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The Journal of Immunology, 2000, 164: 1230-1235.
Copyright © 2000 by The American Association of Immunologists

Induction of CTL Responses by Simultaneous Administration of Liposomal Peptide Vaccine with Anti-CD40 and Anti-CTLA-4 mAb1

Daisuke Ito*,{dagger}, Kazumasa Ogasawara{dagger}, Kazuya Iwabuchi{dagger}, Yukio Inuyama* and Kazunori Onoé2,{dagger}

* Department of Otolaryngology, School of Medicine, Hokkaido University, Sapporo, Japan; and {dagger} Section of Pathology, Institute of Immunological Science, Hokkaido University, Sapporo, Japan


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Activation of APC via CD40-CD40 ligand pathway induces up-regulation of costimulatory molecules such as B7 and production of IL-12. Interaction between B7 on APC and CD28 on naive T cells is necessary for priming the T cells. On the other hand, interaction between B7 on APC and CTLA-4 on activated T cells transduces a negative regulatory signal to the activated T cells. In the present study, we attempted to generate tumor-specific CTL by s.c. administration of antigenic peptides encapsulated in multilamellar liposomes (liposomal peptide vaccine) with anti-CD40 mAb and/or anti-CTLA-4 mAb. Liposomal OVA257–264 and anti-CD40 mAb or anti-CTLA-4 mAb were administrated to C57BL/6 mice and the splenocytes were cocultured with OVA257–264 for 4 days. The splenic CD8+ T cells showed a significant cytotoxicity against EL4 cells transfected with cDNA of OVA. In addition, administration of both anti-CD40 and anti-CTLA-4 mAb enhanced the CTL responses. Considerable CTL responses were induced in MHC class II deficient mice by the same procedure. This finding indicated that CTL responses could be generated even in the absence of Th cells. When BALB/c mice were immunized with pRL1a peptide that are tumor-associated Ag of RL1 leukemia cells using the same procedure, significant CTL responses were induced and prolonged survival of the BALB/c mice was observed following RL1 inoculation. These results demonstrate that anti-CD40 mAb and anti-CTLA-4 mAb function as immunomodulators and may be applicable to specific cancer immunotherapy with antitumor peptide vaccine.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
CD40 is a 45- to 50-kDa glycoprotein of 277 aa and a member of the TNF receptor superfamily. The CD40 is expressed on B lymphocytes, dendritic cell (DC),3 follicular DC, hematopoietic progenitor cells, and epithelial cells. CD40 ligand (CD40L) is a 35-kDa glycoprotein of 261 aa and a member of the TNF superfamily. CD40L is expressed on most activated CD4+ T cells and a subset of activated CD8+ T cells (1, 2). Interaction between CD40 and CD40L induces not only B cell growth, isotype switching, and Ig production, but also activation of APC, resulting in up-regulation of costimulatory molecules such as CD80 (B7-1) and CD86 (B7-2), and production of IL-12 (3, 4). Recently, it has been established that helper function of CD4+ T cells for the priming of Ag-specific CD8+ T cell response is performed by activation of DC following CD40-CD40L signaling. Furthermore, it was shown that anti-CD40 mAb replaced a role of CD4+ Th cell in activating DC (5, 6, 7).

In general, naive T cells are activated by two signals from activated APC. One is an Ag-specific signal that is provided when TCR is ligated by the antigenic peptide-MHC molecular complex. The other is a costimulatory signal via the B7-CD28 pathway. On the other hand, activated T cells express CTLA-4 on the cell surface. CTLA-4 is a glycoprotein of 223 aa that belongs to the Ig superfamily. CTLA-4 is the second receptor for B7 family members and binds these members with much higher affinity than CD28. Following interaction with B7 on the APC, CTLA-4 functions as a negative regulator of T cell activation (8, 9). It has been reported that blockade of the B7-CTLA-4 pathway with anti-CTLA-4 mAb enhances antitumor T cell responses and leads to tumor rejection (10, 11).

Recently, genes encoding tumor-associated Ag (TAA) recognized by T lymphocytes have been isolated, and clinical trials by antitumor peptide vaccines consisting of these Ag have been performed especially in melanoma patients (12, 13). In these studies, adjuvants such as IFA were administrated to the patients. Ag-specific CTL were hardly generated by antigenic peptides alone. However, adjuvants lead often to serious side effects, i.e., local tissue damage. Thus, it seems important to develop adjuvants or immunomodulators which are more effective and have less side effects.

In this paper we attempted to induce tumor-specific CTL responses by administration of antigenic peptides encapsulated in multilamellar liposomes (liposomal peptide vaccine) in combination with anti-CD40 and/or anti-CTLA-4 mAb as immunomodulators in murine tumor models. In addition, we evaluated in vivo antitumor effects of the liposomal peptide vaccine with these mAb. The results shown in the present study suggest that anti-CD40 mAb and anti-CTLA-4 mAb can function as potent and safe immunomodulators in specific cancer immunotherapy with antitumor peptide vaccine encapsulated in liposomes.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice

C57BL/6 and BALB/c female mice were purchased from Shizuoka Laboratory Animal Corporation (Hamamatsu, Japan) and used at 6–10 wk of age. C57BL/6 ß2m-/- and C57BL/6 Aßb-/- mice were purchased from The Jackson Laboratory (Bar Harbor, ME) and Taconic Farms (Germantown, NY), respectively.

