T cells, and the purified cells were then employed for assay. A, irradiated spleen cells from BALB/c mice were used as stimulators after pulsation with the SART3315-323 or the HIV peptide. After 36 hours, the supernatant was collected and the level of IFN- was determined. B, the purified CD8+ T cells were employed for CTL assay at an E:T ratio of 20:1. Colon26 cells, which were prepulsed with the SART3315-323 peptide or the HIV peptide, and B16 cells were used as targets. Columns, mean; *, P < 0.05 (statistical significance). These data are representative of two separate experiments.

View larger version (23K): [in this window] [in a new window]   Fig. 3. In vivo antitumor effects of combination therapy together with OK-432. BALB/c mice were treated as shown in Table 1. A, 9 days after colon26 inoculation, tumor size was monitored by measuring the longest diameter (a, mm) and the shorter diameter (b, mm) of the lesion, and the results were scored according to the formula: a x b (mm2). Lines, tumor growth of an individual mouse. B, the mean tumor growth of each group (five mice) and percentage of survival on day 79. These data are representative of two separate experiments.

View larger version (21K): [in this window] [in a new window]   Fig. 4. Peptide-specific and colon26-reactive reactivity of the spleen and lymph nodes from treated mice. BALB/c mice were treated as shown in Table 1. Ten days after the last peritumoral injection of either or both of the SART3315-323 peptide and OK-432, the spleen cells or lymph nodes were harvested and stimulated in vitro with the SART3315-323 peptide, as described in Materials and Methods. After 5 days of culture, the reactivity of the cultured cells in response to irradiated BALB/c spleen cells (A) or colon26 cells (B), both of which were pulsed with either the SART3315-323 peptide or the HIV peptide, was examined by measuring the level of IFN-. *, P < 0.05 (statistical significance). These data are representative of two separate experiments.

View larger version (25K): [in this window] [in a new window]   Fig. 5. Peptide-specific and colon26-reactive cytotoxicity. BALB/c mice were treated as shown in Table 1. Ten days after the last peritumoral injection of either or both of the SART3315-323 peptide and OK-432, the spleen cells and lymph nodes were harvested and stimulated in vitro with the SART3315-323 peptide, as described in Materials and Methods. Thereafter, cytotoxicity to colon26, P815, and B16 cells that were pulsed with the SART3315-323 peptide or the HIV peptide was examined by 6 hours of 51Cr-release assay. These data are representative of two separate experiments.

View larger version (21K): [in this window] [in a new window]   Fig. 6. Flow cytometric analysis of the TILs. After the sixth peritumoral injection, the solid tumor was removed, and the TILs were analyzed by flow cytometry. FITC-conjugated anti-CD4 and PE-conjugated anti-CD8 monoclonal antibodies were used. Numbers, percentage of each division. These data are representative of three separate experiments.

View larger version (14K): [in this window] [in a new window]   Fig. 7. SART3 peptide-specific IgG in mice treated with combination therapy. A, the sera from eight groups were examined by ELISA for IgG reactive to the SART3315-323 peptide. The Lck486-494 peptide was used as a negative control (absorbance). B, to confirm the specificity of IgG reactive to the SART3315-323 peptide, 100 µL of serum sample was incubated in plates precoated with the SART3315-323 or the Lck486-494 peptide. Thereafter, the level of IgG reactive to the SART3315-323 peptide in the resultant supernatant serum was determined by ELISA. Columns, means; *, P < 0.05, statistical significance. C, to further confirm the specificity of IgG reactive to the SART3315-323 peptide, IgG binding to the SART3315-323 peptide was eluted from the plates, and the level of SART3315-323 peptide-specific IgG was determined by ELISA. Columns, means; *, P < 0.05 (statistical significance).

	Discussion

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In this study, we used the SART3_315-323 peptide in the colon26-BALB/c system. The human SART3_315-323 peptide has the same amino acid sequence as the "mouse" SART3_316-324 peptide (22). We considered that this system would be very useful for examining the in vivo antitumor effects of a human tumor antigen–derived peptide. To date, several HLA class I-transgenic mice lines have been established and used for the identification of antigenic peptides having the potential to generate HLA class I-restricted CTLs (31, 32). However, these transgenic mice systems are not as useful for examining the in vivo antitumor effects because the corresponding HLA class I-expressing murine tumor cell lines are necessary for such an experiment. On the other hand, there is no such issue when we apply the human SART3_315-323 peptide to the colon26-BALB/c mouse system. This constitutes a distinct advantage of the present experimental system.

