Cancer Gene Therapy (2003) 10, 509–517 r 2003 Nature Publishing Group All rights reserved 0929-1903/03 $25.00 www.nature.com/cgt Safety of intravenous administration of a canarypox virus encoding the human wild-type p53 gene in colorectal cancer patients Anand G Menon,1 Peter JK Kuppen,1 Sjoerd H van der Burg,2 Rienk Offringa,2 Marie Claude Bonnet,5 Bert IJ Harinck,1 Rob AEM Tollenaar,1 Anke Redeker,2 Hein Putter,4 Philippe Moingeon,5 Hans Morreau,3 Cornelis JM Melief,2 and Cornelis JH van de Velde1 1Department of Surgery, Leiden University Medical Center, Leiden, The Netherlands; 2Department of Immunohematology and Bloodbank, Leiden University Medical Center, Leiden, The Netherlands; 3Department of Pathology, Leiden University Medical Center, Leiden, The Netherlands; 4Department of Medical Statistics, Leiden University Medical Center, Leiden, The Netherlands; and 5Aventis Pasteur, Lyon, France. Overexpression of p53 occurs in more than 50% of colorectal cancers. Therefore, p53 represents an attractive target antigen for immunotherapy. We assessed the safety of a canarypox virus encoding the human wild-type p53 gene given intravenously to end- stage colorectal cancer patients in a three-step dose escalation study aimed at inducing p53 immune responses. Patients with metastatic disease of p53-overexpressing colorectal cancers were vaccinated three times at 3-week intervals, each time with 106.5 7.0 7.5 CCID50 (CCID50 ¼ cell culture infectious dose 50%; group 1, n ¼ 5), 10 CCID50 (group 2, n ¼ 5) or 10 CCID50 (group 3, n ¼ 6). Vital signs and the occurrence of adverse events were monitored and blood was analyzed for biochemical and hematological parameters as well as signs of auto-immune safety. In all, 16 patients were enrolled and 15 patients completed three vaccinations. No anaphylactic reaction or unwanted auto-immune reactions were observed. A total of 16 serious adverse events (SAEs) occurred: 10 in group 1, three in group 2 and three in group 3. All SAEs were tumor-related complications. There was no difference in the frequency of adverse events between the three groups, except for fever. Fever was the only vaccination-related adverse event consistently observed and was most frequent and outspoken in the group 3 patients. The majority was a grade 1 or 2 fever (93%) and grade 3 fever (7%) was observed in three patients of group 3. Some patients showed humoral and cellular responses against p53, following vaccinations. After having completed his initial treatment cycle, one patient (group 2) received a second treatment cycle 7.5 of three doses of 10 CCID50 and subsequently showed stable disease. All other patients showed progressive disease. We conclude that ALVAC-p53 can be administered intravenously to colorectal cancer patients without serious toxicity or pathological autoimmunity and can induce immune responses against p53. Cancer Gene Therapy (2003) 10, 509–517. doi:10.1038/sj.cgt.7700600 Keywords: colorectal cancer; P53; immunotherapy; canarypox virus umors are characterized by numerous changes in a antigens), viral antigens (e.g. HPV E6/E7), protein Tvariety of genes, resulting in altered products that can antigens (e.g. CEA, HER-2/neu, Ep-Cam) and mutated be unique targets for the immune system. Although the oncogene products (e.g. ras, p53, c-myc).1 majority are expressed on both tumor cells and normal The p53 gene is a tumor suppressor gene located on the cells, the expression of these tumor-associated antigens short arm of chromosome 17 that plays a pivotal role in (TAAs) has been found to differ significantly between ensuring genetic homeostasis.2 Mutations in the p53 gene normal cells and tumor cells, and thus may provide an are the most common genetic alteration in many tumors immunotherapeutic window. Potential targets include and result in the production of a defective protein with a carbohydrate antigens (e.g. gangliosides, blood groups significantly longer than normal half-life of p53.3 More than 50% of colorectal cancers demonstrate p53 over- expression, potentially allowing the tumor cells to be Received January 25, 2003. processed and the immunogenic epitopes to be presented Address correspondence and reprint requests to: Professor Dr CJH 4 van de Velde, Department of Surgery, K6-R, Leiden University to the immune system. Although p53 is an auto-antigen, it Medical Center, PO Box 9600, 2300 RC Leiden, The Netherlands. has been demonstrated that humoral and cellular E-mail: [email protected] immune responses against p53 can be detected in cancer Safety of ALVAC-p53 in colorectal cancer AG Menon et al 510 patients.5–10 Enhancing these responses could result in ALVAC-p53 used in the present study, with doses up to effective antitumor responses, but also induce a hazar- 10 times the proportional human dose, revealed no dous reaction to normal cells that have lowexpression toxicity or auto-immune phenomena upon clinical or on levels of this auto-antigen. histopathological evaluation.14 The safety of ALVAC Vaccination against p53 can