Cells

The following tumor cell lines were used: RL1 (radiation-induced leukemia; H-2d), P815 (mastocytoma; H-2d), YAC-1 (NK-sensitive leukemia), EL4 (thymoma; H-2b), and EG7 (EL4 transfected with cDNA of chicken OVA).

Peptides

The following antigenic peptides were used: OVA257–264 (SIINFEKL; Kb binding) and pRL1a (IPGLPLSL; Ld binding). These peptides were made using an automatic peptide synthesizer (model 430A, Applied Biosystems, Foster City, CA) as described elsewhere (14). After separation from phenylacetamide methyl-resin by treatment with trifluoromethane sulfonic acid, the peptides were purified to >95% by reverse-phase HPLC on Vydac C18 columns (Waters Japan, Tokyo, Japan).

Liposome

The multilamellar liposome containing antigenic peptides was prepared by mixing with the peptide (100 µg/mouse), 1.25 µmol/mouse phosphatidylserine (Avanti Polar Lipids, Alabaster, AL), and 2.75 µmol/mouse phosphatidylcholine (Avanti Polar Lipids) in chloroform. The mixture in a glass tube was blown by N2 gas, evaporated at 40°C, and aspirated for 3 h. The peptides incorporated in the multilamellar liposome adhered to the inside of the glass tube were dissolved in 200 µl PBS by vortex mixing and sonication washer.

Monoclonal Ab

Anti-CD40 mAb (3/23), anti-CTLA-4 mAb (UC10.4F10.11), rat IgG 2a {kappa}, and hamster IgG were purchased from PharMingen (San Diego, CA). Rat IgG 2a {kappa} and hamster IgG were used as isotype control mAb. Anti-CD8 mAb (Lyt-2.2) and anti-CD4 mAb (GK1.5) were purchased from Meiji (Tokyo, Japan).

Cytotoxicity assays

Mice were s.c. administrated peptides (100 µg/mouse) encapsulated in liposomes with anti-CD40 mAb (50 µg/mouse) and/or anti-CTLA-4 mAb (50 µg/mouse), or with isotype control mAb (50 µg/mouse of rat IgG 2a {kappa} and 50 µg/mouse of hamster IgG). One week later, the primed splenocytes (2 x 106/ml) were cocultured with antigenic peptides (100 ng/ml) in RPMI 1640 medium supplemented with 10% FCS, 10 mM HEPES, 1 mM sodium pyruvate, 0.1 mM nonessential amino acids, 2 mM L-glutamine, and 50 µM 2-ME at 37°C for 4 days and then used as effector cells. In some experiments, the harvested effector cells (2.5 x 107/ml) were incubated with optimally diluted anti-CD4 mAb or anti-CD8 mAb on ice for 30 min. After extensive washing, cells were incubated with 1:10 diluted rabbit C purchased from Cedarlane (Ontario, Canada) at 37°C for 40 min and extensively washed. Target cells were radioactively labeled with Na251CrO4 and added to serially diluted effector cells in 96-well microtiter plates. After 6 h incubation at 37°C, 100 µl of the supernatants was harvested and counted in a gamma counter. The specific lysis of target cells was determined as follows: specific lysis (%) = [(experimental release - spontaneous release)/(maximum release - spontaneous release)] x 100 (7). Spontaneous release was consistently less than 10% of maximum release of the experimental group. Cytotoxicity valures (±SD) at a 100:1 E:T ratio, calculated from those obtained with three or four separate experiments, are illustrated in the figures. The data of mean cytotoxicity were analyzed with Student’s t test. A p value <0.05 was considered significant.

Tumor challenge experiments

For evaluation of protective effects of peptide vaccines, seven groups of five BALB/c mice each were administrated anti-CD40 mAb (50 µg/mouse) and/or anti-CTLA-4 mAb (50 µg/mouse) or isotype control mAb (50 µg/mouse of rat IgG 2a {kappa} and 50 µg/mouse of hamster IgG) with liposomal pRL1a (100 µg/mouse), liposomal pRL1a alone, anti-CD40 mAb and anti-CTLA-4 mAb with empty liposome, or PBS s.c. into the left flank twice at 7-day intervals. One week after the last immunization, mice were implanted s.c. with RL1 cells (2 x 106/mouse) into the right flank, and the survival was monitored.

For evaluation of curative effects, two groups of five BALB/c mice each were implanted s.c. with RL1 cells (2 x 106/mouse) into the right flank on day 0. Mice of each group were administrated both anti-CD40 mAb (50 µg/mouse) and anti-CTLA-4 mAb (50 µg/mouse) with liposomal pRL1a (100 µg/mouse), or PBS into the left flank s.c. beginning on day 13 and subsequently on days 18, 23, and 28. The survival was monitored. The data of mean survival time were analyzed with Student’s t test, and the data of survival ratio were analyzed with Wilcoxon test. A p value <0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
CTL induction by liposomal peptide vaccine and anti-CD40 mAb

