Volker Huppert - Academia.edu (original) (raw)

Papers by Volker Huppert

Research paper thumbnail of Editorial: Translating NK cell scientific research to clinical product manufacturing

Research paper thumbnail of Process Development and Manufacturing: A FULLY AUTOMATED CD34+ CELLS ENRICHMENT FROM FRESH AND FROZEN UMBILICAL CORD BLOOD FOR CLINICAL SCALE MANUFACTURING OF NK CELLS USING A CLINIMACS PRODIGY PLATFORM

Research paper thumbnail of Novel approach for depletion of TCRab+ and CD45RA+ cells from apheresis products products using the CliniMACS Prodigy

Cytotherapy, 2018

DLI resulted in dominant donor T & B cell chimerism (Figure 1A,B) unlike other subsets (Figure 1C... more DLI resulted in dominant donor T & B cell chimerism (Figure 1A,B) unlike other subsets (Figure 1C,D). BAL values exhibited different kinetics (Figure 1). T & B cells, TCR/BCR repertoires, & TREC exceeded pre-BMT values by 3-6m (Figure 2A, B). BK virus specific T cells cleared viremia(Figure 2C). Mild skin GVHD post-DLI cleared in 2w with <1 mg/kg prednisone. Serial lung biopsies have been negative for rejection at last f/up (Nov17; 5m off ISD). At 18m post-BMT (2m post-ISD withdrawal), circulating donor T cells were unresponsive to host DC while responded to 3 rd party APC (Figure 3), fulfilling a critical tenet of tolerance. Tolerance towards host APC was Treg & Tr1 independent(Figure 3). Case 2: 38y/o woman with CVID on ECMO underwent BOLT. She had 3 episodes of lung rejection before becoming eligible for BMT (1/8 match) 14 months later(Nov17) (3E6 CD34+cells/kg, 2E3 CD3+cells/kg infused). She engrafted by D+12 with 95% whole blood chimerism but T cells of host origin that is treated with DLI. Case 1 is the first in human to demonstrate 1) durable engraftment, 2) immune competence, and 3) acquisition of tolerance from deceased donor VB marrow 4) matched only at a single class I MHC allele, providing proof of principle, that is currently also tested in adults.

Research paper thumbnail of Umbilical cord blood NK cells offer multiple advantages for cancer immunotherapy: lessons learned from Glycostem’s orphan drug oNKord®

Cell and Gene Therapy Insights, 2021

Research paper thumbnail of A bright commercial future for off-the-shelf NK cell therapy

Cell and Gene Therapy Insights, 2021

Research paper thumbnail of Technology for purifying NK cells and other cell types by concurrent gravity sedimentation and magnetic separation

Research paper thumbnail of HLA-Bw 4 80 ( T ) and multiple HLA-Bw 4 copies combined with KIR 3 DL 1 are associated with superior immune control of HCV infection in people who inject drugs

Background and aims: Natural killer T (NKT) cells represent a subset of immune cells that share c... more Background and aims: Natural killer T (NKT) cells represent a subset of immune cells that share characteristics of the innate and adaptive immunity. Invariant NKT cells recognize glycolipid antigens such as α galactosylceramide (αGalCer) presented by the non classical MHC molecule CD1d. Decreased NKT cell frequencies have been reported in chronically HCV infected patients, however, contradicting reports exist. We therefore aimed to comparatively study NKT cell frequencies and function in people who inject drugs (PWID) with chronic and spontaneously resolved HCV infection.

Research paper thumbnail of Adapter Chimeric Antigen Receptor Expressing Cells for Targeting of Multiple Antigens

Research paper thumbnail of Design of artifical tumor cells to induce Natural Killer cell proliferation

Research paper thumbnail of Method for cell separation

Research paper thumbnail of Next Level of Automated Cell Processing System for Flexible Manufacturing of Cellular Products

Blood, 2010

1186 Introduction: Currently, manufacturing of cellular products for cellular therapies is done m... more 1186 Introduction: Currently, manufacturing of cellular products for cellular therapies is done manually or semi-automated. To make cellular therapies applicable for routine use, a standardized production of cellular therapeutic agents is necessary. Therefore closed and highly automated manufacturing procedures are required. Solution: A new integrated cell processing device has been developed to automate and standardize the manufacturing process of cellular therapeutic agents and to handle several cell handling procedures in a fully automated and unified way. These procedures are filtering, centrifugation, temperature-controlled centrifugation, magnetic separation and cell culture. A functionally closed tubing system was developed to allow the application of specific combinations of the manufacturing steps listed. A new type of centrifugation chamber was designed to enable in-process liquid exchange and cell fractionation. Integrated ports allow controlled adding and removal of liqu...

