- Email: [email protected]
574. Magnetic Colocalization of Viral Vectors and Target Cells Improves Transduction Efficiency in Human Hematopoietic Cells
CELL PROCESSING AND VECTOR MANUFACTURE increased from 7% in the absence of PMA to greater than 20% in the presence of 1nM PMA, and cell division was hindered. The lack of enhanced viral transduction with sca-1+ cells may be due to the different PKC isoform found in murine sca-1+ cells compared to those in human cells. Delineation of the mechanism whereby PMA is acting, specifically focusing on which PKC isoform is most affected by the presence of PMA, is currently underway.
574. Magnetic Colocalization of Viral Vectors and Target Cells Improves Transduction Efficiency in Human Hematopoietic Cells
Constanze Lehmann,1 Sigrid Espenlaub,2 Florian Kreppel,2 Ian C. D. Johnston.1 1 Miltenyi Biotec GmbH, Bergisch Gladbach, Germany; 2 Department of Gene Therapy, University of Ulm, Ulm, Germany.
A major factor limiting viral transduction of target cells in cell culture is the diffusion of virus particles to the cell surface. Polycationic reagents, recombinant fibronectin, spinoculation or the use of polycationic magnetic particles and a magnetic field can be used to increase vector concentration at the cell surface. In a further refinement of this latter method, we have labeled the target cells with standard magnetic cell separation reagents (MACS®MicroBeads) in addition to magnetic labeling of the viral vector particles before placing these on a magnetic cell separation column. Magnetic labeling of both virus particles and the target cells ensures an efficient colocalization within the high-gradient magnetic field of a MACS Separator leading to reduced viral reagent requirements and specific transduction of only the magnetically-labeled target cells (Blood: 117: e171–181). In this study, both polycationic magnetic particles and antibody-conjugated superparamagnetic nanoparticles (MicroBeads) were assessed for their ability to bind to viral vector particles and transduce human cell lines and primary hematopoietic cells. Polycationic magnetic particles complexed GFP-encoding adenoviral (AdV) and lentiviral (LV) vector particles effectively. After AdV transduction (pMOI, physical particles per cell=200-500) of human cells of low permissivity (K562, M-07e, HuT78), up to 15fold more cells were transduced after magnetic colocalization with increases in target gene expression of over 40-fold. Similarly, CD34+ cells isolated from peripheral blood with CD34 MicroBeads could be transduced efficiently (pMOI=2000, 15% GFP+ cells (3 fold over control) and 8-fold higher MFI). Transduction of human T cells and CD34+ cells with LV vectors was also supported using this protocol. Both numbers of transduced cells and the expression intensity of the transgene were increased (T cells, MOI=0.5, 30% GFP+; CD34+ cells, MOI=50, 75% GFP+). During LV budding from the cell membrane, host membrane proteins are also incorporated into the viral membrane. MicroBeads that bind these molecules can be used to isolate wildtype HIV from patient samples, but can also be used to magnetically label recombinant LV vectors (efficiency >90%) and direct them to a target cell. Using this approach, vector particles could be directed to target cells expressing the same surface molecule. An enhanced transduction was observed (3-fold) when both magnetically labeled vector and cells were combined in MACS columns as described above. (T cells, MOI=0.5, 30% GFP+; CD34+ cells, MOI=25, 69% GFP+). These novel transduction reagents and protocols enable a fast, flexible and reproducible transduction of target cells to be performed that is independent of vector titer. As current cell therapy and gene therapy approaches require many manual handling steps between collecting the patient or donor cell sample and returning the modified cell product to the patient, these protocols are currently being assessed for their suitability for incorporation into a functionally closed and fully automated cell processing device for the manufacture of gene therapeutic cellular products.
Molecular Therapy Volume 20, Supplement 1, May 2012 Copyright © The American Society of Gene & Cell Therapy
575. Optimizing the Manufacture of CAR-T Cells for Clinical Applications
Roopa Mucharla,1 Usanarat Anurathapan,2 Natalia Lapteva,3 Ann M. Leen,4 Cliona Rooney,5 Juan F. Vera.6 1 Center for Cell and Gene Therapy, Baylor College of Medicine, Houston, TX.
