- Email: [email protected]
174. B Cells Can Function as Tumor Antigen Presenting Cells In Vivo
as part ofa multi site trial in 33 advancedand 10early stage NSCLC patients (Nemunaitis et ai, 2004 JNCI 96(4): 326-331). No significant acute treatment related toxicity was observed. Three patients achieved complete and durable response. Survival was suggested to be improved in patients vaccinated with GVAX demonstrating ~ 40 ng ofGMCSF protein production in vitro ofthe vaccine over 24 hours per 106transfectedirradiatedcells (p '=' 0.028). Manufacturing ofGVAX for all site patients (n'='83) was done at MCCRC. Wenow report 5 year follow up of the 61 patients harvested at MCCRC. Median survival of the vaccinated (n'='29) patients was 363 (792422) days compared to the non vaccinated (n'='32) patients which was 151 (3-2484) days. Two of the 3 patients achieving complete response remain alive and without evidence of disease at 2253 and 2283 days. No long term treatment related toxic efTect has been observed. Long term follow up is being continued on all surviving patients who received vaccination (n'='4). In conclusion, no long term toxic effect related to the adenoviral vector or GMCSF gene deliverywas observed, supportingconfidencein the longterm safety of gene based vaccines.
173. Her-2/neu DNA Immunization through Constant-Current Electroporation Provides LongTerm Protection from Autochthonous Mammary Carcinomas Amir S. Khan,' Claudia Curcio,' Elena Quaglino,' Federica Cavallo.? Guido Forni.' Ruxandra Draghia-Akli.'
IVGX Pharmaceuticals, Immune Therapeutics Division, The Woodlands, TX; 2Molecular Biology Center; Department a/Clinical and Biological Sciences, University a/Turin, Turin, Italy.
I3ALl3/c mice transgenic for the transforming rat Her-2/neu oncogene (I3ALI3-neuT66-Iv.E) develop invasive mammary gland carcinomasand all animalsdisplay at leasta palpabletumor as early as 20 weeks of age. Intramuscular injection of plasmids coding for the extracellular and transmembrane domains (EC-TM plasmids) of the protein product of the Her-2/neu oncogene followed by voltage-based electroporation induced a delay in tumor onset, with all mice eventually developing a palpable tumour In the present study, we have used a novel constant current electroporation(CCE) technique to deliver EC-TM plasmids in two different models of Her-2/neudriven tumorigenesis: mice implanted with a lethal dose of Her-Z'neu' TU130tumor cells, and I3ALI3-neu·l"66-lv.Emice. Electroporation conditions, as well as the dose of EC-TM plasmids and the timing of initial vaccination, were varied to set-up the optimum conditions of vaccination. Different groups of BALB/c mice were electroporated with 5, 10,25 and 50 ug of EC-TM plasmid. After 42 days they were challenged with a lethal, 105 dose ofTUBO cells. One hundred and fitly days post-challengeall mice treated with 10, 25, or 50 ug were completely tumor free. These tumor-free mice were re-challenged with TUBO cells 206 days after the initial vaccination and these animals also remained tumor free until the end of the experiment (Table I). Remarkably, when 50 Ilg of EC-TM plasmid were delivered by CCE under optimum conditions, the BALB- neuT66-1v.E mice remained tumour free for more than a year. Similar results were obtained using 100ug of EC-TM plasmid delivered with voltage-based methods. This study demonstrates that vaccination using EC-TM plasmid vaccine delivered by CCE can prevent tumour formation in both I3ALl3/c and I3ALI3- neuT(.6-IV.E mice using a clinically relevant dose of plasmid. These improvements in the efficacyof this cancer vaccine regimen vastly enhance its chances of clinical success. Optimal DNA dose that induces ....j ection of tumors after tumor cell implantation in IIALll/c mice
Plasmid dose 10 I~ 1 1; ~ ~2 S [mlcrogra ms] I -:-:--+('~ _+: I ' First T UllO cell challenge T umo rs/lolal ~4 / 4 12f3 0/4 ,Of4 [Second T UllO cell challenge Tumors/lolal mice 4f4 Of l Of4 Of4 r-r- '
f=:- -
S66
so 0/4 Of4
174. B Cells Can Function as Tumor Antigen Presenting Cells In Vivo
James F. Curtin,' Tamer Fakhouri,' Chunyan Liu,' Pedro R. Lowenstein,' Maria G. Castro.' 'Gene Therapeutics Research Institute, Cedars-Sinai Medical Center; Los Angeles, CA.
