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Canarypox expressing wild type p53 for gene therapy in murine tumors mutated in p53 Laurence Odin,1 Marie Favrot,1 Dominique Poujol,1 Jean-Philippe Michot,2 Philippe Moingeon,3 James Tartaglia,4 and Isabelle Puisieux1

1DeÂpartement de Biologie des Tumeurs, Centre LeÂon BeÂrard, Lyon, France; 2DeÂpartement d'Anatomie-Cytologie Pathologiques, Centre LeÂon BeÂrard, Lyon, France; 3Pasteur-MeÂrieux Connaught, Marcy l'Etoile, France; and 4Virogenetics, Troy, New York.

The antitumor activity of a recombinant virus expressing wild type murine p53 (ALVAC-p53)was investigated in two murine syngeneic tumors harboring an endogenous p53 mutation (CMS4 and TS/A). Direct intratumor injections of ALVAC-p53 in

CMS4 pre-established subcutaneous tumors induced total tumor regression in 66% of mice. Furthermore, 100% of the cured mice was protected against a contralateral subsequent challenge with the parental tumor cells. The intravenous treatment of experimental metastasis by ALVAC-p53 also induced significant tumor growth inhibition in both models. The antitumor effect of ALVAC-p53 was only observed in immunocompetent animals and was associated with the generation of a specific antitumor immune response. ALVAC-p53 induced the expression of a functional p53 wild type protein as demonstrated by up-regulation of p21waf1 and induction of apoptosis. A strategy using intravenous or subcutaneous ALVAC-p53/NYVAC-p53 prime boost protocol failed to induce CTL against p53 wild type used as target tumor , and failed to protect mice against challenge with the mutated tumor cells. The mechanism of the curative and protective effects observed after direct intratumor injections results from the induction of a specific antitumor response directed against other than p53. Our results suggest that the local induction of tumor apoptosis, combined with the adjuvant effect of ALVAC vector, enhances the immunogenicity of the intratumor environment and allows induction of specific antitumor immune response. Gene Therapy (2001) 8, 87±98

Key words: Canarypox virus; ALVAC; p53; gene therapy.

isruption of the function of the tumor suppressor gene nonsmall cell lung carcinoma.7 The mechanism of the Dp53 through mutation or deletion occurs very fre- antitumor response includes the direct induction of cell death quently in human cancer (in more than 50% of all human or growth arrest through a transcriptional activation of tumors).1,2 Loss of p53 function has been linked with p21waf1,8,9 or indirect processes such as a higher sensitivity unfavorable prognosis in terms of more aggressive tumors, to radiotherapy or chemotherapy and the inhibition of tumor early metastasis, and decreased survival rates. Current angiogenesis through the transcriptional activation of progress in the understanding of the key role of p53 in the thrombospondin I.10 However, the exact molecular basis of regulation of apoptosis and cell cycle arrest provides novel the bystander effect is unclear at present. strategy using gene therapy replacement with p53 wild type The induction of tumor-specific immunity against p53 (p53wt). It is now clear, even in cells which contain multiple is also an attractive therapeutic approach because muta- genetic abnormalities, that cell transformation and tumor cell tions of this tumor suppressor gene result in the growth can be suppressed by the re-introduction of the stabilisation of the mutant protein and its overexpression p53wt gene into tumor cells harboring either a mutant p53 in tumor cells.2,11 Despite the presence of mutations, the or a deletion of the p53 gene.3,4 Roth et al 5 and Fujiwara et antibodies and CTLprecursors in cancer patients appear to al6 have shown that retrovirus-mediated gene transfer of be directed against wild type epitopes of the protein.12 ± 14 p53wt into both human lung tumors and xenograft tumor Therefore, p53, as a majority of the human tumor- models could lead to the inhibition of tumor cell growth. associated antigens already described, derives from an The low efficiency of the in vivo gene transfer with overexpressed self-protein.15 The detection of CTL retrovirus vector suggests the possibility of a ``bystander precursors against p53wt epitopes in cancer patients effect''. Adenovirus-mediated transfer of the p53wt has suggests that active immunization can be used to increase also indicated promising results in a preliminary trial in the number of CTLs. However, the in vivo efficiency of these CTLs is still unknown, and no correlation between Received July 12, 2000; accepted November 9, 2000. their presence and the induction of a clinical response has Address correspondence and reprint requests to Dr. Isabelle Puisieux, been demonstrated yet. Furthermore, the rational of such DeÂpartement de Biologie des Tumeurs, Centre LeÂon BeÂrard, 28 rue p53wt vaccination trial is not yet well demonstrated in LaeÈnnec, 69373 Lyon, France. E-mail address: [email protected] syngeneic tumor model.

Cancer Gene Therapy, Vol 8, No 2, 2001: pp 87±98 87 88 ODIN, FAVROT, POUJOL, ET AL: ALVAC MEDIATED WILD-TYPE P53 GENE TRANSFER