At first we attempted to induce CTL activity in C57BL/6 mice by administration of liposomal peptide (OVA257–264) vaccine and anti-CD40 mAb. Because our preliminary experiments showed that 50–500 µg/ml of the anti-CD40 mAb generated almost the same level of enhancement effect, we used 50 µg/ml anti-CD40 mAb throughout the present study. Spleen cells from the immunized C57BL/6 mice were cultured with OVA257–264 for 4 days and evaluated for their cytotoxicity. Fig. 1Goa shows a representative result from three separate experiments. The effector cells from C57BL/6 mice immunized with liposomal OVA257–264 and anti-CD40 mAb exhibited a significant cytotoxic activity against EG7 in the E:T ratio-dependent manner. No or negligible lytic activity was observed against parental EL4 or NK-sensitive YAC-1, respectively (Fig. 1Gob). Thus, the effector cells appeared to contain a NK cell population. By contrast, the effector cells from mice that had been administrated free OVA257–264 with anti-CD40 mAb, empty liposomes with anti-CD40 mAb, or liposomal OVA257–264 without anti-CD40 mAb showed negligible cytotoxic activity (Fig. 1Goa). When the effector cells from mice immunized with liposomal OVA257–264 and anti-CD40 mAb were treated with anti-CD8 mAb and rabbit C before analysis, the cytotoxic activity was considerably decreased (Fig. 1Goc). A slight cytotoxicity seen in this population appeared to be induced by the remaining CD8+ CTL. On the other hand, CD4+ T cell-depleted effector cells showed the same killing activity against EG7 as that seen with the C control. These results demonstrated that the major effector cells were EG7-specific CD8+ CTL.



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  		FIGURE 1. CTL induction by s.c. administration of liposomal peptide vaccine and anti-CD40 mAb. a, C57BL/6 mice were injected s.c. with liposomal OVA257–264 and anti-CD40 mAb ({square}), free OVA257–264 and anti-CD40 mAb ({circ}), empty liposomes and anti-CD40 mAb ({diamond}), or liposomal OVA257–264 ({triangleup}). One week after immunization, primed splenocytes were cultured with OVA257–264 (100 ng/ml) for 4 days, and the CTL activity against EG7 cells was determined by 51Cr release assay. b, CTL activity of the effector cells from C57BL/6 mice immunized with liposomal OVA257–264, and anti-CD40 mAb was examined against EG7 ({square}), EL4 ({triangleup}), or YAC-1 cells ({circ}). c, The effector cells from C57BL/6 mice immunized with liposomal OVA257–264 and anti-CD40 mAb were treated with anti-CD8 mAb and C ({square}), anti-CD4 mAb and C ({triangleup}), or C alone ({circ}) before 51Cr release assay. Each line represents cytotoxicity obtained with one individual mouse.

 
Fig. 2Go shows mean cytotoxicity at 100:1 E/T ratio in each experimental group calculated from three separate experiments. It was again demonstrated that administration of 1iposomal OVA257–264 and anti-CD40 mAb generated EG7 specific CD8+ CTL, although NK cells killing YAC-1 cells might be present in the splenocytes.



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  		FIGURE 2. Mean cytotoxicity of splenocytes from C57BL/6 mice administrated liposomal OVA257–264 and anti-CD40 mAb. a, C57BL/6 mice were administrated liposomal OVA257–264 or free OVA257–264 with or without anti-CD40 mAb. b, Cytotoxicity of splenocytes from C57BL/6 mice administrated liposomal OVA257–264 and anti-CD40 mAb was determined for EG7, EL4, or YAC-1 targets. c, The splenocytes were treated with anti-CD8 mAb plus C, anti-CD4 mAb plus C, or C alone before analysis. Statistical analysis is indicated in the figure. Cytotoxicity values (mean ± SD) at a 100:1 E/T ratio in each experimental group, calculated from three separate experiments, are shown.

 
CTL induction by liposomal peptide vaccine and anti-CTLA-4 mAb

Next we attempted to prime CTL precursors by administration of liposomal peptide vaccine and anti-CTLA-4 mAb. Because our preliminary experiments showed that 50–500 µg/ml of the anti-CTLA-4 mAb exerted the same level of influence, we used 50 µg/ml anti-CTLA-4 mAb throughout this study. Spleen cells from C57BL/6 mice immunized with liposomal OVA257–264 and anti-CTLA-4 mAb were cultured with OVA257–264 and then quantitated for their cytotoxicity. Fig. 3Go shows mean cytotoxicity at 100:1 E:T ratio in each experimental group calculated from three separate experiments. The effector cells recovered from the culture showed significant cytotoxic activity against EG7 (Fig. 3Goa). Although at this high E:T ratio, control groups treated with free OVA257–264 with anti-CTLA-4 mAb, empty liposomes with anti-CTLA-4 mAb or liposomal OVA257–264 without anti-CTLA-4 mAb exhibited a slight cytotoxicity against EG7, the experimental group (Fig. 3Goa, top line) showed significantly higher cytotoxicity than those in other groups. These effector cells showed no or negligible cytotoxicity against EL4 or YAC-1 cells (Fig. 3Gob). Thus, it seemed that a small number of NK cells was present in the effector cell population. When the effector cells from mice immunized with liposomal OVA257–264 and anti-CTLA-4 mAb were treated with anti-CD8 mAb and rabbit C, the cytotoxic activity was considerably decreased (Fig. 3Goc). Treatment with anti-CD4 plus rabbit C showed no influence. These results indicate that administration of liposomal peptide vaccine and anti-CTLA-4 mAb generates precursors of Ag-specific CD8+ CTL in vivo.