The role of B cells as antigen presenting cells (APC) in overcoming immune ignorance against brain tumor antigen (Ag) has not yet been determined. In this study, we demonstrate that B cells arc necessary to overcome immune ignorance in the brain and mount effective, tumor specific T cell immune responses in vivo. Intratumoral expression of Fms-like tyrosine kinase 3 ligand (Flt3 ligand) and Herpes Simplex Virus I Thymidine Kinase (TK) induced B cell dependent clonal expansion ofTrp2( 180-188)(tumor Ag) specificT cells and improved long-term survival in C5713L16 mice bearing syngeneic GL26 intracranialbrain tumors. Wedid not observe any increase in tumor opsonizing immunoglobulins,or in the number of B cells infiltrating into the tumor mass. We found that total numbersof B cells and expression of co-stimulatory molecules CD80 and CD86 were increased in draining lymph nodes 7 days after treatment with Flt3 ligand and HSVI-TK. Moreover, higher numbers of'B cells containing CellTracker Green™-labeled tumor Ag were detected in the lymph nodes of tumor bearing mice treated with Flt3Land HSVI-TK compared with saline 7 days after treatment. Our data demonstrate a role for B cells as tumor APC in surmounting immune ignorance by propagating afferent immune responses against brain tumor Ag. Funded by NINDS IROI NS 42893.0 I, U54 NS045309-0I, IR21 NS047298-0I; and I3ram & Elaine Goldsmith Chair in Gene Therapeutics to P.R.L.; NINDS IROI NS44556.01, and NIDDK I R03 TW006273-01 to MGC; The Linda Tallen & David Paul Kane Foundationand the Board of Governors at CSMS.
175. Single Dosage Surface Functionalized Microparticles for Combinatorial DNA and Oligonucleotide/Adjuvant Immunization
Ankur Singh,' l3ilal Ghosn,' Kassandra Thomson,' Krishnendu Roy.' 'Department 0/ Biomedical Engineering, The University of Texas at Austin, Austin, TX.
Introduction: Earlierstudies by our group have demonstrated the potential of branched Polyethyleneimine (PEl, 70Kd) conjugated poly(lactide-co-glycolide)PLGAmicroparticles forenhanced in vitro transfection in antigen presenting cells (APCs) and significantantitumor immunity response in a mouse model ofB cell lymphoma's, Our goal here is to further improve the adjuvancy of these cationic microparticulate systems throughcombinatorial deliveryof plasmid DNA (pDNA), Oligonucleotide (ODN) and lipid-based adjuvants withinthe same microparticles. Wepresentherea novel multi-tiered system of branched PEl conjugated PLGA microparticles encapsulating Monophosphoryl Lipid-A (MPL) with surface adsorbed pDNA/CpG oligonucleotides. Our results indicate successful and efficient co-loading of adjuvants and pDNA in same particle formulations. Materials and Methods: MPL was encapsulated in the organic phase ofa w/o/w double emulsion of Resomer® RG502H (50:50, II Kd). Following adjuvant encapsulation, branched PEI (70Kd) was conjugated to the microparticle surface through EDCI NHS chemistry', MPLencapsulation, beforeand after PEl modification,was quantified usinga colorimetric assay for phospholipids with AmmoniumFerrothiocyanate', pDNA(pgWiz-betaGalactosidase or pgWiz-Luciferase) or CpG ODN was loadcd on the surface ofMPL loaded cationic microparticles at 1.2 w/w % target loading under conditions specifiedby Kasturi et al.' pDNAloadingon the cationic microparticleswas evaluatedby determiningthe unloadedpDNAor Molecular The....4lpy Volume 15.Supplement t• •\ br 2007 Co pyright © "111e American SOI;ic;ty o f Gene "l1u:mpr
173. Her-2/neu DNA Immunization through Constant-Current Electroporation Provides LongTerm Protection from Autochthonous Mammary Carcinomas Amir S. Khan,' Claudia Curcio,' Elena Quaglino,' Federica Cavallo.? Guido Forni.' Ruxandra Draghia-Akli.'
IVGX Pharmaceuticals, Immune Therapeutics Division, The Woodlands, TX; 2Molecular Biology Center; Department a/Clinical and Biological Sciences, University a/Turin, Turin, Italy.
I3ALl3/c mice transgenic for the transforming rat Her-2/neu oncogene (I3ALI3-neuT66-Iv.E) develop invasive mammary gland carcinomasand all animalsdisplay at leasta palpabletumor as early as 20 weeks of age. Intramuscular injection of plasmids coding for the extracellular and transmembrane domains (EC-TM plasmids) of the protein product of the Her-2/neu oncogene followed by voltage-based electroporation induced a delay in tumor onset, with all mice eventually developing a palpable tumour In the present study, we have used a novel constant current electroporation(CCE) technique to deliver EC-TM plasmids in two different models of Her-2/neudriven tumorigenesis: mice implanted with a lethal dose of Her-Z'neu' TU130tumor cells, and I3ALI3-neu·l"66-lv.Emice. Electroporation conditions, as well as the dose of EC-TM plasmids and the timing of initial vaccination, were varied to set-up the optimum conditions of vaccination. Different groups of BALB/c mice were electroporated with 5, 10,25 and 50 ug of EC-TM plasmid. After 42 days they were challenged with a lethal, 105 dose ofTUBO cells. One hundred and fitly days post-challengeall mice treated with 10, 25, or 50 ug were completely tumor free. These tumor-free mice were re-challenged with TUBO cells 206 days after the initial vaccination and these animals also remained tumor free until the end of the experiment (Table I). Remarkably, when 50 Ilg of EC-TM plasmid were delivered by CCE under optimum conditions, the BALB- neuT66-1v.E mice remained tumour free for more than a year. Similar results were obtained using 100ug of EC-TM plasmid delivered with voltage-based methods. This study demonstrates that vaccination using EC-TM plasmid vaccine delivered by CCE can prevent tumour formation in both I3ALl3/c and I3ALI3- neuT(.6-IV.E mice using a clinically relevant dose of plasmid. These improvements in the efficacyof this cancer vaccine regimen vastly enhance its chances of clinical success. Optimal DNA dose that induces ....j ection of tumors after tumor cell implantation in IIALll/c mice
Plasmid dose 10 I~ 1 1; ~ ~2 S [mlcrogra ms] I -:-:--+('~ _+: I ' First T UllO cell challenge T umo rs/lolal ~4 / 4 12f3 0/4 ,Of4 [Second T UllO cell challenge Tumors/lolal mice 4f4 Of l Of4 Of4 r-r- '
f=:- -
S66
so 0/4 Of4
174. B Cells Can Function as Tumor Antigen Presenting Cells In Vivo
James F. Curtin,' Tamer Fakhouri,' Chunyan Liu,' Pedro R. Lowenstein,' Maria G. Castro.' 'Gene Therapeutics Research Institute, Cedars-Sinai Medical Center; Los Angeles, CA.
