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The Journal of Immunology, 2003, 170: 3401-3407.
Copyright © 2003 by The American Association of Immunologists

CTLA-4 Blockade Enhances the Therapeutic Effect of an Attenuated Poxvirus Vaccine Targeting p53 in an Established Murine Tumor Model1

Jonathan Espenschied*, Jeffrey Lamont*, Jeff Longmate{dagger}, Solange Pendas*, Zhongde Wang{ddagger}, Don J. Diamond{ddagger} and Joshua D. I. Ellenhorn2,*

Divisions of * General and Oncologic Surgery, and {dagger} Information Sciences, City of Hope National Medical Center, and {ddagger} Laboratory of Vaccine Research, Beckman Research Institute of the City of Hope, Duarte, CA 91010


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
p53 is overexpressed by half of all cancers, and is an attractive target for a vaccine approach to immunotherapy. p53 overexpression is frequently the result of point mutations, which leaves the majority of the protein in its wild-type form. Therefore, the majority of p53 sequence is wild type, making it a self-protein for which tolerance plays a role in limiting immune responses. To overcome tolerance to p53, we have expressed wild-type murine p53 in the nonpathogenic attenuated poxvirus, modified vaccinia virus Ankara (recombinant modified vaccinia virus Ankara expressing wild-type murine p53 (rMVAp53)). Mice immunized with rMVAp53 vaccine developed vigorous p53-specific CTL responses. rMVAp53 vaccine was evaluated for its ability to inhibit the outgrowth of the syngeneic murine sarcoma Meth A, which overexpresses mutant p53. Mice were inoculated with a lethal dose (5 x 105 cells injected s.c.) of Meth A tumor cells and vaccinated by i.p. injection 3 days later with 5 x 107 PFU of rMVAp53. The majority of mice remained tumor free and resistant to rechallenge with Meth A tumor cells. We wished to determine whether rMVAp53 immunization could effect the rejection of an established, palpable Meth A tumor. In subsequent experiments, mice were injected with 106 Meth A tumor cells, and treated 6 days later with anti-CTLA-4 Ab (9H10) and rMVAp53. The majority of treated mice had complete tumor regression along with lasting tumor immunity. In vivo Ab depletion confirmed that the antitumor effect was primarily CD8 and to a lesser extent CD4 dependent. These experiments demonstrate the potential of a novel cell-free vaccine targeting p53 in malignancy.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Mutations of p53, which abrogate its function as a suppresser of cell division, are associated with a high nuclear and cytoplasmic concentration of the p53 protein. What makes the p53 gene product an attractive target for an adaptive immune response is that the intracellular concentration of nonmutated p53 is normally very low, and normal cells have a low p53 turnover rate (1). Cells expressing normal p53 at low levels will most likely escape an enhanced immune response to overexpressed mutant p53 (2). Mutations in p53 occur at many sites, most of which do not correspond to immunologic epitopes (3). To be widely applicable, p53-directed immunotherapy would need to target wild-type p53 (wtp53)3 epitopes. Several groups have generated human CTL against HLA class I-binding motif peptides from wtp53. Multiple in vitro stimulations were required to elicit wtp53-specific CTL derived from human PBMC, capable of lysing human tumor cells that overexpress p53 (4). In addition, by immunizing HLA-A2-transgenic mice, we (5, 6) and others (7) have been able to generate CTL that can recognize and lyse HLA-A2+ and p53-overexpressing human tumor cells.

