- Email: [email protected]
177. Cytokine-Based Log-Scale Expansion of Functional Murine Dendritic Cells
Cancer - Immunotherapy: Cytokine Gene Therapy, Dendritic Cells, and Modified Effector Cells impairment of antitumor activity. These results indicate the essential role of RIG-I-independent signaling on antitumor immunity induced by rSeV-activated DCs, and may provide important insights to DCbased immunotherapy for advanced malignancies.In an interesting thing, the anti-metastatic effect of rSeV/dF-DCs was clearly canceled by anti-asiaro GM1 antibody administration. As for prevention of lung metastasis by rSeV/dF-DCs therapy, the innate NK cell system may play a important role.
175. Adenovirus-Mediated Dendritic Cell Transduction for Melanoma Immunotherapy in a Canine Model
Erin E. Thacker,1 Masaharu Nakayama,1 Theresa V. Strong,2 Bruce F. Smith,3,4 R. Curtis Bird,4 David T. Curiel.1 1 Division of Human Gene Therapy, Departments of Medicine, Obstetrics and Gynecology, Pathology, Surgery and The Gene Therapy Center, University of Alabama at Birmingham, Birmingham, AL; 2Division of Hematology and Oncology, Department of Medicine, University of Alabama at Birmingham, Birmingham, AL; 3Scott-Richey Research Center, College of Veterinary Medicine, Auburn University, Auburn, AL; 4Department of Pathobiology, College of Veterinary Medicine, Auburn University, Auburn, AL. Dendritic cell (DC)-based vaccines are currently being investigated as immunotherapeutic treatments for melanoma, a highly invasive metastatic cancer. DCs play a critical role in activating immune responses through the capture, processing and direct presentation of antigens to T-cells, making them attractive immunotherapeutic targets. Vaccination strategies employing DCs transduced with tumor-associated antigens (TAA) and matured ex vivo have yielded promising results in experimental models, but results from clinical trials suggest these vaccines are not yet optimal. Indeed, recent evidence indicates DCs matured ex vivo may not accurately mimic DCs matured in vivo. Therefore, we hypothesize that a method allowing DC transduction in vivo will stimulate proper DC maturation and allow a more robust systemic immune response to TAA. Adenoviruses (Ad) represent promising gene therapy delivery vehicles that are well-suited to transfer TAA in vivo, yet DCs are normally refractory to Ad transduction due to low CAR expression. To circumvent this, we have developed strategies to target Ad5 vectors to DCs in vivo. To this end, we previously evaluated modification of Ad5 to bind CD40 expressed on the DC cell surface. CD40-targeted Ad5 mediated efficient transduction of otherwise refractory DCs, leading to expression of TAA as well as DC maturation, in vitro. Preliminary in vivo experiments in healthy dogs resulted in a qualitative enhancement in antigen-specific T cell proliferation following vaccination with CD40-targeted Ad5 compared to vaccination with untargeted Ad5. Together, these results highlight that our in vivo DC-targeting strategy can indeed generate a tumor antigen specific systemic immune response. We further hypothesize that targeting Ad5 to proteins with a more DC-restricted expression profile, such as the receptor DC-SIGN, will enhance the TAA-specific immune response. Our lab previously demonstrated that DC-SIGN-targeted Ad5 vectors transduce human DCs in vitro, resulting in transgene expression levels comparable to levels induced by CD40-targeted Ad5. Canine DC-SIGN-targeted Ad5 vectors are currently being developed in order to evaluate and compare the effectiveness of CD40- and DC-SIGN-targeted Ad5 vaccines against canine melanoma, a spontaneous and aggressive tumor which provides a stringent study system for the evaluation of antitumor immunotherapies. As canine patients are immunocompetent and outbred, the canine melanoma model represents a crucial bridge between murine and human immunotherapy studies. This effort will thus allow us to test our in vivo Ad5-targeted DC immunotherapy approach in a melanoma model with a high level of analogy to its human counterpart. S66
176. Enhanced Dendritic Cell-Based Immunotherapy for Cancer