In the present study, conducted on two syngeneic murine Viral vectors tumors harboring an endogenous p53 mutation (CMS4 and Two types of viral vectors were used: ALVAC, a canarypox TS/A), we evaluate the antitumor effect of a direct gene virus of the Avipox virus family, capable of replication in therapy approach using a recombinant canarypox virus avian only, and NYVAC, a derivative of Copenhagen expressing murine wild type p53 (ALVAC-p53). Recombi- virus with multiple deletions and impaired replica- nant avian pox (Avipox) such as canarypox tion in human cells.21 The ALVAC and NYVAC-based (ALVAC) are of special interest for gene therapy protocols 16 recombinant viruses were generated by in vitro recombination for three main reasons. First, they are safe vectors because using standard techniques.22,23 ALVAC-p53 and NYVAC- their replication is restricted to avian cells. Second, they p53 contained the murine wild type p53 coding sequence infect mammalian cells and express the inserted transgene under the control of the early/late vaccinia virus H6 with high efficiency. Third, clinical studies have already .23,24 Vectors expressing the -galactosidase shown that ALVAC-expressing foreign antigens (i.e., rabies gene (ALVAC- Gal and NYVAC- Gal) and Photinus glycoprotein) are well tolerated and can induce both pyralis luciferase gene (ALVAC-luciferase) were used as humoral and strong cellular immune response against controls. infectious diseases.17 More recently, ALVAC-expressing carcinoembryonic antigen (ALVAC-CEA) was evaluated in a phase I study on cancer patients, demonstrating a Characterization of p53 mutation in TS/A and CMS4 significant increase of CTLprecursors specific for CEA in Total cellular RNA was extracted from subconfluent tumor the PBMC in seven of nine patients after three intramuscular cells with Trizol reagent (Gibco BRL) according to the vaccinations.18 manufacturer's instructions. cDNA synthesis was per- In this study, we evaluated whether ALVAC-p53 could formed using M-MLV Reverse Transcriptase (Gibco induced tumor regression by a mechanism of gene replace- BRL) as described in the manufacturer's protocol. The ment with functional p53wt and/or induction of CTL cDNA was amplified with different sets of primers specific response against p53wt epitopes. We observed an antitumor for murine p53, using TaqDNA polymerase transcriptase effect of ALVAC-p53 using either intratumor injections of (Boehringer Mannheim, Meylan, France). The first set of ALVAC-p53 into pre-established subcutaneous (s.c.) primers used for bp 41±283 amplification (exons 4 to 8) tumors or intravenous (i.v.) treatment for lung metastases. included F41 (50 -ATGGACGATCTGTTGCTGC-30 ) and The mechanism of this antitumor response involved R283 (50 -TTCTTCTGTACGGCGGTCTC-30 ); the second induction of apoptosis and a memory T-cell response against set of primers used for bp 102±283 amplification (exons 4 the parental tumor. This immune response was not directed to 8) included F102 (50 -GCAACTATGGCTTC- against p53wt epitopes. CACCTG-30 ) and R283. Samples were amplified for 35 cycles in a DNA Thermocycler Mastercycler Gradient (Eppendorf, Le Pecq, France) under the following MATERIALS AND METHODS conditions: 948C for 1 minute, 588C for 1 minute, Mice 728C for 3 minutes, followed by 728C for 15 minutes. d The PCR products were purified in Microspin S-300HR Six- to 8-week-old female Balb-c (H-2 ) mice and 6- columns (Pharmacia Biotech, Orsay, France) before week-old female Nu/Nu (Swiss) mice were purchased sequencing. Double-stranded DNA was sequenced with from IFFA CREDO laboratory (l'Arbresle, France). Abi prism Dye Terminator and ampliTaq DNa polymer- ase kit (Perkin Elmer, Courtaboeuf, France) according to Murine tumor model and culture conditions the manufacturer's instructions. Samples amplified with TS/A is a highly aggressive and metastatic cell line F41/R283 primers were sequenced in front with primer established from the first in vivo transplant of a mammary F102 or primer F234 (50 -TGTGTAATAGCTCCTG- adenocarcinoma that arose spontaneously in a 20-month- CATGG-30 ), and in reverse with primer R251 (50 - old Balb-c female mouse.19 TS/A expresses major class I GGAGTCTTCCAGTGTGATGATG-30 ) or primer R102 histocompatibility complex but not class II molecules, and (50 -AGGTGGAAGCCATAGTTGCC-30 ). Samples ampli- does not stimulate a syngeneic antitumor response in vitro fied with F102/R283 primers were sequenced in front or in vivo. The minimal 100% tumor-inducing dose in with primer F114 (50 -GACAGCCAAGTCTGTTATG- 4 0 syngeneic Balb-c mice is 4Â10 TS/A cells. CMS4,a TCC-3 ) and F234 and in reverse with primer R251 Balb-c sarcoma induced by methylcholanthrene, has been and primer R200 (50 -TGCCTGTCTTCCAGATACTCG- described previously.20 TS/A cells were maintained in 30 ). Amplification was performed in a Thermocycler RPMI 1640 medium (Gibco BRL, Cergy Pontoise, Mastercycler Gradient (Eppendorf) for 35 cycles under France) supplemented with 10% heat-inactivated fetal the following conditions: 968C for 30 seconds, 588C for bovine serum (Gibco BRL), 100 U/mL penicillin, 100 30 seconds, and 688C for 4 minutes. After amplification, U/mLstreptomycin, 50 mg/mLgentamycin, and samples were precipitated, washed, diluted in forma- À 5 2.5Â10 M -mercaptoethanol. CMS4 cells were mide±EDTA, denaturated at 908C for 5 minutes, and maintained in Dulbecco's modified Eagle's medium placed on ice. The samples were loaded on a 6% (Gibco BRL) supplemented with 10% heat-inactivated acrylamide sequencing gel and analyzed on an automated fetal bovine serum, 100 U/mLpenicillin, and 100 U/mL sequencer (Applied Biosystems±Perkin Elmer, Courta- streptomycin. boeuf, France).

