The Experts below are selected from a list of 9 Experts worldwide ranked by ideXlab platform
Eliana Ruggiero - One of the best experts on this subject based on the ideXlab platform.
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Generation of lentivirus-induced dendritic cells under GMP-compliant conditions for adaptive immune reconstitution against Cytomegalovirus after stem cell transplantation
Journal of Translational Medicine, 2015Co-Authors: Bala Sai Sundarasetty, Sonja Naundorf, Klaus Kuehlcke, Stephan Kloess, Olaf Oberschmidt, Anusara Daenthanasanmak, Laura Gerasch, Constanca Figueiredo, Rainer Blasczyk, Eliana RuggieroAbstract:Hintergrund Die Reaktivierung latenter Viren wie das humane Cytomegalovirus (HCMV) führt zu einer hohen Morbidität und Mortalität nach allogener Stammzelltransplantation (allo-HSZT). Aufgrund verzögerter T-Zell-Entwicklung nach allo-HSZT ist eine wirksame Immunisierung der Patienten gegen HCMV von großer klinischer Bedeutung. Dabei spielt die Immunrekonstitution Dendritischer Zellen (DCs) eine wichtige Rolle. Frühere Verfahren zur ex vivo Generierung von DCs zur klinischen Anwendung sind komplex und wenig reproduzierbar, insbesondere im Hinblick auf die Vitalität und Potenz der Zellen nach der Kryopreservierung. In früheren Arbeiten konnten wir in humanisierten Stammzelltransplantations-Maus-Modellen eine neue Methode mittels Lentivirus-induzierten DCs (“SmyleDCPp65”) vorstellen, die zu einer beschleunigten Entwicklung Antigen-spezifischer T-Zellen führt. Verfahren In der vorliegenden Arbeit zeigen wir die Möglichkeit, Monozyten mit einem Integrase-defekten lentiviralen Vektor (IDLV) unter guter Herstellungspraxis (GMP) zu transduzieren zur Ko-expression von GM-CSF, IFN-α und Pp65 Zytomegalovirus Antigen. Nach Transduktion wurden die Zellen kryokonserviert. Ergebnisse Die standardisierte Produktion des IDLVs und die Herstellung von SmyleDCPp65 (n=3) unter GMP-konformen Bedingungen konnte demonstriert werden. Analytische Parameter zur Qualitätskontrolle der SmyleDCPp65 Identität nach dem Auftauen und Potenz nach der Kultivierung wurden definiert. Zellgewinnung, Uniformität der Zellen, Effizienz des Gentransfers, Reinheit und Vitalität waren hoch und konsistent. SmyleDCPp65 Zellen zeigten geringe IDLV Integrationen im Genom und ein polyklonales Integrationsmuster ohne Präferenz zu Protoonkogenen. Letztendlich wurde ein Verfahren zur Stimulation autologer T-Zellen durch GMP-SmyleDCPp65 validiert. Fazit Die weitere Entwicklung dieser individuellen Zellvakzine für klinische Studien ist von hoher Relevanz, um die Immunrekonstitution gegen Zytomegalovirus nach allo-HSZT zu beschleunigen. Background Reactivation of latent viruses such as human Cytomegalovirus (HCMV) after allogeneic hematopoietic stem cell transplantation (HSCT) results in high morbidity and mortality. Effective immunization against HCMV shortly after allo-HSCT is an unmet clinical need due to delayed adaptive T cell development. Donor-derived dendritic cells (DCs) have a critical participation in stimulation of naïve T cells and immune reconstitution, and therefore adoptive DC therapy could be used to protect patients after HSCT. However, previous methods for ex vivo generation of adoptive donor-derived DCs were complex and inconsistent, particularly regarding cell viability and potency after thawing. We have previously demonstrated in humanized mouse models of HSCT the proof-of-concept of a novel modality of lentivirus-induced DCs (“SmyleDCPp65”) that accelerated Antigen-specific T cell development. Methods Here we demonstrate the feasibility of good manufacturing practices (GMP) for production of donor-derived DCs consisting of monocytes from peripheral blood transduced with an integrase-defective lentiviral vector (IDLV, co-expressing GM-CSF, IFN-α and the Cytomegalovirus Antigen Pp65) that were cryopreserved and thawed. Results Upscaling and standardized production of one lot of IDLV and three lots of SmyleDCPp65 under GMP-compliant conditions were feasible. Analytical parameters for quality control of SmyleDCPp65 identity after thawing and potency after culture were defined. Cell recovery, uniformity, efficacy of gene transfer, purity and viability were high and consistent. SmyleDCPp65 showed only residual and polyclonal IDLV integration, unbiased to proto-oncogenic hot-spots. Stimulation of autologous T cells by GMP-grade SmyleDCPp65 was validated. Conclusion These results underscore further developments of this individualized donor-derived cell vaccine to accelerate immune reconstitution against HCMV after HSCT in clinical trials.
