- Email: [email protected]
Adenovirus mediated gene transfer in dendritic cells
162
23 June I997 - Posterpresentations
Novel therapy in immunodeficiencies and cancer
and expressed recombinant cytokines in mycobacteria. The levels of cytokine expression as measured by specific ELISA were as follows; IL-7 (10 wml), IL-8 (50 @ml), IL-15 (300 pg/ml), RANTES (20 @ml), MCP-1 (50 @ml) and TNFa (see Haley &al). Using gfF to study the binding of mycobacterfa to tumour cells, we demonstrated the mobllisation of cr-actinin. Concluslonr: High level cytoktne expression has been achieved in mycobactertal clones. The unique difficulties of recombinant gene expression in mycobacterta, our initial results on the biological activities of mycobactertalderived cytokines, and the future uses for this system to express tumour antigens will be discussed. Lastly, we shall present our initial data on the biological potential of cytokine secreting mycobacterta in the context of bladder cancer.
P.5.03.11
Efficacy of gene therapy of cancer in aglng: Ill-transfected tumor cells are rejected but do not Induce lona-lasting immune memow In old mice
M. Provinciali, G. Di Stefano, S. Stronati, A. Tibaldi, G. Fomi ‘. /mmuno/ogy Center, INRCA Gerontology Research Department, Ancona, /ta/x 1lmmunoganetics and Histocompatibility Center, CNR, Turin, /ta/y Transfer and expression of cytokine genes into tumor cells is now regarded as a valuable approach for investigating antiiumor activities of cytokines in experimental models. The injection of tumor cells genetically modified for the constitutive expression of cytokines into syngeneic immunocompetent mice results in tumor rejection and specific memory acquisition by activation of hostdeoendent antitumor resoonses. The oblective of the study was to evaluate the effectiveness of gene therapy of cancer with tumor ceils transfected with cytoktnes in aging, pertod of lie charactertsed by a progressive immune detertoration particularty evident at the level of thymus-dependent immunity. Young (2-3 mo) and old (24 mo) Balb/c inbred mice were injected with TS/A (mammary adenocarcinoma) parental cells transfected with IL-2 gene. Three clones of TS/A cells were used producing low (30 U, B1.30) intermediate (3800 U, 88.3800) or high (8008 U, 84.8000) 11-2.Young and old mice injected with untransfected TS/A cells were used as controls. After 30 days, mice with no tumors after the challenge with lL2-tranfected clones received a lethal challenge of the TS/A parental cells. While the B1.30 clone grew in 100% of mice with a kinetics similar to that of TS/A p.c. in both young and old mice, the 86.3800 and 84.8000 clones were promptly rejected in both aged groups. A slower kinetics of rejection was observed in old than young mice. In young mice, rejection was associated with a large neutrophil and macrophage Infiltration, with a minor number of CD4+ and CD8+ lymphocytes. In old mice, neutrophils and macrophages were also the main cells involved in tumor rejection, through they were less represented than In young animals. Both CD4+ and CD8+ lymphocytes were nearly absent in perl- and intratumoml infiltrate. To test whether the injection of tumor clones producing IL-2 induced a long-lasting, tumor specific, immune memory, mice with no tumors after the challenge with 88.3800 and B4.8CO8clones perfoned 30 days earlier received a lethal challenge of the TS/A parental cells. Good protection was found in young mice that had been injected with the B4.8000 clone since 80% of them rejected the TS/A p.c. cells. Rejection was observed only in 30% of young mice that were challenged with the B8.3800 clone. In old mice, no protection was found in animals that had been injected with B4.8ooO clone whereas only 10% of mice which received B8.3800 cells were able to reject TS/A pc. These data demonstrate that the injection of IL-2 tmnsfected tumor cells in old mice is effective in inducing tumor rejection through the intervention of neutrophils and macrophages, but is not able to elicit a long-lasting immune memory because of the scarce induction of CD4+ and CD8+ lymphocytes.