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Western blot analysis of murine p53 and p21 proteins with TUNEL(terminal deoxynucleotidyltransferase Adherent cells were infected in culture with ALVAC-p53 [TdT] -mediated dUTP-X nick end labeling) reaction or with ALVAC- Gal at different multiplicities of mixture 90 minutes at 378C, then rinsed in PBS. infection (MOI) for 2 hours at 378C in RPMI 1640 Fluorescence was analyzed on a FACScan flow cyt- containing 1% fetal calf serum (FCS). After 24 hours of ometer (Beckton Dickinson, Pont de Claix, France). culture in medium containing 10% FCS, the cell mono- Luciferase assay layers were collected with a scraper, then rinsed with phosphate-buffered saline (PBS). Cells were lysed in TS/A or CMS4 s.c. tumors were established in Balb-c buffer containing 50 mmol/LTris±HCl pH 7.4, 250 mice and injected at day 14 with a unique dose, or at mmol/LNaCl, 5 mmol/LEDTA, 50 mmol/LNaF, 0.1% days 14, 17, and 21 with multiple doses of ALVAC- 5 6 NP-40, 1 mmol/LPMSF, 10 g/mLleupeptin, and 10 luciferase at 2.5Â10 TCID50 or 2.5Â10 TCID50. g/mLaprotinin. After centrifugation, protein concentra- Twenty-four hours following the last injection, mice were tion was evaluated with Folin reagent (Biorad kit, Ivry euthanized and tumors were removed for luciferase assay. 5 sur Seine, France) and normalized for total protein Naive mice were injected i.v. with 2Â10 CMS4 cells, 7 content. Proteins were separated on sodium dodecyl then with 10 TCID50 ALVAC-luciferase. Twenty-four sulfate polyacrylamide gel electrophoresis at 10% acryla- hours later, mice were euthanized and were mide, then transferred to PVDF membranes (Millipore, removed for luciferase assay. Tumors and lungs were Saint Quentin en Yvelines, France). The membrane was homogenized in cell lysis reagent (100 Lfor 20 mg of blocked with I-Block solution (Tropix±Perkin Elmer, tissue) (Kit Promega, France) containing 1% Triton and Courtaboeuf, France) and 0.1% Tween 20 (Sigma protease inhibitor. After incubation for 15 minutes at Aldrich, Saint Quentin Fallavier, France) in PBS, then room temperature and centrifugation, 20 Lof the blotted with primary antibodies: sheep anti-p53 poly- supernatant was mixed with 100 Lof luciferase assay clonal antibody Ab7 (Calbiochem, Meudon, France), or reagent (Promega) containing beetle luciferin. Emission of rabbit anti-p21 polyclonal antibody Ab-5 (Calbiochem), light was measured with a luminometer in the integrated and secondary antibodies: biotinylated rabbit antisheep mode time for 10 seconds. Results are expressed as rlu/10 s/ IgG (Calbiochem) or biotinylated swine antirabbit tumor or /lung. (Dako, Trappes, France), respectively. p53 and p21 proteins were detected by enhanced chemiluminescence Intratumoral treatment of tumor-bearing animals according to the Tropix Chemiluminescent detection Six-week-old female Balb-c mice were injected (day 0) system protocol. s.c. in the left flank with the lethal dose of 105 TS/A 6 3 cells or 2Â10 CMS4 cells. All along the study, the s.c. [ H ]Thymidine incorporation tumors were measured twice a week with callipers. The Non-adherent cells were infected at different MOIs (0±40) doses and schedule of administration of the ALVAC-p53 with serial dilutions of ALVAC virus in RPMI 1640 gene therapy protocol were based on the in vitro containing 1% FCS. After 2 hours of infection at 378C, the transgene expression (maximal for 3 days) and on a cells were washed, then plated in triplicate in 96-well plates previous study using intratumor injections of ALVAC- (4Â103 cells/well) with fresh medium containing 10% IL12.25 ALVAC-p53 vector was injected twice a week for 3 5 FCS. Eighteen hours before harvest, 1 Ci of [ H] 3 weeks, with a constant virus dose of 2.5Â10 TCID50 thymidine (Amersham, Les Ulis, France) was added to in 50 Lof PBS buffer. Pre-established s.c. TS/A tumors each well. Cells were harvested onto glass fiber filters or CMS4 tumors were treated by intratumor injections at (Filtermate, Packard Instruments, Rungis, France) 42 hours days 7, 10, 14, 17, 21, and 24. For each experiment, after the ALVAC virus infection, and total [3H]thymidine control groups were injected with 50 Lof PBS buffer or incorporation was detected using liquid scintillation (Top- the same dose of ALVAC- Gal. A preliminary experi- Count, Packard Instruments). Incorporated [ 3H]thymidine ment showed no difference in terms of s.c. tumor (cpm/well) at each MOI was expressed as a percentage of inhibition between an empty ALVAC-vector and the untreated control cells. ALVAC- Gal (data not shown). The induction of long- term antitumor immune response was appreciated by a TUNEL assay second contralateral challenge at day 28 with 105 TS/A 6 Adherent cells were infected in culture with ALVAC-p53 parental cells or 2Â10 CMS4 parental cells. These doses or with ALVAC- Gal at different MOIs for 2 hours at induced tumors in 100% of naõÈve mice. 378C in RPMI 1640 containing 1% FCS. After 24 to 60 hours of culture in medium containing 10% FCS, the The i.v. treatment of pre-established lung metastases cell monolayers were collected with PBS±EDTA buffer. Six-week-old female Balb-c mice were injected (day 0) 5 5 Cells were fixed in 4% paraformaldehyde in PBS for 30 i.v. with 10 TS/A cells or 2Â10 CMS4 cells. ALVAC- minutes at room temperature, then permeabilized in 0.1% p53 vector was injected at days 3, 10, and 18 in the tail 7 Triton X-100, 0.1% sodium citrate for 8 to 15 minutes vein with a constant virus dose of 10 TCID50 in 200 L at room temperature. The labeling was carried out of PBS buffer. For each experiment, control groups were according to the in situ cell death detection kit protocol injected with the same dose of ALVAC- Gal or were (Boehringer Mannheim). Briefly, cells were incubated left untreated. On day 21, mice were euthanized and

Cancer Gene Therapy, Vol 8, No 2, 2001 90 ODIN, FAVROT, POUJOL, ET AL: ALVAC MEDIATED WILD-TYPE P53 GENE TRANSFER lungs were collected for analysis of lung metastasis in six-well plates. After 5 days, viable cells were harvested invasion. and tested in a 51Cr release assay for their ability to lyse CMS4 parental cell, CMS4 loaded with Gal-peptide, CMS4 Immunohistochemistry of paraffin-embedded tissue loaded with p53 232±240wt peptide, or YAC cells (a target The s.c. tumors or lungs with metastatic tumors were for NK cells). resected at a specified time, immediately fixed in formalin 10%, embedded in paraffin, and sectioned at Determination of specific CTLs against gal or p53wt

4 m. Sections of metastatic lung tumors were For CTLassay, CMS 4 cells were incubated with peptide deparaffinized and treated with 3% hydrogen peroxide for 1 hour at 10 g/mL. CMS4, YAC, Gal-peptide± for 10 minutes to exhaust endogenous peroxidase. Tissue pulsed CMS4 cells, and p53 232±240wt peptide±pulsed 51 sections were placed in sodium citrate solution (0.01 M, CMS4 cells were radiolabeled with 100 Ci Na2 CrO4 / pH 6) and incubated three times for 5 minutes in a 4Â106 cells for 1 hour at 378C (Amersham). The 104 750-W microwave oven. After preincubation in blocking target cells were dispensed into each well of a 96-well serum for 30 minutes (0.2% bovine serum albumin, round bottom plate. Effector cells were added at different 1.5% rabbit serum in PBS), the tissue sections were concentrations, plates were incubated for 4 hours at 378C, incubated 90 minutes at room temperature with Ab7 then the supernatant was harvested and counted. The sheep anti-p53 polyclonal antibody diluted 1:50 (Cal- percentage of specific 51Cr release was calculated using biochem). Sections were washed and incubated with the the formula: percent release=100Â(cpm maximum relea- secondary antibody, biotinylated rabbit antisheep IgG seÀcpm spontaneous release) / (cpm maximum releaseÀ (Calbiochem), for 10 minutes at room temperature. Cells cpm spontaneous release), where spontaneous release was were stained according to the streptavidine-peroxidase obtained from target cells in RPMI+10% FCS alone, and reagents (ABC/HRP, Dako) and diaminobenzidine maximum release was obtained from target cells incubated (DAB) chromogen. Sections were slightly counterstained in RPMI+10% FCS+HCl 0.1 N. with hematoxylin. Statistical analysis Immunization of mice Data are expressed as mean‹SEM. For comparison of 7 Two groups of mice received i.v. injections of 10 TCID50 of different treatment groups, one-way analysis of variance ALVAC virus at day 0, and 107 pfu of NYVAC virus at day (ANOVA) was performed. An unpaired Student's t test 18. One group was treated with p53 constructs and the other was also used to compare the tumor growth between with Gal constructs. Seven days after the last injection, one the ALVAC- Gal group and the untreated control mouse from each group was killed and its was group. In all cases, statistical significance is designated removed for in vitro restimulation. Other mice were at the 95% confidence interval using a two-tailed test 6 5 challenged s.c. with 2Â10 CMS4 cells or 10 TS/A cells, (P<.05). 5 5 or i.v. with 2Â10 CMS4 cell or 10 TS/A cells. The s.c. tumor growth was monitored twice a week. The tumor volume was calculated according to the formula V=ab 2(/ RESULTS 8) (a=largest tumor diameter and b=smallest tumor Identification of p53 mutation in CMS and TS/A cell lines diameter). Mice with tumor diameter >3 mm were classified 4 as tumor bearers. Mice were euthanized for ethical reasons Using direct sequencing of p53 PCR products, we when the tumor was too necrotic or exceeded 20 mm in determined that CMS4 harbored a mutation of p53 at codon diameter. Lung metastases were analyzed 21 days after i.v. 239 (Cys to Tyr), whereas in TS/A, p53 was mutated at implantation. codon 270 (Arg to His).