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  		FIGURE 3. CTL induction by s.c. administration of liposomal peptide vaccine and anti-CTLA-4 mAb. a, C57BL/6 mice were injected s.c. with liposomal OVA257–264 and anti-CTLA-4 mAb, free OVA257–264 and anti-CTLA-4 mAb, empty liposomes and anti-CTLA-4 mAb, or liposomal OVA257–264. One week after immunization, primed splenocytes were cultured with OVA257–264 (100 ng/ml) for 4 days, and the CTL activity against EG7 cells was determined by 51Cr release assay. b, CTL activity of the effector cells from C57BL/6 mice immunized with liposomal OVA257–264 and anti-CTLA-4 mAb was determined against EG7, EL4, or YAC-1 cells. c, The effector cells from C57BL/6 mice immunized with liposomal OVA257–264 and anti-CTLA-4 mAb were treated with anti-CD8 mAb and C, anti-CD4 mAb and C, or C alone before 51Cr release assay. Cytotoxicity values (mean ± SD) at a 100:1 E:T ratio in each experimental group, calculated from three separate experiments, are shown.

 
CTL priming in the absence of Th cell

It has been shown that anti-CD40 mAb can replace a function of Th cell which activates APC, especially DC (5, 6, 7). We then asked whether the combination of liposomal OVA257–264 and anti-CD40 mAb induced CTL responses in CD4+ T cell-deficient mice. Liposomal OVA257–264 and anti-CD40 mAb were administrated to C57BL/6 Aßb-/- mice and the spleen cells were cultured with OVA257–264 for 4 days. Fig. 4Goa shows mean cytotoxicity ± SD at a 100:1 E:T ratio in each experimental group calculated from three separate experiments. The effector cells recovered from the culture exhibit comparable cytotoxic activity against EG7 to those from C57BL/6 Aßb+/+ mice treated in the same manner. The slightly lower cytotoxicity seen in C57BL/6 Aßb-/- mice, however, suggested that Th cells might exert some influences. By contrast, effector cells from C57BL/6 ß2m-/- mice immunized using the same procedure showed no significant cytotoxicity. When C57BL/6 Aßb-/- mice were immunized with liposomal OVA257–264 and anti-CTLA-4 mAb, effector cells from these C57BL/6 Aßb-/- mice also showed comparable cytotoxic activity against EG7 to those from C57BL/6 Aßb+/+ mice treated in the same manner (Fig. 4Gob). Effector cells from C57BL/6 ß2m-/- mice treated with liposomal OVA257–264 and anti-CTLA-4 mAb showed a slight cytotoxicity against EG7 cells. This finding cannot be explained at present. These results on the whole demonstrate that CTL priming by liposomal peptide vaccine with either anti-CD40 mAb or anti-CTLA-4 mAb is Th cell-independent.



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  		FIGURE 4. Induction of CTL responses in the absence of Th cell. C57BL/6, C57BL/6 ß2m-/- and C57BL/6 Aßb-/- mice were s.c. administrated liposomal OVA257–264 with either anti-CD40 mAb (a) or anti-CTLA-4 mAb (b). One week after immunization, primed splenocytes were cultured with OVA257–264 (100 ng/ml) for 4 days, and the CTL activity against EG7 cells was determined by 51Cr release assay. Cytotoxicity values (mean ± SD) at a 100:1 E:T ratio in each experimental group, calculated from three separate experiments, are shown.

 
CTL induction by liposomal peptide vaccine with both anti-CD40 and anti-CTLA-4 mAb

Next we attempted to enhance cytotoxic activity by simultaneous administration of both anti-CD40 and anti-CTLA-4 mAb with liposomal peptide vaccine. Fig. 5Go summarizes four separate experiments. The effector cells from C57BL/6 mice administrated both anti-CD40 and anti-CTLA-4 mAb with liposomal OVA257–264 showed markedly higher lytic activity against EG7 than those administrated either anti-CD40 mAb or anti-CTLA-4 mAb with liposomal OVA257–264 (Fig. 5Goa). However, the effector cells from C57BL/6 mice treated with isotype control mAb and liposomal OVA257–264 showed negligible lytic activity against EG7. Similar augmentation was obtained with another peptide-tumor system. When BALB/c mice were administrated anti-CD40 mAb or anti-CTLA-4 mAb with liposomal pRL1a that is an antigenic peptide derived from BALB/c-derived leukemia, RL1, and the spleen cells were restimulated with pRL1a in vitro, a significant cytotoxic activity against RL1 cells was demonstrated (Fig. 5Gob). Furthermore, the effector cells from BALB/c mice given liposomal pRL1a with both anti-CD40 mAb and anti-CTLA-4 mAb showed considerably higher level of cytotoxic activity against RL1 than those treated with liposomal pRL1a with either anti-CD40 mAb or anti-CTLA-4 mAb alone. These effector cells killed neither syngeneic P815 nor YAC-1 cells (Fig. 5Goc). When the effector cells from BALB/c mice immunized with liposomal pRL1a and both anti-CD40 mAb and anti-CTLA-4 mAb were treated with anti-CD8 mAb and rabbit C, the cytotoxic activity was significantly decreased (Fig. 5God). Depletion of CD4+ T cells from effector cells showed no influence on the cytotoxicity. Thus, it was again demonstrated that the major effector cells induced by immunization with liposomal pRL1a and both anti-CD40 mAb and anti-CTLA-4 mAb were RL1-specific CD8+ CTL. These results also indicate that the simultaneous administration of both anti-CD40 mAb and anti-CTLA-4 mAb with liposomal peptide vaccine enhances markedly the cytotoxic activity as compared with that of liposomal peptide vaccine and either mAb alone.



