Intracellular Cytokines Produced by Stimulated CD3+ Cells from Chronic Myeloid Leukemia Patients.

Our work examined the production of intracellular interferon (INF)-γ, tumor necrosis factor (TNF)-α, interleukin (IL)-2, and IL-4 by in vitro stimulated CD3+ cells from 38 chronic myeloid leukemia (CML) patients. At the time of diagnosis the percentages of cells producing INF-γ, TNF-α, and IL-2 were strongly suppressed compared to those in healthy control subjects. Hematological remission achieved through treatment with tyrosine-kinase inhibitors was associated with a highly significant increase in the ratio of cells producing all 4 cytokines. The percentages of CD3+ cells producing cytokines were dependent on age, more so in CML patients than in healthy controls, and they negatively correlated with the number of leukocytes. Patients with an optimal therapy outcome possessed higher percentages of cytokine-producing CD3+ cells at diagnosis than those with nonoptimal outcomes. This difference was statistically significant in the case of INF-γ-producing cells, and it was on the brink of significance in the case of IL-2-producing cells.

Predictive value of serum cytokine levels in chronic myeloid leukemia patients.

Serum samples taken at diagnosis in 28 chronic myeloid leukemia patients were tested for the presence of 20 cytokines by a magnetic bead-based Bio-plex immunoassay. According to complete cytogenetic remission achieved at 12 months of treatment, patients were divided into groups with either optimal or non-optimal outcome. Patients with increased cytokine levels tended to react optimally to the therapy more frequently than those others. TGF-ß3 was a notable exception; its levels were significantly higher in patients with non-optimal outcomes. Further analysis enabled us to define two combinations of cytokine cut-off levels - namely low TGF-ß3 and either high IL-8 or high MCP-1-each of which corresponded to therapy outcome better than either Sokal or EUTOS scores.

Combined Cancer Immunotherapy Against Aurora Kinase A.

Aurora kinase A (AURKA) is a centrosomal protein that is overexpressed in a number of human malignancies and can contribute to tumor progression. As we used this protein as a target of DNA immunization, we increased its immunogenicity by the addition of the PADRE helper epitope and decreased its potential oncogenicity by mutagenesis of the kinase domain. For in vitro analysis of induced immune responses in mice, we identified the Aurka(220-228) nonapeptide representing an H-2Kb epitope. As DNA vaccination against the Aurka self-antigen by a gene gun did not show any antitumor effect, we combined DNA immunization with anti-CD25 treatment that depletes mainly regulatory T cells. Whereas 1 anti-CD25 dose injected before DNA vaccination did not enhance the activation of Aurka-specific splenocytes, 3 doses administered on days of immunizations augmented about 10-fold immunity against Aurka. However, an opposite effect was found for antitumor immunity-only 1 anti-CD25 dose combined with DNA vaccination reduced tumor growth. Moreover, the administration of 3 doses of anti-CD25 antibody alone accelerated tumor growth. Analysis of tumor-infiltrating cells showed that 3 anti-CD25 doses not only efficiently depleted regulatory T cells but also activated helper T cells and CD3(-)CD25(+) cells. Next, we found that blockade of the PD-1 receptor initiated 1 week after the first immunization was necessary for significant inhibition of tumor growth with therapeutic DNA vaccination against Aurka combined with depletion of CD25 cells. Our results suggest that combined cancer immunotherapy should be carefully evaluated to achieve the optimal antitumor effect.

Kynurenine and uric acid levels in chronic myeloid leukemia patients.