Because p53 is an autoantigen widely expressed throughout development, tolerance to p53 will limit the effectiveness of p53-directed immunotherapies. Both functional (8, 9) and tetramer studies (10) in mice have clearly demonstrated tolerance to p53 at the CTL level. To achieve successful p53-directed immunotherapy, it will be necessary to break immunological tolerance to p53. The murine system provides an excellent preclinical model for evaluating the ability of immunologic approaches to overcoming tolerance to p53. Murine and human p53 are physiologically analogous, with 80% sequence homology. Using an immunization strategy in mice, it has been possible to generate wtp53-specific CTL that can lyse murine p53-overexpressing tumor cells (11, 12). In addition, some immunotherapy approaches in the murine model have been successful at preventing the outgrowth of p53-overexpressing tumors (13, 14, 15, 16, 17). Treatment of established p53-overexpressing tumors in mice has required intratumoral immunization with ALVAC/p53 (18), infusion of epitope-specific CTL (12, 19), infusion of epitope-pulsed or adenovirus-infected dendritic cells (DC) (11, 20), or infusion of mutant p53 epitope with IL-12 (21). In these reports, tumor rejection occurred with the noticeable absence of autoreactivity toward autologous noncancerous murine cells that express normal levels of p53 (11, 19).

An optimal immunotherapy approach to p53 would involve an immunization strategy which generates vigorous effector and memory T cell responses, without the requirement for a haplotype (HLA-matched)- or patient-specific vaccine. An ideal viral vector candidate for such a vaccine is modified vaccinia virus Ankara (MVA). rMVA-based vaccines elicit systemic immunity or protection against viral (22) and tumor-associated Ags (23, 24, 25) in murine models. Successes in limiting disease to several different pathogens motivated us to investigate whether rMVA expressing murine wtp53 (rMVAp53) enhances p53-specific immunity. We found that immunization with rMVAp53 promotes p53-specific CTL and results in the rejection of established p53-overexpressing tumors in vivo following a large tumor inoculum. Furthermore, the addition of CTLA-4 mAb blockade enhances the effects of rMVAp53 immunization resulting in rejection of palpable p53-overexpressing tumor cells and lasting tumor immunity.


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

Female 6- to 8-wk-old BALB/c and IFN-{gamma} knockout (IFN-{gamma}KO) mice on the BALB/c background, were obtained from The Jackson Laboratory (Bar Harbor, ME) and maintained in a specific pathogen-free environment. All studies were approved by the Research Animal Care Committee of City of Hope National Medical Center, and performed under the American Association for the Accreditation of Laboratory Animal Care guidelines.

Cell lines

CV-1 (26), TK- (27), and baby hamster kidney cells (BHK-21) (28) were purchased from American Type Culture Collection (Manassas, VA) and grown in MEM supplemented with nonessential amino acids, L-glutamine, and 10% FCS. Meth A sarcoma cells (Meth A) (29) were a kind gift of Dr. L. J. Old (Memorial Sloan-Kettering Cancer Center, New York, NY). Meth A was passaged as an ascitic tumor. Cells were harvested, counted, and washed with PBS before use. HEK-293 cells (30) and p53null 10.1 cells were kind gifts from Drs. K. K. Wong and S. Kane, respectively (City of Hope National Medical Center).

Antibodies

Anti-CD4 (GK1.5) (31) and anti-NK1.1 (PK136) (32) were purchased from American Type Culture Collection. Anti-CD8 (H35) (33) and anti-CTLA-4 (9H10) (34) were a kind gift from J. Allison (University of California, Berkeley, CA). Abs were produced using a CELLine device (BD Biosciences, Mountain View, CA). IgG Abs were purified by absorbance over protein G-Sepharose (Amersham, Uppsala, Sweden) followed by elution with 0.1 M glycine-HCl (pH 2.7). The product was then dialyzed against phosphate-buffered normal saline and concentrated using a Centriplus centrifugal filter device (Millipore, Bedford, MA). Control Syrian Hamster IgG was obtained from Jackson ImmunoResearch (West Grove, PA).

