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Comparison of Dfferent Suicide Gene Strategies for the Safety Improvement of Genetically Manipulated T Cells.

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Zeitschriftentitel: Blood
Personen und Körperschaften: Marin, Virna, Pizzitola, Irene, Biondi, Andrea, Biagi, Ettore, Pule, Martin
In: Blood, 116, 2010, 21, S. 3753-3753
Format: E-Article
Sprache: Englisch
veröffentlicht:
American Society of Hematology
Schlagwörter:
Cell Biology
Hematology
Immunology
Biochemistry
author_facet Marin, Virna
Pizzitola, Irene
Biondi, Andrea
Biagi, Ettore
Pule, Martin
Marin, Virna
Pizzitola, Irene
Biondi, Andrea
Biagi, Ettore
Pule, Martin
author Marin, Virna
Pizzitola, Irene
Biondi, Andrea
Biagi, Ettore
Pule, Martin
spellingShingle Marin, Virna
Pizzitola, Irene
Biondi, Andrea
Biagi, Ettore
Pule, Martin
Blood
Comparison of Dfferent Suicide Gene Strategies for the Safety Improvement of Genetically Manipulated T Cells.
Cell Biology
Hematology
Immunology
Biochemistry
author_sort marin, virna
spelling Marin, Virna Pizzitola, Irene Biondi, Andrea Biagi, Ettore Pule, Martin 0006-4971 1528-0020 American Society of Hematology Cell Biology Hematology Immunology Biochemistry http://dx.doi.org/10.1182/blood.v116.21.3753.3753 <jats:title>Abstract</jats:title> <jats:p>Abstract 3753</jats:p> <jats:p>Immunotherapy with TCR or chimeric receptor-genetically modified T cells may result in toxicity related to direct target effects, unanticipated off-target effects or lymphoproliferation due to insertional mutagenesis. Therefore it is required to include a suicide gene in the viral vector. We compared different suicide genes in vitro in Epstein Barr Virus-specific cytotoxic T cells (EBV-CTL). Herpes Simplex Virus Thymidine Kinase (HSV-tk), human inducible Caspase 9 (iCasp9), human CD20 and mutant human thymidylate kinase (mTMPK) genes were cloned in frame with truncated CD34 (dCD34, marker gene), separated by the 2A peptide in a SFG-based vector. We previously reported with the iCasp9-coding construct, a lower transduction efficiency and instable expression of the dCD34 marker gene. We therefore codon-optimized the iCasp9 sequence (iCasp9opt) and repeated the cloning in frame with 2A-dCD34 in the SFG vector. EBV-CTLs could be similarly and efficiently transduced with iCasp9opt-2A-dCD34, HSV-TK-2A-dCD34, mTMPK-2A-dCD34 and CD20-2A-dCD34 (mean % CD34+, 80%, n=5), similarly to the control vector containing dCD34 alone. Expression of the marker gene was stable up to 3 weeks. Expression of the suicide genes was not associated with alterations in the expansion rate, immunophenotype and capacity to kill autologous lymphoblastoid cell lines. Transduced and CD34-selected EBV-CTLs have been tested for their sensibility to the corresponding activator in vitro by evaluating residual CD34+ cells. iCasp9opt- transduced cells were rapidly killed with high efficiency by CID, (mean survival, 11% after 24 hours, and 5% after 7 days; n=7). Gancyclovir treated HSV-tk expressing cells showed similar levels of efficacy only after 3 days and CD20 and mTMPK-transduced cells showed only minimal killing at all time points (mean survival after 7 days,84% and 32% respectively).The same results were obtained by analyzing apoptosis induction through Annexin-7AAD staining. In fact, after 24 hours of incubation with CID, nearly 100% iCasp9opt+ cells were apoptotic, whereas a significant lower % of apoptotic cells was observed with the other suicide genes. Altogether our results suggest that the faster activity of iCasp9 might be advantageous in case of occurring severe toxicity, and, together with its lack of immunogenicity and the absence of side-effects of CID, support the clinical applicability of iCap9-based suicide strategy.</jats:p> <jats:sec> <jats:title>Disclosures:</jats:title> <jats:p>No relevant conflicts of interest to declare.</jats:p> </jats:sec> Comparison of Dfferent Suicide Gene Strategies for the Safety Improvement of Genetically Manipulated T Cells. Blood
doi_str_mv 10.1182/blood.v116.21.3753.3753
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title Comparison of Dfferent Suicide Gene Strategies for the Safety Improvement of Genetically Manipulated T Cells.