In vitro restimulation of splenocytes from In vitro dose-dependent ALVAC-p53 induction of immunized animals functional p53wt Groups of mice immunized with ALVAC/NYVAC virus Canarypox virus±mediated wild type p53 expression was were euthanized 1 week after the last injection. Their analyzed in two murine cell lines containing a mutation of were removed for in vitro restimulation on mitomycin- p53 (CMS4 and TS/A) and in a human lung carcinoma treated CMS4 cells or on P815 cells transfected with the harboring a deletion of the p53 gene (H358). Cells were B7.1-encoding cDNA loaded with a Gal peptide or p53 either not infected or infected in vitro with ALVAC- Gal 232±240wt peptide. The peptides were synthesized by at MOI of 5 or ALVAC-p53 at MOIs of 1 and 5. Twenty- Neosystem (Strasbourg, France). The H-2Ld±binding four hours after infection, cells were harvested and 50 g gal-encoded peptide has already been described; its of total proteins obtained from cell lysates was analyzed 26 sequence is TPHPARIGL( gal876 ±884 ). The p53 232± by Western blot using a specific antibody for p53 240wt peptide KYMCNSSCM has been described as (recognising both human and murine p53) and a specific resented by H-2Kd.27 Splenocytes obtained from each antibody for p21waf1 proteins (recognising only the murine experimental group were restimulated at a cell density of p21). As shown in Figure 1, the infection of the p53-null 6 6 6 6Â10 splenocytes/0.6Â10 CMS4 or 0.6 10 peptide- H358 human cell line with ALVAC-p53 induced the pulsed P815 B7.1 cells per 3 mLRPMI containing 10% FCS expression of the p53wt protein. In both murine cell lines

Cancer Gene Therapy, Vol 8, No 2, 2001 ODIN, FAVROT, POUJOL, ET AL: ALVAC MEDIATED WILD-TYPE P53 GENE TRANSFER 91

CMS4 and TS/A, the basal level of p53 expression was Induction of apoptosis by in vitro infection with high, resulting from the mutant form of the p53 protein ALVAC-p53 (the Ab7 anti-p53 antibody recognized both mutant and The CMS4 cell line was more sensitive to apoptosis mediated p53wt proteins from human or murine species). The level by p53wt expression than TS/A cell line. As demonstrated in of p53 expression increased with the infected dose. The a TUNELassay, 24 hours after infection with ALVAC-p53 functionality of the transduced p53wt was confirmed by at MOI 20, 11% of CMS cells was in apoptosis compared the induction of p21waf1 24 hours after infection with 4 waf1 with only 3.6% of TS/A cells. Concerning CMS4 cells, the ALVAC-p53. The p21 was obviously not detected in different rate of induction of apoptosis between cells infected the human cell line H358 infected with ALVAC-p53wt with ALVAC- Gal and ALVAC-p53 at MOI 20 evidenced expressing a murine p53wt (and anti-p21 antibody was waf1 the specific effect of p53wt expression (2.5% vs. 11%, only specific for murine p21 ). Interestingly, at MOI of respectively, 24 hours postinfection). 5, the infection with ALVAC- Gal also induced the waf1 expression of p21 . In vivo canarypox gene transfer efficiency In vitro dose-dependent ALVAC-p53 tumor Efficiency of gene transfer in vivo was evaluated with growth inhibition measurement of luciferase . The expression of the transgene in s.c. tumors was very low in the CMS4 6 The p53-mutated cell lines, CMS4 and TS/A, were infected model (0.1Â10 ‹0.02 rlu/10 s/tumor for multiple injec- 6 in vitro with ALVAC- Gal or ALVAC-p53wt at different tions of ALVAC-luciferase at 2.6Â10 TCID50 ) and higher MOIs (0, 0.2, 1, 5, 10, 20, and 40). A [ 3H]thymidine in the TS/A model (1.2Â106‹0.6 rlu/10 s/tumor for incorporation assay was performed 24 hours postinfection. multiple injections of ALVAC-luciferase at 2.6Â106 CMS4 and TS/A cell lines were equally transduced by the TCID50 ) (Fig 3). This low efficiency was mostly due to a ALVAC vector, as determined by the percentage of X-gal± leak outside of the tumor during the intratumor injection. We positive cells after ALVAC- Gal infection. Twenty-four were not able to inject more than 50 Lof viral suspension in hours postinfection, 80±100% of CMS4 and TS/A particular in the CMS4 sarcoma, which is a very dense tumor. 7 expressed the Gal gene at MOI=2 (data not shown). For After i.v. injection with ALVAC-luciferase at 10 TCID50, both cell lines, we observed a dose-dependent antiprolifera- the transgene expression was analyzed 24 hours postinjec- tive effect of ALVAC-p53 (Fig 2). This antiproliferative tion in different organs (lungs, spleen, liver, lymph nodes, effect was more pronounced for CMS4 cells than for TS/A , heart, blood, serum). The distribution of ALVAC- cells. An antiproliferative effect was also observed after the luciferase was predominantly found in lungs (0.4Â106 rlu/ ALVAC- Gal infection for the highest MOI, resulting from 10 s/lungs) and otherwise only detected in the spleen (6273 the direct cytopathic effect of the ALVAC vector and the rlu/10 s/spleen). We verified that the luciferase transgene induction of p21waf1 as previously shown in the Western blot expression was also detected in lungs bearing metastases 5 analysis (Fig 1). (day 0: i.v. injection of 2Â10 CMS4 cells; day 10: i.v.