Research paper thumbnail of Fully automated concentration of large scale cultured cells for immune therapy using the clinimacs prodigy ®

Research paper thumbnail of Optimization of Human NK Cell Manufacturing: Fully Automated Separation, Improved Ex Vivo Expansion Using IL-21 with Autologous Feeder Cells, and Generation of Anti-CD123-CAR-Expressing Effector Cells

Human Gene Therapy, 2017

The administration of ex vivo expanded natural killer (NK) cells as potential antitumor effector ... more The administration of ex vivo expanded natural killer (NK) cells as potential antitumor effector cells appears to be suitable for effector cell-based immunotherapies in high-risk cancer patients. However, good manufacturing practice (GMP)-compliant manufacturing of clinical-grade NK cells at sufficiently high numbers represents a great challenge. Therefore, previous expansion protocols for those effector cells were improved and optimized by using newly developed culture medium, interleukin (IL)-21, and autologous feeder cells (FCs). Separation of primary human NK cells (CD56(+)CD3(-)) was carried out with the CliniMACS Prodigy(®) in a single process, starting with approximately 1.2 × 10(9) leukocytes collected by small-scale lymphapheresis or from buffy coats. Enriched NK cells were adjusted to starting cell concentrations within approximately 1 × 10(6) effector cells/mL and cultured in comparative expansion experiments for 14 days with IL-2 (1,000 IU/mL) in different GMP-compliant media (X-VIVO(™)10, CellGro(®), TexMACS(™), and NK MACS(®)). After medium optimization, beneficial effects for functionality and phenotype were investigated at the beginning of cell expansion with irradiated (25 Gy) autologous FCs at a ratio of 20:1 (feeder: NK) in the presence or absence of IL-21 (100 ng/mL). Additionally, expanded NK cells were gene modified to express chimeric antigen receptors (CARs) against CD123, a common marker for acute myeloid leukemia (AML). Cytotoxicity, degranulation, and cytokine release of transduced NK cells were determined against KG1a cells in flow cytometric analysis and fluorescent imaging. The Prodigy manufacturing process revealed high target cell viabilities (median 95.4%), adequate NK cell recovery (median 60.4%), and purity of 95.4% in regard to CD56(+)CD3(-) target cells. The process in its early phase of development led to a median T-cell depletion of log 3.5 after CD3 depletion and log 3.6 after the whole process, including CD3 depletion and CD56 enrichment steps. Manually performed experiments to test different culture media demonstrated significantly higher NK cell expansion rates and an approximately equal distribution of CD56(dim)CD16(pos) and CD56(bright)CD16(dim&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;neg) NK subsets on day 14 with cells cultivated in NK MACS(®) media. Moreover, effector cell expansion in manually performed experiments with NK MACS(®) containing IL-2 and irradiated autologous FCs and IL-21, both added at the initiation of the culture, induced an 85-fold NK cell expansion. Compared to freshly isolated NK cells, expanded NK cells expressed significantly higher levels of NKp30, NKp44, NKG2D, TRAIL, FasL, CD69, and CD137, and showed comparable cell viabilities and killing/degranulation activities against tumor and leukemic cell lines in vitro. NK cells used for CAR transduction showed the highest anti-CD123 CAR expression on day 3 after gene modification. These anti-CD123 CAR-engineered NK cells demonstrated improved cytotoxicity against the CD123(pos) AML cell line KG1a and primary AML blasts. In addition, CAR NK cells showed higher degranulation and enhanced secretion of tumor necrosis factor alpha, interferon gamma, and granzyme A and B. In fluorescence imaging, specific interactions that initiated apoptotic processes in the AML target cells were detected between CAR NK cells and KG1a. After the fully automated NK cell separation process on Prodigy, a new NK cell expansion protocol was generated that resulted in high numbers of NK cells with potent antitumor activity, which could be modified efficiently by novel third-generation, alpha-retroviral SIN vector constructs. Next steps are the integration of the manual expansion procedure in the fully integrated platform for a standardized GMP-compliant overall process in this closed system that also may include gene modification of NK cells to optimize target-specific antitumor activity.