Viral constructs

Wild-type MVA was obtained from Drs. B. Moss and L. Wyatt (National Institutes of Health, Bethesda, MD). The entire cDNA of murine wtp53 was amplified by PCR from mRNA obtained from murine splenocytes. The murine p53 PCR product was ligated into the cloning site of the MVA expression vector pMCO3 (obtained from Drs. B. Moss and L. Wyatt). This vector contains sequences that insert into deletion III of the MVA genome, and contains the GUS (Escherichia coli {beta}-glucuronidase) gene used for screening purposes (35). Generation of rMVA was achieved on monolayers of BHK-21 (BHK) cells. Briefly, BHK cells were transfected with 20 µg of plasmid DNA using Lipofectin (Invitrogen, Carlsbad, CA) and infected with wild-type MVA at a multiplicity of infection of 0.01. The infected cells were incubated for 48 h, and then harvested, pelleted, and subjected to three cycles of freeze/thaw and sonication to lyse the cells. The rMVAp53 was screened for GUS expression by adding X-GlcA (5-bromo-4-chloro-3-indolyl {beta}-D-glucuronide; Sigma-Aldrich, St. Louis, MO). After 10 rounds of purification, the rMVAp53 was expanded on BHK monolayers. The rMVA titer was determined by immunostaining infected cultures using the Vectastain Elite ABC (avidin/biotin complex) kit (Vector Laboratories, Burlingame, CA). An rMVA expressing pp65 (rMVApp65), a CMV tegument protein, was also constructed, using similar techniques (Z. Wang, C. LaRosa, S. F. Lacey, M. Villacres, R. Maas, S. Markel, J. Brewer, S. Mekhoubad, H. Ly, W. Britt, and D. J. Diamond, manuscript in preparation). Recombinant Western Reserve (WR) strain vaccinia virus expressing murine wild-type p53 (rVVp53) or pp65 (rVVpp65) was constructed using published techniques (36).

Recombinant adenovirus expressing wtp53 (rAdp53) was constructed using the pAd Easy system (37). Both pAd Track-CMV and pAd Easy-1 plasmids were kindly provided by Dr. B. Vogelstein (Johns Hopkins Oncology Center, Baltimore, MD). Murine wtp53 cDNA was cloned into the BglII and XbaI site of the pAd Track-CMV shuttle vector containing green fluorescent protein with a CMV promoter (p53-pAd Track-CMV). The p53-pAd Track-CMV was cotransformed into BJ5183 cells with the pAd Easy-1 plasmid to generate the p53 recombinant adenoviral construct by homologous recombination (37). The presence of the p53 gene in the recombinants was confirmed by DNA sequencing. The p53 recombinant adenoviral construct was cleaved with PacI and transfected into HEK-293 cells. rAdp53 was harvested 5 days after transfection, and p53 protein expression was confirmed by Western blot. The adenovirus was expanded on HEK-293 cells and purified by cesium chloride gradient. The purified virus was dialyzed in PBS, titered on HEK-293 cells, and stored at -80°C in 20% glycerol.

Expression of p53 protein by rMVA viruses

Standard Western blotting techniques were performed using ECL Western blot kit (Amersham Pharmacia Biotech, Buckinghamshire, U.K.). Lysates were prepared from Meth A cells or infected BHK or HEK-293 cells and subjected to SDS-PAGE and Western blotting. The samples were incubated with a purified mouse anti-p53 mAb, PAb 122 (38), followed by incubation with a peroxidase-labeled goat anti-mouse secondary Ab provided in the ECL Western blot kit.

In vivo tumor challenge experiments

rMVA. Six-week-old female BALB/c mice were injected by s.c. route in the left flank with 5 x 105 Meth A cells. On day 3, the mice were treated with 5 x 107 PFU of rMVAp53 by i.p. injection. Negative control mice were injected with 5 x 107 rMVApp65 or PBS. The s.c. tumors were measured twice weekly in three dimensions with calipers.

rMVA plus CTLA-4 mAb. BALB/c mice were injected s.c. in the left flank with 106 Meth A cells. This tumor dose was shown to produce a rapidly lethal tumor in the majority of mice despite CTLA-4 mAb treatment (Fig. 4). On day 7, mice were injected i.p. with 5 x 107 PFU of rMVAp53. Controls were the same as above. Anti-CTLA-4 mAb Ab or control hamster Ab were injected i.p. on days 6, 9, and 12 at doses of 100, 50, and 50 µg, respectively.