Figure 1. In vitro infection with recombi- nant canarypox virus ALVAC-p53 mediated the expression of functional p53wt. The ALVAC-p53 infection of H358cells (harboring a deletion of p53 gene) restored the expression of p53

protein. In the mutated tumors (CMS4 and TS/A), the basal level of p53 in parental cells was high, resulting from the mutant form of p53. After in vitro infection with ALVAC-p53, the level of p53 ex- pression increased with the infection dose. ALVAC-p53 mediated the produc- tion of a functionally active p53 as shown by the induction of p21waf1 expression. The p21waf1 was obviously not detected in the human cell line H358infected with ALVAC-p53wt expressing a murine p53wt. Interestingly, at an MOI of 5, the infection with ALVAC- Gal also induced the expression of p21waf1.

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AB CMS4 24H Post-infection TS/A 24H Post-infection

120 120

100 100

80 80

ALVAC- βGal β 60 60 ALVAC- Gal ALVAC-p53 ALVAC-p53 40 40 % Proliferation/Control % Proliferation/Control Media 20 20 % Proliferation % Proliferation /Control Media

0 0 0 0,2 1 5 10 20 40 0 0,2 1 5 10 20 40 m.o.i m.o.i 3 Figure 2. Inhibition of CMS4 and TS/A cell growth was determined in a [ H]thymidine incorporation assay. After 2 hours of infection at different MOIs (0±40) with ALVAC-p53 (&) or ALVAC- Gal (5), cells were plated in triplicate in 96-well plates at 4Â103 cells/well. After 24 hours of culture and 18hours pulse with [ 3H]thymidine, cells were harvested and counted on a liquid scintillation counter. Incorporated [ 3H]thymidine

(cpm/well) at each MOI was expressed as a percentage of untreated control cells. A: Proliferation of CMS4 cell line 24 hours postinfection. B: Proliferation of TS/A cell line 24 hours postinfection.

7 injection of ALVAC-luciferase at 10 TCID50 ). We found in with the PBS group by unpaired t test, P=.0408). this condition 1.4Â106‹0.1 rlu/10 s/lung. However, a curative effect was only observed for 16% of mice (2 of 12). As shown in Figure 4B, the ALVAC- Antitumor effects of direct intratumor injections of ALVAC- p53 was totally ineffective in the CMS4 tumor model p53 in s.c. tumors established in nude mice (Fig 4B), which demonstrated Intratumor injections of ALVAC-p53 inhibited the growth that the cooperation of T cells was absolutely necessary to obtain the rejection of the primary tumor. In the TS/A s.c. of CMS4 s.c. tumors established in Balb-c mice (Fig 4A). In this model, multiple injections of low doses of ALVAC- model, no antitumor effect was observed. This model was p53 (days 7, 10, 14, 17, 21, 24) induced significant tumor more tumorigenic than CMS4 and less sensitive to the growth inhibition (on day 14 P=.031; on day 21 P=.017; ALVAC-p53 antiproliferative effect and to the induction of and on day 28 P=.002) and resulted in a curative effect apoptosis (as previously shown). for 66% of animals (8 of 12 mice). The animals remained tumor-free all along the experiment (more than 3 Antitumor effect of i.v. administrations of ALVAC-p53 on months). Furthermore, the ALVAC-p53 treatment induced experimental lung metastases long-term antitumor immunity in 100% of treated animals, Experimental lung metastases were established by i.v. as demonstrated by the rejection of a subsequent s.c. administration of CMS4 or TS/A in syngeneic Balb-c mice. contralateral challenge with the parental CMS4 cells on ALVAC-p53 i.v. treatment on days 3, 10, and 17 induced a day 35. In the group treated with the same dose of significant inhibition of tumor growth in both tumor models. ALVAC- Gal, a beneficial effect was observed with a ALVAC-p53 injections induced a significant decrease of the significant decrease in the tumor growth (in comparison number of pulmonary metastases on day 21 in the CMS4

2000000

1800000

1600000

Figure 3. In vivo canarypox gene transfer 1400000 efficiency. Established CMS or TS/A 4 1200000 s.c. tumors were directly injected on day 2,5.105 14 for the unique treatment or on days 1000000 2,6.106 14, 17, and 21 for multiple treatments 107 with 2.5Â105 TCID (5) or 2.5Â106 800000 50 rlu/10s/organ TCID (&) of ALVAC-luciferase virus. 50 600000 A group of mice with 9 days CMS4 established lung metastases were in- 400000 7 jected i.v. with 10 TCID50 of ALVAC- luciferase virus (9). Twenty hours after 200000 the last injection, tumors or lungs were 0 removed and luciferase activity was CMS Uniq CMS Multi TS/A Uniq TS/A Multi Lung iv. measured. Activity is reported in rlu/ 4 4 10 s/organ. Intra-tumour or iv. injection of ALVAC-Luciferase

Cancer Gene Therapy, Vol 8, No 2, 2001 ODIN, FAVROT, POUJOL, ET AL: ALVAC MEDIATED WILD-TYPE P53 GENE TRANSFER 93

AB Day 7 Day 7 Day 14 Day 14 1600 3500 Day 21 Day 21 1400 Day 28 3000 1200 2500 Volume Volume (mm3) 1000 2000 800 1500 600 ** 1000 400 * * 200 Tumour 500 Tumour Volume (mm3) Volume Tumour 0 0 PBS ALVAC- βGal ALVAC-p53 PBS ALVAC- βGal ALVAC-p53 Treatment Treatment Balb-c (n=12) Nude (n=5) Figure 4. Antitumor effect of ALVAC-p53 after repeated intratumor injections in subcutaneous tumors. Groups of mice ( n=12) were injected 6 subcutaneously with 2Â10 CMS4 tumor cells (day 0). On day 7, all mice have developed visible s.c. solid tumors, 5 to 6 mm in diameter. 5 Repeated intratumor injections with 2.5Â10 TCID50 of ALVAC- Gal or ALVAC-p53 were achieved on days 7, 10, 14, 17, 21, and 24 post-tumor implantation. A control group was similarly injected with the same volume (50 L) of PBS buffer. A: Treatment of tumors in immunocompetent animals (groups of 12 Balb-c mice). On day 28, total tumor regression was observed in 8 of 12 Balb-c mice treated with ALVAC-p53; a significant tumor growth inhibition was observed in the ALVAC-p53 group compared with the PBS group and the ALVAC- Gal group (on day 14, P=.031; on day 21, P=.017; and on day 28, P=.002). In the group treated with ALVAC- Gal, a significant reduction of the tumor growth was observed on day 28in comparison with the PBS group (unpaired t test, P=.0408), but only 2 of 12 mice were cured. B: Treatment of tumors in athymic animals (groups of five nude mice) on days 7, 10, 14, 17, and 21. Even though on day 7 the size of the tumor in nude mice was comparable to the size observed in the immunocompetent animals, the CMS4 tumor grew more rapidly in nude mice and the animals had to be euthanized for ethical reasons on day 21. In the T cell depleted system, no tumor growth inhibition was observed (in any group of nude mice). This experiment was repeated twice with similar results. model (15‹5, P=.0013), compared with the untreated A model (Fig 5B). The numeration of TS/A metastases was group (71‹12) or with the group injected with ALVAC- not valuable in this model because of the diffuse aspect of the Gal (52‹9) (Fig 5A). The i.v. injections of ALVAC-p53 tumors. Tumor invasion was estimated by measuring the also induced a significant tumor growth inhibition in the TS/ total lung weight. The mean lung weight in the ALVAC-p53