Natalia Lapteva,1 Priya Narayanan,1 Rama D. Gangula,1 Mamatha R. Seethammagari,1 Kevin M. Slawin,2 David M. Spencer.1 1 Immunology, Baylor College of Medicine, Houston, TX; 2 Vanguard Urologic Institute, Houston, TX. Despite the predominant role of dendritic cells (DCs) in priming an immune response to tumor or pathogen-derived antigens, vaccines based on pulsing ex vivo-matured DCs with antigens have been almost universally disappointing, suggesting that scientists must rethink vaccine design. We recently demonstrated that augmenting DC survival or enhancing their activation state could significantly improve in vivo expansion of tumor-specific T cells and anti-tumor responses, likely due to improved DC migration to lymph nodes and prolonged T cell interactions that favor Th1-biased over immunosuppresive responses. Our initial approaches relied on synthetic activation of an inducible allele of the co-stimulatory molecule CD40, iCD40, in antigen-pulsed DCs (HanksB et al (05) Nat Med 11, 130), or overexpression of a highly activated, lipid raft-targeted Akt allele, S*Akt (ParkD et al (06) Nat Biotec 24, 1581). Inducible alleles have the advantage over constitutive or systemic activation of genes by permitting activation in vivo in a more physiologically relevant location and the capacity for rapid reversibility, improving safety. More recently, we observed that for optimum DC activation and maturation, iCD40-expressing DCs needed to be treated ex vivo with adjuvants, such as toll-like receptor 4 (TLR4) ligands, prior to injection; however, TLR4 ligands, such as LPS, would not be ideal for in vivo administration. Therefore, as a targeted, “portable” adjuvant, we have been developing chemically inducible alleles of not only TLR4 but also a panel of “pattern recognition receptors (PRRs)”, such as NOD2 and RIG-I, and are working towards a “unified” adenoviral vector that combines adjuvant properties with costimulation. This vector should permit not only amplified DC activation in vivo, but also temporally extended activation, as well, without triggering systemic inflammation. Moreover, we have also developed powerful, tightly regulated, inducible forms of the antiapoptotic protein Akt, called iS*Akt. Together, these new reagents expand the gene therapist’s armamentarium and permit manipulation of apoptosis residence, costimulation and danger signals in DCs in vivo using synthetic ligands that target only genetically modiffied cells. In addition to describing these novel, and broadly applicable, reagents, our upcoming iCD40-DC-based, phase I/II (3+3), doseescalation study against metastatic androgen-independent prostate cancer will be discussed along with the preclinical pharmacology and toxicology results utilizing the clinical reagents. If successful, these enhanced DC-based approaches should be applicable to both a wide variety of tumors, as well as exogenous pathogens.
177. Cytokine-Based Log-Scale Expansion of Functional Murine Dendritic Cells
Yui Harada,1,2 Yasuji Ueda,1 Takashi Sugimoto,4 Hiroaki Kinoh,1 Atsushi Komaru,1,2 Makoto Inoue,3 Mamoru Hasegawa,3 Naohiko Seki,4 Tomohiko Ichikawa,2 Yoshikazu Yonemitsu.1 1 Department of Gene Therapy, Graduate School of Medicine, Chiba University, Chiba, Japan; 2Department of Urology, Graduate School of Medicine, Chiba University, Chiba, Japan; 3 DNAVEC Corporation, Thukuba, Ibaraki, Japan; 4Department of Functional Genomics, Graduate School of Medicine, Chiba University, Chiba, Japan. PURPOSE Dendritic cells (DCs) play a crucial role in maintaining the immune system. Though DC-based cancer immunotherapy has been suggested to hold potential to treat various kinds of malignancies, clinical efficacies are still insufficient in many human trials. We proved that DCs pulsed with Sendai virus (SeV) showed antitumor effects on established tumors in vivo mouse models (S. Shibata et al., Molecular Therapy Volume 16, Supplement 1, May 2008 Copyright © The American Society of Gene Therapy
Cancer - Immunotherapy: Cytokine Gene Therapy, Dendritic Cells, and Modified Effector Cells J. Immunol, 2006; Y. Yoneyama et al., BBRC, 2007). This antitumor effect depends on the number of DCs, and it is necessary to prepare an enough number of DCs for effective treatments of tumors. In some clinical cases, it is predicted that insufficient number of DC progenitors can be obtained. In this study, therefore, we attempted to expand DC progenitors ex vivo with cytokine cocktail, and to differentiate them into DCs. Materials and Methods DC progenitor cells were obtained from mouse (C3H) bone marrow by a negative selection. These progenitor cells were expanded in the presence of mFlt-3 ligand, mSCF, mIL-3, and mIL-6 (FS36) for several weeks, then differentiated into DCs by following 7 days culture with GMCSF and IL-4. Expanded DC properties such as surface marker’s expression, inflammatory cytokines production, phagocytotic activity, antigen presentating ability in vitro, and anti-metastasis efficacies in vivo were analyzed, and compared with those of conventional DC’s. RESULTS In this study, the