title_unstemmed Comparison of Dfferent Suicide Gene Strategies for the Safety Improvement of Genetically Manipulated T Cells.
title_full Comparison of Dfferent Suicide Gene Strategies for the Safety Improvement of Genetically Manipulated T Cells.
title_fullStr Comparison of Dfferent Suicide Gene Strategies for the Safety Improvement of Genetically Manipulated T Cells.
title_full_unstemmed Comparison of Dfferent Suicide Gene Strategies for the Safety Improvement of Genetically Manipulated T Cells.
title_short Comparison of Dfferent Suicide Gene Strategies for the Safety Improvement of Genetically Manipulated T Cells.
title_sort comparison of dfferent suicide gene strategies for the safety improvement of genetically manipulated t cells.
topic Cell Biology
Hematology
Immunology
Biochemistry
url http://dx.doi.org/10.1182/blood.v116.21.3753.3753
publishDate 2010
physical 3753-3753
description <jats:title>Abstract</jats:title> <jats:p>Abstract 3753</jats:p> <jats:p>Immunotherapy with TCR or chimeric receptor-genetically modified T cells may result in toxicity related to direct target effects, unanticipated off-target effects or lymphoproliferation due to insertional mutagenesis. Therefore it is required to include a suicide gene in the viral vector. We compared different suicide genes in vitro in Epstein Barr Virus-specific cytotoxic T cells (EBV-CTL). Herpes Simplex Virus Thymidine Kinase (HSV-tk), human inducible Caspase 9 (iCasp9), human CD20 and mutant human thymidylate kinase (mTMPK) genes were cloned in frame with truncated CD34 (dCD34, marker gene), separated by the 2A peptide in a SFG-based vector. We previously reported with the iCasp9-coding construct, a lower transduction efficiency and instable expression of the dCD34 marker gene. We therefore codon-optimized the iCasp9 sequence (iCasp9opt) and repeated the cloning in frame with 2A-dCD34 in the SFG vector. EBV-CTLs could be similarly and efficiently transduced with iCasp9opt-2A-dCD34, HSV-TK-2A-dCD34, mTMPK-2A-dCD34 and CD20-2A-dCD34 (mean % CD34+, 80%, n=5), similarly to the control vector containing dCD34 alone. Expression of the marker gene was stable up to 3 weeks. Expression of the suicide genes was not associated with alterations in the expansion rate, immunophenotype and capacity to kill autologous lymphoblastoid cell lines. Transduced and CD34-selected EBV-CTLs have been tested for their sensibility to the corresponding activator in vitro by evaluating residual CD34+ cells. iCasp9opt- transduced cells were rapidly killed with high efficiency by CID, (mean survival, 11% after 24 hours, and 5% after 7 days; n=7). Gancyclovir treated HSV-tk expressing cells showed similar levels of efficacy only after 3 days and CD20 and mTMPK-transduced cells showed only minimal killing at all time points (mean survival after 7 days,84% and 32% respectively).The same results were obtained by analyzing apoptosis induction through Annexin-7AAD staining. In fact, after 24 hours of incubation with CID, nearly 100% iCasp9opt+ cells were apoptotic, whereas a significant lower % of apoptotic cells was observed with the other suicide genes. Altogether our results suggest that the faster activity of iCasp9 might be advantageous in case of occurring severe toxicity, and, together with its lack of immunogenicity and the absence of side-effects of CID, support the clinical applicability of iCap9-based suicide strategy.</jats:p> <jats:sec> <jats:title>Disclosures:</jats:title> <jats:p>No relevant conflicts of interest to declare.</jats:p> </jats:sec>
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author Marin, Virna, Pizzitola, Irene, Biondi, Andrea, Biagi, Ettore, Pule, Martin