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Generation of lentivirus-induced dendritic cells under GMP-compliant conditions for adaptive immune reconstitution against Cytomegalovirus after stem cell transplantation
Journal of translational medicine, 2015Co-Authors: Bala Sai Sundarasetty, Sonja Naundorf, Klaus Kuehlcke, Stephan Kloess, Olaf Oberschmidt, Anusara Daenthanasanmak, Laura Gerasch, Constanca Figueiredo, Rainer Blasczyk, Eliana RuggieroAbstract:Reactivation of latent viruses such as human Cytomegalovirus (HCMV) after allogeneic hematopoietic stem cell transplantation (HSCT) results in high morbidity and mortality. Effective immunization against HCMV shortly after allo-HSCT is an unmet clinical need due to delayed adaptive T cell development. Donor-derived dendritic cells (DCs) have a critical participation in stimulation of naive T cells and immune reconstitution, and therefore adoptive DC therapy could be used to protect patients after HSCT. However, previous methods for ex vivo generation of adoptive donor-derived DCs were complex and inconsistent, particularly regarding cell viability and potency after thawing. We have previously demonstrated in humanized mouse models of HSCT the proof-of-concept of a novel modality of lentivirus-induced DCs (“SmyleDCPp65”) that accelerated Antigen-specific T cell development. Here we demonstrate the feasibility of good manufacturing practices (GMP) for production of donor-derived DCs consisting of monocytes from peripheral blood transduced with an integrase-defective lentiviral vector (IDLV, co-expressing GM-CSF, IFN-α and the Cytomegalovirus Antigen Pp65) that were cryopreserved and thawed. Upscaling and standardized production of one lot of IDLV and three lots of SmyleDCPp65 under GMP-compliant conditions were feasible. Analytical parameters for quality control of SmyleDCPp65 identity after thawing and potency after culture were defined. Cell recovery, uniformity, efficacy of gene transfer, purity and viability were high and consistent. SmyleDCPp65 showed only residual and polyclonal IDLV integration, unbiased to proto-oncogenic hot-spots. Stimulation of autologous T cells by GMP-grade SmyleDCPp65 was validated. These results underscore further developments of this individualized donor-derived cell vaccine to accelerate immune reconstitution against HCMV after HSCT in clinical trials.
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456. Pharmacodynamics and GMP Development of a Lentivirus-Induced DC Vaccine for Accelerated Adaptive Immune Reconstitution Against Cytomegalovirus after Stem Cell Transplantation
Molecular Therapy, 2015Co-Authors: Bala Sai Sundarasetty, Sonja Naundorf, Klaus Kuehlcke, Candida Deves Roth, Valery Volk, Eliana Ruggiero, Manfred G. Schmidt, Ulrike Koehl, Arnold Ganser, Renata StripeckeAbstract:Effective immunization of patients shortly after allogeneic hematopoietic stem cell transplantation (allo-HSCT) is an unmet clinical need due to delayed de novo development of T and B cells. We demonstrated a recombinant, donor-derived cell vaccine against Cytomegalovirus that can be easily manufactured, cryopreserved and, upon thaw and administration, turn themselves into highly-viable dendritic cells (DCs). The cell source consisted of monocytes from peripheral blood or cord blood (CB), transduced with an integrase-defective lentiviral vector (IDLV, co-expressing GM-CSF, IFN-α and the Cytomegalovirus Antigen Pp65). The pharmacodynamics of the frozen/thawed vaccine (“SmyleDCPp65”) was evaluated by immunization of NOD.Rag1–/–.IL2rγ–/– mice transplanted with human CD34+ CB stem cells. SmyleDCPp65 stimulated in vivo thymic and extrathymic expansion of human cytotoxic T cells (CTL) reactive against Pp65, and high levels of human immunoglobulins and immune-stimulatory cytokines in plasma. Standardized production of the IDLV and SmyleDCPp65 was established using Good Manufacturing Practices (GMP). Analytical parameters for quality control demonstrated high recovery, uniformity, purity and viability of SmyleDCPp65 after thawing. Stimulation of autologous T cells by GMP-grade SmyleDCPp65 was validated. SmyleDCPp65 showed only residual and polyclonal IDLV integration, unbiased to protooncogenic hot-spots. These results underscore further developments of this individualized cell vaccine to accelerate immune reconstitution against Cytomegalovirus after allo-HSCT.
Bala Sai Sundarasetty - One of the best experts on this subject based on the ideXlab platform.