Research paper thumbnail of Influence of Irradiated Peripheral Blood Mononuclear Cells on Both Ex Vivo Proliferation of Human Natural Killer Cells and Change in Cellular Property

Frontiers in Immunology, 2017

Research paper thumbnail of Cell Separation Method

Research paper thumbnail of Automated in vitro generation of tri-virus-specific CD4+ and CD8+ T-lymphocytes for adoptive immunotherapy

Research paper thumbnail of A large-scale method for the selective depletion of αβ T lymphocytes from PBSC for allogeneic transplantation

Cytotherapy, 2007

... A total of seven large-scale experiments was performed. ... Remaining RBC were lysed with 3 m... more ... A total of seven large-scale experiments was performed. ... Remaining RBC were lysed with 3 mL ammonium chloride solution, centrifuged and washed once with staining buffer (Dulbecco's PBS ... As the Ab used for the depletion (clone BMA031) and the detection of αβ T cells after ...

Research paper thumbnail of Development of Automated Separation, Expansion, and Quality Control Protocols for Clinical-Scale Manufacturing of Primary Human NK Cells and Alpharetroviral Chimeric Antigen Receptor Engineering

Human Gene Therapy Methods, 2019

Research paper thumbnail of Novel process for clinical scale purification of NK cells using a two step separation strategy within a single automated procedure

Research paper thumbnail of Isolation of CD56+ Natural Killer Cells Using the CliniMACS® MARRS Tubing Set, a Newly Developed Tubing Set Including a Cross-Flow Filtration Module for Removal of Unbound Reagent

Blood

Use of Natural Killer (NK) cells in cellular immunotherapy requires processing of large numbers o... more Use of Natural Killer (NK) cells in cellular immunotherapy requires processing of large numbers of NK cells in a closed system consisting of clinical grade components. We have previously developed a two step NK cell isolation process in which the CliniMACS® plus Instrument and CliniMACS® CD3 and CD56 Reagents are used for CD3+ cell depletion and subsequent CD56+ cell enrichment, respectively. We have recently optimized the CD3 depletion step for very large scale cell processing by development of the CliniMACS® Depletion Tubing Set (“DTS”) and appropriate cell separation software, reducing the processing time of the CD3 depletion step by 50%. We now have developed the CliniMACS® MARRS Tubing Set (MARRS = MAgnetic Reagent Removal System), a tubing set which includes a cross-flow filtration module for removal of unbound magnetic reagent after magnetic labelling. The cross-flow filtration module allows to directly connect a magnetically labelled cell product to the tubing set without ce...

Research paper thumbnail of Editorial: Translating NK cell scientific research to clinical product manufacturing

Research paper thumbnail of Process Development and Manufacturing: A FULLY AUTOMATED CD34+ CELLS ENRICHMENT FROM FRESH AND FROZEN UMBILICAL CORD BLOOD FOR CLINICAL SCALE MANUFACTURING OF NK CELLS USING A CLINIMACS PRODIGY PLATFORM

Research paper thumbnail of Novel approach for depletion of TCRab+ and CD45RA+ cells from apheresis products products using the CliniMACS Prodigy

Cytotherapy, 2018

DLI resulted in dominant donor T & B cell chimerism (Figure 1A,B) unlike other subsets (Figure 1C... more DLI resulted in dominant donor T & B cell chimerism (Figure 1A,B) unlike other subsets (Figure 1C,D). BAL values exhibited different kinetics (Figure 1). T & B cells, TCR/BCR repertoires, & TREC exceeded pre-BMT values by 3-6m (Figure 2A, B). BK virus specific T cells cleared viremia(Figure 2C). Mild skin GVHD post-DLI cleared in 2w with <1 mg/kg prednisone. Serial lung biopsies have been negative for rejection at last f/up (Nov17; 5m off ISD). At 18m post-BMT (2m post-ISD withdrawal), circulating donor T cells were unresponsive to host DC while responded to 3 rd party APC (Figure 3), fulfilling a critical tenet of tolerance. Tolerance towards host APC was Treg & Tr1 independent(Figure 3). Case 2: 38y/o woman with CVID on ECMO underwent BOLT. She had 3 episodes of lung rejection before becoming eligible for BMT (1/8 match) 14 months later(Nov17) (3E6 CD34+cells/kg, 2E3 CD3+cells/kg infused). She engrafted by D+12 with 95% whole blood chimerism but T cells of host origin that is treated with DLI. Case 1 is the first in human to demonstrate 1) durable engraftment, 2) immune competence, and 3) acquisition of tolerance from deceased donor VB marrow 4) matched only at a single class I MHC allele, providing proof of principle, that is currently also tested in adults.