Indoleamine 2,3-dioxygenase 1 (IDO1), IDO2 and tryptophan 2,3-dioxygenase (TDO) represent some of the key immune regulators. Their increased activity has been demonstrated in a number of human malignancies but not yet in chronic myeloid leukemia (CML). In the present study, the activity of these enzymes was tested in 29 CML patients and 28 healthy subjects by monitoring the kynurenine (KYN)/tryptophan ratio. Serum samples taken prior to the therapy displayed a highly significant difference in KYN levels between the patient and control groups. However, increased KYN levels were detected in only 13 (44.8%) of these CML patients. The KYN levels in pretreatment sera of the patients correlated with the tumor burden. There was also a strong correlation between KYN levels and uric acid levels (UA). This suggests but does not prove the possible involvement of UA in activating IDO family of enzymes. Whenever tested, the increased KYN levels normalized in the course of the therapy. Patients with normal KYN levels in their pretreatment sera and subsequently treated with interferon-α, showed a transitory increase in their KYN levels. The present data indicate that CML should be added to the malignancies with an increased activity of the IDO family of enzymes and suggest that IDO inhibitors may be used in the treatment of CML patients.

Enhancement of DNA vaccine potency against legumain.

The asparaginyl endopeptidase legumain that is overexpressed in M2-polarized tumor-associated macrophages has been identified as a suitable target for elimination of these cells supporting tumor progression. To enhance the efficacy of DNA immunization against legumain, we performed several modifications in this protein that could improve induction of immune responses. First, we mutated the RGD motif into GGD or RGG sequences. This alteration resulted in diminished maturation of legumain and impaired cellular localization. Then, as tolerance to self-antigens can be broken by the activation of CD4 T-cell help, we tried to enhance the immunogenicity of legumain by the insertion of a foreign helper epitope, namely the p30 epitope from the tetanus toxin. Finally, the 2 modifications were combined. After gene gun DNA immunization of C57BL/6 mice with these constructs, we identified the Lgmn111-119 CD8 T-cell epitope that binds to H-2D molecules. Furthermore, we showed that mutagenesis in the RGD motif significantly enhanced the immune response against legumain. The addition of the p30 helper epitope induced the specific production of IFN-γ by T cells, but did not significantly increase legumain-specific immunity activated after mutagenesis in the RGD motif which might be caused by simultaneous activation of a Th2 response demonstrated by the production of IL-4. However, the beneficial effect of the helper epitope on legumain-specific response was proved after the depletion of regulatory T cells by antibody against CD25 that preferentially stimulated Th1 immunity. The antitumor effect of the modified legumain gene was shown in the immunization against tumors induced by MK16 cells.

Antitumor DNA vaccination against the Sox2 transcription factor.

As cancer stem cells (CSCs) are resistant to chemotherapy, radiotherapy and targeted molecular therapy, immunotherapy of tumors could be aimed at their elimination. Markers specific for CSCs have not been identified to date, but microarray analyses have shown that CSCs and embryonic stem cells use similar transcriptional programs, thus suggesting the production of shared transcription factors. In this study, we developed an experimental DNA vaccine against the transcription factor Sox2 that is important for self-renewal of stem cells and is overexpressed in numerous human cancers. The Sox2 gene was codon optimized for the expression in human cells, its sequences encoding two nuclear localization signals (NLSs) were mutagenized, and the sequence coding for the PADRE helper epitope was fused with its 5' terminus. While codon optimization did not increase Sox2 production and mutagenesis in NLSs only partially reduced nuclear localization of Sox2, the addition of the PADRE epitope was crucial for the enhancement of Sox2 immunogenicity. The antitumor effect was shown after immunization against mouse oncogenic TC-1/B7 cells derived from the lung cancer cell line TC-1 and characterized by high Sox2 production. Sox2-specific reactivity in an ELISPOT assay was further augmented by the depletion of regulatory T (Treg) cells, but this depletion did not enhance the antitumor effect. These data demonstrated the induction of immune responses against the Sox2 self-antigen, but did not confirm the usefulness of Treg depletion when combined with antitumor vaccination.

DNA vaccine against human papillomavirus type 16: modifications of the E6 oncogene.

