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Antitumor effect of ganciclovir on HPV16 transformed HSV-TK gene modified hamster cells
164
I..P 5
23 June 1997 - Poster presentations
Novel therapy in immunodeficiencies and cancer 03 .19
) induction of intracellular immunity to HIV infection in CD4+ T lymphocytes by transfection of an endopiasmic reticulum-retained CD4 chimera
E. San Jo&‘, M.A. Mufioz-Femandez *, E. Femandez-Cruz *, B. Alar&. 1Centro de Biokwla Molecular Sever0 Ochoa, CSIC, UAM, Madrid, Spain, *Department of lkmunollosv; Gregorio Ma&i& Univetsitary Hospital, Madrid, Spain Introduction: The human immunodeficiency virus (HIV) is the causal agent of the acquired immunodeficiency syndrome. CD4 is the main cellular receptor for HIV and, consequently, its major cellular targets are CD4+cells: mostly a subset of T lymphocytes and macrophages. The interaction between CD4 and gp120, one of the two glycoproteins of the viral envelope, is essential for the binding and entry of the viral partides. The viral glycoproteins, gp120 and gp41, are synthesized in the endoplasmic reticulum (ER) as a 160 kD precursor. Only a small percentage (5-15%) of the gp160 precursor is proteolytically processed to gp120 and gp41. The remaining is degraded in lysosomes. It has been reported that the CD4 molecule is associated with gp160 in the ER. These complex not only prevent the transport of CD4 to the cell membrane but the processing of gp160 to gp120 and gp41 as well. We have obtained a CD4 molecule with an ER retention signal; the association of this molecule with gp160 could result in a complex that would have its transport outside of the ER inhibited. The ER-retained CD4 chimera could be useful for intracellular immunization of T cells from HIV seropositive patients. Materials and Methods:Stable transfectants with the chimera of CD4 (CD4tag) were obtained in Jurkat cells and theclones which express the chimera were infected with the HIV virus at low multiplicity of infection. The production of p24 antigen, formation of syncytia, induction of apoptosis and PCR of the integrated viral DNA were utilized to assess antireplicative effect of the chimera. To evaluate the capability to induce resistance in non-transformed T cells, we have constructed a retrovirus-based expression vector (pLNCX) under the control of the LTR of HIV, that has been used to transfect blast cells obtained by PHA stimulation of peripheral blood lymphocytes from normal donors. These transfectants have been infected with HIV and the replication of the virus was monitored by the production of p24 antigen in the supematants. Results: The parental Jurkat cells produced considerable amounts of p24 antigen after the infection with HIV, whereas in the clone that express the chimera (clone 36) the production of p24 antigen was practically negligible. It was also shown that in clone 36 the induction of apoptosis was prevented. PCR experiments showed that the proviral DNA is not detected in clone 36 at late times of the infection. The experiments done in blast transduced with the retroviral construction that encodes for the CD4 chimera, showed that after the infection, p24 release was prevented in the presence of the chimera. Conoluslons: First we have demonstrate in Tcell lines that the presence of a CD4 chimera that is retained in the ER, prevents the replication of the HIV virus. Second we have also seen that in a more physiological system it is also reproducible. It would be a model system to evaluate the possibility of transfecting T cells from AIDS patients to make them resistant to HIV infection. Therefore, the chimera could be used for gene therapy of AIDS patients. In a clinical setting, cells from the patients’ bone marrow will have to be transfected ex viva with an appropriate vector and reinjected into the patients aiming to obtain a high percentage of the T cell progeny resistant to infection.