P.5.03.12
Regression of established P815 tumors by intra-tumoral delivery of the IL-2 cDNA using adenoviral vectors carrying dlfferent promoters
Philippe Slos, Pierre Leroy, Cecile Doderer, Ludovic Ruet, Majid Mehtali, Bruce Acres. Tmnsgdne S.A., 11, rue de Molsheim, 67082 Sttasbourg Cedax, France Introduction: The local production of various cytokines in the tumor or at the periphery of the tumor has been shown in different murtne models to activate the immune system leading to the control of tumor growth or ultimately, to tumor rejection. The potential of replication-defective Adenovlral vectors (AEI AE3) to deliver the human IL-2 cDNA into the weakly immunogenic mudne P815 mastocytoma tumor was investigated. Moreover, the influence of the promoter driving the transcription of the IL-2 cDNA on Its capacity to promote tumor growth inhibition or rejection was compared. Materlal and Methods: 5 x 1Os P815 cells were implanted subcutaneously on the right flank of B8D2 mice. Approximately 10 days after tumor cells injection, palpable tumors with an average diameter of 3 to 4 mm were detected. The following vectors were injected directly into the tumors in a volume of 100 ~1 containing 5 x 108 P.F.U.: AdTG5327 (pMLP-IL-2); AdTG8822 (pRSV-IL-P);
AcfTG8824 (pCMV-IL-2) and Ad-IacZ as a control. A minimum of ten mice per group was used. If mice rejected their tumor following the Adenoviral treatment, they were challenged with the same dose of tumor cells on the opposite flank. The cellular immune response against P815 cells was subsequently analyzed in the mice resistant to this challenge. Raeults: a single intra-tumoral injection of the adenoviral vectors described above gave the fotlowing % of tumor rejection: Ad-IacZ (0%); Ad-pMLP-IL-2 (10%); Ad-pCMV-IL-2 (10%); Ad-pRSV-IL-2 (30%). Nevertheless, the best tmnsient increase in survival was obtained in the group of mice treated with AdpCMV-IL-2 (P < 0.02 compared to Ad-IacZ). In the same experiment, multiple sequential injections of Ad-pCMV-IL-2 were performed at day 0,2 and 4 resulting in 40% of tumor rejection. All the mice that rejected their tumor survived to a subsequent challenge and remained tumor-free for more than two months after the challenge. The majority of the mice developed a strong CTL response in vitm against P815 cells. Conclusions: the intra-tumoral delivery of the IL-2 cDNA into P815 tumors using Adenoviral vectors carrying different promoters leads to a variable 1) increase in survtval compared to control groups andlor 2) percentage of tumor rejection. The correlation between the expression level of IL-2 together with the persistence of IL-2 expression both in vitro and in viw is currently under investigation. The analysis of the mechanism(s) responsible for the observed tumor rejection will also be analyzed.
P5.03.13
A,$Iovirus
mediated gene transfer In dendrltlc
J.M. Cuillerot‘, A.I. Michou *, M.P. Kieny2, Fr. Oberling ‘, P. Fenand ‘, A. Bohbot ‘, B. Acres*. ’ Service d’Onco-H&matologie, H6piWx Unive&aires da Sttasbourg, ’ Tmnsgene S.A, 11 rue de Molshei&, Stra&ourg, Francs