author_facet Marin, Virna, Pizzitola, Irene, Biondi, Andrea, Biagi, Ettore, Pule, Martin, Marin, Virna, Pizzitola, Irene, Biondi, Andrea, Biagi, Ettore, Pule, Martin
author_sort marin, virna
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description <jats:title>Abstract</jats:title> <jats:p>Abstract 3753</jats:p> <jats:p>Immunotherapy with TCR or chimeric receptor-genetically modified T cells may result in toxicity related to direct target effects, unanticipated off-target effects or lymphoproliferation due to insertional mutagenesis. Therefore it is required to include a suicide gene in the viral vector. We compared different suicide genes in vitro in Epstein Barr Virus-specific cytotoxic T cells (EBV-CTL). Herpes Simplex Virus Thymidine Kinase (HSV-tk), human inducible Caspase 9 (iCasp9), human CD20 and mutant human thymidylate kinase (mTMPK) genes were cloned in frame with truncated CD34 (dCD34, marker gene), separated by the 2A peptide in a SFG-based vector. We previously reported with the iCasp9-coding construct, a lower transduction efficiency and instable expression of the dCD34 marker gene. We therefore codon-optimized the iCasp9 sequence (iCasp9opt) and repeated the cloning in frame with 2A-dCD34 in the SFG vector. EBV-CTLs could be similarly and efficiently transduced with iCasp9opt-2A-dCD34, HSV-TK-2A-dCD34, mTMPK-2A-dCD34 and CD20-2A-dCD34 (mean % CD34+, 80%, n=5), similarly to the control vector containing dCD34 alone. Expression of the marker gene was stable up to 3 weeks. Expression of the suicide genes was not associated with alterations in the expansion rate, immunophenotype and capacity to kill autologous lymphoblastoid cell lines. Transduced and CD34-selected EBV-CTLs have been tested for their sensibility to the corresponding activator in vitro by evaluating residual CD34+ cells. iCasp9opt- transduced cells were rapidly killed with high efficiency by CID, (mean survival, 11% after 24 hours, and 5% after 7 days; n=7). Gancyclovir treated HSV-tk expressing cells showed similar levels of efficacy only after 3 days and CD20 and mTMPK-transduced cells showed only minimal killing at all time points (mean survival after 7 days,84% and 32% respectively).The same results were obtained by analyzing apoptosis induction through Annexin-7AAD staining. In fact, after 24 hours of incubation with CID, nearly 100% iCasp9opt+ cells were apoptotic, whereas a significant lower % of apoptotic cells was observed with the other suicide genes. Altogether our results suggest that the faster activity of iCasp9 might be advantageous in case of occurring severe toxicity, and, together with its lack of immunogenicity and the absence of side-effects of CID, support the clinical applicability of iCap9-based suicide strategy.</jats:p> <jats:sec> <jats:title>Disclosures:</jats:title> <jats:p>No relevant conflicts of interest to declare.</jats:p> </jats:sec>
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spelling Marin, Virna Pizzitola, Irene Biondi, Andrea Biagi, Ettore Pule, Martin 0006-4971 1528-0020 American Society of Hematology Cell Biology Hematology Immunology Biochemistry http://dx.doi.org/10.1182/blood.v116.21.3753.3753 <jats:title>Abstract</jats:title> <jats:p>Abstract 3753</jats:p> <jats:p>Immunotherapy with TCR or chimeric receptor-genetically modified T cells may result in toxicity related to direct target effects, unanticipated off-target effects or lymphoproliferation due to insertional mutagenesis. Therefore it is required to include a suicide gene in the viral vector. We compared