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Generation of lentivirus-induced dendritic cells under GMP-compliant conditions for adaptive immune reconstitution against Cytomegalovirus after stem cell transplantation
Journal of Translational Medicine, 2015Co-Authors: Bala Sai Sundarasetty, Sonja Naundorf, Klaus Kuehlcke, Stephan Kloess, Olaf Oberschmidt, Anusara Daenthanasanmak, Laura Gerasch, Constanca Figueiredo, Rainer Blasczyk, Eliana RuggieroAbstract:Hintergrund Die Reaktivierung latenter Viren wie das humane Cytomegalovirus (HCMV) führt zu einer hohen Morbidität und Mortalität nach allogener Stammzelltransplantation (allo-HSZT). Aufgrund verzögerter T-Zell-Entwicklung nach allo-HSZT ist eine wirksame Immunisierung der Patienten gegen HCMV von großer klinischer Bedeutung. Dabei spielt die Immunrekonstitution Dendritischer Zellen (DCs) eine wichtige Rolle. Frühere Verfahren zur ex vivo Generierung von DCs zur klinischen Anwendung sind komplex und wenig reproduzierbar, insbesondere im Hinblick auf die Vitalität und Potenz der Zellen nach der Kryopreservierung. In früheren Arbeiten konnten wir in humanisierten Stammzelltransplantations-Maus-Modellen eine neue Methode mittels Lentivirus-induzierten DCs (“SmyleDCPp65”) vorstellen, die zu einer beschleunigten Entwicklung Antigen-spezifischer T-Zellen führt. Verfahren In der vorliegenden Arbeit zeigen wir die Möglichkeit, Monozyten mit einem Integrase-defekten lentiviralen Vektor (IDLV) unter guter Herstellungspraxis (GMP) zu transduzieren zur Ko-expression von GM-CSF, IFN-α und Pp65 Zytomegalovirus Antigen. Nach Transduktion wurden die Zellen kryokonserviert. Ergebnisse Die standardisierte Produktion des IDLVs und die Herstellung von SmyleDCPp65 (n=3) unter GMP-konformen Bedingungen konnte demonstriert werden. Analytische Parameter zur Qualitätskontrolle der SmyleDCPp65 Identität nach dem Auftauen und Potenz nach der Kultivierung wurden definiert. Zellgewinnung, Uniformität der Zellen, Effizienz des Gentransfers, Reinheit und Vitalität waren hoch und konsistent. SmyleDCPp65 Zellen zeigten geringe IDLV Integrationen im Genom und ein polyklonales Integrationsmuster ohne Präferenz zu Protoonkogenen. Letztendlich wurde ein Verfahren zur Stimulation autologer T-Zellen durch GMP-SmyleDCPp65 validiert. Fazit Die weitere Entwicklung dieser individuellen Zellvakzine für klinische Studien ist von hoher Relevanz, um die Immunrekonstitution gegen Zytomegalovirus nach allo-HSZT zu beschleunigen. Background Reactivation of latent viruses such as human Cytomegalovirus (HCMV) after allogeneic hematopoietic stem cell transplantation (HSCT) results in high morbidity and mortality. Effective immunization against HCMV shortly after allo-HSCT is an unmet clinical need due to delayed adaptive T cell development. Donor-derived dendritic cells (DCs) have a critical participation in stimulation of naïve T cells and immune reconstitution, and therefore adoptive DC therapy could be used to protect patients after HSCT. However, previous methods for ex vivo generation of adoptive donor-derived DCs were complex and inconsistent, particularly regarding cell viability and potency after thawing. We have previously demonstrated in humanized mouse models of HSCT the proof-of-concept of a novel modality of lentivirus-induced DCs (“SmyleDCPp65”) that accelerated Antigen-specific T cell development. Methods Here we demonstrate the feasibility of good manufacturing practices (GMP) for production of donor-derived DCs consisting of monocytes from peripheral blood transduced with an integrase-defective lentiviral vector (IDLV, co-expressing GM-CSF, IFN-α and the Cytomegalovirus Antigen Pp65) that were cryopreserved and thawed. Results Upscaling and standardized production of one lot of IDLV and three lots of SmyleDCPp65 under GMP-compliant conditions were feasible. Analytical parameters for quality control of SmyleDCPp65 identity after thawing and potency after culture were defined. Cell recovery, uniformity, efficacy of gene transfer, purity and viability were high and consistent. SmyleDCPp65 showed only residual and polyclonal IDLV integration, unbiased to proto-oncogenic hot-spots. Stimulation of autologous T cells by GMP-grade SmyleDCPp65 was validated. Conclusion These results underscore further developments of this individualized donor-derived cell vaccine to accelerate immune reconstitution against HCMV after HSCT in clinical trials.
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Generation of lentivirus-induced dendritic cells under GMP-compliant conditions for adaptive immune reconstitution against Cytomegalovirus after stem cell transplantation
Journal of translational medicine, 2015Co-Authors: Bala Sai Sundarasetty, Sonja Naundorf, Klaus Kuehlcke, Stephan Kloess, Olaf Oberschmidt, Anusara Daenthanasanmak, Laura Gerasch, Constanca Figueiredo, Rainer Blasczyk, Eliana RuggieroAbstract:Reactivation of latent viruses such as human Cytomegalovirus (HCMV) after allogeneic hematopoietic stem cell transplantation (HSCT) results in high morbidity and mortality. Effective immunization against HCMV shortly after allo-HSCT is an unmet clinical need due to delayed adaptive T cell development. Donor-derived dendritic cells (DCs) have a critical participation in stimulation of naive T cells and immune reconstitution, and therefore adoptive DC therapy could be used to protect patients after HSCT. However, previous methods for ex vivo generation of adoptive donor-derived DCs were complex and inconsistent, particularly regarding cell viability and potency after thawing. We have previously demonstrated in humanized mouse models of HSCT the proof-of-concept of a novel modality of lentivirus-induced DCs (“SmyleDCPp65”) that accelerated Antigen-specific T cell development. Here we demonstrate the feasibility of good manufacturing practices (GMP) for production of donor-derived DCs consisting of monocytes from peripheral blood transduced with an integrase-defective lentiviral vector (IDLV, co-expressing GM-CSF, IFN-α and the Cytomegalovirus Antigen Pp65) that were cryopreserved and thawed. Upscaling and standardized production of one lot of IDLV and three lots of SmyleDCPp65 under GMP-compliant conditions were feasible. Analytical parameters for quality control of SmyleDCPp65 identity after thawing and potency after culture were defined. Cell recovery, uniformity, efficacy of gene transfer, purity and viability were high and consistent. SmyleDCPp65 showed only residual and polyclonal IDLV integration, unbiased to proto-oncogenic hot-spots. Stimulation of autologous T cells by GMP-grade SmyleDCPp65 was validated. These results underscore further developments of this individualized donor-derived cell vaccine to accelerate immune reconstitution against HCMV after HSCT in clinical trials.