total cell number was increased approximately 100.000 fold during expansion culture with FS36 for 3 weeks. After following differentiation culture, about 80% of total cells were CD11b+/CD11c+. Thus, by this system, 1.000 times more DCs in number were obtained than by usual method. As are seen in conventional DCs, expanded DCs showed dendrites after maturation, and endocytotic activities. Expanded DCs also expressed MHC Class II, adhesion molecules, and co-stimulatory molecules and produced inflammatory cytokines with their respective stimulation factors as well as conventional DCs did. Functionally, allo-mixed leukocyte reaction (MLR) assay revealed that both conventional/ expanded immature DCs and DCs activated by LPS could stimulate allogenic T-cell proliferation. Furthermore, expanded DCs showed significant prevention of experimental lung metastasis in vivo, as well as conventional DCs did. CONCLUSIONS] Murine CD11c+ cells could be effectively expanded by culture with cytokine cocktail (FS36). Expanded DC had properties which were required to obtain therapeutic gain. We expect that this technology will be able to contribute largely to both basic and clinical research of human cancer immunotherapy. Enough amount of DC will improve therapeutic gain of cancer and decrease load of apheresis. Key Words : dendritic cell (DC), antitumor immunity, cytokine cocktail.
178. Improved T Cell Receptor (TCR) Assembly and Stability through Genetic Modification for Melanoma Immunotherapy
Richard C. Koya,1 Stephen Mok,1 Antoni Ribas.1,2 Dept. Surgery, UCLA, Los Angeles, CA; 2Dept. Medicine, UCLA, Los Angeles, CA.
1
Metastatic melanoma is a poor prognostic disease refractory to current therapies, warranting intense research for new treatments. Immunotherapy with adoptive transfer of melanoma associated antigen-specific T cells has shown anti-tumor effects. We engineered lymphocytes to express a transgenic TCR specific to a melanoma antigen and a PET reporter gene to be used in an adoptive transfer model to study TCR transgenic cells targeting melanoma tumors in vivo. A tetracistronic lentiviral vector was engineered to express the α and β chains of a hybrid murine/human TCR specific for the endogenous melanoma antigen tyrosinase (Tyr), along with a modified HSV1-tk with higher affinity for nucleotide analogues (sr39tk) used as PET reporter gene, which was fused to the GFP marker. The transgenes were interspaced with picornavirus-derived highly efficient self-cleaving 2A-like sequences to allow balanced protein expression. The hybrid TCR chains contained murine constant regions and human variable regions specific for human HLA-A2.1, allowing the specific recognition of HLA-A2.1 restricted peptides. Transduction of ex vivo activated murine splenocytes with concentrated lentivirus stocks (titer range of 1.3-2.4 x 10e8 infection units/ml) demonstrated high transduction efficiency, with 31-78% of GFP positive cells at M.O.I.=20 by flow-cytometry. However, the surface expression of Molecular Therapy Volume 16, Supplement 1, May 2008 Copyright © The American Society of Gene Therapy
the assembled Tyr-specific TCR was only 2-7%, as assessed by Tyr (368-376) tetramer assays. We hypothesized that the correct pairing of the introduced α and β chains may be hindered by their competition with the corresponding endogenous chains. To address this problem, we introduced changes in the TCR sequence to allow an advantageous and more stable pairing of the chains. First we created different constructs with extended murine constant regions from one with intracellular and transmembrane regions to a full murinized constant region till the extracellular segment and to allow more stable chain pairing, we introduced cysteine in both chains to form an extra interchain disulfide bond. These changes increased the TCR expression 1.5 to 2 times in primary T cells. Furthermore, extension segments to the carboxyl termini of both chains were added to form a stable leucine zipper, enhancing the pairing of the α and β subunits and increasing expression to 22-25% Tyr(368-376) tetramer positive T cells after lentiviral transduction. We have confirmed that transduced murine splenocytes stimulated with EL4 murine lymphoma stably transduced with HLA-A2.1/Kb pulsed with Tyr (368-376) showed higher IFN-γ producing cells in comparison to control cells in an ELISPOT functional assay. In conclusion, improved TCR chain pairing through genetic engineering approaches results in improved surface expression and function. These modifications will be tested in TCRs developed for future clinical translational studies.