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  		FIGURE 4. rMVAp53 immunization and CTLA-4 blockade in Meth A tumor-bearing mice. Mice were injected s.c. in the flank with 106 Meth A cells. On days 6, 9, and 12, mice were injected i.p. with either anti-CTLA-4 or control mAb. Mice were treated with rMVAp53 or rMVApp65 on day 7. Tumor size was measured biweekly, and the proportion of surviving mice was plotted. The survival advantage of the rMVAp53 plus CTLA-4-treated animals (n = 14) over the control animals receiving rMVApp65 plus CTLA-4 (n = 14), rMVAp53 plus control Ab (n = 14), or rMVApp65 plus control (n = 6) is statistically significant (p < 0.001) as determined by the log rank test.

 
In vivo mAb injections. Mice treated with rMVA plus CTLA-4 mAb were depleted of T and NK cells by i.p. injection of 200 µg of the relevant mAb or control mAb on days -1, 1, and 3, with maintenance dose every 7 days until sacrifice of the animals. This regimen was shown to deplete (>95%) BALB/c mice of CD4, CD8, or NK 1.1 cells based on flow cytometry of peripheral blood from treated animals (data not shown).

Cytotoxicity assays

Mice were immunized with either 5 x 107 PFU of rMVAp53 or rMVApp65. After 2 wk, spleens were harvested and disassociated, and splenocytes were washed and counted. Splenocytes were stimulated in vitro for 6 days with syngeneic LPS blasts infected with rAdp53 or LPS blasts infected with rMVAp53. Na51CrO4-labeled target cells were added to the 96-well plates with the effectors, in triplicate, at various E:T ratios, in 200 µl of complete medium. The plates were incubated for 4 h at 37°C, and the supernatant was harvested and analyzed. Percent specific lysis was calculated using the formula: percent specific release = (experimental release - spontaneous release)/(total release - spontaneous release) x 100.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Characterization of p53 expression

Expression of p53 protein following infection with rMVAp53 was analyzed to determine the fidelity and extent of its expression from recombinant virus. Western blot analysis of BHK cells infected with rMVAp53 demonstrates abundant p53 expression (Fig. 1). The remarkable level of expression exhibited by rMVAp53 compared with other viral and cellular forms suggests that it will be suitable for use as a vaccine. Note the volume on the MVA lane is between 80- and 160-fold less than what was applied to the gel in the other lanes. We used Meth A cells as a positive control and human CMV (HCMV) IE1 exon 4-rMVA-infected BHK as negative controls. Meth A is a BALB/c-derived, tumorigenic 3-methylcholanthrene-induced sarcoma that overexpresses mutated p53 (39). A 53-kDa band was observed in both the p53-overexpressing Meth A sarcoma and the rMVAp53-infected BHK cells (Fig. 1). This contrasts with the absence of detectable p53 expression in the HCMV IE1 exon 4-rMVA-infected BHK cells. Strong p53 expression was also observed by fluorescence microscopy in BHK cells infected with rMVAp53 (data not shown).



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  		FIGURE 1. Murine p53 protein expression. Cell lysates were subjected to SDS-PAGE and Western blotting. Lane 1, Meth A, unmanipulated Meth A sarcoma cells; lane 2, HCMV IE1 exon 4-rMVA-infected BHK cells; lanes 3 and 4, rMVAp53-infected BHK cells (loaded 0.125 and 0.25 µl cell lysates, respectively); lane 5, rAdp53; and lane 6, rAdpp65-infected HEK-293 cells. All lanes were loaded with 20 µl of sample unless indicated specifically.

 
CTL responses from rMVAp53-immunized mice

Immunization of mice with rMVA expressing viral and tumor-associated Ags (24) results in enhanced Ag-specific CTL responses. We wanted to determine whether immunization with rMVAp53 could break tolerance, resulting in the generation of p53-specific CTL. Splenocytes were harvested from mice following a single i.p. immunization with rMVAp53, and restimulated in vitro with p53-overexpressing cells. The splenocytes recognized and lysed wtp53-overexpressing targets (Fig. 2). In contrast, splenocytes from mice immunized with rMVApp65, which stimulates vigorous pp65-specific CTL responses (Z. Wang, C. LaRosa, S. F. Lacey, M. Villacres, R. Maas, S. Markel, J. Brewer, S. Mekhoubad, H. Ly, W. Britt, and D. J. Diamond, manuscript in preparation), did not recognize the p53-overexpressing targets (Fig. 2B). We also evaluated the ability of rMVAp53 immunization to stimulate CTL recognition of a cell line bearing mutated p53, Meth A. Restimulated splenocytes from rMVAp53-immunized mice, but not control rMVApp65-immunized mice, recognized mutant p53-overexpressing Meth A (Fig. 2C).