A B 90 80 600 70 500 60 400 50 * 40 300

30 ** 200 Lung Weight (mg) Weight Lung

Lung Metastases Number 20 100 10 0 0 β Control untreated ALVAC- βGal ALVAC-P53 Control untreated ALVAC- Gal ALVAC-p53 IV Treatment IV Treatment

CMS4 (n=10) TS/A (n=5) Figure 5. The i.v. ALVAC-p53 treatment induced growth inhibition of pre-established lung metastases. Lung metastases were established by i.v. 5 5 7 injection of 2Â10 CMS4 cells or 10 TS/A cells at day 0. On days 3, 10, and 17, i.v. injections of 10 TCID50 ALVAC-p53 or ALVAC- Gal were achieved. A positive control group was left untreated. On day 21, mice were euthanized and lungs were collected for analysis. A: The i.v. treatment of pre-established lung metastases in the CMS4 tumor model. Results are reported as the mean number of lung metastases in each group‹SEM. On day 21, a significant decrease of the number of pulmonary metastases was noted for the group treated with ALVAC-p53 (15‹5) ( P=.0013), in comparison with the untreated group (71‹12) and with the group treated with ALVAC- Gal (52‹9). B: The i.v. treatment of pre-established lung metastases in the TS/A tumor model. Results are reported as the mean lung weight in each group‹SEM. On day 21, a significant decrease of the mean lung weight was noted in the group treated with ALVAC-p53 (200 mg‹17) ( P=.0111), in comparison with the untreated group (425‹52 mg), or with the group treated with ALVAC- Gal (327‹40 mg).

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Figure 6. In vivo p53 gene transfer in CMS4 lung metastases after i.v. injections of ALVAC-p53. Mice were injected i.v. with CMS4 cells and either left untreated (A, B), or treated by i.v. injections on days 3, 10, and 17 with ALVAC- gal (C, D) or with ALVAC-p53 (E, F). Mice were euthanized on day 21 for immunoperoxidase detection of p53 in lung sections. After i.v. injection of CMS4, lung metastases localized in the pulmonary parenchyma and perivascular area. In mice from the untreated group (A,B) or in mice from the group treated with ALVAC- Gal (C,

D), note the heterogeneous intranuclear staining of p53 in the tumor resulting from the mutant status of p53 in CMS4. In the group treated with ALVAC-p53 (E, F), we observe an intense intranuclear and intracytoplasmic overexpression of p53 in several tumor areas, in particular in perivascular zones (F) resulting from the in vivo gene transfer of p53wt. Original magnification (Â100: A, C, E; Â400: B, D, F). group (200‹17 mg) was significantly lower (P=.0111) Representative experiments of the immunohistochemical than in the untreated group (425‹52 mg) and in the analysis of lung sections for evaluation of p53 expression are ALVAC- Gal group (327‹40 mg). shown in Figure 6. After i.v. injection of CMS4, lung

Cancer Gene Therapy, Vol 8, No 2, 2001 ODIN, FAVROT, POUJOL, ET AL: ALVAC MEDIATED WILD-TYPE P53 GENE TRANSFER 95 metastases localized in the pulmonary parenchyma and the protocol using i.v. injections of ALVAC- Gal/NYVAC- perivascular area. In mice from the untreated group or in Gal (days 0 and 18) efficiently induced a CTLpriming mice from the group treated with ALVAC- Gal, we against a Gal epitope (peptide 876±884). Splenocytes observed a heterogeneous intranuclear staining of p53 in from immunized mice were taken 7 days after the last the tumor metastases resulting from the mutant status of p53 injection then restimulated 5 days in vitro on peptide Gal- in CMS4. In the group treated with ALVAC-p53 (Fig 6E), pulsed P815-B7.1. These splenocytes killed 45% of Gal- we observed an in vivo p53 gene transfer in CMS4 lung pulsed CMS4 targets (E/T=40) and not CMS4 parental metastases with increased intranuclear and intracytoplasmic cells (8% at E/T=40), nor the YAC cells used as NK target expressions of p53 in several tumor areas, in particular in (5% at E/T=40). These results confirmed that the prime/ perivascular zones (Fig 6F). boost immunization with ALVAC- Gal/NYVAC- Gal was efficient in a context of ``nonself'' target antigen to generate CTLpriming. In contrast, using ALVAC-p53/NYVAC-p53 Preventive approaches using s.c. or i.v. immunization with prime/boost vaccination and restimulation of splenocytes by ALVAC-p53wt failed to protect mice against tumor CMS4 tumor cells or peptide p53-232±240wt±pulsed challenge with CMS4 or TS/A P815-B7.1, we were able to kill only 25% of CMS4 parental We investigated the efficiency of different prime/boost cells (at E/T=40), which mainly corresponded to the protocols of vaccination (days 0, 18), using ALVAC-p53/ induction of an NK activity, as shown by the identical range ALVAC-p53, ALVAC-p53/DNA-p53, or ALVAC-p53/ of YAC killing (Fig 7B). These results demonstrated that NYVAC-p53. None of these protocols administered i.v. or no CTLpriming against a natural p53wt peptide processed s.c. was able to protect mice against further s.c. or i.v. tumor and presented on the surface of CMS4 cells in association challenge with CMS4 or TS/A (Fig 7). The prime/boost with MHC class I molecules was induced by vaccination A B IV Vaccination: ALVAC/NYVAC- βGal IV Vaccination: ALVAC/NYVAC-p53