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456. Pharmacodynamics and GMP Development of a Lentivirus-Induced DC Vaccine for Accelerated Adaptive Immune Reconstitution Against Cytomegalovirus after Stem Cell Transplantation
Molecular Therapy, 2015Co-Authors: Bala Sai Sundarasetty, Sonja Naundorf, Klaus Kuehlcke, Candida Deves Roth, Valery Volk, Eliana Ruggiero, Manfred G. Schmidt, Ulrike Koehl, Arnold Ganser, Renata StripeckeAbstract:Effective immunization of patients shortly after allogeneic hematopoietic stem cell transplantation (allo-HSCT) is an unmet clinical need due to delayed de novo development of T and B cells. We demonstrated a recombinant, donor-derived cell vaccine against Cytomegalovirus that can be easily manufactured, cryopreserved and, upon thaw and administration, turn themselves into highly-viable dendritic cells (DCs). The cell source consisted of monocytes from peripheral blood or cord blood (CB), transduced with an integrase-defective lentiviral vector (IDLV, co-expressing GM-CSF, IFN-α and the Cytomegalovirus Antigen Pp65). The pharmacodynamics of the frozen/thawed vaccine (“SmyleDCPp65”) was evaluated by immunization of NOD.Rag1–/–.IL2rγ–/– mice transplanted with human CD34+ CB stem cells. SmyleDCPp65 stimulated in vivo thymic and extrathymic expansion of human cytotoxic T cells (CTL) reactive against Pp65, and high levels of human immunoglobulins and immune-stimulatory cytokines in plasma. Standardized production of the IDLV and SmyleDCPp65 was established using Good Manufacturing Practices (GMP). Analytical parameters for quality control demonstrated high recovery, uniformity, purity and viability of SmyleDCPp65 after thawing. Stimulation of autologous T cells by GMP-grade SmyleDCPp65 was validated. SmyleDCPp65 showed only residual and polyclonal IDLV integration, unbiased to protooncogenic hot-spots. These results underscore further developments of this individualized cell vaccine to accelerate immune reconstitution against Cytomegalovirus after allo-HSCT.
Sonja Naundorf - One of the best experts on this subject based on the ideXlab platform.
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Generation of lentivirus-induced dendritic cells under GMP-compliant conditions for adaptive immune reconstitution against Cytomegalovirus after stem cell transplantation
Journal of Translational Medicine, 2015Co-Authors: Bala Sai Sundarasetty, Sonja Naundorf, Klaus Kuehlcke, Stephan Kloess, Olaf Oberschmidt, Anusara Daenthanasanmak, Laura Gerasch, Constanca Figueiredo, Rainer Blasczyk, Eliana RuggieroAbstract:Hintergrund Die Reaktivierung latenter Viren wie das humane Cytomegalovirus (HCMV) führt zu einer hohen Morbidität und Mortalität nach allogener Stammzelltransplantation (allo-HSZT). Aufgrund verzögerter T-Zell-Entwicklung nach allo-HSZT ist eine wirksame Immunisierung der Patienten gegen HCMV von großer klinischer Bedeutung. Dabei spielt die Immunrekonstitution Dendritischer Zellen (DCs) eine wichtige Rolle. Frühere Verfahren zur ex vivo Generierung von DCs zur klinischen Anwendung sind komplex und wenig reproduzierbar, insbesondere im Hinblick auf die Vitalität und Potenz der Zellen nach der Kryopreservierung. In früheren Arbeiten konnten wir in humanisierten Stammzelltransplantations-Maus-Modellen eine neue Methode mittels Lentivirus-induzierten DCs (“SmyleDCPp65”) vorstellen, die zu einer beschleunigten Entwicklung Antigen-spezifischer T-Zellen führt. Verfahren In der vorliegenden Arbeit zeigen wir die Möglichkeit, Monozyten mit einem Integrase-defekten lentiviralen Vektor (IDLV) unter guter Herstellungspraxis (GMP) zu transduzieren zur Ko-expression von GM-CSF, IFN-α und Pp65 Zytomegalovirus Antigen. Nach Transduktion wurden die Zellen kryokonserviert. Ergebnisse Die standardisierte Produktion des IDLVs und die Herstellung von SmyleDCPp65 (n=3) unter GMP-konformen Bedingungen konnte demonstriert werden. Analytische Parameter zur Qualitätskontrolle der SmyleDCPp65 Identität nach dem Auftauen und Potenz nach der Kultivierung wurden definiert. Zellgewinnung, Uniformität der Zellen, Effizienz des Gentransfers, Reinheit und Vitalität waren hoch und konsistent. SmyleDCPp65 Zellen zeigten geringe IDLV Integrationen im Genom und ein polyklonales Integrationsmuster ohne Präferenz zu Protoonkogenen. Letztendlich wurde ein Verfahren zur Stimulation autologer T-Zellen durch GMP-SmyleDCPp65 validiert. Fazit Die weitere Entwicklung dieser individuellen Zellvakzine für klinische Studien ist von hoher Relevanz, um die Immunrekonstitution gegen Zytomegalovirus nach allo-HSZT zu beschleunigen. Background Reactivation of latent viruses such as human Cytomegalovirus (HCMV) after allogeneic hematopoietic stem cell transplantation (HSCT) results in high morbidity and mortality. Effective immunization against HCMV shortly after allo-HSCT is an unmet clinical need due to delayed adaptive T cell development. Donor-derived dendritic cells (DCs) have a critical participation in stimulation of naïve T cells and immune reconstitution, and therefore adoptive DC therapy could be used to protect patients after HSCT. However, previous methods for ex vivo generation of adoptive donor-derived DCs were complex and inconsistent, particularly regarding cell viability and potency after thawing. We have previously demonstrated in humanized mouse models of HSCT the proof-of-concept of a novel modality of lentivirus-induced DCs (“SmyleDCPp65”) that accelerated Antigen-specific T cell development. Methods Here we demonstrate the feasibility of good manufacturing practices (GMP) for production of donor-derived DCs consisting of monocytes from peripheral blood transduced with an integrase-defective lentiviral vector (IDLV, co-expressing GM-CSF, IFN-α and the Cytomegalovirus Antigen Pp65) that were cryopreserved and thawed. Results Upscaling and standardized production of one lot of IDLV and three lots of SmyleDCPp65 under GMP-compliant conditions were feasible. Analytical parameters for quality control of SmyleDCPp65 identity after thawing and potency after culture were defined. Cell recovery, uniformity, efficacy of gene transfer, purity and viability were high and consistent. SmyleDCPp65 showed only residual and polyclonal IDLV integration, unbiased to proto-oncogenic hot-spots. Stimulation of autologous T cells by GMP-grade SmyleDCPp65 was validated. Conclusion These results underscore further developments of this individualized donor-derived cell vaccine to accelerate immune reconstitution against HCMV after HSCT in clinical trials.