Since its discovery, DNA vaccination has become an effective strategy for the development of vaccines against cancer including cervical carcinoma (CC). The formation of CC is associated with human papillomavirus (HPV) infection. Viral E6 and E7 oncoproteins are suitable targets for therapeutic vaccination. To adapt the HPV16 E6 oncogene for DNA immunisation, we performed several modifications. First we fused the E6 gene with the 5' or 3'-terminus of the Escherichia coli beta-glucuronidase (GUS) gene and showed enhanced immunogenicity of the 3' fusion (GUS.E6). Then, as the E6 oncogene contains two alternative introns that result in the production of truncated forms of the E6 protein, we abolished the 5' splice site in the E6 gene. This modification completely eliminated the expression of the truncated E6 transcripts and thus increased the production of the full-length E6 protein. At the same time, it moderately reduced the immunogenicity of the modified non-fused (E6cc) or fused (GUS.E6cc) genes, probably as a consequence of the substitution in the immunodominant E6 epitope following the abolishment of the splice site. Furthermore, we reduced the oncogenicity of the E6 protein by two point mutations (E6GT) that, together, prevented E6-mediated p53 degradation. Finally, we constructed the GUS.E6GT gene characterized by enhanced safety and immunogenicity when compared with the wild-type E6 gene.

DNA vaccination against bcr-abl-positive cells in mice.

A series of DNA vaccines based on the bcr-abl fusion gene were developed and tested in mice. Two mouse (BALB/c) bcr-abl-transformed cell lines, B210 and 12B1, which both expressed p210bcr-abl and were oncogenic for syngeneic animals but differed in some other respects, were used as a model system. In the first series of experiments, plasmids carrying either the complete bcr-abl fusion gene or a fragment thereof coding for a 25-amino acid-long junction zone (bcr-abl25aa) linked with genes coding for a variety of immunostimulatory factors were used as the DNA vaccines. A plasmid carrying the complete bcr-abl gene was capable of inducing protection against challenge with either B210 or 12B1 cells. However, the DNA vaccines based on the gene fragment coding for p25aabcr-abl did not induce significant protection. To localize the immunizing epitopes on the p210bcr-abl protein, the whole fusion gene was split into nine overlapping fragments and these, individually or in various combinations, were used for immunization. Although none of the vaccines based on any single fragment provided potent protection, some combinations of these fragment-based vaccines were capable of eliciting protection comparable to that seen after immunization with the whole-gene vaccine. Surprisingly, a mixture of six fragment-vaccines was more immunogenic than the complete set of fragment DNA vaccines. To analyze this phenomenon, the three fragments missing from the hexavaccine were either individually or in various combinations mixed with the hexavaccine. The results obtained suggested that the product of the fragment coding for 197 amino acids forming the N-terminal of the BCR protein was involved in the decreased immunogenicity. However, further experiments are needed to clarify the point. Additional experiments revealed that all the important epitopes were located in the ABL portion of the p210bcr-abl protein. The livers, spleens and bone marrows of the successfully immunized animals were tested for the presence of bcr-abl-positive cells by RT-PCR. The results were negative, this suggesting that these animals were free of any residual disease.

Effects of endostatin production on oncogenicity and metastatic activity of HPV16-transformed mouse cells: role of interleukin 1alpha.

Two mouse HPV16-transformed cell lines, viz. MK16 cells, which induce metastasizing tumors, and TC-1 cells, which induce non-metastasizing tumors were transduced with the gene for mouse endostatin. Two clones constitutively expressing endostatin were isolated from each of them. They were denoted ME3 and ME9, and TE2 and TE5, respectively. When inoculated into mice, ME3 cells were non-oncogenic. Nearly all mice inoculated with ME9 cells developed tumors, but considerably later than did the parental MK16 cells and metastasis formation was strongly reduced in these animals. On the other hand, TE2 and TE5 cells displayed oncogenic potential similar to that of the parental cells. To provide more information on these different effects of endostatin production, cell lysates of all six lines studied were tested for the content of 25 factors known to be involved in angiogenesis. The parental MK16 cells differed from the parental TC-1 cells and also from all endostatin producing sublines by a markedly higher production of interleukin 1alpha (IL-1alpha) and, to a lesser extent, by a higher production of several other factors tested. Additional experiments indicated that the suppression of the production of IL-1alpha by the parental MK16 caused by endostatin was due to an autocrine mechanism.