P.5.03.20 ) Antitumor effect of ganciciovir on HPV16 transformed HSV-TK gene modified hamster ceils Eva Sobotkovd, Michal Smahel, Hider0 Kitasato, Eva HamSikov& Hana SainerovB, Vladimir Vonka. Dept.of fxp-etimental Virology, institute of Hematology and Blood Transfusion, Prague, Czech Republic Introduction: HPVs are apparently invdved in human cancer. Immune reactions against HPV-induced tumors are in the focus of interest. MaterlaIr and Methods: Syrian hamster kidney cells were transformed by co-transfection with plasmids carrying EWE7 genes of HPV16, activated Ha-ras oncogene and neomycin resistence gene. From one of highly oncogenic cell lines denoted K3/ll, a subline was isolated deficient in cellular thymidlne kinase (TK) (TK- cells). These cells were transfected with a plasmid carrying herpes simplex virus TK gene (HSV-TK). The growth of the successfully transformed cells (HSV TK+) was inhibited by ganciclovir (GCV) as well as by other antivirals phosphotylated by HSV-TK. One of these HSV TK+ cell lines denoted KL1/6 was used in the subsequent experiments. Reeufte: The development of tumors by KL1/6 cells, but not K3/ll cells, was prevented when GCV therapy was started at the time of inoculation of the cells. When GCV therapy was initiated after small tumors developed, they regressed either temporarily or permanently. Simultaneous administration of KL1/6 and K3/ll cells (in mixtures or given separately) resulted in partial or complete suppression of the tumor gmwth, depending on GCV therapy, cell dose and the way of administration of the cells. By-stander effect as well as concomitant immunity were demonstrated in these experiments.
Conclusion: A system was developed in Syrian hamsters suitable for studying various aspects of immunity against HPVlG-transformed cells.
P.5.03.21
Poiycistronic retrovirai vectors for immune-gene therapy of human melanoma
M. Wiznerowicz, A.C. Fong ‘, P. Wysocki, A. Mackiewicz, R.G. Hawley ‘. 1Department of Cancer Immunolcg~ University Schwl of Medica/ Sciences, Poznah, Poland, Oncology Research Laboratories, University of Toronto, Canada Introduction: Paracrfne cytokine adjuvant therapy is one version of genetic cancer vaccines to generate tumor-specific immunity and is now being evaluated in human clinical trials. The general principle of this approach is to increase the immunogenic&y of tumor cells by genetically engineering them to express high local concetrations of cytokines at the tumor site. Retroviral vectors are widely used in delivering of cytokines genes to tumor cell in number ofclinicalpmtccds. However, their efficiency and effectiveness are still not satisfactory. Material and Method% To ensure expression of foreign gene in target cells La&Z-marker and Neo-resistance genes were linked with IRES sequence and such dicistmnic cassette was inserted into three different retmviral vectors. Moreover double-copy vector (DCCMV), containing dicistronic cassette under control of HCMV-IE promoter was constructed. For expression of three genes from vital LTR tricistronic vector was constructed in which GFP as a second marker was used (GFP-IRES-LacZ-IRES-Neo). Human melanoma cell lines were transduced with obtained recombinant viruses. Number of LacZ positive cells was evaluated after selection in geneticin and level of mRNA expression was assayed using Northern blot. In order to create human allogenic melanoma vaccine for clinical trials. DCCMV vector carrying hlL-6 and its soluble a-receptor cDNAs was constructed and used to transduce human melanoma cell lines. Moreover ttfcistronic vector containing shlL-GR-IRES-IL-6-IRES-Neo cassette was constructed and used to transduce melanoma cells. mRNA expression and protein production was determined by Northern blotting and ELISA. Results: Almost 100% of melanoma cells transduced with dicistmnic vectors were LacZ positive after selection in geneticin. Level of LacZ expression was the highest in case of DCCMV vector. Level of expression of GFP and LacZ in triplecistronic vector was relatively lower. Transduction of melanoma cells with, double-copy dicistmnic vector carrying hlL-6 and slL-6R cDNAs resulted in high mRNA and protein expression (hlL-6: 300 ng/ml/106 cells; shlL_GR:100 ng/ml106 cells). Conclusions: Double-copy dicistmnic vector provides new and effective tool for high level of ex vivo cytokines transduction Into human cancer cells. Trfcistmnic retmviral vectors may be usefull in simultaneous expression of multiple genes in human melanoma cells.
