Allogeneic Gene-Modified Tumour Cells in Metastatic Kidney

In a limited study, comprising only ten patients, we have previously reported that allogeneic irradiated RCC-cell-line cells, engineered to produce IL-2 (ACHN-IL-2), admixed with autologous metastatic formalin-treated tumour cells were used to vaccinate MRCC patients in progression of disease and also receiving IL-2 immunotherapy. The cells, admixed to autologous TC, were administered subcutaneously. We now report an extended study on thirty patients and one hundred thirty-one controls. Patients received 4ñ20 injections (mean 10 ± 4), containing an average of 92 × 106 ± 45 × 106 ACHN-IL-2 transfected cells (a minimum of 25 × 106, and a maximum of 200 × 106). Autologous TC, admixed to allogeneic, were also administered by 4ñ16 s.c. injections (mean 7 ± 3), i.e. a total of 12 × 106ñ160 × 106 cells. Vaccination was administered during 73ñ1451 (307 ± 316) days, and the follow-up continued for 1122 ± 1240 days (106ñ5137). Throughout this period, the patients continued receiving the previously set immunotherapy treatment. No adverse side effects related to the treatment were noticed. One complete and four partial tumour responses were observed, as well as nine cases of stable disease. Thirteen patients died in the treated group (43%) and 63 (44%) in the control group. Responding patients resumed progression in 4ñ11 months and died 18 and 36 months after beginning the vaccine therapy. The Gehan Wilcoxonís test showed a significantly (P < 0.01) better survival in the vaccinated patients compared to that of the controls. Thus, we confirm, in an increased number of patients and an extensive followup, that our vaccination protocol is safe, devoid of adverse side effects, and promising. It is well known that patients suffering from metastatic renal cell cancer (MRCC) have a poor prognosis and the treatment represents a very difficult challenge in urological oncology (Hrushsky and Murphy, 1977; Elson et al., 1988). It is also commonly accepted that, because the response rate is in average 20% (Bukowski et al., 1997, 2000; Vogelzang et al., 1998; Pizza et al. 2001, 2002), the most promising therapy for MRCC is immunotherapy. However, since 80% of patients go in progression, new therapeutic approaches are needed; an ìoldî tool is now emerging reshaped: tumour vaccination. The concept of tumour vaccines is not new and although no definite proof is offered concerning the Original Articles Allogeneic Gene-Modified Tumour Cells in Metastatic Kidney Cancer. Report II ( vaccine / gene-modified / immunotherapy / IL-2 / MRCC / renal cancer ) G. PIZZA1, C. DE VINCI1, G. LO CONTE1, A. MAZZUCA1, V. DI MAIO1, S. RATINI1, G. SEVERINI1, L. BUSUTTI2, A. P. PALARETI3, A. GULINO4, A. VACCA4, L. MELCHIORRI5, M. FERRARI6, L. GIACOMELLI7, O. R. BARICORDI5, S. FORZINI8, R. CAPANNA8 1Immunotherapy Module, Operative Unit (O. U.) of Urology, Department of Urology and Nephrology, and 2O.U. of Radiotherapy, Department of Oncology, S. Orsola-Malpighi Hospital, Bologna, Italy 3Department of Computer Science, University of Bologna, Bologna, Italy 4Experimental Medicine, University ìLa Sapienzaî, Rome, Italy 5Department of Experimental and Diagnostic Medicine, Section of Medical Genetics, University of Ferrara, Ferrara, Italy 6Experimental Institute of Zooprophylaxis, Emilia-Romagna and Lombardia Regions, Brescia, Italy 7Department of Surgical Science University of Rome ÑLa Sapienzaì, Rome, Italy 82nd Division of Orthopaedics and Reconstructive Surgery Centre, Florence, Italy Received November 11, 2004. Accepted November 12, 2004. Corresponding author: Giancarlo Pizza, Immunotherapy Module, Operative Unit of Urology, Department of Urology and Nephrology, S. Orsola-Malpighi Hospital, Via P. Palagi, 9, 40138 Bologna, Italy. Tel.: +39-051-6362478; Fax: +39-0516362476; e-mail: [email protected]. Abbreviations: ATC ñ autologous tumour cells, CR ñ complete response, IL-2 ñ interleukin-2, i.m. ñ intramuscularly, LAK ñ lymphokine-activated killer cells, MRCC ñ metastatic renal cell cancer, PB ñ peripheral blood, PBL ñ peripheral blood lymphocytes, PR ñ partial response, PROG ñ tumour progression, PS ñ performance status, pt(s) ñ patient(s), s.c. ñ subcutaneous, sd ñ standard deviation, TAA ñ tumour-associated antigens, TNM ñ tumour, node, metastasis, U ñ unit, y ñ year(s). Folia Biologica (Praha) 50, 175-183 (2004) presence of tumour-specific antigens in kidney cancer cells (Ueda et al., 1981; Oosterwijk et al., 1986), many different approaches have been suggested with some encouraging observations (Belldegrun et al., 1993; Pizza et al., 1999, 2003; Schwaab et al., 2000; Dillman et al., 2001). We report here a continuation of our previous studies describing observations obtained with 30 patients and a larger group of control patients. The treatment was administered following a 3-year clinical experimental protocol authorized by the Ministry of Health and the members of the local Ethics Committee. Material and Methods Transfected allogeneic cell line (ACHN) The ACHN tumour cell line from ATCC, American TissueType Culture Collection, established from a kidney cancer was obtained from the EmiliaRomagna and Lombardia Regions Experimental Institute of Zooprophylaxis. The transfection and its ability to produce IL-2 after irradiation have been already reported (Pizza et al. 1999; 2003). Briefly, a humanIL-2 expression vector, pcDNA-I Neo-IL-2 (7683 bp length), has been prepared by insertion of IL-2 cDNA (683 bp length) in HindIII/BamHI sites of the plasmid of the polylinker pcDNAINeo (InVitrogenTM, Invitrogen S.R.L., San Donato Milanese, Italy). The amount of 107 ACHN cells has been transfected with 10 μg of pcDNAhIL2 using the CaPO4 technique (Graham and Vanderb, 1973; Cavallo et al., 1993). Resistant clones were isolated, and IL-2 production was evaluated using the CTLL-2 line (Gillis and Watson, 1981), or the PHA(Difco, Detroit, MI) and IL-2-conditioned human blasts (Pizza et al., 1984). Cells were irradiated with a 60-cobalt bomb at 100 cGy/min to a total of 40ñ60 Gy. They were unable to replicate, but the in vitro IL-2 production appeared to continue for 32 days, with a mean concentration of 230 pg/day/106 cells as assessed using the ELISA kit ìBiotrakîÆ (Amersham, Life Science, Little Chalfont, UK) (Pizza et al., 2003). Tumorigenesis in nude mice was negative and their in vitro ability to replicate nil during an observation period of 60 days. At the end of this period all cells were dead. Autologous metastatic tumour cells (ATC) ATC were obtained from patientsí metastases during surgery carried out mainly because of pathologic bone fractures as described by Pizza et al. (2003). Briefly, G. Pizza et al. 176 Vol. 50 Parameter No. treated pts No. control pts