Vaccination with human papillomavirus type 16-derived peptides using a tattoo device.

Tattooing has been shown to be very efficient at inducing immunity by vaccination with DNA vaccines. In this study, we examined the usability of tattooing for delivery of peptide vaccines. We compared tattooing with subcutaneous (s.c.) needle injection using peptides derived from human papillomavirus type 16 (HPV16) proteins. We observed that higher peptide-specific immune responses were elicited after vaccination with the simple peptides (E7(44-62) and E7(49-57)) and keyhole limpet hemocyanin-(KLH)-conjugated peptides (E7(49-57), L2(18-38) and L2(108-120)) with a tattoo device compared to s.c. inoculation. The administration of the synthetic oligonucleotide containing immunostimulatory CpG motifs (ODN1826) enhanced the immune responses developed after s.c. injection of some peptides (E7(44-62), KLH-conjugated L2(18-38) and L2(108-120)) to levels close to or even comparable to those after tattoo delivery of identical peptides with ODN1826. The highest efficacy of tattooing was observed in combination with ODN1826 for the vaccination with the less immunogenic E6(48-57) peptide and KLH-conjugated and non-conjugated E7(49-57) peptides which form the visible aggregates that could negatively influence the development of immune responses after s.c. injection but probably not after tattooing. In summary, we first evidenced that tattoo administration of peptide vaccines that might be useful in some cases efficiently induced both humoral and cell-mediated immune responses.

Combined chemo- and immunotherapy of tumors induced in mice by bcr-abl-transformed cells.

For our experiments we selected two oncogenic, bcr-abl-transformed mouse cell lines, viz. B210 and 12B1. Both cell types are capable of inducing leukemia-like disease in syngeneic BALB/c mice after intravenous inoculation. 12B1 cells can moreover form solid tumors after subcutaneous injection. Since immunotherapy would expectedly be most effective in animals in which the tumor mass had been reduced by other therapeutic means, we attempted to develop a combined therapeutic system for suppressing tumor growth. In the present study, mice inoculated with the aggressive 12B1 cells were treated with imatinib mesylate (IM), mouse interferon alpha (IFNalpha) and cyclophosphamide (Cy) in combination with genetically modified tumor cells engineered to produce various cytokines. These cell vaccines had been derived from B210 cells. Therapy with IM or IFNalpha alone or cell immunotherapy alone resulted in partial suppression of tumor growth. Of the different therapeutic regimens tested, a combination of repeated doses of IM, IFNalpha and cell vaccines with one relatively high dose of Cy (200 mg/kg) was the most effective, resulting in tumor-free survival of a large portion of mice. The spleens, livers and bone marrows of the successfully treated animals were tested for the presence of bcr-abl-positive cells by means of RT-PCR technique. Results were negative, this suggesting that the animals had been cleared of residual disease.

Efficacy of reovirus therapy combined with cyclophosphamide and gene-modified cell vaccines on tumors induced in mice by HPV16-transformed cells.

Oncolytic virotherapy is a novel approach to cancer treatment. In the present study we tested the ability of reovirus type 3, strain Dearing, to suppress the growth of tumors induced in mice by HPV16-transformed TC-1 cells. In vitro, these cells are highly susceptible to the virus. In repeated in vivo tests the intratumoral inoculation of the virus resulted in only a minor slow-down of tumor growth, never in a complete cure. The effect of the treatment was not enhanced by the simultaneous administration of non-oncogenic, genetically modified TC-1 cells expressing either IL-2, IL-12 or GM-CSF, and, in fact, the oncolytic effect of the virus was even less expressed in some instances. When cyclophosphamide was used in combination with the viral treatment, a synergistic effect resulting in tumor suppression was observed. In most instances the tumor regression was transitory, however, and was followed by tumor progression. The outcome of these experiments was dependent on the timing of the two treatments.