The role of B cells as antigen presenting cells (APC) in overcoming immune ignorance against brain tumor antigen (Ag) has not yet been determined. In this study, we demonstrate that B cells arc necessary to overcome immune ignorance in the brain and mount effective, tumor specific T cell immune responses in vivo. Intratumoral expression of Fms-like tyrosine kinase 3 ligand (Flt3 ligand) and Herpes Simplex Virus I Thymidine Kinase (TK) induced B cell dependent clonal expansion ofTrp2( 180-188)(tumor Ag) specificT cells and improved long-term survival in C5713L16 mice bearing syngeneic GL26 intracranialbrain tumors. Wedid not observe any increase in tumor opsonizing immunoglobulins,or in the number of B cells infiltrating into the tumor mass. We found that total numbersof B cells and expression of co-stimulatory molecules CD80 and CD86 were increased in draining lymph nodes 7 days after treatment with Flt3 ligand and HSVI-TK. Moreover, higher numbers of'B cells containing CellTracker Green™-labeled tumor Ag were detected in the lymph nodes of tumor bearing mice treated with Flt3Land HSVI-TK compared with saline 7 days after treatment. Our data demonstrate a role for B cells as tumor APC in surmounting immune ignorance by propagating afferent immune responses against brain tumor Ag. Funded by NINDS IROI NS 42893.0 I, U54 NS045309-0I, IR21 NS047298-0I; and I3ram & Elaine Goldsmith Chair in Gene Therapeutics to P.R.L.; NINDS IROI NS44556.01, and NIDDK I R03 TW006273-01 to MGC; The Linda Tallen & David Paul Kane Foundationand the Board of Governors at CSMS.
175. Single Dosage Surface Functionalized Microparticles for Combinatorial DNA and Oligonucleotide/Adjuvant Immunization
Ankur Singh,' l3ilal Ghosn,' Kassandra Thomson,' Krishnendu Roy.' 'Department 0/ Biomedical Engineering, The University of Texas at Austin, Austin, TX.
Introduction: Earlierstudies by our group have demonstrated the potential of branched Polyethyleneimine (PEl, 70Kd) conjugated poly(lactide-co-glycolide)PLGAmicroparticles forenhanced in vitro transfection in antigen presenting cells (APCs) and significantantitumor immunity response in a mouse model ofB cell lymphoma's, Our goal here is to further improve the adjuvancy of these cationic microparticulate systems throughcombinatorial deliveryof plasmid DNA (pDNA), Oligonucleotide (ODN) and lipid-based adjuvants withinthe same microparticles. Wepresentherea novel multi-tiered system of branched PEl conjugated PLGA microparticles encapsulating Monophosphoryl Lipid-A (MPL) with surface adsorbed pDNA/CpG oligonucleotides. Our results indicate successful and efficient co-loading of adjuvants and pDNA in same particle formulations. Materials and Methods: MPL was encapsulated in the organic phase ofa w/o/w double emulsion of Resomer® RG502H (50:50, II Kd). Following adjuvant encapsulation, branched PEI (70Kd) was conjugated to the microparticle surface through EDCI NHS chemistry', MPLencapsulation, beforeand after PEl modification,was quantified usinga colorimetric assay for phospholipids with AmmoniumFerrothiocyanate', pDNA(pgWiz-betaGalactosidase or pgWiz-Luciferase) or CpG ODN was loadcd on the surface ofMPL loaded cationic microparticles at 1.2 w/w % target loading under conditions specifiedby Kasturi et al.' pDNAloadingon the cationic microparticleswas evaluatedby determiningthe unloadedpDNAor Molecular The....4lpy Volume 15.Supplement t• •\ br 2007 Co pyright © "111e American SOI;ic;ty o f Gene "l1u:mpr