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  		FIGURE 2. Immunization with rMVAp53 enhances the p53-specific CTL responses. A, Splenocytes from mice treated with rMVAp53 were harvested at 14 days and restimulated in vitro for 6 days with rAdp53-infected syngeneic LPS blasts. CTL activity was evaluated in a standard 4-h 51Cr release assay using rVVp53 (solid line)- or rVVpp65 (dashed line)-infected 10.1 cells. B, Splenocytes from rMVAp53 (solid line)- or rMVApp65 (dashed line)-immunized mice were harvested at 14 days following immunization and restimulated in vitro for 6 days with rAdp53-infected syngeneic LPS blasts. Cytotoxicity was measured against rVVp53-infected 10.1 cells. C, Splenocytes harvested 14 days following rMVAp53 (solid line) or rMVApp65 (dashed line) immunization were restimulated in vitro for 6 days using syngeneic LPS blasts infected with rMVAp53. Cytotoxicity was measured against Meth A cells by a standard 4-h 51Cr release assay.

 
Effect of immunization on early Meth A tumor-bearing mice

Because a single immunization with rMVAp53 resulted in enhanced CTL responses, there was sufficient justification to examine the effect of rMVAp53 immunization on the growth of Meth A tumor cells in vivo. Animals inoculated s.c. with 5 x 105 Meth A cells develop a rapidly growing fibrosarcoma that kills the majority of mice within 21 days (Fig. 3). Groups of mice were inoculated s.c. with 5 x 105 Meth A tumor cells in the flank. Three days later, separate groups of mice were immunized i.p. with rMVAp53, control rMVApp65, or PBS alone. Tumors in rMVAp53-treated animals grew at a much slower rate than those in controls. At 14 days, the mean s.c. tumor volume for the rMVAp53-treated group (n = 16) was significantly lower than that of both the rMVApp65 (n = 16) and PBS (n = 12) controls (22 vs 348 mm3, p < 0.001, and 22 vs 252 mm3, p < 0.001 by Student's t test). Survival of rMVAp53-treated animals was significantly prolonged compared with either control group (Fig. 3). In fact, 12 of the 16 rMVAp53-immunized mice failed to develop tumors. The 12 tumor-free rMVAp53-treated animals were rechallenged at day 52 with 5 x 105 Meth A tumor cells. All animals remained tumor free for the duration of a 30-day observation period (data not shown).



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  		FIGURE 3. Effect of rMVAp53 on the survival of Meth A tumor-bearing mice. Three days following s.c. injection of 5 x 105 Meth A sarcoma cells, groups of mice were treated with rMVAp53 (n = 16), rMVApp65 (n = 16), or PBS (n = 12). The survival plot shows the proportion of surviving animals in each group as a function of days post-tumor challenge. The improvement in survival of the rMVAp53-treated mice over both control groups is statistically significant (p < 0.001) as determined by the log rank test.