Enhancement of T cell-mediated and humoral immunity of beta-glucuronidase-based DNA vaccines against HPV16 E7 oncoprotein.

Therapeutic DNA vaccines against oncogenic infection with human papillomavirus type 16 (HPV16) are mostly targeted against viral oncoproteins E7 and E6. To adapt the E7 oncoprotein for DNA immunization, we have previously reduced its oncogenicity by modification of the Rb-binding site and enhanced immunogenicity of the modified E7GGG gene by the fusion with the 5'-terminus of the gene encoding E. coli beta-glucuronidase (GUS). In this study, we attempted to improve immunogenicity of the GUS-based anti-E7 vaccines by increasing the steady-state level of fusion proteins. We fused deletion mutants of E7GGG and codon-optimized E7GGG with the 5'-terminus of GUS and unaltered E7GGG with the 3'-terminus of GUS. Furthermore, we mutated the initiation codon of the GUS gene in the E7GGG.GUS construct, as GUS alone was produced from this fusion gene. We found that only the fusion of E7GGG with the 3'-terminus of GUS (GUS.E7GGG) and deletion mutants of E7GGG with the 5'-terminus of GUS increased the steady-state level of fusion proteins in transfected human 293T cells. Analysis of immune reactions induced in mice by vaccination via a gene gun showed that the increased steady-state level of fusion proteins resulted in augmented production of E7-specific antibodies, but did not enhance cell-mediated anti-tumor immunity. Finally, we joined the signal sequence of the adenoviral E3 protein with GUS.E7GGG. This modification led to the predominant localization of the fusion protein in the endoplasmic reticulum and enhancement of CD8+ T-cell response, while antibody production was reduced. In conclusion, we found modifications of the E7GGG.GUS fusion gene that augmented either humoral or cell-mediated immune responses.

Live cell vaccines expressing B7.1, monocyte chemoattractant protein 1 and granulocyte-macrophage colony stimulation factor derived from mouse HPV16-transformed cells.

One of the gene therapy strategies in oncology is immunization with cancer cells that express various cytokines. We used a thymidine-kinase deficient (cTK-) cell line designated 123IA, which had been derived from HPV16-transformed mouse (C57BL/6) cells MK16/I/III/ABC (MK16). To obtain genetically modified cells, 123IA cells were transfected with bicistronic plasmid vectors carrying the herpes simplex type 1 thymidine kinase (HSV TK) gene and either the gene for the mouse B7.1 (CD80) co-stimulatory molecule or the gene for the monocyte-chemoattractant protein 1 (MCP-1). For control purposes, a plasmid vector carrying only the HSV TK gene was used. The transfected cells were cultivated in medium supplemented with hypoxanthine, aminopterin and thymidine. For comparative purposes we also used B9 cells, which express the granulocyte-macrophage colony stimulation factor (GM-CSF) and had been derived from 123A cells by transduction with the recombinant adeno-associated virus carrying the HSV TK gene and the mouse GM-CSF gene. All of the cell lines isolated were found to be sensitive to minute amounts of ganciclovir, revealing the production of HSV TK, and to express the respective transgenes. When inoculated into 5-week-old female syngeneic mice, cells expressing either GM-CSF or B7.1 were non-oncogenic. On the other hand, nearly all mice inoculated with MCP-1-producing cells developed tumours, though considerably later than animals inoculated with the same dose of the parental MK16 cells. Animals injected with GM-CSF- or B7.1-producing cells were protected against challenge with the parental MK16 cells. When another mouse (C57BL/6) HPV16-transformed oncogenic cell line, TC-1, which differs from the MK16 cells in a number of properties such as MHC class I and B7.1 expression, was used for the challenge, the protective effect was much less pronounced.

DNA vaccines based on chimeric potyvirus-like particles carrying HPV16 E7 peptide (aa 44-60).