Introduction:dendrttic cells are professional antigen presenting cells that can initiate primary T cell responses. Therefore, large quantities of genetically modified dendritic cells would provide a powerful tool for immunotherapy, especially for malignant diseases. Two critical issues must be investigated before any clinical application based on the use of gene modiied dendritic cells: (1) production of large batches of dendrftic cells using procedures suitable for a clinical purpose and (2) an efficient gene transfer protocol, in order to obtain a sustained expression of the transgene. These considerations are explored in this work. Msterlalsand Methods:peripheral blood mononuclear cells are harvested from normal healthy volunteers with the use of an automated continuous flow cell separator COBE SPECTRA using standard procedures. Monocytes are then separated by counter-flow centtifugation elutdatton using a J-8M elutriation centrifuge. Monocytes are then incubated at 3P and 5% C# in a 50 ml Teflon bag at a cell density of 1 x 10s cells/ml in RPM1complemented with fetal calf serum 5%. Hepes 20 mM. GM-CSF 50 @ml (Shedng Plough), and IL4 200 U/ml (Pmmeoa). After 7 davs of culture, cells are harvested. Then, cell viability and phenotype’ were evaluated by Trypan Blue exclusion and the expression level of CDla and CD14. Cells are then seeded in 24 wells plates (I x lo6 cells per wells) and allow to adhere overnight. First generation adenovimses encoding for nls-IacZ or human factor IX under the control of Rous Sarcoma Virus promoter were used at several MOI ranging from 25 to 100. Transgenes expressions were evaluated by X-Gal staining or by an Elisa assay (factor IX) using a monoclonal antibody. Results:2 x IO’ viable cells are routinely obtained from an initial inoculum of 5 x 10’ monocytes. More than 50% of this population express CDla and CD14 expression was always below 30%. Sustained transgene expression has been demonstrated with a first generation adenovlrus at an MOI of 100, using human factor IX as a gene reporter. Conclusion: the up-scaling of culture conditions and the use of clinically relevant transgenes are actually under investigation. Resub will be presented and discussed. P.5.03.14
Perspective of immunotherapy and lmmunoprophylaxis
N.A. Mikhailova, T.N. Kyznetsova. lmmunopreparat Research, Pmduction Association, Uk. Russia A new product, Pseudomonas aeruginosa Toxoid, was developed by us. It is intended for prophylaxis of Pseudomonas infection and for donor vaccination to produce antipseudomonas immune plasma. Immunologically Toxoid is a three component system including the protecttve antioens of A exotoxin. slime. and omtease-three main factors of the microbe ”
.
.
”
,
and protein synthesis inhibition. It has been established that the preparation was well tolerated by donors and patients, produced no pyrogenic, allergic, and anaphylactic reactions. Toxoid was found to prevent AIDS development In bum and surgical intervention. It was worth noting that the product stimulated specific antibody formation. increased G immunoglobulin content and B rosette-farming lymphocytes as well.
23 June I997 - Posterpresentations
Novel therapy in immunodeficiencies and cancer
and expressed recombinant cytokines in mycobacteria. The levels of cytokine expression as measured by specific ELISA were as follows; IL-7 (10 wml), IL-8 (50 @ml), IL-15 (300 pg/ml), RANTES (20 @ml), MCP-1 (50 @ml) and TNFa (see Haley &al). Using gfF to study the binding of mycobacterfa to tumour cells, we demonstrated the mobllisation of cr-actinin. Concluslonr: High level cytoktne expression has been achieved in mycobactertal clones. The unique difficulties of recombinant gene expression in mycobacterta, our initial results on the biological activities of mycobactertalderived cytokines, and the future uses for this system to express tumour antigens will be discussed. Lastly, we shall present our initial data on the biological potential of cytokine secreting mycobacterta in the context of bladder cancer.