 
Additive effect of CTLA-4 blockade on rMVAp53 immunization

One potent strategy for optimizing tumor vaccines involves manipulating negative regulation of T cell responsiveness by Ab that blocks CTLA-4 engagement with ligand. This phenomenon has been referred to as CTLA-4 blockade (40). Application of CTLA-4 mAb in combination with cancer vaccines expressing tumor-associated autoantigens in some cases results in tumor rejection along with breaking of tolerance and induction of autoimmunity (41, 42). Therefore, mAb specific to CTLA-4 was added to rMVAp53 immunization to determine whether it would augment the antitumor activity against Meth A in vivo. A more rigorous tumor model was designed to overcome the potent antitumor effect of CTLA-4 blockade alone. By increasing the Meth A tumor challenge to 106 cells and postponing treatment until a palpable tumor nodule was identified (day 6), the effect of CTLA-4 blockade was overcome (Fig. 4). Most (11 of 14) animals treated with rMVAp53 in the presence of CTLA-4 mAb rejected tumors, resulting in tumor-free survival for the duration of the 60-day observation period (Fig. 4). Delaying treatment beyond day 6 was not feasible, because untreated control animals begin to die by day 8 following challenge with 106 Meth A tumor cells. Mice treated with rMVApp65 and control Ab died rapidly of progressive tumor (Fig. 4), as did PBS-treated controls (data not shown). In this more aggressive tumor model, the effects of rMVAp53 and CTLA-4 blockade when administered separately were modest. Tumor-free rMVAp53 plus CTLA-4-treated mice (11 of 14) also rejected a rechallenge with 106 Meth A tumor cells at 60 days, and remained tumor free for the duration of a 30-day observation period (data not shown).

Contribution of CD4, CD8, and NK cells to vaccine effect

To determine the relative contribution of NK and T cell subsets on rejection of Meth A sarcoma following rMVAp53 immunization and CTLA-4 blockade, mice were treated with additional mAb at doses which depleted CD4+ or CD8+ T cells or NK1.1+ cells before vaccination. As shown in Fig. 5A, mice depleted of CD8+ cells, or of CD4+ and CD8+ T cells simultaneously, develop rapidly lethal tumors, resistant to rMVAp53 immunization with CTLA-4 mAb. In contrast, CD4+ T cell depletion resulted in only partial abrogation of the response to the vaccine. NK1.1 cell depletion had little effect on the ability of immunized mice to reject Meth A (Fig. 5A). Similar results were observed when depleting mAbs were administered following vaccine and CTLA-4 treatment (data not shown).



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  		FIGURE 5. Mechanism of vaccine effect. The murine tumor model with rMVAp53 and CTLA-4 Ab treatment was constructed as in Fig. 4. Groups of four mice were treated with depleting doses of anti-CD4, anti-CD8, anti-NK1.1, or control mAb on days -1, 1, 3, and 10, and weekly thereafter. A, Mean tumor growth was calculated for each group with error bars illustrating SD. The last data point for each line represents the first mortality. B, IFN-{gamma}KO BALB/c mice were injected s.c. in the flank with 106 Meth A cells. On days 6, 9, and 12, groups of five mice were injected i.p. with anti-CTLA-4 mAb or control (PBS) and treated with rMVAp53 or rMVApp65 on day 7. The proportion of surviving mice is plotted.

 
Contribution of IFN-{gamma}

The contribution of IFN-{gamma} secretion to the effect of CTLA-4 blockade and rMVAp53 immunization was evaluated in IFN-{gamma}KO mice. Unvaccinated mice or mice treated with CTLA-4 mAb blockade with rMVApp65 vaccine developed lethal tumors at a rate similar to that seen in normal BALB/c mice (Fig. 5B). In contrast to the vaccine effect in normal mice, the majority of IFN-{gamma}KO mice (3 of 5) vaccinated with rMVAp53 along with CTLA-4 mAb developed lethal tumor growth (p = 0.04 by log rank test for survival difference in normal over IFN{gamma}KO mice), confirming a contribution of IFN-{gamma} to the vaccine/CTLA-4 blockade effect.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
There has been considerable interest in the development of immunotherapy strategies targeting p53 overexpression. Success in preclinical murine tumor studies have led to the initiation of a number of clinical trials involving stimulation of p53-specific immune responses using defined CTL epitopes from p53. In murine models, a number of immune-based strategies have been effective at preventing outgrowth or eliminating established murine p53-overexpressing tumors. There have been three general strategies used. These include adoptive transfer of epitope-specific CTL, immunization with epitope-pulsed DC, and direct immunization with recombinant vaccinia virus, adenovirus, or ALVAC expressing p53. Each of these strategies has considerable drawbacks with regard to clinical applicability. CTL infusion is expensive and entirely dependent on the ability to generate and propagate high affinity CTL, which may be limited as a result of tolerance (8). Infusion of epitope-pulsed DC requires mutation-specific or HLA haplotype-specific vaccination and would therefore have limited applicability. In addition, this immunization strategy might not stimulate responses to cryptic epitopes or stimulate a p53-specific Th response (43). To effectively target murine p53 in a syngeneic murine model, the viral immunization strategies previously used required either intratumoral injections or immunization before tumor challenge. Recently, Nikitina et al. (11) described an effective tumor treatment regimen in mice using rAdp53-infected and activated DCs. Effective control of Meth A sarcoma was achieved using multiple immunizations in a maintenance pattern for the entire duration of observation. Modest p53-specific CTL activity in immunized mice was seen only after exposure to a Meth A tumor challenge. In addition, lasting tumor immunity was not demonstrated.