179. Generation of FK506 Resistant EBV CTL for Treatment of Post Transplant Lymphoproliferative Disease
Jennifer Brewin,1,2 Anna Donaldson,1 Persis J. Amrolia,1 Martin A. Pule.2 1 Molecular Immunology, Institute of Child Health, University College London, London, United Kingdom; 2Dept of Haematology, University College London, London, United Kingdom.
Post Transplant Lymphoproliferative Disease (PTLD), an EBVdriven lymphoma, is a significant complication of solid organ transplantation. This is consequent to cellular immunosuppression by calcineurin (Cn) inhibitors - cyclosporin(CsA) or tacrolimus (FK506). Clinical studies have shown efficacy of either patient derived or third party HLA matched EBV specific Cytotoxic T Lymphocytes (CTL). However, these adoptively transferred EBV-CTLs rapidly disappear in the face of continued calcineurin inhibitors. Reducing this immunosuppression is difficult as it may endanger the graft. We report the generation of both Calcineurin A (CnA) and Calcineurin B (CnB) mutants which renders expressing cells resistant to either or both Cyclosporin and Tacrolimus. Based on structural data we generated 22 CnA mutants and 32 CnB mutants. Screening with an NFAT-luciferase reporter in ionomycin stimulated 293T cells, we identified 6 of the CnA mutants giving >15% resistance to FK506 (best gave 70% resistance), and 5 CnA mutants giving >15% resistance to CsA (best giving 100% resistance in this assay). With CnB mutants, 4 were identified to give >15% resistance to FK506 (best giving 60% resistance), and 5 giving >15% resistance to CsA (best giving 40% resistance). Four of these CnB mutants overlapped, in that they resulted in resistance to both FK504 and CsA. No CnA mutants resulted in overlapping resistance. Next we tested the effects of expression of these mutants in Jurkat T-cells. Retroviral expression resulted in high, maintained expression in Jurkat T-cells as determined by Western blotting. Resistance to FK506 and CsA was determined by measuring IL-2 output in response to PMA/ionomycin stimulation, in the face of increasing concentrations of these immunosuppressants. Resistance was found to be equivalent to and correlated with that found in 293T cells. Significantly, with most of the mutants, resistance plateaued above 60% despite supra-therapeutic doses of FK506 (>40ng/ml) and CsA (>400ng/ml). Some of the mutants rendered the Jurkat T-cells more responsive to stimulation with increased output of IL-2. EBV-CTLs were generated from healthy donors. S67
175. Adenovirus-Mediated Dendritic Cell Transduction for Melanoma Immunotherapy in a Canine Model
Erin E. Thacker,1 Masaharu Nakayama,1 Theresa V. Strong,2 Bruce F. Smith,3,4 R. Curtis Bird,4 David T. Curiel.1 1 Division of Human Gene Therapy, Departments of Medicine, Obstetrics and Gynecology, Pathology, Surgery and The Gene Therapy Center, University of Alabama at Birmingham, Birmingham, AL; 2Division of Hematology and Oncology, Department of Medicine, University of Alabama at Birmingham, Birmingham, AL; 3Scott-Richey Research Center, College of Veterinary Medicine, Auburn University, Auburn, AL; 4Department of Pathobiology, College of Veterinary Medicine, Auburn University, Auburn, AL. Dendritic cell (DC)-based vaccines are currently being investigated as immunotherapeutic treatments for melanoma, a highly invasive metastatic cancer. DCs play a critical role in activating immune responses through the capture, processing and direct presentation of antigens to T-cells, making them attractive immunotherapeutic targets. Vaccination strategies employing DCs transduced with tumor-associated antigens (TAA) and matured ex vivo have yielded promising results in experimental models, but results from clinical trials suggest these vaccines are not yet optimal. Indeed, recent evidence indicates DCs matured ex vivo may not accurately mimic DCs matured in vivo. Therefore, we hypothesize that a method allowing DC transduction in vivo will stimulate proper DC maturation and allow a more robust systemic immune response to TAA. Adenoviruses (Ad) represent promising gene therapy delivery vehicles that are well-suited to transfer TAA in vivo, yet DCs are normally refractory to Ad transduction due to low CAR expression. To circumvent this, we have developed strategies to target Ad5 vectors to DCs in vivo. To this end, we previously evaluated modification of Ad5 to bind CD40 expressed on the DC cell surface. CD40-targeted Ad5 