P.5.03.11
Efficacy of gene therapy of cancer in aglng: Ill-transfected tumor cells are rejected but do not Induce lona-lasting immune memow In old mice
M. Provinciali, G. Di Stefano, S. Stronati, A. Tibaldi, G. Fomi ‘. /mmuno/ogy Center, INRCA Gerontology Research Department, Ancona, /ta/x 1lmmunoganetics and Histocompatibility Center, CNR, Turin, /ta/y Transfer and expression of cytokine genes into tumor cells is now regarded as a valuable approach for investigating antiiumor activities of cytokines in experimental models. The injection of tumor cells genetically modified for the constitutive expression of cytokines into syngeneic immunocompetent mice results in tumor rejection and specific memory acquisition by activation of hostdeoendent antitumor resoonses. The oblective of the study was to evaluate the effectiveness of gene therapy of cancer with tumor ceils transfected with cytoktnes in aging, pertod of lie charactertsed by a progressive immune detertoration particularty evident at the level of thymus-dependent immunity. Young (2-3 mo) and old (24 mo) Balb/c inbred mice were injected with TS/A (mammary adenocarcinoma) parental cells transfected with IL-2 gene. Three clones of TS/A cells were used producing low (30 U, B1.30) intermediate (3800 U, 88.3800) or high (8008 U, 84.8000) 11-2.Young and old mice injected with untransfected TS/A cells were used as controls. After 30 days, mice with no tumors after the challenge with lL2-tranfected clones received a lethal challenge of the TS/A parental cells. While the B1.30 clone grew in 100% of mice with a kinetics similar to that of TS/A p.c. in both young and old mice, the 86.3800 and 84.8000 clones were promptly rejected in both aged groups. A slower kinetics of rejection was observed in old than young mice. In young mice, rejection was associated with a large neutrophil and macrophage Infiltration, with a minor number of CD4+ and CD8+ lymphocytes. In old mice, neutrophils and macrophages were also the main cells involved in tumor rejection, through they were less represented than In young animals. Both CD4+ and CD8+ lymphocytes were nearly absent in perl- and intratumoml infiltrate. To test whether the injection of tumor clones producing IL-2 induced a long-lasting, tumor specific, immune memory, mice with no tumors after the challenge with 88.3800 and B4.8CO8clones perfoned 30 days earlier received a lethal challenge of the TS/A parental cells. Good protection was found in young mice that had been injected with the B4.8000 clone since 80% of them rejected the TS/A p.c. cells. Rejection was observed only in 30% of young mice that were challenged with the B8.3800 clone. In old mice, no protection was found in animals that had been injected with B4.8ooO clone whereas only 10% of mice which received B8.3800 cells were able to reject TS/A pc. These data demonstrate that the injection of IL-2 tmnsfected tumor cells in old mice is effective in inducing tumor rejection through the intervention of neutrophils and macrophages, but is not able to elicit a long-lasting immune memory because of the scarce induction of CD4+ and CD8+ lymphocytes.
P.5.03.12
Regression of established P815 tumors by intra-tumoral delivery of the IL-2 cDNA using adenoviral vectors carrying dlfferent promoters
Philippe Slos, Pierre Leroy, Cecile Doderer, Ludovic Ruet, Majid Mehtali, Bruce Acres. Tmnsgdne S.A., 11, rue de Molsheim, 67082 Sttasbourg Cedax, France Introduction: The local production of various cytokines in the tumor or at the periphery of the tumor has been shown in different murtne models to activate the immune system leading to the control of tumor growth or ultimately, to tumor rejection. The potential of replication-defective Adenovlral vectors (AEI AE3) to deliver the human IL-2 cDNA into the weakly immunogenic mudne P815 mastocytoma tumor was investigated. Moreover, the influence of the promoter driving the transcription of the IL-2 cDNA on Its capacity to promote tumor growth inhibition or rejection was compared. Materlal and Methods: 5 x 1Os P815 cells were implanted subcutaneously on the right flank of B8D2 mice. Approximately 10 days after tumor cells injection, palpable tumors with an average diameter of 3 to 4 mm were detected. The following vectors were injected directly into the tumors in a volume of 100 ~1 containing 5 x 108 P.F.U.: AdTG5327 (pMLP-IL-2); AdTG8822 (pRSV-IL-P);
AcfTG8824 (pCMV-IL-2) and Ad-IacZ as a control. A minimum of ten mice per group was used. If mice rejected their tumor following the Adenoviral treatment, they were challenged with the same dose of tumor cells on the opposite flank. The cellular immune response against P815 cells was subsequently analyzed in the mice resistant to this challenge. Raeults: a single intra-tumoral injection of the adenoviral vectors described above gave the fotlowing % of tumor rejection: Ad-IacZ (0%); Ad-pMLP-IL-2 (10%); Ad-pCMV-IL-2 (10%); Ad-pRSV-IL-2 (30%). Nevertheless, the best tmnsient increase in survival was obtained in the group of mice treated with AdpCMV-IL-2 (P < 0.02 compared to Ad-IacZ). In the same experiment, multiple sequential injections of Ad-pCMV-IL-2 were performed at day 0,2 and 4 resulting in 40% of tumor rejection. All the mice that rejected their tumor survived to a subsequent challenge and remained tumor-free for more than two months after the challenge. The majority of the mice developed a strong CTL response in vitm against P815 cells. Conclusions: the intra-tumoral delivery of the IL-2 cDNA into P815 tumors using Adenoviral vectors carrying different promoters leads to a variable 1) increase in survtval compared to control groups andlor 2) percentage of tumor rejection. The correlation between the expression level of IL-2 together with the persistence of IL-2 expression both in vitro and in viw is currently under investigation. The analysis of the mechanism(s) responsible for the observed tumor rejection will also be analyzed.