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Generation of lentivirus-induced dendritic cells under GMP-compliant conditions for adaptive immune reconstitution against Cytomegalovirus after stem cell transplantation
Journal of translational medicine, 2015Co-Authors: Bala Sai Sundarasetty, Sonja Naundorf, Klaus Kuehlcke, Stephan Kloess, Olaf Oberschmidt, Anusara Daenthanasanmak, Laura Gerasch, Constanca Figueiredo, Rainer Blasczyk, Eliana RuggieroAbstract:Reactivation of latent viruses such as human Cytomegalovirus (HCMV) after allogeneic hematopoietic stem cell transplantation (HSCT) results in high morbidity and mortality. Effective immunization against HCMV shortly after allo-HSCT is an unmet clinical need due to delayed adaptive T cell development. Donor-derived dendritic cells (DCs) have a critical participation in stimulation of naive T cells and immune reconstitution, and therefore adoptive DC therapy could be used to protect patients after HSCT. However, previous methods for ex vivo generation of adoptive donor-derived DCs were complex and inconsistent, particularly regarding cell viability and potency after thawing. We have previously demonstrated in humanized mouse models of HSCT the proof-of-concept of a novel modality of lentivirus-induced DCs (“SmyleDCPp65”) that accelerated Antigen-specific T cell development. Here we demonstrate the feasibility of good manufacturing practices (GMP) for production of donor-derived DCs consisting of monocytes from peripheral blood transduced with an integrase-defective lentiviral vector (IDLV, co-expressing GM-CSF, IFN-α and the Cytomegalovirus Antigen Pp65) that were cryopreserved and thawed. Upscaling and standardized production of one lot of IDLV and three lots of SmyleDCPp65 under GMP-compliant conditions were feasible. Analytical parameters for quality control of SmyleDCPp65 identity after thawing and potency after culture were defined. Cell recovery, uniformity, efficacy of gene transfer, purity and viability were high and consistent. SmyleDCPp65 showed only residual and polyclonal IDLV integration, unbiased to proto-oncogenic hot-spots. Stimulation of autologous T cells by GMP-grade SmyleDCPp65 was validated. These results underscore further developments of this individualized donor-derived cell vaccine to accelerate immune reconstitution against HCMV after HSCT in clinical trials.
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456. Pharmacodynamics and GMP Development of a Lentivirus-Induced DC Vaccine for Accelerated Adaptive Immune Reconstitution Against Cytomegalovirus after Stem Cell Transplantation
Molecular Therapy, 2015Co-Authors: Bala Sai Sundarasetty, Sonja Naundorf, Klaus Kuehlcke, Candida Deves Roth, Valery Volk, Eliana Ruggiero, Manfred G. Schmidt, Ulrike Koehl, Arnold Ganser, Renata StripeckeAbstract:Effective immunization of patients shortly after allogeneic hematopoietic stem cell transplantation (allo-HSCT) is an unmet clinical need due to delayed de novo development of T and B cells. We demonstrated a recombinant, donor-derived cell vaccine against Cytomegalovirus that can be easily manufactured, cryopreserved and, upon thaw and administration, turn themselves into highly-viable dendritic cells (DCs). The cell source consisted of monocytes from peripheral blood or cord blood (CB), transduced with an integrase-defective lentiviral vector (IDLV, co-expressing GM-CSF, IFN-α and the Cytomegalovirus Antigen Pp65). The pharmacodynamics of the frozen/thawed vaccine (“SmyleDCPp65”) was evaluated by immunization of NOD.Rag1–/–.IL2rγ–/– mice transplanted with human CD34+ CB stem cells. SmyleDCPp65 stimulated in vivo thymic and extrathymic expansion of human cytotoxic T cells (CTL) reactive against Pp65, and high levels of human immunoglobulins and immune-stimulatory cytokines in plasma. Standardized production of the IDLV and SmyleDCPp65 was established using Good Manufacturing Practices (GMP). Analytical parameters for quality control demonstrated high recovery, uniformity, purity and viability of SmyleDCPp65 after thawing. Stimulation of autologous T cells by GMP-grade SmyleDCPp65 was validated. SmyleDCPp65 showed only residual and polyclonal IDLV integration, unbiased to protooncogenic hot-spots. These results underscore further developments of this individualized cell vaccine to accelerate immune reconstitution against Cytomegalovirus after allo-HSCT.