Immunization with rMVAp53 overcomes many of the theoretical limitations to effective immunotherapy. The immunization does not target individual p53 mutations and is not MHC haplotype specific. In addition, it does not require the ex vivo propagation of epitope-specific CTL. Vigorous CTL responses are observed following a single immunization, and lasting tumor immunity is achieved in all effectively treated mice. This cell-free immunization strategy does not require the ex vivo generation and propagation of any cells, including DC, which facilitates a relatively straightforward immunotherapy approach.

MVA was derived by 570 passages of the Ankara strain of vaccinia virus on primary chick embryo fibroblast, during which viral genes involved in host immunoregulation (44) and host range were lost (45). Although able to efficiently replicate its DNA in mammalian cells, MVA is avirulent and does not produce infectious progeny. Several properties of MVA as an attenuated poxvirus makes it ideal for the generation of a therapeutic response to overexpressed p53. In contrast to NYVAC (attenuated Copenhagen strain) and ALVAC (host range-restricted avipox), both early and late transcription are unimpaired, allowing for continuous gene expression throughout the viral life cycle, a favorable property of a vaccine candidate (46). In fact, preclinical mouse studies have shown it to be a more potent vaccine vector than the vaccinia virus WR strain. Unlike the WR strain, MVA can cause robust anti-insert immunity in conditions of pre-existing poxvirus immunity (47). In animal models, rMVA-based vaccines have elicited systemic immunity or protection against viral (22) and tumor-associated Ags (23, 24, 25). In primate studies, MVA used alone as a prime and subsequent boost elicited immune responses as effective as those seen with DNA priming followed by an MVA boost (48). Its favorable clinical profile is buttressed by results of a trial in which MVA was administered to over 120,000 high-risk individuals, including the aged and very young, as a smallpox vaccine without serious side effects (49). More recently, MVA was also administered to immunocompromised nonhuman primates without adverse outcome (50). In summary, MVA has favorable characteristics as a delivery system for cancer genes because of its potency as an expression vector and its safety profile in primates and humans.

Combining MVA with CTLA-4 blockade represents a novel approach to the immunotherapy of p53-overexpressing tumors. CTLA-4 plays a significant role in regulating peripheral T cell tolerance by interfering with T cell activation through both passive and active mechanisms (51). In vitro, CTLA-4 mAb blockade lowers the T cell activation threshold and removes the attenuating effects of CTLA-4. Administration of Abs that block the interaction between CTLA-4 and B7 in vivo results in the rejection of a number of transplantable tumors in mice including colon carcinoma, fibrosarcoma, prostatic carcinoma, lymphoma, and renal carcinoma (51). When combined with GM-CSF-producing tumor cell vaccines, CTLA-4 results in rejection of established poorly immunogenic melanoma, mammary carcinoma, and prostate carcinoma grafts (41, 42, 52). This occurs through a process which involves breaking tolerance to tumor-associated autoantigen. Our discovery that CTLA-4 blockade enhances the effect of a potent p53-expressing MVA vaccine to cause rejection of established tumors extends the work of others (53) who initially found that CTLA-4 blockade enhances CTL responses to p53.