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  		FIGURE 5. CTL induction by s.c. administration of liposomal peptide vaccine with anti-CD40 mAb and anti-CTLA-4 mAb. a, C57BL/6 mice were injected s.c. with liposomal OVA257–264, anti-CD40 mAb and anti-CTLA-4 mAb, liposomal OVA257–264 and anti-CD40 mAb, liposomal OVA257–264 and anti-CTLA-4 mAb, or liposomal OVA257–264 and isotype control mAb. The CTL activity was determined as described in the legend to Fig. 1Go. b, BALB/c mice were injected s.c. with liposomal pRL1a, anti-CD40 mAb and anti-CTLA-4 mAb, liposomal pRL1a and anti-CD40 mAb, liposomal pRL1a and anti-CTLA-4 mAb, or liposomal pRL1a and isotype control mAb. One week after immunization, primed splenocytes were cultured with pRL1a (100 ng/ml) for 4 days, and the CTL activity against RL1 cells was determined by 51Cr release assay. c, CTL activity of the effector cells from BALB/c mice immunized with liposomal pRL1a, anti-CD40 mAb, and anti-CTLA-4 mAb was examined against RL1, P815 or YAC-1 cells. d, The effector cells from BALB/c mice immunized with liposomal pRL1a, anti-CD40 mAb, and anti-CTLA-4 mAb were treated with anti-CD8 mAb and C, anti-CD4 mAb and C, or C alone before 51Cr release assay. Cytotoxicity values (mean ± SD) at a 100:1 E:T ratio in each experimental group, calculated from four separate experiments, are shown.

 
Prolongation of survival of tumor-implanted mice by administration of liposomal peptide vaccine with both anti-CD40 and anti-CTLA-4 mAb

We examined in vivo antitumor effects of liposomal peptide vaccine with anti-CD40 and/or anti-CTLA-4 mAb. BALB/c mice were s.c. administrated liposomal pRL1a with anti-CD40 mAb and/or anti-CTLA-4 mAb twice at 7-day intervals. Our preliminary experiments indicated that this protocol increased the CTL activity specific for RL1 (data not shown). One week after the last immunization, the immunized BALB/c mice were s.c. implanted RL1 cells (2 x 106/mouse). All mice in the control groups that were treated with liposomal pRL1a and isotype control mAb, liposomal pRL1a alone, empty liposome with both anti-CD40 mAb and anti-CTLA-4 mAb, or PBS died within 48 days (Fig. 6Goa). By contrast, 40% mice in the groups administrated either anti-CD40 mAb or anti-CTLA-4 mAb with liposomal pRL1a survived up to 80 days (p < 0.05). It should be noted that 100% mice in the group given both anti-CD40 and anti-CTLA-4 mAb with liposomal pRL1a survived until 80 days (p < 0.01) (Fig. 6Goa). This finding suggests that in mice treated with liposomal peptides and both anti-CD40 and anti-CTLA-4 mAb, potent CTL are generated.



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  		FIGURE 6. Induction of in vivo antitumor activity by s.c. administration of liposomal peptide vaccine with anti-CD40 mAb and/or anti-CTLA-4 mAb. a, Seven groups of five BALB/c mice each were immunized s.c. into the left flank with liposomal pRL1a, anti-CD40 mAb and anti-CTLA-4 mAb ({square}), liposomal pRL1a and anti-CD40 mAb ({circ}), liposomal pRL1a and anti-CTLA-4 mAb ({diamond}) liposomal pRL1a and isotype control mAb ({triangleup}), liposomal pRL1a ({triangledown}), empty liposome, anti-CD40 mAb and anti-CTLA-4 mAb ({blacktriangleup}) or PBS ({blacktriangledown}) twice at 7-day intervals. One week after the last immunization, these mice were implanted s.c. with RL1 cells (2 x 106/mouse) into the right flank. Thereafter, survival of the tumor implanted mice was observed. A representative result from two separate experiments is shown. b, Two groups of five BALB/c mice each were implanted s.c. with RL1 cells (2 x 106/mouse) into the right flank on day 0. These mice were s.c. administrated liposomal pRL1a with both anti-CD40 mAb and anti-CTLA-4 mAb ({square}) or PBS ({blacktriangledown}) into the left flank on days 13, 18, 23, and 28. Thereafter, survival of the mice was observed.