be performed using vector-based vaccines has been well established in various genetically modified viral vectors allowing high expression clinical trials where more than 1500 individuals, including of the transfected gene within antigen-presenting cells, cancer patients, have received ALVAC encoding a variety thereby eliciting strong immune response against p53. The of genes such as the rabies glycoprotein gene,15,16 HIV canarypox virus (ALVAC) is a well-characterized viral envelope genes,17,18 CEA19 and even multiple genes such vector capable of infecting a variety of human cells. This as CEA and B7.1.20 In this study, we show that repeated vector, which cannot replicate in non-avian species, can i.v. administration of ALVAC-p53 in end-stage colorectal nevertheless express inserted genes for up to a fewdays. cancer patients is well tolerated and does not induce This results in the induction of strong humoral and pathological auto-immunity, while stimulating p53-speci- cellular responses against these gene products.11 Hurpin fic immune responses. et al.12 compared subcutaneous, intramuscular, intrader- mal and intravenous (i.v.) administrations of ALVAC- p53 in mice and showed that only the i.v. vaccination Methods route was capable of inducing CTL responses against p53. Subsequent studies showed that i.v. vaccination of mice Patients with ALVAC-p53 resulted in long-term protection against Patients older than 18 years with histologically proven challenges with p53 overexpressing tumors. Although, colorectal cancer and evidence by imaging techniques of this does not resemble the clinical situation in which irresectable disease were eligible for inclusion. Patients tumors are already present, Roth et al.13 also showed that with metastatic disease that was untreatable by conven- established p53-overexpressing tumors were successfully tional therapies and patients with metastatic disease that eradicated with i.v. ALVAC-p53 administrations. was potentially treatable, but who refused conventional The use of viral vectors in humans is associated with therapy were also eligible. The study protocol was safety concerns for the patients and the environment, but approved by the local and national medical ethics as the canarypox virus is unable to replicate in committees as well as by the biological safety committee mammalian cells, these concerns do not apply to and the Dutch Ministry of Health and Environment ALVAC-based vaccines. Studies performed on rhesus before the study started. The inclusion and exclusion macaques that received i.v. injections of the same lot of criteria are summarized in Table 1. Table 1 Inclusion and exclusion criteria for participation in the ALVAC-p53 study Inclusion criteria Histologically proven colorectal cancer Evidence of irresectable or metastatic disease in patients untreatable by conventional therapies, or in patients who are treatable by conventional therapies, but who refuse these Immunohistochemically proven expression of p53 and MHC class I in at least 30% of tumor cells Serum CEA X10 mg/l Age over 18 years Negative pregnancy test (for women of child-bearing age) Use effective contraception during the trial and up to 3 months after the last injection (for men and women) WHO performance status of 0–1 Renal function and bilirubin levels within 1.5 Â the normal upper and lower levels, ASAT and ALAT within 3 Â the normal upper value and alkaline phospahatase within 5x the normal upper value Signed informed consent Exclusion criteria Auto-immune disease Symptomatic viral or other infections HIV seropositivity or refusal to hear the result of the HIV test Organ grafts Life expectancy of less than 3 months History of allergy: severe asthma or atopic eczema or known allergy to egg products or neomycin History of severe neurological, cardiovascular, renal, hepatic, endocrine, respiratory or bone marrow dysfunction Known family history of Li-Fraumeni syndrome Chemotherapy (with the exception of nitrosurea), or radiation therapy within 4 weeks preceding inclusion Immunotherapy, chemotherapy using nitrosurea, or hormonal therapy (other than contraception) within 6 weeks preceding inclusion History of treatment with extracted growth hormone Cancer Gene Therapy Safety of ALVAC-p53 in colorectal cancer AG Menon et al 511 Treatment and follow-up schedule toxicity tests, pyrogen assay identification, viral potency test and a particle count test. Patients were enrolled into three sequential groups of five individuals each and received three i.v. injections of ALVAC-p53 at 3-week intervals. At each of the three Statistical analyses injections, patients in group 1 received a tenth (106.5 The w2 test, Kruskal–Wallis test (CEA) and the ANOVA CCID50) of the total dose of ALVAC-p53, patients in (age and diagnosis metastases) tests were performed to 7.0 group 2 received a third (10 CCID50) of the total dose analyze the differences between clinical and tumor and patients in group 3 received the total dose (107.5 characteristics between groups 1 to 3.