Klaus Kuehlcke - One of the best experts on this subject based on the ideXlab platform.
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Generation of lentivirus-induced dendritic cells under GMP-compliant conditions for adaptive immune reconstitution against Cytomegalovirus after stem cell transplantation
Journal of Translational Medicine, 2015Co-Authors: Bala Sai Sundarasetty, Sonja Naundorf, Klaus Kuehlcke, Stephan Kloess, Olaf Oberschmidt, Anusara Daenthanasanmak, Laura Gerasch, Constanca Figueiredo, Rainer Blasczyk, Eliana RuggieroAbstract:Hintergrund Die Reaktivierung latenter Viren wie das humane Cytomegalovirus (HCMV) führt zu einer hohen Morbidität und Mortalität nach allogener Stammzelltransplantation (allo-HSZT). Aufgrund verzögerter T-Zell-Entwicklung nach allo-HSZT ist eine wirksame Immunisierung der Patienten gegen HCMV von großer klinischer Bedeutung. Dabei spielt die Immunrekonstitution Dendritischer Zellen (DCs) eine wichtige Rolle. Frühere Verfahren zur ex vivo Generierung von DCs zur klinischen Anwendung sind komplex und wenig reproduzierbar, insbesondere im Hinblick auf die Vitalität und Potenz der Zellen nach der Kryopreservierung. In früheren Arbeiten konnten wir in humanisierten Stammzelltransplantations-Maus-Modellen eine neue Methode mittels Lentivirus-induzierten DCs (“SmyleDCPp65”) vorstellen, die zu einer beschleunigten Entwicklung Antigen-spezifischer T-Zellen führt. Verfahren In der vorliegenden Arbeit zeigen wir die Möglichkeit, Monozyten mit einem Integrase-defekten lentiviralen Vektor (IDLV) unter guter Herstellungspraxis (GMP) zu transduzieren zur Ko-expression von GM-CSF, IFN-α und Pp65 Zytomegalovirus Antigen. Nach Transduktion wurden die Zellen kryokonserviert. Ergebnisse Die standardisierte Produktion des IDLVs und die Herstellung von SmyleDCPp65 (n=3) unter GMP-konformen Bedingungen konnte demonstriert werden. Analytische Parameter zur Qualitätskontrolle der SmyleDCPp65 Identität nach dem Auftauen und Potenz nach der Kultivierung wurden definiert. Zellgewinnung, Uniformität der Zellen, Effizienz des Gentransfers, Reinheit und Vitalität waren hoch und konsistent. SmyleDCPp65 Zellen zeigten geringe IDLV Integrationen im Genom und ein polyklonales Integrationsmuster ohne Präferenz zu Protoonkogenen. Letztendlich wurde ein Verfahren zur Stimulation autologer T-Zellen durch GMP-SmyleDCPp65 validiert. Fazit Die weitere Entwicklung dieser individuellen Zellvakzine für klinische Studien ist von hoher Relevanz, um die Immunrekonstitution gegen Zytomegalovirus nach allo-HSZT zu beschleunigen. Background Reactivation of latent viruses such as human Cytomegalovirus (HCMV) after allogeneic hematopoietic stem cell transplantation (HSCT) results in high morbidity and mortality. Effective immunization against HCMV shortly after allo-HSCT is an unmet clinical need due to delayed adaptive T cell development. Donor-derived dendritic cells (DCs) have a critical participation in stimulation of naïve T cells and immune reconstitution, and therefore adoptive DC therapy could be used to protect patients after HSCT. However, previous methods for ex vivo generation of adoptive donor-derived DCs were complex and inconsistent, particularly regarding cell viability and potency after thawing. We have previously demonstrated in humanized mouse models of HSCT the proof-of-concept of a novel modality of lentivirus-induced DCs (“SmyleDCPp65”) that accelerated Antigen-specific T cell development. Methods Here we demonstrate the feasibility of good manufacturing practices (GMP) for production of donor-derived DCs consisting of monocytes from peripheral blood transduced with an integrase-defective lentiviral vector (IDLV, co-expressing GM-CSF, IFN-α and the Cytomegalovirus Antigen Pp65) that were cryopreserved and thawed. Results Upscaling and standardized production of one lot of IDLV and three lots of SmyleDCPp65 under GMP-compliant conditions were feasible. Analytical parameters for quality control of SmyleDCPp65 identity after thawing and potency after culture were defined. Cell recovery, uniformity, efficacy of gene transfer, purity and viability were high and consistent. SmyleDCPp65 showed only residual and polyclonal IDLV integration, unbiased to proto-oncogenic hot-spots. Stimulation of autologous T cells by GMP-grade SmyleDCPp65 was validated. Conclusion These results underscore further developments of this individualized donor-derived cell vaccine to accelerate immune reconstitution against HCMV after HSCT in clinical trials.