The systemic immunization and subsequent local injection of the SART3_315-323 peptide together with OK-432 efficiently suppressed s.c.-inoculated colon26. The systemic immunization of the SART3_315-323 peptide and subsequent local injection of OK-432 alone also suppressed the tumor growth, but the survival rate on day 79 after inoculation differed markedly. That is, the survival rate of mice treated with the combination therapy was 100%, whereas that of mice treated by systemic immunization and the subsequent local injection of OK-432 alone was 20%. In addition, the survival rate of the mice treated by the systemic immunization and subsequent local injection of the SART3_315-323 peptide alone was 20%. The survival rate of the mice that had not received systemic immunization but that had received local injections of both the SART3 peptide and OK-432 alone was 60%. There seemed to be a reverse correlation between the size of the tumor, survival (Fig. 3B), and the percentage of tumor-infiltrating T cells (Fig. 6), whereas such a correlation was not clear in the other groups. Taken together, these results indicate that the optimal antitumor effect was induced in colon26-bearing mice only when the mice had been immunized in advance and then subsequently administered local injections of both the SART3 peptide and OK-432.

We examined the TILs in colon26 carcinoma after different types of immunotherapy protocols, and we found that combination therapy together with OK-432 led to the most efficient accumulation of not only CD8⁺ T cells, but also CD4⁺ T cells. In the mice which had not been systemically immunized, but which had been treated by local injection with both the OK-432 and the SART3 peptide, CD8⁺ T cells predominantly infiltrated into the tumor sites (Fig. 6, G-4). A similar infiltration of CD8⁺ T cells was observed in mice that had been systemically immunized and subsequently treated with both the OK-432 and the SART3 peptide (Fig. 6, G-8). In order to examine the peptide-specific reactivity of the TILs, we tried to purify and expand the TILs from colon26, using anti-CD3 monoclonal antibody and IL-2, but we were not successful because the contaminated colon26 cells rapidly proliferated, which in turn disrupted the in vitro proliferation of the TILs in culture with IL-2.⁵ Although we could not directly show that the combination therapy increased peptide-specific CTLs in the TILs, we consider that the combination therapy could lead the accumulation of peptide-specific and tumor-reactive CTLs into the tumor site based on the following reasons. First, the combination therapy increased peptide-specific CTLs in the spleen and lymph nodes (Fig. 4). Second, in spite of a nonspecific manner, the local injection of OK-432 could promote the infiltration of such lymphocytes to the tumor sites (Fig. 6).

We have recently been examining the levels of IgGs reactive to CTL-directed peptides administered to patients with various types of cancer, and we have found that increases in the level of IgGs reactive to the administered peptides correlate well with overall survival (27, 28). In this study, increased levels of IgG reactive to the SART3_315-323 peptide were observed in mice that had received local injections with both the SART3 peptide and OK-432 (Fig. 7). The level of peptide-specific IgG was higher among those mice treated with combination therapy among those treated only by local therapy. This finding indicates that systemic immunization with the SART3 peptide was essential to induce high levels of IgG reactive to the administered peptide. Importantly, the mice treated with the combination therapy exhibited a better survival rate than those of the other seven groups. Although we have not yet obtained a clear answer regarding the precise role of peptide-specific IgG in the antitumor effects observed following peptide-based immunotherapy, it is considered likely that such IgGs could exert antitumor effects via antibody-dependent cell-mediated or compliment-dependent cytotoxicity.

OK-432 is well known to elicit local immune responses in vivo (14, 15). On the other hand, recent clinical trials of peptide-based anticancer immunotherapy revealed that although vaccination could increase the frequency of peptide-specific CTL precursors in the periphery, associated clinical responses are rarely observed (13). Insufficient accumulation of such CTLs into tumor sites may account for the lack of antitumor effects. On the other hand, it has been reported that the local injection of OK-432 could facilitate the accumulation of adoptively transferred CTLs into the tumor site (20). In this study, we examined TILs, and we found that the infiltration of T cells with either CD8 or CD4 molecules was augmented when the mice were locally injected with both the SART3 peptide and OK-432. The local injection of either the SART3 peptide or OK432 did not necessarily augment the infiltration of T cells. Systemic immunization and subsequent local injection with both the SART3 peptide and OK-432 did elicit a strong immune response and promoted the accumulation of T cells into tumor sites. There remains the possibility that the SART3 peptide-specific IgG that was induced by systemic immunization could form immune complexes with the locally injected SART3 peptide, thereby resulting in an augmentation of the local immune response.

In conclusion, we showed here that antitumor effects could be efficiently induced by combination therapy involving systemic and local immunization with a CTL-directed peptide together with the local injection of OK-432. This novel protocol may provide a new treatment modality to facilitate the accumulation into tumor sites of vaccine-induced peptide-specific CTLs in the periphery.

	Footnotes

 
Grant support: Supported in part by Grants-in-Aid from the Ministry of Education, Science, Sports, and Culture of Japan (no. 12213134 to K. Itoh and Research Center of Innovative Cancer Therapy of 21st Century COE Program for Medical Science to K. Itoh and A. Yamada), and from the Ministry of Health, Labor and Welfare, Japan (no. H14-trans-002, 11-16, and H12-cancer-004 to K. Itoh, and 15-17 to M. Harada).

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.

⁵ Unpublished observation.

Received 6/21/05; revised 11/11/05; accepted 11/30/05.

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