different suicide genes in vitro in Epstein Barr Virus-specific cytotoxic T cells (EBV-CTL). Herpes Simplex Virus Thymidine Kinase (HSV-tk), human inducible Caspase 9 (iCasp9), human CD20 and mutant human thymidylate kinase (mTMPK) genes were cloned in frame with truncated CD34 (dCD34, marker gene), separated by the 2A peptide in a SFG-based vector. We previously reported with the iCasp9-coding construct, a lower transduction efficiency and instable expression of the dCD34 marker gene. We therefore codon-optimized the iCasp9 sequence (iCasp9opt) and repeated the cloning in frame with 2A-dCD34 in the SFG vector. EBV-CTLs could be similarly and efficiently transduced with iCasp9opt-2A-dCD34, HSV-TK-2A-dCD34, mTMPK-2A-dCD34 and CD20-2A-dCD34 (mean % CD34+, 80%, n=5), similarly to the control vector containing dCD34 alone. Expression of the marker gene was stable up to 3 weeks. Expression of the suicide genes was not associated with alterations in the expansion rate, immunophenotype and capacity to kill autologous lymphoblastoid cell lines. Transduced and CD34-selected EBV-CTLs have been tested for their sensibility to the corresponding activator in vitro by evaluating residual CD34+ cells. iCasp9opt- transduced cells were rapidly killed with high efficiency by CID, (mean survival, 11% after 24 hours, and 5% after 7 days; n=7). Gancyclovir treated HSV-tk expressing cells showed similar levels of efficacy only after 3 days and CD20 and mTMPK-transduced cells showed only minimal killing at all time points (mean survival after 7 days,84% and 32% respectively).The same results were obtained by analyzing apoptosis induction through Annexin-7AAD staining. In fact, after 24 hours of incubation with CID, nearly 100% iCasp9opt+ cells were apoptotic, whereas a significant lower % of apoptotic cells was observed with the other suicide genes. Altogether our results suggest that the faster activity of iCasp9 might be advantageous in case of occurring severe toxicity, and, together with its lack of immunogenicity and the absence of side-effects of CID, support the clinical applicability of iCap9-based suicide strategy.</jats:p> <jats:sec> <jats:title>Disclosures:</jats:title> <jats:p>No relevant conflicts of interest to declare.</jats:p> </jats:sec> Comparison of Dfferent Suicide Gene Strategies for the Safety Improvement of Genetically Manipulated T Cells. Blood
spellingShingle Marin, Virna, Pizzitola, Irene, Biondi, Andrea, Biagi, Ettore, Pule, Martin, Blood, Comparison of Dfferent Suicide Gene Strategies for the Safety Improvement of Genetically Manipulated T Cells., Cell Biology, Hematology, Immunology, Biochemistry
title Comparison of Dfferent Suicide Gene Strategies for the Safety Improvement of Genetically Manipulated T Cells.
title_full Comparison of Dfferent Suicide Gene Strategies for the Safety Improvement of Genetically Manipulated T Cells.
title_fullStr Comparison of Dfferent Suicide Gene Strategies for the Safety Improvement of Genetically Manipulated T Cells.
title_full_unstemmed Comparison of Dfferent Suicide Gene Strategies for the Safety Improvement of Genetically Manipulated T Cells.
title_short Comparison of Dfferent Suicide Gene Strategies for the Safety Improvement of Genetically Manipulated T Cells.
title_sort comparison of dfferent suicide gene strategies for the safety improvement of genetically manipulated t cells.
title_unstemmed Comparison of Dfferent Suicide Gene Strategies for the Safety Improvement of Genetically Manipulated T Cells.
topic Cell Biology, Hematology, Immunology, Biochemistry
url http://dx.doi.org/10.1182/blood.v116.21.3753.3753