 
We then attempted to treat tumor-bearing mice by administration of liposomal peptide vaccine with anti-CD40 mAb and anti-CTLA-4 mAb. BALB/c mice were s.c. implanted RL1 cells (2 x 106/mouse) on day 0. On day 13 these mice had palpable tumors. The tumor-bearing mice were then s.c. administrated liposomal pRL1a with anti-CD40 mAb and anti-CTLA-4 mAb. In the control group treated with PBS alone, mean survival time was 32.8 days. However, in the group of mice treated with liposomal pRL1a and both mAb, mean survival time of the dead mice was 44.5 days (p < 0.002), and complete tumor rejection was observed in one of the five mice (Fig. 6Gob). Thus, CTL specific for RL1 appeared to be generated in the tumor-bearing mice. These results demonstrate that antitumor immunity is indeed induced by liposomal peptide vaccine with anti-CD40 mAb and anti-CTLA-4 mAb.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Since human gene MAGE-1 encoding an Ag recognized by CTL was first reported on a human melanoma cell, various TAA have been identified on a variety of tumor cell lines (15, 16). Using these TAA, many different methods have been contemplated to generate CTL specific for the TAA. It has become evident that it is difficult to prime antitumor CTL by administration of the antigenic peptide alone. Thus far, it has been considered that administration of DC that are pulsed by antigenic peptides deduced from TAA is the most promising method for priming potent Ag-specific CTL (17). The peptide-pulsed DC express consistently a high level of costimulatory molecules and present efficiently the peptide-MHC complexes to T cells. However, this approach includes an intricacy in that DC must be generated in vitro by culturing bone marrow cells or peripheral blood cells of the subject in the presence of GM-CSF and IL-4 (18). To circumvent the intricacy, carriers of antigenic peptides and/or appropriate adjuvants have been developed. Among the carriers, heat shock protein (HSP) is a potent one (19, 20). However, HSP must be isolated from the autologous tumor cells and has not been applied to humans, yet.

In the present study, we used another carrier, liposome, to induce potent CTL specific for peptide Ag. It is well established that the liposome is safe and applicable to the human case (21). In addition, it has been reported that antigenic peptides encapsulated in liposome can be phagocytosed by APC, especially DC, and transferred into the classical MHC class I pathway via phagosome-to-cytosol pathway (22, 23). This pathway seems to be similar to that for processing apoptotic cells. DC preferentially engulf the apoptotic cells using scavenger receptors which bind phosphatidyl serine present in outer membrane of the apoptotic cells (24).

In addition to liposome, we employed anti-CD40 mAb and anti-CTLA-4 mAb as immunomodulators. Thus far, IFA, keyhole limpet hemocyanin and Bacille Calmette-Guérin (BCG) have been used as the adjuvants in clinical trials of tumor immunotherapy. However, these adjuvants showed various side effects such as transient erythema and induration as delayed-type hypersensitivity reactivity, skin breakdown at the injection site, fever, chills, and fatigue (12, 17, 25). Thus, development of adjuvants or immunomodulators, which are more effective and have less side effects, has been required.

We could demonstrate that administration of anti-CD40 and anti-CTLA-4 mAb enhanced considerably the peptide-specific CTL priming. Indeed, the CTL showed a prominent antitumor activity in vitro. It seems that anti-CD40 mAb that substitutes the roles of CD40L on Th cell activates DC and induces up-regulation of B7 on the DC, which results in efficient priming of CTL precursors. On the other hand, anti-CTLA-4 mAb may block the inhibitory pathway through interaction between the increased B7 on DC and CTLA-4 on activated CTL and maintain the activation stage of the CTL (5, 6, 7, 10). We consider that in this way anti-CD40 mAb and anti-CTLA-4 mAb function synergistically and enhance priming CTL specific for the antigenic peptide encapsulated in liposome.

In the present study, using C57BL/6 Aßb-/- mice, we demonstrated that CTL induction by liposomal peptide vaccine and anti-CD40 mAb was Th cell-independent. Furthermore, it was shown that the CTL induction by liposomal peptide vaccine and anti-CTLA-4 mAb was also Th cell-independent. The exact mechanism underlying the antitumor T cell responses following CTLA-4 blockade remains unclear. The CTLA-4 blockade might not only sustain proliferation of activated T cells by interfering with negative signaling through B7-CTLA-4 interactions, but also lower the level of overall threshold required for the T cell activation, which might result in activation of otherwise unreactive CTL. In this connection, it should be noted that splenocytes from C57BL/6 ß2m-/- mice treated with anti-CTLA-4 mAb alone showed a substantial cytotoxicity against EG7 cells (Fig. 4Gob). This point should be pursued in future studies.

In the last experiment, we were able to demonstrate protective effects of liposomal pRL1a peptide with anti-CD40 mAb and anti-CTLA-4 mAb against RL1 tumor cell inoculation in the preimmunized mice. However, we were unable to induce sufficiently curative effects in the tumor-bearing mice by the same method (Fig. 6Go). This result seems to be explained by the rapid tumor growth (11). Thus, routes, dosages, and intervals of vaccination for the treatment of tumor-bearing animals should be reconsidered. In addition, the insufficient antitumor effect in the tumor-bearing mice may be attributed to the tumor-induced T cell apoptosis (26), development of peripheral tolerance to the antigenic peptides (27, 28), alterations in T cell signal transduction of tumor-bearing hosts (29, 30, 31), or the generation of tumor-induced suppressor cells (32).