mediated efficient transduction of otherwise refractory DCs, leading to expression of TAA as well as DC maturation, in vitro. Preliminary in vivo experiments in healthy dogs resulted in a qualitative enhancement in antigen-specific T cell proliferation following vaccination with CD40-targeted Ad5 compared to vaccination with untargeted Ad5. Together, these results highlight that our in vivo DC-targeting strategy can indeed generate a tumor antigen specific systemic immune response. We further hypothesize that targeting Ad5 to proteins with a more DC-restricted expression profile, such as the receptor DC-SIGN, will enhance the TAA-specific immune response. Our lab previously demonstrated that DC-SIGN-targeted Ad5 vectors transduce human DCs in vitro, resulting in transgene expression levels comparable to levels induced by CD40-targeted Ad5. Canine DC-SIGN-targeted Ad5 vectors are currently being developed in order to evaluate and compare the effectiveness of CD40- and DC-SIGN-targeted Ad5 vaccines against canine melanoma, a spontaneous and aggressive tumor which provides a stringent study system for the evaluation of antitumor immunotherapies. As canine patients are immunocompetent and outbred, the canine melanoma model represents a crucial bridge between murine and human immunotherapy studies. This effort will thus allow us to test our in vivo Ad5-targeted DC immunotherapy approach in a melanoma model with a high level of analogy to its human counterpart. S66
176. Enhanced Dendritic Cell-Based Immunotherapy for Cancer
Natalia Lapteva,1 Priya Narayanan,1 Rama D. Gangula,1 Mamatha R. Seethammagari,1 Kevin M. Slawin,2 David M. Spencer.1 1 Immunology, Baylor College of Medicine, Houston, TX; 2 Vanguard Urologic Institute, Houston, TX. Despite the predominant role of dendritic cells (DCs) in priming an immune response to tumor or pathogen-derived antigens, vaccines based on pulsing ex vivo-matured DCs with antigens have been almost universally disappointing, suggesting that scientists must rethink vaccine design. We recently demonstrated that augmenting DC survival or enhancing their activation state could significantly improve in vivo expansion of tumor-specific T cells and anti-tumor responses, likely due to improved DC migration to lymph nodes and prolonged T cell interactions that favor Th1-biased over immunosuppresive responses. Our initial approaches relied on synthetic activation of an inducible allele of the co-stimulatory molecule CD40, iCD40, in antigen-pulsed DCs (HanksB et al (05) Nat Med 11, 130), or overexpression of a highly activated, lipid raft-targeted Akt allele, S*Akt (ParkD et al (06) Nat Biotec 24, 1581). Inducible alleles have the advantage over constitutive or systemic activation of genes by permitting activation in vivo in a more physiologically relevant location and the capacity for rapid reversibility, improving safety. More recently, we observed that for optimum DC activation and maturation, iCD40-expressing DCs needed to be treated ex vivo with adjuvants, such as toll-like receptor 4 (TLR4) ligands, prior to injection; however, TLR4 ligands, such as LPS, would not be ideal for in vivo administration. Therefore, as a targeted, “portable” adjuvant, we have been developing chemically inducible alleles of not only TLR4 but also a panel of “pattern recognition receptors (PRRs)”, such as NOD2 and RIG-I, and are working towards a “unified” adenoviral vector that combines adjuvant properties with costimulation. This vector should permit not only amplified DC activation in vivo, but also temporally extended activation, as well, without triggering systemic inflammation. Moreover, we have also developed powerful, tightly regulated, inducible forms of the antiapoptotic protein Akt, called iS*Akt. Together, these new reagents expand the gene therapist’s armamentarium and permit manipulation of apoptosis residence, costimulation and danger signals in DCs in vivo using synthetic ligands that target only genetically modiffied cells. In addition to describing these novel, and broadly applicable, reagents, our upcoming iCD40-DC-based, phase I/II (3+3), doseescalation study against metastatic androgen-independent prostate cancer will be discussed along with the preclinical pharmacology and toxicology results utilizing the clinical reagents. If successful, these enhanced DC-based approaches should be applicable to both a wide variety of tumors, as well as exogenous pathogens.