Research paper thumbnail of Umbilical cord blood NK cells offer multiple advantages for cancer immunotherapy: lessons learned from Glycostem’s orphan drug oNKord®

Cell and Gene Therapy Insights, 2021

Research paper thumbnail of A bright commercial future for off-the-shelf NK cell therapy

Cell and Gene Therapy Insights, 2021

Research paper thumbnail of Technology for purifying NK cells and other cell types by concurrent gravity sedimentation and magnetic separation

Research paper thumbnail of HLA-Bw 4 80 ( T ) and multiple HLA-Bw 4 copies combined with KIR 3 DL 1 are associated with superior immune control of HCV infection in people who inject drugs

Background and aims: Natural killer T (NKT) cells represent a subset of immune cells that share c... more Background and aims: Natural killer T (NKT) cells represent a subset of immune cells that share characteristics of the innate and adaptive immunity. Invariant NKT cells recognize glycolipid antigens such as α galactosylceramide (αGalCer) presented by the non classical MHC molecule CD1d. Decreased NKT cell frequencies have been reported in chronically HCV infected patients, however, contradicting reports exist. We therefore aimed to comparatively study NKT cell frequencies and function in people who inject drugs (PWID) with chronic and spontaneously resolved HCV infection.

Research paper thumbnail of Adapter Chimeric Antigen Receptor Expressing Cells for Targeting of Multiple Antigens

Research paper thumbnail of Design of artifical tumor cells to induce Natural Killer cell proliferation

Research paper thumbnail of Method for cell separation

Research paper thumbnail of Next Level of Automated Cell Processing System for Flexible Manufacturing of Cellular Products

Blood, 2010

1186 Introduction: Currently, manufacturing of cellular products for cellular therapies is done m... more 1186 Introduction: Currently, manufacturing of cellular products for cellular therapies is done manually or semi-automated. To make cellular therapies applicable for routine use, a standardized production of cellular therapeutic agents is necessary. Therefore closed and highly automated manufacturing procedures are required. Solution: A new integrated cell processing device has been developed to automate and standardize the manufacturing process of cellular therapeutic agents and to handle several cell handling procedures in a fully automated and unified way. These procedures are filtering, centrifugation, temperature-controlled centrifugation, magnetic separation and cell culture. A functionally closed tubing system was developed to allow the application of specific combinations of the manufacturing steps listed. A new type of centrifugation chamber was designed to enable in-process liquid exchange and cell fractionation. Integrated ports allow controlled adding and removal of liqu...

Research paper thumbnail of Fully automated concentration of large scale cultured cells for immune therapy using the clinimacs prodigy ®

Research paper thumbnail of Optimization of Human NK Cell Manufacturing: Fully Automated Separation, Improved Ex Vivo Expansion Using IL-21 with Autologous Feeder Cells, and Generation of Anti-CD123-CAR-Expressing Effector Cells