In conclusion, we demonstrated in this paper that anti-CD40 mAb and anti-CTLA-4 mAb were applicable as the immunomodulators when administrated with antitumor peptide vaccine encapsulated in liposome. Our procedure, in which limited materials are used, induces tumor-specific CTL without detrimental side effects. Moreover, the CTL responses induced by our method were Th cell-independent. Thus, this approach may be not only a useful strategy for clinical treatment of human cancer, but also applicable to patients with HIV infection who have generally severe deficiency of CD4+ T cells.


   Footnotes
 
1 This study was supported by Grant-in-Aid for Scientific Research by the Ministry of Education, Science, Sports and Culture, Japan (20169163, 09470059); the Ministry of Health and Welfare, Japan; and the Sagawa Cancer Foundation. Back

2 Address correspondence and reprint requests to Dr. Kazunori Onoé, Section of Pathology, Institute of Immunological Science, Hokkaido University, Sapporo 060-0815, Japan. E-mail address: Back

3 Abbreviations used in this paper: DC, dendritic cell; CD40L, CD40 ligand; TAA, tumor-associated Ag. Back

Received for publication May 20, 1999. Accepted for publication November 16, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Banchereau, J., F. Bazan, D. Blanchard, F. Briére, J. P. Galizzi, C. van Kooten, Y. J. Liu, F. Rousset, S. Saeland. 1994. The CD40 antigen and its ligand. Annu. Rev. Immunol. 12:881.[Medline]
  2. Lane, P., A. Traunecker, S. Hubele, S. Inui, A. Lanzavecchia, D. Gray. 1992. Activated human T cells express a ligand for the human B cell-associated antigen CD40 which participates in T cell-dependent activation of lymphocytes. Eur. J. Immunol. 22:2573.[Medline]
  3. Caux, C., C. Massacrier, B. Vanbervliet, B. Dubois, C. Van Kooten, I. Durand, J. Banchereau. 1994. Activation of human dendritic cells through CD40 cross-linking. J. Exp. Med. 180:1263.[Abstract/Free Full Text]
  4. Shu, U., M. Kiniwa, C. Y. Wu, C. Maliszewski, N. Vezzio, J. Hakimi, M. Gately, G. Delespesse. 1995. Activated T cells induce interleukin-12 production by monocytes via CD40-CD40 ligand interaction. Eur. J. Immunol. 25:1125.[Medline]
  5. Ridge, J. P., F. D. Rosa, P. Matzinger. 1998. A conditioned dendritic cell can be a temporal bridge between a CD4+ T-helper and T-killer cell. Nature 393:474.[Medline]
  6. Bennett, S. R. M., F. R. Carbone, F. Karamalis, R. A. Flavell, J. F. A. P. Miller, W. R. Heath. 1998. Help for cytotoxic-T-cell responses is mediated by CD40 signalling. Nature 393:478.[Medline]
  7. Schoenberger, S. P., R. E. M. Toes, E. I. H. van der Voort, R. Offringa, C. J. M. Melief. 1998. T-cell help for cytotoxic T lymphocytes is mediated by CD40-CD40L interactions. Nature 393:480.[Medline]
  8. Burnet, J. F., F. Denizot, M. F. Luciani, M. Roux-Dosseto, M. Suzan, M. G. Mattei, P. Golstein. 1987. A new member of the immunoglobulin superfamily: CTLA-4. Nature 328:267.[Medline]
  9. Walunas, T. L., D. J. Lenschow, C. Y. Bakker, P. S. Linsley, G. J. Freeman, J. M. Green, C. B. Thompson, J. A. Bluestone. 1994. CTLA-4 can function as a negative regulator of T cell activation. Immunity 1:405.[Medline]
  10. Leach, D. R., M. F. Krummel, J. P. Allison. 1996. Enhancement of antitumor immunity by CTLA-4 blockade. Science 271:1734.[Abstract]
  11. Yang, Y., J. Zou, J. Mu, R. Wijesuriya, S. Ono, T. Walunas, J. Bluestone, H. Fujiwara, T. Hamaoka. 1997. Enhanced induction of antitumor T-cell responses by cytotoxic T lymphocyte-associated molecule-4 blockade: the effect is manifested only at the restricted tumor-bearing stages. Cancer Res. 57:4036.[Abstract/Free Full Text]
  12. Rosenberg, S. A., J. C. Yang, D. J. Schwartzentruber, P. Hwu, F. M. Marincola, S. L. Topalian, N. P. Restifo, M. E. Dudley, S. L. Schwarz, P. J. Spiess, et al 1998. Immunologic and therapeutic evaluation of a synthetic peptide vaccine for the treatment of patients with metastatic melanoma. Nat. Med. 4:321.[Medline]
  13. Boon, T.. 1993. Tumor antigens recognized by cytolytic T lymphocytes: present perspectives for specific immunotherapy. Int. J. Cancer 54:117.
  14. Ogasawara, K., H. Naruse, Y. Itoh, T. Gotohda, J. Arikawa, H. Kida, R. A. Good, K. Onoé. 1992. A strategy for making synthetic peptide vaccines. Proc. Natl. Acad. Sci. USA 89:8995.[Abstract/Free Full Text]