P.5.03.22
The role of immune co-stimulation in K1735 and B16FlO mouse melanoma treatment
A.L. Barnard. D. Darling, J. G&ken, F. Farzaneh. Department of Molecular Medicine, The Rayne Institute, 123 Coldharbour Lane, London, England Introduction: Many mouse models with autologous or totally allogeneic tumour cells are used in melanoma vaccine design. Using mice that are semi-syngeneic with respect to the tumour vaccine, we can assess whether it is necessary for tumour vaccine cells to present tumour antigen themselves or whether pmfessional antigen presenting cells (APCs) am essential for efficient anti-tumour responses. Materlals and Methoda: K1735 cells were transfected with retmviral vectors to express combinations of 87.1, an immune co-stimulatory molecule or cytokines (IL-2 or 11-4). Tumorfgenicity studies were carded out in syngeneic C3WHe mice and semi-syngeneic C57BU6 x C3H/He (B6C3) Fl hybrid mice. 106 modified or wild-type K1735 cells were injected (SC)into the flanks of 6-6 week old mice. Growth of tumours was monitored over 5 months. Similar exoeriments were done to demonstrate whether irradiated K1735 cells could treat established wild-type turnouts. B6C3 mice were injected with 3 x l@ wild-type K1735 cells or lo5 wild-type B16FlO melanoma cells (syngeneic with C57BU6 mice. known to exoress &iaens wmmon to K1735 cells). 7 and 14 days after inocilation, the animals we; injected (ip) with 5 x 106 imdiated wild-&e or modified K1735 cells in HBSS. Control groups received HBSS alone. Results: Similar tumorfgenicity was observed in C3H/He mice and the B6C3 Fl hybrids. Expression of the immune co-stimulatoty molecule, 87.1 alone did not lead to tumour rejection, but with co-expression of IL-2 or IL-4 less than 10% of animals formed tumours. B6C3 mice inoculated with wfld-type K1735 and then treated with intrapetftoneal injections of irradiated modified K1735 cells formed tumours in all but 2 cases. Tumour formation was delayed by 5 to 10 days in animals treated with K1735 cells producing only one of the cytokines with or without 87.1, treatment with K1735 secreting IL-2 and IL-4 together having no effect on K1735 tumour formation. However with the same treatment protocol 40% of the B16FlO inoculated mice remained tumour free after 50 days, the control mice having all formed turnouts after 21 days.
I..P 5
23 June 1997 - Poster presentations
Novel therapy in immunodeficiencies and cancer 03 .19
) induction of intracellular immunity to HIV infection in CD4+ T lymphocytes by transfection of an endopiasmic reticulum-retained CD4 chimera
E. San Jo&‘, M.A. Mufioz-Femandez *, E. Femandez-Cruz *, B. Alar&. 1Centro de Biokwla Molecular Sever0 Ochoa, CSIC, UAM, Madrid, Spain, *Department of lkmunollosv; Gregorio Ma&i& Univetsitary Hospital, Madrid, Spain Introduction: The human immunodeficiency virus (HIV) is the causal agent of the acquired immunodeficiency syndrome. CD4 is the main cellular receptor for HIV and, consequently, its major cellular targets are CD4+cells: mostly a subset of T lymphocytes and macrophages. The interaction between CD4 and gp120, one of the two glycoproteins of the viral envelope, is essential for the binding and entry of the viral partides. The viral glycoproteins, gp120 and gp41, are synthesized in the endoplasmic reticulum (ER) as a 160 kD precursor. Only a small percentage (5-15%) of the gp160 precursor is proteolytically processed to gp120 and gp41. The remaining is degraded in lysosomes. It has been reported that the CD4 molecule is associated with gp160 in the ER. These complex not only prevent the transport of CD4 to the cell membrane but the processing of gp160 to gp120 and gp41 as well. We have obtained a CD4 molecule with an ER retention signal; the association of this molecule with gp160 could result in a complex that would have its transport outside of the ER inhibited. The ER-retained CD4 chimera could be useful for intracellular immunization of T cells from HIV