177. Cytokine-Based Log-Scale Expansion of Functional Murine Dendritic Cells
Yui Harada,1,2 Yasuji Ueda,1 Takashi Sugimoto,4 Hiroaki Kinoh,1 Atsushi Komaru,1,2 Makoto Inoue,3 Mamoru Hasegawa,3 Naohiko Seki,4 Tomohiko Ichikawa,2 Yoshikazu Yonemitsu.1 1 Department of Gene Therapy, Graduate School of Medicine, Chiba University, Chiba, Japan; 2Department of Urology, Graduate School of Medicine, Chiba University, Chiba, Japan; 3 DNAVEC Corporation, Thukuba, Ibaraki, Japan; 4Department of Functional Genomics, Graduate School of Medicine, Chiba University, Chiba, Japan. PURPOSE Dendritic cells (DCs) play a crucial role in maintaining the immune system. Though DC-based cancer immunotherapy has been suggested to hold potential to treat various kinds of malignancies, clinical efficacies are still insufficient in many human trials. We proved that DCs pulsed with Sendai virus (SeV) showed antitumor effects on established tumors in vivo mouse models (S. Shibata et al., Molecular Therapy Volume 16, Supplement 1, May 2008 Copyright © The American Society of Gene Therapy
Cancer - Immunotherapy: Cytokine Gene Therapy, Dendritic Cells, and Modified Effector Cells J. Immunol, 2006; Y. Yoneyama et al., BBRC, 2007). This antitumor effect depends on the number of DCs, and it is necessary to prepare an enough number of DCs for effective treatments of tumors. In some clinical cases, it is predicted that insufficient number of DC progenitors can be obtained. In this study, therefore, we attempted to expand DC progenitors ex vivo with cytokine cocktail, and to differentiate them into DCs. Materials and Methods DC progenitor cells were obtained from mouse (C3H) bone marrow by a negative selection. These progenitor cells were expanded in the presence of mFlt-3 ligand, mSCF, mIL-3, and mIL-6 (FS36) for several weeks, then differentiated into DCs by following 7 days culture with GMCSF and IL-4. Expanded DC properties such as surface marker’s expression, inflammatory cytokines production, phagocytotic activity, antigen presentating ability in vitro, and anti-metastasis efficacies in vivo were analyzed, and compared with those of conventional DC’s. RESULTS In this study, the total cell number was increased approximately 100.000 fold during expansion culture with FS36 for 3 weeks. After following differentiation culture, about 80% of total cells were CD11b+/CD11c+. Thus, by this system, 1.000 times more DCs in number were obtained than by usual method. As are seen in conventional DCs, expanded DCs showed dendrites after maturation, and endocytotic activities. Expanded DCs also expressed MHC Class II, adhesion molecules, and co-stimulatory molecules and produced inflammatory cytokines with their respective stimulation factors as well as conventional DCs did. Functionally, allo-mixed leukocyte reaction (MLR) assay revealed that both conventional/ expanded immature DCs and DCs activated by LPS could stimulate allogenic T-cell proliferation. Furthermore, expanded DCs showed significant prevention of experimental lung metastasis in vivo, as well as conventional DCs did. CONCLUSIONS] Murine CD11c+ cells could be effectively expanded by culture with cytokine cocktail (FS36). Expanded DC had properties which were required to obtain therapeutic gain. We expect that this technology will be able to contribute largely to both basic and clinical research of human cancer immunotherapy. Enough amount of DC will improve therapeutic gain of cancer and decrease load of apheresis. Key Words : dendritic cell (DC), antitumor immunity, cytokine cocktail.
178. Improved T Cell Receptor (TCR) Assembly and Stability through Genetic Modification for Melanoma Immunotherapy
Richard C. Koya,1 Stephen Mok,1 Antoni Ribas.1,2 Dept. Surgery, UCLA, Los Angeles, CA; 2Dept. Medicine, UCLA, Los Angeles, CA.