50 50

40 40

β CMS 30 CMS4-pep- Gal 30 4 YAC CMS4 20 YAC 20 CMS4 % Specific Lysis % Specific Lysis 10 -pep p53 10

0 0 1102040 1102040 E/T Ratio E/T Ratio

C 1000 900 800 700 600 500 400 300

Lung Weight Lung Weight (mg) 200 100 0 Normal Lung Unvaccinated Vaccination Vaccination ALV/NYV- βGal ALV/NYV-p53 7 7 Figure 7. On day 0, mice were vaccinated i.v. with 10 TCID50 of ALVAC-p53 virus and on day 18with 10 pfu of NYVAC-p53. A control group was injected with the same protocol using ALVAC/NYVAC- Gal constructs. On day 24, one mice per group was euthanized, spleens were removed, and cells were tested for cytotoxic activity (A,B) against CMS4,CMS4 loaded with Gal peptide, CMS4 loaded with p53-234wt peptide, 5 or YAC cells (as NK specific targets). Other mice were challenged by i.v. injection with 2Â10 CMS4 tumor cells (C). A group of naive mice was challenged as control group (unvaccinated group). A: Splenocytes of mice vaccinated with ALVAC/NYVAC- Gal were restimulated in vitro by peptide Gal-pulsed P815 B7.1 cells. These effector cells specifically lysed peptide Gal-pulsed CMS4 target cells and not parental CMS4 cells. This lysis was CTL-mediated, as shown by minimal lysis of the YAC cells (target for NK cells). B: Splenocytes of mice vaccinated with ALVAC/

NYVAC-p53 were stimulated in vitro by peptide p53-234wt±pulsed P815 B7.1 cells. These effector cells showed only 25% of CMS4 target lysis at E/T=40. This CMS4 lysis was in the same range than the YAC target lysis, demonstrating only a NK response. The 234wt-p53 peptide was not a target epitope, as shown by the absence of specific lysis of peptide p53-234wt±pulsed CMS4 target cells. C: The i.v. vaccination by ALVAC/ NYVAC-p53 failed to protect mice against i.v. CMS4 challenge. The lungs were similarly highly invaded by CMS4 in all three groups of mice (n=5). Results are presented as total lung weight (mg); the lung weight of a healthy mouse of the same age was also represented. This experiment was repeated twice with similar results.

Cancer Gene Therapy, Vol 8, No 2, 2001 96 ODIN, FAVROT, POUJOL, ET AL: ALVAC MEDIATED WILD-TYPE P53 GENE TRANSFER using ALVAC-p53/NYVAC-p53. No CTL against the potential target antigens for the because they already described epitope (peptide p53-232±240wt) 27 was are specifically overexpressed in tumor cells. Antibodies and identified. CTLprecursors have been found in patients with p53- mutated ; they recognize nonmutated regions of the protein.12,14,31,32 However, little is known about the DISCUSSION antitumor efficiency of autoreactive T cells that recognize these nonmutated self-antigens. First, we validated the We have evaluated the therapeutic efficiency and mechanism efficiency of our vaccine strategy for a ``nonself'' target of action of p53wt gene therapy in p53-mutated murine antigen ( Gal) using ALVAC- Gal/NYVAC- Gal and we tumors by delivery of a recombinant canarypox observed the induction of anti- Gal CTLs. In contrast, encoding the murine p53wt gene. Multiple intratumor ALVAC-p53/NYVAC-p53 injected s.c. or i.v. was not able injections of low-dose ALVAC-p53 resulted both in the to elicit a CTLresponse against p53wt epitopes nor to protect growth inhibition of s.c. established CMS4 tumors and mice against further tumor challenge with the mutated cell subsequent protection against s.c. contralateral challenge lines, CMS4 or TS/A. We also tested other vaccine with parental tumor cells. In contrast to adenovirus, which approaches using ALVAC-p53/DNA-p53 s.c. or intramus- induced neutralizing antibodies,7,28 multiple injections of cular routes of administration, but we obtained no more canarypox virus were possible and allowed to enhance the success in terms of protection against tumor challenge (data efficiency of transgene expression as shown after multiple not shown). These results may suggest that immunotoler- injections of ALVAC-luciferase (Fig 3) or ALVAC-IL12.25 ance against p53wt prevents the induction of an efficient Interestingly, multiple i.v. administrations of ALVAC-p53 CTLpriming in syngeneic tumors harboring an endogenous were associated with the growth inhibition of lung p53 mutation. Hurpin et al24 have shown a partial protection metastases derived from i.v. injections of CMS4 and TS/A against transfected human p53 tumors (3T3 or P815) in a cells, and followed by increased p53 cellular staining in xenogenic context (human p53 in mice) using i.v. or particular sites of residual lung metastases. After i.v. intrasplenic injections with ALVAC-hp53 (expressing administration of ALVAC-luciferase, most of the virus human p53wt). They have reported the induction of CTLs vectors localized in the lungs and a small amount in the that killed these tumor cells. Human and murine p53 share spleen (as shown by us and by Hurpin et al24 ). This i.v. only 81% of homology.32 This structural difference makes biodistribution of the vector was then favorable for the likely the generation of ``nonself'' epitopes. Similarly, Roth targeting of lung metastases. ALVAC-p53 mediated the et al 33 have reported a 75% protection using s.c. injections of production of a functionally active p53, as shown by the ALVAC-hp53 in a mice model transfected with xenogenic induction of p21WAF1 expression. More importantly, p53wt human p53. However, they did not show any CTLand only overexpression induced tumor cell growth inhibition and reported humoral response against p53wt. Our results are in 34 apoptosis, particularly in CMS4 cells. p53 gene replacement agreement with Theobald et al, who clearly demonstrated a in tumor cells harboring a p53 mutation was considered a p53 self-tolerance in p53+ / + mice. They reported that proper way of reversing the malignant phenotype. However, CTLs with high avidity for a naturally processed p53wt self- this strategy required a very high efficiency of in vivo gene epitopes were obtained only in p53À / À mice, whereas they transfer. In our approach, the in vivo gene transfer efficiency were completely aborted in p53+ / + mice. A response in was weak, as shown after the intratumor injections of the Balb-c mice to a p53wt sequence was only reported using ALVAC-luciferase reporter vector. This low efficiency immunization with dendritic cells pulsed with p53 232± 27 suggested the existence of a bystander effect, which was 240wt peptide. This strategy protected mice against CMS4 only observed for tumors established in immunocompetent challenge. We also investigated the generation of CTLs animals, highly suggesting the involvement of the immune against this 232±240 p53wt peptide after ALVAC-p53/ system in the antitumor mechanism of ALVAC-p53. Our NYVAC-p53 vaccination by restimulating splenocytes from results are in agreement with Li et al29 using multiple immunized mice with P815-B7.1 cells loaded with this administrations of a recombinant adenovirus expressing wild peptide and using p53 peptide-pulsed CMS4 target. Tumor- type p53 in an immunocompetent mouse tumor model reactive CTLs were not detectable. It is important to note that (RENCA). They showed tumor regression in 30% of the the position of the p53 mutation, which we found in the mice. They observed a protection against a second tumor CMS4 cell line, was different from that of Mayordomo et challenge in cured mice and the induction of apoptosis by al.27 They described a mutation in position 194 and we found overexpression of p53. a mutation in position 239, which possibly modified the Therefore, we investigated whether the antitumor immune 232±240wt epitope and its processing and binding to the response was directed against p53wt as potential target tumor H2-Kd class I molecules. In this context, we were not able to antigen. We tried a preventive vaccinal approach using benefit from the help of known p53 peptide epitopes to prime/boost with ALVAC-p53/NYVAC-p53. The concept evidence CTLinduction using in vitro cytotoxic assay. of a diversified prime/boost immunization using avian pox/ In the TS/A tumor model, no antitumor effect was vaccinia virus has already been validated in mice, in a observed after direct intratumor injections of ALVAC-p53 in protocol using CEA as target antigen.26,30 Host immune s.c. tumors. TS/A cells were less susceptible to the induction responses to the first vector have been shown to reduce the of apoptosis by ALVAC-p53. Furthermore, this model was effectiveness of subsequent boost inoculations with the same very tumorigenic and became immunogenic only with the vector. Self-epitopes derived from p53 theoretically provide local secretion of cytokines such as IL2 or IL12.35 We have

