Eva Sobotkova

Metastatic MHC class I-negative mouse cells derived by transformation with human papillomavirus type 16

In the endeavour to develop a model for studying gene therapy of cancers associated with human papillomaviruses (HPVs), mouse cells were transformed with the HPV type 16 (HPV16) and activated H-ras oncogenes. This was done by contransfection of plasmid p16HHMo, carrying the HPV16 E6/E7 oncogenes, and plasmid pEJ6.6, carrying the gene coding for human H-ras oncoprotein activated by G12V mutation, into secondary C57BL/6 mouse kidney cells. An oncogenic cell line, designated MK16/1/IIIABC, was derived. The epithelial origin of the cells was confirmed by their expression of cytokeratins. No MHC class I and class II molecules were detected on the surface of MK16/1/IIIABC cells. Spontaneous metastases were observed in lymphatic nodes and lungs after prolonged growth of MK16/1/IIIABC-induced subcutaneous tumours. Lethally irradiated MK16/1/IIIABC cells induced protection against challenge with 105 homologous cells, but not against a higher cell dose (5 × 105). Plasmids p16HHMo and pEJ6.6 were also used for preventive immunization of mice. In comparison with a control group injected with pBR322, they exhibited moderate protection, in terms of prolonged survival, against MK16/1/IIIABC challenge (P < 0.03). These data suggest that MK16/1/IIIABC cells may serve as a model for studying immune reactions against HPV16-associated human tumours.

Systemic Administration of CpG Oligodeoxynucleotide and Levamisole as Adjuvants for Gene-Gun-Delivered Antitumor DNA Vaccines

DNA vaccines showed great promise in preclinical models of infectious and malignant diseases, but their potency was insufficient in clinical trials and is needed to be improved. In this study, we tested systemic administration of two conventional adjuvants, synthetic oligodeoxynucleotide carrying immunostimulatory CpG motifs (CpG-ODN) and levamisole (LMS), and evaluated their effect on immune reactions induced by DNA vaccines delivered by a gene gun. DNA vaccination was directed either against the E7 oncoprotein of human papillomavirus type 16 or against the BCR-ABL1 oncoprotein characteristic for chronic myeloid leukemia. High doses of both adjuvants reduced activation of mouse splenic CD8+ T lymphocytes, but the overall antitumor effect was enhanced in both tumor models. High-dose CpG-ODN exhibited a superior adjuvant effect in comparison with any combination of CpG-ODN with LMS. In summary, our results demonstrate the benefit of combined therapy with gene-gun-delivered antitumor DNA vaccines and systemic administration of CpG-ODN or LMS.

Induction of anti-tumour immunity by suicide-gene-modified HPV-16-transformed hamster cells

From K3/II, which is a highly oncogenic HPV16‐transformed Syrian hamster cell line, thymidine‐kinase(TK)‐less cells, denoted B 49, were derived. B49 cells were transfected by a plasmid containing the herpes‐simplex‐virus TK gene (HSV TK) and several sub‐lines expressing this gene were isolated from the transfected cultures. The HSV TK+ cells were highly sensitive to ganciclovir (GCV) and other anti‐viral substances whose inhibitory effect is based on their phosphorylation by HSV TK. One of the cell lines, denoted KL1/6, exhibited relatively high stability of the HSV TK+ phenotype and was used in subsequent experiments. When KL1/6 cells were co‐cultivated in the presence of GCV with various other cell lines of hamster, mouse or monkey origin, the by‐stander effect (BE) was observed. GCV treatment of hamsters prevented development of tumours after the administration of KL1/6 cells but not K3/II cells. The treatment of animals with already established KL1/6‐induced tumours resulted in tumour regression in all instances, but complete regression was observed only in animals carrying small tumours. The BE of KL1/6 cells on K3/II cells was also seen in vivo. In addition, concomitant immunity was observed in animals simultaneously inoculated with KL1/6 cells and K3/II cells at 2 separate sites of the body. This effect was evident not only in animals in which KL1/6 tumours developed, but also in those in which tumour outgrowth was prevented by GCV treatment. In other experiments it was demonstrated that one KL1/6 + GCV treatment resulted in partial resistance, 2 such treatments in complete resistance to the challenge with K3/II cells. Int. J. Cancer 77:470–475, 1998.