seropositive patients. Materials and Methods:Stable transfectants with the chimera of CD4 (CD4tag) were obtained in Jurkat cells and theclones which express the chimera were infected with the HIV virus at low multiplicity of infection. The production of p24 antigen, formation of syncytia, induction of apoptosis and PCR of the integrated viral DNA were utilized to assess antireplicative effect of the chimera. To evaluate the capability to induce resistance in non-transformed T cells, we have constructed a retrovirus-based expression vector (pLNCX) under the control of the LTR of HIV, that has been used to transfect blast cells obtained by PHA stimulation of peripheral blood lymphocytes from normal donors. These transfectants have been infected with HIV and the replication of the virus was monitored by the production of p24 antigen in the supematants. Results: The parental Jurkat cells produced considerable amounts of p24 antigen after the infection with HIV, whereas in the clone that express the chimera (clone 36) the production of p24 antigen was practically negligible. It was also shown that in clone 36 the induction of apoptosis was prevented. PCR experiments showed that the proviral DNA is not detected in clone 36 at late times of the infection. The experiments done in blast transduced with the retroviral construction that encodes for the CD4 chimera, showed that after the infection, p24 release was prevented in the presence of the chimera. Conoluslons: First we have demonstrate in Tcell lines that the presence of a CD4 chimera that is retained in the ER, prevents the replication of the HIV virus. Second we have also seen that in a more physiological system it is also reproducible. It would be a model system to evaluate the possibility of transfecting T cells from AIDS patients to make them resistant to HIV infection. Therefore, the chimera could be used for gene therapy of AIDS patients. In a clinical setting, cells from the patients’ bone marrow will have to be transfected ex viva with an appropriate vector and reinjected into the patients aiming to obtain a high percentage of the T cell progeny resistant to infection.
P.5.03.20 ) Antitumor effect of ganciciovir on HPV16 transformed HSV-TK gene modified hamster ceils Eva Sobotkovd, Michal Smahel, Hider0 Kitasato, Eva HamSikov& Hana SainerovB, Vladimir Vonka. Dept.of fxp-etimental Virology, institute of Hematology and Blood Transfusion, Prague, Czech Republic Introduction: HPVs are apparently invdved in human cancer. Immune reactions against HPV-induced tumors are in the focus of interest. MaterlaIr and Methods: Syrian hamster kidney cells were transformed by co-transfection with plasmids carrying EWE7 genes of HPV16, activated Ha-ras oncogene and neomycin resistence gene. From one of highly oncogenic cell lines denoted K3/ll, a subline was isolated deficient in cellular thymidlne kinase (TK) (TK- cells). These cells were transfected with a plasmid carrying herpes simplex virus TK gene (HSV-TK). The growth of the successfully transformed cells (HSV TK+) was inhibited by ganciclovir (GCV) as well as by other antivirals phosphotylated by HSV-TK. One of these HSV TK+ cell lines denoted KL1/6 was used in the subsequent experiments. Reeufte: The development of tumors by KL1/6 cells, but not K3/ll cells, was prevented when GCV therapy was started at the time of inoculation of the cells. When GCV therapy was initiated after small tumors developed, they regressed either temporarily or permanently. Simultaneous administration of KL1/6 and K3/ll cells (in mixtures or given separately) resulted in partial or complete suppression of the tumor gmwth, depending on GCV therapy, cell dose and the way of administration of the cells. By-stander effect as well as concomitant immunity were demonstrated in these experiments.
Conclusion: A system was developed in Syrian hamsters suitable for studying various aspects of immunity against HPVlG-transformed cells.