Vaccine strategies for the treatment of human papillomavirus-induced cervical cancer are based mainly on the human papillomavirus 16 E7 (HPV16 E7) oncoprotein. The immunogenicity of the E7 gene has been enhanced by its fusion to many different genes. Here, we linked a short sequence coding for the E7 peptide (aa 44-60) containing immunodominant epitopes for B and T cells to the 3' end of the gene coding for the whole coat protein (CP) of the poty-virus, potato virus A (PVA), and its deleted form (CPdel) with a short C-terminal deletion of 5 amino acids (LGVKG). CP-E7 and CPdel-E7 fusion proteins, just like CP alone, spontaneously assembled into virus-like particles in both procaryotic and eucaryotic cells. The CP-E7 and CPdel-E7 fusion genes induced slightly stronger E7-specific cytotoxic T-lymphocyte responses than the whole E7 gene, although they were still lower than those elicited by the previously constructed fusion gene, Sig/E7GGG/LAMP-1. The E7- and CP-specific antibody responses were not detected in mice vaccinated with CP-E7 and CPdel-E7 fusion genes. The CP-E7 and CPdel-E7 fusion genes protected mice against the development of tumors induced by TC-1 cells producing the E7 antigen and were also effective in the therapeutic setting, i.e. when the vaccination was performed after tumor cell administration. Their antitumor effect was comparable to those of the whole E7 gene and Sig/E7GGG/LAMP-1 fusion gene. There was no relevant difference between immune responses elicited by CP-E7 and CPdel-E7 DNA vaccination.

Combined immunization with fusion genes of mutated E7 gene of human papillomavirus type 16 did not enhance antitumor effect.

BACKGROUND: The E7 oncoprotein of human papillomavirus type 16 (HPV16) is frequently used as a model tumor-associated antigen. Its immunogenicity has been substantially enhanced by fusion with several proteins of various origins and functions. Different mechanisms have been responsible for increased vaccination efficacy of fusion proteins. METHODS AND RESULTS: We linked E7 and its mutated form (E7GGG) with the mouse heat-shock protein 70.1 (HSP70.1). Enhanced immunogenicity of both fusion genes administered via a gene gun was demonstrated by protection of C57BL/6 mice against oncogenic MHC class I positive TC-1 cells producing the HPV16 E7 oncoprotein but not against the MHC class I negative TC-1/A9 subline. To assess if the efficacy of E7-based DNA vaccines could be increased by combination of various fusion genes, we combined the HSP70.1 fusion genes (i.e. E7HSP or E7GGGHSP) with the fusion construct linking E7GGG with targeting signals of lysosome-associated membrane protein 1 (Sig/E7GGG/LAMP-1). Treatment of mice 4 days after TC-1 cell inoculation showed moderately higher immunization potency of HSP70.1 fusion genes in comparison with the Sig/E7GGG/LAMP-1 gene. Any combination of two fusion genes given in the same gene gun shot neither was more effective compared with single genes nor protected mice against TC-1/A9 cells. As fusion of E7GGG with E. coli glucuronidase (E7GGG.GUS) had been previously proven to provide partial protection from TC-1/A9-induced tumors, we also combined E7GGGHSP with E7GGG.GUS. The genes were inoculated either in mix in two gene gun shots or separately each gene in one shot into opposite sides of the abdomen. Neither mode of combined immunization induced higher protection than E7GGG.GUS alone. However, doubling the DNA dose considerably enhanced the antitumor efficacy of E7GGG.GUS. CONCLUSIONS: We constructed highly immunogenic fusions of HPV16 E7 and E7GGG with mouse HSP70.1. Furthermore, we substantially enhanced protection against TC-1/A9 cells with downregulated MHC class I expression by doubling the pBSC/E7GGG.GUS dose, but we failed to demonstrate a beneficial effect of any combination of two fusion genes with different mechanisms causing enhancement of HPV16 E7 immunogenicity.

Combined immunization with DNA and transduced tumor cells expressing mouse GM-CSF or IL-2.