Cancer Gene Therapy, Vol 8, No 2, 2001 ODIN, FAVROT, POUJOL, ET AL: ALVAC MEDIATED WILD-TYPE P53 GENE TRANSFER 97 previously reported that tumor suppression and long-term orthotopic lung cancer model. J Natl Cancer Inst. 1994; antitumor immunity could be achieved by intratumor 86:1458±1462. injections of ALVAC-IL12 in this TS/A model.25 However, 7. Yen N, Ioannides CG, Xu K, et al. Cellular and humoral despite its inefficiency in the s.c. tumor model, ALVAC-p53 immune responses to adenovirus and p53 protein antigens in injected i.v. into pre-established TS/A lung metastases was patients following intratumoral injection of an adenovirus vector expressing wild-type p53 (Ad-p53). Cancer Gene effective and induced significant tumor cell growth inhibi- Ther. 2000;7:530±536. tion. This discrepancy between s.c. and i.v. tumor models 8. Harris C. Structure and function of the p53 tumor suppressor was probably due to a higher transfection efficiency and gene: clues for rational cancer therapeutic strategies. J Natl more favorable microenvironment in the lungs. Cancer Inst. 1996;88:1442±1455. 9. Hamada K, Alemany R, Zhang WW, et al. Adenovirus- mediated transfer of a wild-type p53 gene and induction of CONCLUSION apoptosis in cervical cancer. Cancer Res. 1996;56:3047±3054. 10. Xu M, Kumar D, Srinivas S, et al. Parental gene therapy with Our results suggest that a direct gene therapy approach using p53 inhibits human breast tumors in vivo through a bystander intratumor injections of canarypox virus expressing wild mechanism without evidence of toxicity. Hum Gene Ther. 1997;8:177±185. type p53 may be a way to increase the immunogenicity of 11. Levine AJ, Momand J, Finlay CA. The p53 tumor suppressor tumors mutated in p53. This strategy induces an antitumor gene. Nature. 1991;6:453±456. immune response directed to antigens other than p53. This 12. Tilkin AF, Lubin R, Soussi T, et al. Primary proliferative T-cell observation is in accordance with a recent clinical study response to wild-type p53 protein in patients with breast using intratumor injections of adenovirus p53wt in patients cancer. Eur J Immunol. 1995;25:1765±1769. with advanced nonsmall cell lung carcinoma.7 They reported 13. Houbiers J, Nijman H, Van der Burg S, et al. In vitro induction antitumor effect with prolonged tumor stability or regression; of human cytotoxic T-lymphocyte responses against peptides however, neither antibodies to p53 protein nor increased of mutant and wild-type p53. Eur J Immunol. 1993;23:2072± lymphocytes' proliferative response to p53wt or mutant 2077. peptides have been detected. We suggest in our approach that 14. Ropke M, Hald J, Guldberg P, et al. Spontaneous squamous cell carcinomas are killed by a human cytotoxic T-lymphocyte local induction of apoptosis mediated by p53wt gene transfer clone recognizing a wild-type p53-derived peptide. Proc Natl combined with the adjuvant effect of the viral vector itself Acad Sci USA. 1996;93:14704±14707. (as shown by some antitumoral effects of ALVAC- Gal), 15. Boon T, Van Der Bruggen, P. Human tumor antigens probably makes it possible to liberate tumor antigens and recognized by T lymphocytes. J Exp Med. 1996;183:725±729. increase intratumor cytokines secretion which conduct the 16. Paoletti E. Applications of pox virus vectors to vaccination: an generation of a potent, specific antitumor immunity. update. Proc Natl Acad Sci USA. 1996;93:11349±11353. 17. Fries LF, Tartaglia J, Taylor J, et al. Human safety and immunogenicity of a canarypox±rabies glycoprotein recombi- nant vaccine: an alternative poxvirus vector system. Vaccine. ACKNOWLEDGMENTS 1996;14:428±434. 18. Marshall JL, Hawkins MJ, Tsang KY, et al. Phase I study in This work was supported, in part, by grants from the ARC cancer patients of a replication-defective avipox recombinant ``Association de Recherche contre le Cancer'', France and vaccine that expresses human carcinoembryonic antigen. J Clin through the obtention of a CRTG (Centre de Recherche en Oncol. 1999;17:332±337. TheÂrapie GeÂnique). L.O. was supported by a grant from the 19. Nanni P, De Giovanni C, Lollini PL, et al. TS/A: a new ``Ligue Nationale contre le Cancer'', France. We thank Dr G. metastasizing cell line originated from balb-c spontaneous Forni for his kind gift of the TS/A cells, Dr A. Deleo for the mammary adenocarcinoma. Clin Exp Metastasis. 1983;1:373± CMS4 cells, and Dr L. Zitvogel for the P815-B7.1 cells. 376. 20. De Leo AB, Shuku H, Takahashi T, et al. Cell surface antigens of chemically induced sarcomas of the mouse: I. 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