  15. van der Bruggen, P., C. Traversari, P. Chomez, C. Lurquin, E. De Plaen, B. van den Eynde, A. Knuth, T. Boon. 1991. A gene encoding an antigen recognized by cytolytic T lymphocytes on a human melanoma. Science 254:1643.[Abstract/Free Full Text]
  16. Traversari, C., P. van der Bruggen, I. F. Luescher, C. Lurquin, P. Chomez, A. Van Pel, E. De Plaen, A. Amar-Costesec, T. Boon. 1992. A nonapeptide encoded by human gene MAGE-1 is recognized on HLA-A1 by cytolytic T lymphocytes directed against tumor antigen MZ2-E. J. Exp. Med. 176:1453.[Abstract/Free Full Text]
  17. Nestle, F. O., S. Alijagic, M. Gilliet, Y. Sun, S. Grabbe, R. Dummer, G. Burg, D. Schadendorf. 1998. Vaccination of melanoma patients with peptide or tumor lysate-pulsed dendritic cells. Nat. Med. 4:328.[Medline]
  18. Romani, N., S. Gruner, D. Brang, E. Kämpgen, A. Lenz, B. Trockenbacher, G. Konwalinka, P. O. Fritsch, R. M. Steinman, G. Schuler. 1994. Proliferating dendritic cell progenitors in human blood. J. Exp. Med. 180:83.[Abstract/Free Full Text]
  19. Suto, R., P. K. Srivastava. 1995. A mechanism for the specific immunogenicity of heat shock protein-chaperoned peptides. Science 269:1585.[Abstract/Free Full Text]
  20. Tamura, Y., P. Peng, K. Liu, M. Daou, P. K. Srivastava. 1997. Immunotherapy of tumors with autologous tumor-derived heat shock protein preparations. Science 278:117.[Abstract/Free Full Text]
  21. Fries, L. F., D. M. Gordon, R. L. Richards, J. E. Egan, M. R. Hollingdale, M. Gross, C. Silverman, C. R. Alving. 1992. Liposomal malaria vaccine in humans: A safe and potent adjuvant strategy. Proc. Natl. Acad. Sci. USA 89:358.[Abstract/Free Full Text]
  22. Alving, C. R.. 1991. Liposome as carriers of antigens and adjuvants. J. Immunol. Methods 140:1.[Medline]
  23. Shen, Z., G. Reznikoff, G. Dranoff, K. L. Rock. 1997. Cloned dendritic cells can present exogenous antigens on both MHC class I and class II molecules. J. Immunol. 158:2723.[Abstract]
  24. Albert, M. L., B. Sauter, N. Bhardwaj. 1998. Dendritic cells acquire antigen from apoptotic cells and induce class I-restricted CTLs. Nature 392:86.[Medline]
  25. Goydos, J. S., E. Elder, T. L. Whiteside, O. J. Finn, M. T. Lotze. 1996. A phase I trial of a synthetic mucin peptide vaccine. J. Surg. Res. 63:298.[Medline]
  26. Hahne, M., D. Rimoldi, M. Schröter, P. Romero, M. Schreier, L. E. French, P. Schneider, T. Bornand, A. Fontana, D. Lienard, et al 1996. Melanoma cell expression of Fas (Apo-1/CD95) ligand: implications for tumor immune escape. Science 274:1363.[Abstract/Free Full Text]
  27. Toes, R. E. M., R. Offringa, R. J. J. Blom, C. J. M. Melief, W. M. Kast. 1996. Peptide vaccination can lead to enhanced tumor growth through specific T-cell tolerance induction. Proc. Natl. Acad. Sci. USA 93:7855.[Abstract/Free Full Text]
  28. Staveley-O’Carroll, K., E. Sotomayor, J. Montgomery, I. Borrello, L. Hwang, S. Fein, D. Pardoll, H. Levitsky. 1998. Induction of antigen-specific T cell anergy: an early event in the course of tumor progression. Proc. Natl. Acad. Sci. USA 95:1178.[Abstract/Free Full Text]
  29. Mizoguchi, H., J. J. O’Shea, D. L. Longo, C. M. Loeffler, D. W. McVicar, A. C. Ochoa. 1992. Alterations in signal transduction molecules in T lymphocytes from tumor-bearing mice. Science 258:1795.[Abstract/Free Full Text]
  30. Nakagomi, H., M. Petersson, I. Magnusson, C. Juhlin, M. Matsuda, H. Mellstedt, J. L. Taupin, E. Vivier, P. Anderson, R. Kiessling. 1993. Decreased expression of the signal-transducing {zeta} chains in tumor-infiltrating T-cells and NK cells of patients with colorectal carcinoma. Cancer Res. 53:5610.[Abstract/Free Full Text]
  31. Renner, C., S. Ohnesorge, G. Held, S. Bauer, W. Jung, J. P. Pfitzenmeier, M. Pfreundschuh. 1996. T cells from patients with Hodgkin’s disease have a defective T-cell receptor {zeta} chain expression that is reversible by T-cell stimulation with CD3 and CD28. Blood 88:236.[Abstract/Free Full Text]
  32. Tamada, K., M. Harada, M. Abex, M. Lix, H. Tada, Y. Onoe, K. Nomoto. 1997. Immunosuppressive activity of cloned natural killer (NK1.1+) T cells established from murine tumor-infiltrating lymphocytes. J. Immunol. 158:4846.[Abstract]



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