Human Gene Therapy, 2017

The administration of ex vivo expanded natural killer (NK) cells as potential antitumor effector ... more The administration of ex vivo expanded natural killer (NK) cells as potential antitumor effector cells appears to be suitable for effector cell-based immunotherapies in high-risk cancer patients. However, good manufacturing practice (GMP)-compliant manufacturing of clinical-grade NK cells at sufficiently high numbers represents a great challenge. Therefore, previous expansion protocols for those effector cells were improved and optimized by using newly developed culture medium, interleukin (IL)-21, and autologous feeder cells (FCs). Separation of primary human NK cells (CD56(+)CD3(-)) was carried out with the CliniMACS Prodigy(®) in a single process, starting with approximately 1.2 × 10(9) leukocytes collected by small-scale lymphapheresis or from buffy coats. Enriched NK cells were adjusted to starting cell concentrations within approximately 1 × 10(6) effector cells/mL and cultured in comparative expansion experiments for 14 days with IL-2 (1,000 IU/mL) in different GMP-compliant media (X-VIVO(™)10, CellGro(®), TexMACS(™), and NK MACS(®)). After medium optimization, beneficial effects for functionality and phenotype were investigated at the beginning of cell expansion with irradiated (25 Gy) autologous FCs at a ratio of 20:1 (feeder: NK) in the presence or absence of IL-21 (100 ng/mL). Additionally, expanded NK cells were gene modified to express chimeric antigen receptors (CARs) against CD123, a common marker for acute myeloid leukemia (AML). Cytotoxicity, degranulation, and cytokine release of transduced NK cells were determined against KG1a cells in flow cytometric analysis and fluorescent imaging. The Prodigy manufacturing process revealed high target cell viabilities (median 95.4%), adequate NK cell recovery (median 60.4%), and purity of 95.4% in regard to CD56(+)CD3(-) target cells. The process in its early phase of development led to a median T-cell depletion of log 3.5 after CD3 depletion and log 3.6 after the whole process, including CD3 depletion and CD56 enrichment steps. Manually performed experiments to test different culture media demonstrated significantly higher NK cell expansion rates and an approximately equal distribution of CD56(dim)CD16(pos) and CD56(bright)CD16(dim&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;neg) NK subsets on day 14 with cells cultivated in NK MACS(®) media. Moreover, effector cell expansion in manually performed experiments with NK MACS(®) containing IL-2 and irradiated autologous FCs and IL-21, both added at the initiation of the culture, induced an 85-fold NK cell expansion. Compared to freshly isolated NK cells, expanded NK cells expressed significantly higher levels of NKp30, NKp44, NKG2D, TRAIL, FasL, CD69, and CD137, and showed comparable cell viabilities and killing/degranulation activities against tumor and leukemic cell lines in vitro. NK cells used for CAR transduction showed the highest anti-CD123 CAR expression on day 3 after gene modification. These anti-CD123 CAR-engineered NK cells demonstrated improved cytotoxicity against the CD123(pos) AML cell line KG1a and primary AML blasts. In addition, CAR NK cells showed higher degranulation and enhanced secretion of tumor necrosis factor alpha, interferon gamma, and granzyme A and B. In fluorescence imaging, specific interactions that initiated apoptotic processes in the AML target cells were detected between CAR NK cells and KG1a. After the fully automated NK cell separation process on Prodigy, a new NK cell expansion protocol was generated that resulted in high numbers of NK cells with potent antitumor activity, which could be modified efficiently by novel third-generation, alpha-retroviral SIN vector constructs. Next steps are the integration of the manual expansion procedure in the fully integrated platform for a standardized GMP-compliant overall process in this closed system that also may include gene modification of NK cells to optimize target-specific antitumor activity.

Research paper thumbnail of Influence of Irradiated Peripheral Blood Mononuclear Cells on Both Ex Vivo Proliferation of Human Natural Killer Cells and Change in Cellular Property

Frontiers in Immunology, 2017

Research paper thumbnail of Cell Separation Method

Research paper thumbnail of Automated in vitro generation of tri-virus-specific CD4+ and CD8+ T-lymphocytes for adoptive immunotherapy

Research paper thumbnail of A large-scale method for the selective depletion of αβ T lymphocytes from PBSC for allogeneic transplantation

Cytotherapy, 2007

... A total of seven large-scale experiments was performed. ... Remaining RBC were lysed with 3 m... more ... A total of seven large-scale experiments was performed. ... Remaining RBC were lysed with 3 mL ammonium chloride solution, centrifuged and washed once with staining buffer (Dulbecco's PBS ... As the Ab used for the depletion (clone BMA031) and the detection of αβ T cells after ...

Research paper thumbnail of Development of Automated Separation, Expansion, and Quality Control Protocols for Clinical-Scale Manufacturing of Primary Human NK Cells and Alpharetroviral Chimeric Antigen Receptor Engineering

Human Gene Therapy Methods, 2019

Research paper thumbnail of Novel process for clinical scale purification of NK cells using a two step separation strategy within a single automated procedure

Research paper thumbnail of Isolation of CD56+ Natural Killer Cells Using the CliniMACS® MARRS Tubing Set, a Newly Developed Tubing Set Including a Cross-Flow Filtration Module for Removal of Unbound Reagent

Blood

Use of Natural Killer (NK) cells in cellular immunotherapy requires processing of large numbers o... more Use of Natural Killer (NK) cells in cellular immunotherapy requires processing of large numbers of NK cells in a closed system consisting of clinical grade components. We have previously developed a two step NK cell isolation process in which the CliniMACS® plus Instrument and CliniMACS® CD3 and CD56 Reagents are used for CD3+ cell depletion and subsequent CD56+ cell enrichment, respectively. We have recently optimized the CD3 depletion step for very large scale cell processing by development of the CliniMACS® Depletion Tubing Set (“DTS”) and appropriate cell separation software, reducing the processing time of the CD3 depletion step by 50%. We now have developed the CliniMACS® MARRS Tubing Set (MARRS = MAgnetic Reagent Removal System), a tubing set which includes a cross-flow filtration module for removal of unbound magnetic reagent after magnetic labelling. The cross-flow filtration module allows to directly connect a magnetically labelled cell product to the tubing set without ce...