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Generation of lentivirus-induced dendritic cells under GMP-compliant conditions for adaptive immune reconstitution against Cytomegalovirus after stem cell transplantation
Journal of translational medicine, 2015Co-Authors: Bala Sai Sundarasetty, Sonja Naundorf, Klaus Kuehlcke, Stephan Kloess, Olaf Oberschmidt, Anusara Daenthanasanmak, Laura Gerasch, Constanca Figueiredo, Rainer Blasczyk, Eliana RuggieroAbstract:Reactivation of latent viruses such as human Cytomegalovirus (HCMV) after allogeneic hematopoietic stem cell transplantation (HSCT) results in high morbidity and mortality. Effective immunization against HCMV shortly after allo-HSCT is an unmet clinical need due to delayed adaptive T cell development. Donor-derived dendritic cells (DCs) have a critical participation in stimulation of naive T cells and immune reconstitution, and therefore adoptive DC therapy could be used to protect patients after HSCT. However, previous methods for ex vivo generation of adoptive donor-derived DCs were complex and inconsistent, particularly regarding cell viability and potency after thawing. We have previously demonstrated in humanized mouse models of HSCT the proof-of-concept of a novel modality of lentivirus-induced DCs (“SmyleDCPp65”) that accelerated Antigen-specific T cell development. Here we demonstrate the feasibility of good manufacturing practices (GMP) for production of donor-derived DCs consisting of monocytes from peripheral blood transduced with an integrase-defective lentiviral vector (IDLV, co-expressing GM-CSF, IFN-α and the Cytomegalovirus Antigen Pp65) that were cryopreserved and thawed. Upscaling and standardized production of one lot of IDLV and three lots of SmyleDCPp65 under GMP-compliant conditions were feasible. Analytical parameters for quality control of SmyleDCPp65 identity after thawing and potency after culture were defined. Cell recovery, uniformity, efficacy of gene transfer, purity and viability were high and consistent. SmyleDCPp65 showed only residual and polyclonal IDLV integration, unbiased to proto-oncogenic hot-spots. Stimulation of autologous T cells by GMP-grade SmyleDCPp65 was validated. These results underscore further developments of this individualized donor-derived cell vaccine to accelerate immune reconstitution against HCMV after HSCT in clinical trials.
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456. Pharmacodynamics and GMP Development of a Lentivirus-Induced DC Vaccine for Accelerated Adaptive Immune Reconstitution Against Cytomegalovirus after Stem Cell Transplantation
Molecular Therapy, 2015Co-Authors: Bala Sai Sundarasetty, Sonja Naundorf, Klaus Kuehlcke, Candida Deves Roth, Valery Volk, Eliana Ruggiero, Manfred G. Schmidt, Ulrike Koehl, Arnold Ganser, Renata StripeckeAbstract:Effective immunization of patients shortly after allogeneic hematopoietic stem cell transplantation (allo-HSCT) is an unmet clinical need due to delayed de novo development of T and B cells. We demonstrated a recombinant, donor-derived cell vaccine against Cytomegalovirus that can be easily manufactured, cryopreserved and, upon thaw and administration, turn themselves into highly-viable dendritic cells (DCs). The cell source consisted of monocytes from peripheral blood or cord blood (CB), transduced with an integrase-defective lentiviral vector (IDLV, co-expressing GM-CSF, IFN-α and the Cytomegalovirus Antigen Pp65). The pharmacodynamics of the frozen/thawed vaccine (“SmyleDCPp65”) was evaluated by immunization of NOD.Rag1–/–.IL2rγ–/– mice transplanted with human CD34+ CB stem cells. SmyleDCPp65 stimulated in vivo thymic and extrathymic expansion of human cytotoxic T cells (CTL) reactive against Pp65, and high levels of human immunoglobulins and immune-stimulatory cytokines in plasma. Standardized production of the IDLV and SmyleDCPp65 was established using Good Manufacturing Practices (GMP). Analytical parameters for quality control demonstrated high recovery, uniformity, purity and viability of SmyleDCPp65 after thawing. Stimulation of autologous T cells by GMP-grade SmyleDCPp65 was validated. SmyleDCPp65 showed only residual and polyclonal IDLV integration, unbiased to protooncogenic hot-spots. These results underscore further developments of this individualized cell vaccine to accelerate immune reconstitution against Cytomegalovirus after allo-HSCT.
Stephan Kloess - One of the best experts on this subject based on the ideXlab platform.
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Generation of lentivirus-induced dendritic cells under GMP-compliant conditions for adaptive immune reconstitution against Cytomegalovirus after stem cell transplantation
Journal of translational medicine, 2015Co-Authors: Bala Sai Sundarasetty, Sonja Naundorf, Klaus Kuehlcke, Stephan Kloess, Olaf Oberschmidt, Anusara Daenthanasanmak, Laura Gerasch, Constanca Figueiredo, Rainer Blasczyk, Eliana RuggieroAbstract:Reactivation of latent viruses such as human Cytomegalovirus (HCMV) after allogeneic hematopoietic stem cell transplantation (HSCT) results in high morbidity and mortality. Effective immunization against HCMV shortly after allo-HSCT is an unmet clinical need due to delayed adaptive T cell development. Donor-derived dendritic cells (DCs) have a critical participation in stimulation of naive T cells and immune reconstitution, and therefore adoptive DC therapy could be used to protect patients after HSCT. However, previous methods for ex vivo generation of adoptive donor-derived DCs were complex and inconsistent, particularly regarding cell viability and potency after thawing. We have previously demonstrated in humanized mouse models of HSCT the proof-of-concept of a novel modality of lentivirus-induced DCs (“SmyleDCPp65”) that accelerated Antigen-specific T cell development. Here we demonstrate the feasibility of good manufacturing practices (GMP) for production of donor-derived DCs consisting of monocytes from peripheral blood transduced with an integrase-defective lentiviral vector (IDLV, co-expressing GM-CSF, IFN-α and the Cytomegalovirus Antigen Pp65) that were cryopreserved and thawed. Upscaling and standardized production of one lot of IDLV and three lots of SmyleDCPp65 under GMP-compliant conditions were feasible. Analytical parameters for quality control of SmyleDCPp65 identity after thawing and potency after culture were defined. Cell recovery, uniformity, efficacy of gene transfer, purity and viability were high and consistent. SmyleDCPp65 showed only residual and polyclonal IDLV integration, unbiased to proto-oncogenic hot-spots. Stimulation of autologous T cells by GMP-grade SmyleDCPp65 was validated. These results underscore further developments of this individualized donor-derived cell vaccine to accelerate immune reconstitution against HCMV after HSCT in clinical trials.