P.5.03.21
Poiycistronic retrovirai vectors for immune-gene therapy of human melanoma
M. Wiznerowicz, A.C. Fong ‘, P. Wysocki, A. Mackiewicz, R.G. Hawley ‘. 1Department of Cancer Immunolcg~ University Schwl of Medica/ Sciences, Poznah, Poland, Oncology Research Laboratories, University of Toronto, Canada Introduction: Paracrfne cytokine adjuvant therapy is one version of genetic cancer vaccines to generate tumor-specific immunity and is now being evaluated in human clinical trials. The general principle of this approach is to increase the immunogenic&y of tumor cells by genetically engineering them to express high local concetrations of cytokines at the tumor site. Retroviral vectors are widely used in delivering of cytokines genes to tumor cell in number ofclinicalpmtccds. However, their efficiency and effectiveness are still not satisfactory. Material and Method% To ensure expression of foreign gene in target cells La&Z-marker and Neo-resistance genes were linked with IRES sequence and such dicistmnic cassette was inserted into three different retmviral vectors. Moreover double-copy vector (DCCMV), containing dicistronic cassette under control of HCMV-IE promoter was constructed. For expression of three genes from vital LTR tricistronic vector was constructed in which GFP as a second marker was used (GFP-IRES-LacZ-IRES-Neo). Human melanoma cell lines were transduced with obtained recombinant viruses. Number of LacZ positive cells was evaluated after selection in geneticin and level of mRNA expression was assayed using Northern blot. In order to create human allogenic melanoma vaccine for clinical trials. DCCMV vector carrying hlL-6 and its soluble a-receptor cDNAs was constructed and used to transduce human melanoma cell lines. Moreover ttfcistronic vector containing shlL-GR-IRES-IL-6-IRES-Neo cassette was constructed and used to transduce melanoma cells. mRNA expression and protein production was determined by Northern blotting and ELISA. Results: Almost 100% of melanoma cells transduced with dicistmnic vectors were LacZ positive after selection in geneticin. Level of LacZ expression was the highest in case of DCCMV vector. Level of expression of GFP and LacZ in triplecistronic vector was relatively lower. Transduction of melanoma cells with, double-copy dicistmnic vector carrying hlL-6 and slL-6R cDNAs resulted in high mRNA and protein expression (hlL-6: 300 ng/ml/106 cells; shlL_GR:100 ng/ml106 cells). Conclusions: Double-copy dicistmnic vector provides new and effective tool for high level of ex vivo cytokines transduction Into human cancer cells. Trfcistmnic retmviral vectors may be usefull in simultaneous expression of multiple genes in human melanoma cells.
P.5.03.22
The role of immune co-stimulation in K1735 and B16FlO mouse melanoma treatment
A.L. Barnard. D. Darling, J. G&ken, F. Farzaneh. Department of Molecular Medicine, The Rayne Institute, 123 Coldharbour Lane, London, England Introduction: Many mouse models with autologous or totally allogeneic tumour cells are used in melanoma vaccine design. Using mice that are semi-syngeneic with respect to the tumour vaccine, we can assess whether it is necessary for tumour vaccine cells to present tumour antigen themselves or whether pmfessional antigen presenting cells (APCs) am essential for efficient anti-tumour responses. Materlals and Methoda: K1735 cells were transfected with retmviral vectors to express combinations of 87.1, an immune co-stimulatory molecule or cytokines (IL-2 or 11-4). Tumorfgenicity studies were carded out in syngeneic C3WHe mice and semi-syngeneic C57BU6 x C3H/He (B6C3) Fl hybrid mice. 106 modified or wild-type K1735 cells were injected (SC)into the flanks of 6-6 week old mice. Growth of tumours was monitored over 5 months. Similar exoeriments were done to demonstrate whether irradiated K1735 cells could treat established wild-type turnouts. B6C3 mice were injected with 3 x l@ wild-type K1735 cells or lo5 wild-type B16FlO melanoma cells (syngeneic with C57BU6 mice. known to exoress &iaens wmmon to K1735 cells). 7 and 14 days after inocilation, the animals we; injected (ip) with 5 x 106 imdiated wild-&e or modified K1735 cells in HBSS. Control groups received HBSS alone. Results: Similar tumorfgenicity was observed in C3H/He mice and the B6C3 Fl hybrids. Expression of the immune co-stimulatoty molecule, 87.1 alone did not lead to tumour rejection, but with co-expression of IL-2 or IL-4 less than 10% of animals formed tumours. B6C3 mice inoculated with wfld-type K1735 and then treated with intrapetftoneal injections of irradiated modified K1735 cells formed tumours in all but 2 cases. Tumour formation was delayed by 5 to 10 days in animals treated with K1735 cells producing only one of the cytokines with or without 87.1, treatment with K1735 secreting IL-2 and IL-4 together having no effect on K1735 tumour formation. However with the same treatment protocol 40% of the B16FlO inoculated mice remained tumour free after 50 days, the control mice having all formed turnouts after 21 days.