A combination of different types of vaccines usually induces enhanced immune responses in comparison to immunization with single vaccines. The highest efficacy of a heterologous prime-boost strategy is mostly achieved after priming with a DNA vaccine and boosting with a recombinant virus or a protein vaccine. The aim of this study was to determine whether the combination of a DNA and cellular vaccine elicits stronger antitumor immune responses than vaccines used alone and to find out whether the efficacy of this combined immunization depends on the sequence in which the vaccines were applied. We utilized experimental vaccines that proved to be partially effective in protection against mouse tumor cells representing models of human papillomavirus-induced malignancies. The fusion gene Sig/E7GGG/LAMP-1, inoculated via a gene gun, was used for DNA immunization. As cellular vaccines, HPV16 E6/E7 and H-ras transformed B9 or 181 cells transduced with the gene coding for GM-CSF or IL-2, respectively, were applied. In both preventive and therapeutic immunizations, inoculation first with the DNA vaccine followed by application of a cellular vaccine induced the best protection from tumor growth. These results were confirmed by detection of immune reactions with in vitro tests. We failed to enhance immune reactions by utilization of an equivalent mix of B9 and 181 cells, but the addition of the second DNA-vaccine dose applied simultaneously with a cellular-vaccine boost moderately increased antitumor response. Our findings suggest the benefit of the heterologous prime-boost strategy based on combination of a DNA vaccine with a cellular vaccine and importance of sequence in which the vaccines are administered.

Chemotherapy and immunotherapy of tumours induced by gene-modified HPV16-transformed cells.

HPV16 E6/E7 transformed mouse kidney cells designated MK16/1/IIIABC (MK16) were modified by the insertion of a suicide gene, viz. the thymidine-kinase gene of herpes simplex virus (HSV TK). Tumour induction by these cells, designated N2A, was suppressed by ganciclovir (GCV). The growth of already established tumours was partially inhibited by GCV. This effect was markedly potentiated by a single dose of cyclophosphamide (Cy). Ganciclovir- or GCV+ Cy-cured mice were not protected against challenge with MK16 cells. N2A tumour growth was suppressed by simultaneous administration of MK16-derived, non-oncogenic B9 and 181 cells, which express either mouse GM-CSF or mouse IL2, respectively, in addition to HSV TK. The animals treated were protected against challenge with MK16 cells. Animals with already established N2A tumours were treated with GCV and/or repeated doses of B9 or 181 cells. Ganciclovir treatment alone and immunotherapy alone resulted in partial suppression of tumour growth but not in tumour cure. On the other hand, combined chemo- and immunotherapy resulted in tumour rejection by nearly all animals. Similar results were obtained if the immunotherapy with homologous gene-modified cells was substituted by treatment with anti-CD4 antibody. The animals cured of tumours with GCV combined with cell-based vaccine therapy but not those cured by GCV and anti-CD4 antibody treatment were found resistant to challenge with MK16 cells. The present results suggest that combined specific and non-specific chemo- and immunotherapy of tumours induced by appropriately gene-modified cells might provide a special advantage in the treatment of established tumours.

Immunization with live HPV-16-transformed mouse cells expressing the herpes simplex thymidine kinase and either GM-CSF or IL-2.

From mouse (C57BL/6) HPV-16 transformed cells denoted MK16/1/IIIABC (MK16) a cellular thymidine kinase deficient (cTK-) cell line was isolated. These cTK- cells were transduced by bicistronic recombinant adeno-associated viruses (rAAV) carrying the herpes simplex virus thymidine kinase gene and the gene for either the mouse granulocyte-macrophage colony stimulating factor (GM-CSF) or mouse interleukin-2 (IL-2). Transduced cells were highly sensitive to minute amounts of ganciclovir (GCV) and synthesized moderate amounts of the respective cytokines. A number of cell clones were tested for the cytokine production. The two best producer cell lines, the GM-CSF-producing cells denoted B9 and the IL-2-producing cells denoted 181, were selected for further experiments. Neither B9 nor 181 cells were tumorigenic in syngeneic animals. As inducers of antitumour immunity against challenge with MK16 cells, B9 cells proved superior to the 181 cells. GCV treatment did not markedly influence the level of immunity induced.