1
Metastatic melanoma is a poor prognostic disease refractory to current therapies, warranting intense research for new treatments. Immunotherapy with adoptive transfer of melanoma associated antigen-specific T cells has shown anti-tumor effects. We engineered lymphocytes to express a transgenic TCR specific to a melanoma antigen and a PET reporter gene to be used in an adoptive transfer model to study TCR transgenic cells targeting melanoma tumors in vivo. A tetracistronic lentiviral vector was engineered to express the α and β chains of a hybrid murine/human TCR specific for the endogenous melanoma antigen tyrosinase (Tyr), along with a modified HSV1-tk with higher affinity for nucleotide analogues (sr39tk) used as PET reporter gene, which was fused to the GFP marker. The transgenes were interspaced with picornavirus-derived highly efficient self-cleaving 2A-like sequences to allow balanced protein expression. The hybrid TCR chains contained murine constant regions and human variable regions specific for human HLA-A2.1, allowing the specific recognition of HLA-A2.1 restricted peptides. Transduction of ex vivo activated murine splenocytes with concentrated lentivirus stocks (titer range of 1.3-2.4 x 10e8 infection units/ml) demonstrated high transduction efficiency, with 31-78% of GFP positive cells at M.O.I.=20 by flow-cytometry. However, the surface expression of Molecular Therapy Volume 16, Supplement 1, May 2008 Copyright © The American Society of Gene Therapy
the assembled Tyr-specific TCR was only 2-7%, as assessed by Tyr (368-376) tetramer assays. We hypothesized that the correct pairing of the introduced α and β chains may be hindered by their competition with the corresponding endogenous chains. To address this problem, we introduced changes in the TCR sequence to allow an advantageous and more stable pairing of the chains. First we created different constructs with extended murine constant regions from one with intracellular and transmembrane regions to a full murinized constant region till the extracellular segment and to allow more stable chain pairing, we introduced cysteine in both chains to form an extra interchain disulfide bond. These changes increased the TCR expression 1.5 to 2 times in primary T cells. Furthermore, extension segments to the carboxyl termini of both chains were added to form a stable leucine zipper, enhancing the pairing of the α and β subunits and increasing expression to 22-25% Tyr(368-376) tetramer positive T cells after lentiviral transduction. We have confirmed that transduced murine splenocytes stimulated with EL4 murine lymphoma stably transduced with HLA-A2.1/Kb pulsed with Tyr (368-376) showed higher IFN-γ producing cells in comparison to control cells in an ELISPOT functional assay. In conclusion, improved TCR chain pairing through genetic engineering approaches results in improved surface expression and function. These modifications will be tested in TCRs developed for future clinical translational studies.
179. Generation of FK506 Resistant EBV CTL for Treatment of Post Transplant Lymphoproliferative Disease
Jennifer Brewin,1,2 Anna Donaldson,1 Persis J. Amrolia,1 Martin A. Pule.2 1 Molecular Immunology, Institute of Child Health, University College London, London, United Kingdom; 2Dept of Haematology, University College London, London, United Kingdom.
Post Transplant Lymphoproliferative Disease (PTLD), an EBVdriven lymphoma, is a significant complication of solid organ transplantation. This is consequent to cellular immunosuppression by calcineurin (Cn) inhibitors - cyclosporin(CsA) or tacrolimus (FK506). Clinical studies have shown efficacy of either patient derived or third party HLA matched EBV specific Cytotoxic T Lymphocytes (CTL). However, these adoptively transferred EBV-CTLs rapidly disappear in the face of continued calcineurin inhibitors. Reducing this immunosuppression is difficult as it may endanger the graft. We report the generation of both Calcineurin A (CnA) and Calcineurin B (CnB) mutants which renders expressing cells resistant to either or both Cyclosporin and Tacrolimus. Based on structural data we generated 22 CnA mutants and 32 CnB mutants. Screening with an NFAT-luciferase reporter in ionomycin stimulated 293T cells, we identified 6 of the CnA mutants giving >15% resistance to FK506 (best gave 70% resistance), and 5 CnA mutants giving >15% resistance to CsA (best giving 100% resistance in this assay). With CnB mutants, 4 were identified to give >15% resistance to FK506 (best giving 60% resistance), and 5 giving >15% resistance to CsA (best giving 40% resistance). Four of these CnB mutants overlapped, in that they resulted in resistance to both FK504 and CsA. No CnA mutants resulted in overlapping resistance. Next we tested the effects of expression of these mutants in Jurkat T-cells. Retroviral expression resulted in high, maintained expression in Jurkat T-cells as determined by Western blotting. Resistance to FK506 and CsA was determined by measuring IL-2 output in response to PMA/ionomycin stimulation, in the face of increasing concentrations of these immunosuppressants. Resistance was found to be equivalent to and correlated with that found in 293T cells. Significantly, with most of the mutants, resistance plateaued above 60% despite supra-therapeutic doses of FK506 (>40ng/ml) and CsA (>400ng/ml). Some of the mutants rendered the Jurkat T-cells more responsive to stimulation with increased output of IL-2. EBV-CTLs were generated from healthy donors. S67