Tumor rejection following immunization with MVA in the presence of CTLA-4 blockade may result from the enhancement of p53-specific CTL. These CTL are clearly demonstrated by 51Cr release assay, and by the ability of CD8+ depletion to attenuate the effect of the combined vaccine treatment. The secondary role of CD4+ cells suggests a less critical role for T cell help in the generation or propagation of the p53-specific antitumor response. The noncrucial role of CD4+ cells in CTLA-4 blockade-related tumor rejection is analogous to the findings of others (54). NK+ cell depletion did not affect the combined vaccine, further implicating an Ag-specific CTL mechanism for tumor rejection. It is of interest that the majority of IFN-{gamma}KO mice were not able to reject tumors using the same immunization strategy as with normal BALB/c animals. This highlights that IFN-{gamma} plays a role in the rejection of tumor cells following immunization. An intriguing finding is the success of our vaccination approach in a small proportion of IFN-{gamma}KO mice. This is particularly surprising because the general cytokine profile of splenocytes from mice primed with MVA vaccines is the secretion of IL-2 and IFN-{gamma} (23), and MVA has been shown to enhance Th1 responses (55). Clearly, other mechanisms of tumor rejection independent of IFN-{gamma} are operative in immunized mice.

It is important to use a vigorous tumor model to adequately evaluate the preclinical potential of a p53-based cancer immunotherapy strategy. p53 is an autoantigen subject to tolerance mechanisms (8). Murine studies targeting the xenoantigen, human p53 expressed as a murine tumor transfectant, are of less relevance in a preclinical murine model, although immunization with the xenogeneic rMVA-human p53 might help overcome tolerance to murine p53 (56). The studies reported here use an unmanipulated murine tumorogenic cell line that overexpresses murine p53. The model was made more vigorous by the very high cell number we used, which was over twice that previously described (11). In addition, the delay to treatment used in this study provides evidence of the ability to immunize mice against p53 resulting in regression of well-established tumor deposits. This study supports the need for clinical evaluation of rMVAp53-based vaccines.


   Acknowledgments
 
We acknowledge Saima Ali, Susan Markel, Rebecca Maas, and Veronica Castillo for their assistance in generating recombinant viral constructs and evaluating expression levels of p53, and Corinna La Rosa for her assistance with in vitro assays. We also gratefully acknowledge the assistance of Donna Packer in the preparation of the manuscript. We also warmly recognize the efforts of the staff of the City of Hope animal care facility in overseeing all animal studies that were crucial to the success of these studies.


   Footnotes
 
1 These studies have been partially supported by National Institutes of Health, Division of AIDS, Grants RO1-AI43267 and R21-AI44313, and National Cancer Institute, Grants RO1-CA77544 and PO1-CA30206 (project III) (to D.J.D.). J.D.I.E. is partially supported by R29-CA70819. City of Hope Comprehensive Cancer Center is supported by the National Cancer Institute (CA33572). D.J.D. is partially supported by a Translational Research Award from the LLS and Bea and Edwin Wolfe Foundation. Back

2 Address correspondence and reprint requests to Dr. Joshua D. I. Ellenhorn, Department of General and Oncologic Surgery, City of Hope National Medical Center, 1500 East Duarte Road, Duarte, CA 91010. E-mail address: jellenhorn{at}coh.org Back

3 Abbreviations used in this paper: wtp53, wild-type p53; DC, dendritic cell; MVA, modified vaccinia virus Ankara; rMVAp53, rMVA expressing murine wtp53; rMVApp65, rMVA expressing pp65; rVVp53, recombinant vaccinia virus expressing murine wtp53; rVVpp65, recombinant vaccinia virus expressing murine wild-type pp65; rAdp53, recombinant adenovirus expressing wtp53; IFN-{gamma}KO, IFN-{gamma} knockout; HCMV, human CMV. Back

Received for publication August 22, 2002. Accepted for publication January 3, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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