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Generation of lentivirus-induced dendritic cells under GMP-compliant conditions for adaptive immune reconstitution against Cytomegalovirus after stem cell transplantation
Journal of Translational Medicine, 2015Co-Authors: Bala Sai Sundarasetty, Sonja Naundorf, Klaus Kuehlcke, Stephan Kloess, Olaf Oberschmidt, Anusara Daenthanasanmak, Laura Gerasch, Constanca Figueiredo, Rainer Blasczyk, Eliana RuggieroAbstract:Hintergrund Die Reaktivierung latenter Viren wie das humane Cytomegalovirus (HCMV) führt zu einer hohen Morbidität und Mortalität nach allogener Stammzelltransplantation (allo-HSZT). Aufgrund verzögerter T-Zell-Entwicklung nach allo-HSZT ist eine wirksame Immunisierung der Patienten gegen HCMV von großer klinischer Bedeutung. Dabei spielt die Immunrekonstitution Dendritischer Zellen (DCs) eine wichtige Rolle. Frühere Verfahren zur ex vivo Generierung von DCs zur klinischen Anwendung sind komplex und wenig reproduzierbar, insbesondere im Hinblick auf die Vitalität und Potenz der Zellen nach der Kryopreservierung. In früheren Arbeiten konnten wir in humanisierten Stammzelltransplantations-Maus-Modellen eine neue Methode mittels Lentivirus-induzierten DCs (“SmyleDCPp65”) vorstellen, die zu einer beschleunigten Entwicklung Antigen-spezifischer T-Zellen führt. Verfahren In der vorliegenden Arbeit zeigen wir die Möglichkeit, Monozyten mit einem Integrase-defekten lentiviralen Vektor (IDLV) unter guter Herstellungspraxis (GMP) zu transduzieren zur Ko-expression von GM-CSF, IFN-α und Pp65 Zytomegalovirus Antigen. Nach Transduktion wurden die Zellen kryokonserviert. Ergebnisse Die standardisierte Produktion des IDLVs und die Herstellung von SmyleDCPp65 (n=3) unter GMP-konformen Bedingungen konnte demonstriert werden. Analytische Parameter zur Qualitätskontrolle der SmyleDCPp65 Identität nach dem Auftauen und Potenz nach der Kultivierung wurden definiert. Zellgewinnung, Uniformität der Zellen, Effizienz des Gentransfers, Reinheit und Vitalität waren hoch und konsistent. SmyleDCPp65 Zellen zeigten geringe IDLV Integrationen im Genom und ein polyklonales Integrationsmuster ohne Präferenz zu Protoonkogenen. Letztendlich wurde ein Verfahren zur Stimulation autologer T-Zellen durch GMP-SmyleDCPp65 validiert. Fazit Die weitere Entwicklung dieser individuellen Zellvakzine für klinische Studien ist von hoher Relevanz, um die Immunrekonstitution gegen Zytomegalovirus nach allo-HSZT zu beschleunigen. Background Reactivation of latent viruses such as human Cytomegalovirus (HCMV) after allogeneic hematopoietic stem cell transplantation (HSCT) results in high morbidity and mortality. Effective immunization against HCMV shortly after allo-HSCT is an unmet clinical need due to delayed adaptive T cell development. Donor-derived dendritic cells (DCs) have a critical participation in stimulation of naïve T cells and immune reconstitution, and therefore adoptive DC therapy could be used to protect patients after HSCT. However, previous methods for ex vivo generation of adoptive donor-derived DCs were complex and inconsistent, particularly regarding cell viability and potency after thawing. We have previously demonstrated in humanized mouse models of HSCT the proof-of-concept of a novel modality of lentivirus-induced DCs (“SmyleDCPp65”) that accelerated Antigen-specific T cell development. Methods Here we demonstrate the feasibility of good manufacturing practices (GMP) for production of donor-derived DCs consisting of monocytes from peripheral blood transduced with an integrase-defective lentiviral vector (IDLV, co-expressing GM-CSF, IFN-α and the Cytomegalovirus Antigen Pp65) that were cryopreserved and thawed. Results Upscaling and standardized production of one lot of IDLV and three lots of SmyleDCPp65 under GMP-compliant conditions were feasible. Analytical parameters for quality control of SmyleDCPp65 identity after thawing and potency after culture were defined. Cell recovery, uniformity, efficacy of gene transfer, purity and viability were high and consistent. SmyleDCPp65 showed only residual and polyclonal IDLV integration, unbiased to proto-oncogenic hot-spots. Stimulation of autologous T cells by GMP-grade SmyleDCPp65 was validated. Conclusion These results underscore further developments of this individualized donor-derived cell vaccine to accelerate immune reconstitution against HCMV after HSCT in clinical trials.