P5.03.13
A,$Iovirus
mediated gene transfer In dendrltlc
J.M. Cuillerot‘, A.I. Michou *, M.P. Kieny2, Fr. Oberling ‘, P. Fenand ‘, A. Bohbot ‘, B. Acres*. ’ Service d’Onco-H&matologie, H6piWx Unive&aires da Sttasbourg, ’ Tmnsgene S.A, 11 rue de Molshei&, Stra&ourg, Francs
Introduction:dendrttic cells are professional antigen presenting cells that can initiate primary T cell responses. Therefore, large quantities of genetically modified dendritic cells would provide a powerful tool for immunotherapy, especially for malignant diseases. Two critical issues must be investigated before any clinical application based on the use of gene modiied dendritic cells: (1) production of large batches of dendrftic cells using procedures suitable for a clinical purpose and (2) an efficient gene transfer protocol, in order to obtain a sustained expression of the transgene. These considerations are explored in this work. Msterlalsand Methods:peripheral blood mononuclear cells are harvested from normal healthy volunteers with the use of an automated continuous flow cell separator COBE SPECTRA using standard procedures. Monocytes are then separated by counter-flow centtifugation elutdatton using a J-8M elutriation centrifuge. Monocytes are then incubated at 3P and 5% C# in a 50 ml Teflon bag at a cell density of 1 x 10s cells/ml in RPM1complemented with fetal calf serum 5%. Hepes 20 mM. GM-CSF 50 @ml (Shedng Plough), and IL4 200 U/ml (Pmmeoa). After 7 davs of culture, cells are harvested. Then, cell viability and phenotype’ were evaluated by Trypan Blue exclusion and the expression level of CDla and CD14. Cells are then seeded in 24 wells plates (I x lo6 cells per wells) and allow to adhere overnight. First generation adenovimses encoding for nls-IacZ or human factor IX under the control of Rous Sarcoma Virus promoter were used at several MOI ranging from 25 to 100. Transgenes expressions were evaluated by X-Gal staining or by an Elisa assay (factor IX) using a monoclonal antibody. Results:2 x IO’ viable cells are routinely obtained from an initial inoculum of 5 x 10’ monocytes. More than 50% of this population express CDla and CD14 expression was always below 30%. Sustained transgene expression has been demonstrated with a first generation adenovlrus at an MOI of 100, using human factor IX as a gene reporter. Conclusion: the up-scaling of culture conditions and the use of clinically relevant transgenes are actually under investigation. Resub will be presented and discussed. P.5.03.14
Perspective of immunotherapy and lmmunoprophylaxis
N.A. Mikhailova, T.N. Kyznetsova. lmmunopreparat Research, Pmduction Association, Uk. Russia A new product, Pseudomonas aeruginosa Toxoid, was developed by us. It is intended for prophylaxis of Pseudomonas infection and for donor vaccination to produce antipseudomonas immune plasma. Immunologically Toxoid is a three component system including the protecttve antioens of A exotoxin. slime. and omtease-three main factors of the microbe ”
.
.
”
,
and protein synthesis inhibition. It has been established that the preparation was well tolerated by donors and patients, produced no pyrogenic, allergic, and anaphylactic reactions. Toxoid was found to prevent AIDS development In bum and surgical intervention. It was worth noting that the product stimulated specific antibody formation. increased G immunoglobulin content and B rosette-farming lymphocytes as well.