Chimeric antigen receptors (CARs) are artificial molecules which can be use to redirect T cell immune response against antigens expressed on the surface of tumor cells. Recent encouraging clinical data from our group and others has shown that T cells engineered with these molecules can produce complete clinical response. Although promising, most current protocols expand engineered T cells nonspecifically (using IL2 and OKT3), which often results in a decrease in transgenic populations over time. Additionally, cell expansion using conventional cultureware is complicated and labor intensive, limiting the broader application of this therapy. With the purpose of optimizing and streamlining CAR-T cell manufacture we assessed whether cell expansion could be improved by; (i) substituting non-specific stimuli with an artificial antigen presenting cell (a-APC) expressing cognate antigen, and (ii) culturing cells in a simple and scalable gas permeable culture device (G-Rex). To expand T cells engineered with a CAR targeting the prostate cancer antigen PSCA we first generated an a-APC cell line using K562 cells engineered to express PSCA antigen and different co-stimulatory molecules including CD80, CD86 and 41BB. When co-cultured with CAR-PSCA T cells in vitro we found that a-APCs co-expressing PSCA, CD80 and 41BB in combination were most effective in inducing T cell expansion, with a 1.9 fold increase in cell numbers when compared with CAR T cells cultured in the presence of IL2 alone. In addition this culture condition enriched for engineered T cells as illustrated by a 2.4±1.2 fold increase in the frequency of transgenic cells after only 7 days of culture. To next assess whether we could simply CAR T cell manufacture we transferred our engineered a-APCs and transgenic T cells to a static G-Rex with a surface area of 600cm2, developed by Wilson Wolf Manufacturing as a closed and GMP-compliant culture system. In this flask O2 and CO2 are exchanged across a silicone membrane at the base, allowing an increased depth of medium above the cells, which provides more nutrients while waste products are diluted. These culture conditions resulted in an increase in the cell output when compared with conventional commercial products such as bags, flasks, and 24-well tissue culture plates, without increasing the number of cell doublings. From an initial seeding density of 1.5E+08 T cells (0.25E+06 cells per cm2) we achieved on average a 110 fold cell expansion in 7 days of culture resulting in the production of 1.51.8E+10 CAR-T cells (25-30E+06 T cells per cm2) using only 6L of culture media (without media change). We found that 10ml media/cm2 (volume:surface area) supported maximal cell output and cell viability was maintained at >95% for 3 days. However, while cell output could not be improved by adding additional media the use of an additional 5ml/cm2 sustained cell viability at >95% for 5 days. This optimized bioprocess is GMP compatible and could be adapted for clinical manufacture of CAR-T cells, decreasing the cost and complexity of this technology and making this therapy more accessible.
576. Exploiting the Mechanism of IntronSplicing in Insect Cells To Produce Viral Vectors Harboring Toxin Genes for Cancer Gene Therapy Haifeng Chen.1 VIROVEK Incorporation, Hayward, CA.
1
Suicide gene therapy has been widely investigated for the treatment of human immunodeficiency virus (HIV) infection, for controlling graft-versus-host disease, and also for the treatment of cancer. While the production of viral vectors carrying suicide genes such as the herpes simplex virus thymidine kinase (HSV-TK) or S223
574. Magnetic Colocalization of Viral Vectors and Target Cells Improves Transduction Efficiency in Human Hematopoietic Cells
Constanze Lehmann,1 Sigrid Espenlaub,2 Florian Kreppel,2 Ian C. D. Johnston.1 1 Miltenyi Biotec GmbH, Bergisch Gladbach, Germany; 2 Department of Gene Therapy, University of Ulm, Ulm, Germany.
A major factor limiting viral transduction of target cells in cell culture is the diffusion of virus particles to the cell surface. Polycationic reagents, recombinant fibronectin, spinoculation or the use of polycationic magnetic particles and a magnetic field can be used to increase vector concentration at the cell surface. In a further refinement of this latter method, we have labeled the target cells with standard magnetic cell separation reagents (MACS®MicroBeads) in addition to magnetic labeling of the viral vector particles before placing these on a magnetic cell separation column. Magnetic labeling of both virus particles and the target cells ensures an efficient colocalization within the high-gradient magnetic field of a MACS Separator leading to reduced viral reagent requirements and specific transduction of only the magnetically-labeled target cells (Blood: 117: e171–181). In this study, both polycationic magnetic particles and antibody-conjugated superparamagnetic nanoparticles (MicroBeads) were assessed for their ability to bind to viral vector particles and transduce human cell lines and primary hematopoietic cells. Polycationic magnetic particles complexed GFP-encoding adenoviral (AdV) and lentiviral (LV) vector particles effectively. After AdV transduction (pMOI, physical particles per cell=200-500) of human cells of low permissivity (K562, M-07e, HuT78), up to 15fold more cells were transduced after magnetic colocalization with increases in target gene expression of over 40-fold. Similarly, CD34+ cells isolated from peripheral blood with CD34 MicroBeads could be transduced efficiently (pMOI=2000, 15% GFP+ cells (3 fold over control) and 8-fold higher MFI). Transduction of human T cells and CD34+ cells with LV vectors was also supported using this protocol. Both numbers of transduced cells and the expression intensity of the transgene were increased (T cells, MOI=0.5, 30% GFP+; CD34+ cells, MOI=50, 75% GFP+). During LV budding from the cell membrane, host membrane proteins are also incorporated into the viral membrane. MicroBeads that bind these molecules can be used to isolate wildtype HIV from patient samples, but can also be used to magnetically label recombinant LV vectors (efficiency >90%) and direct them to a target cell. Using this approach, vector particles could be directed to target cells expressing the same surface molecule. An enhanced transduction was observed (3-fold) when both magnetically labeled vector and cells were combined in MACS columns as described above. (T cells, MOI=0.5, 30% GFP+; CD34+ cells, MOI=25, 69% GFP+). These novel transduction reagents and protocols enable a fast, flexible and reproducible transduction of target cells to be performed that is independent of vector titer. As current cell therapy and gene therapy approaches require many manual handling steps between collecting the patient or donor cell sample and returning the modified cell product to the patient, these protocols are currently being assessed for their suitability for incorporation into a functionally closed and fully automated cell processing device for the manufacture of gene therapeutic cellular products.
Molecular Therapy Volume 20, Supplement 1, May 2012 Copyright © The American Society of Gene & Cell Therapy
575. Optimizing the Manufacture of CAR-T Cells for Clinical Applications
Roopa Mucharla,1 Usanarat Anurathapan,2 Natalia Lapteva,3 Ann M. Leen,4 Cliona Rooney,5 Juan F. Vera.6 1 Center for Cell and Gene Therapy, Baylor College of Medicine, Houston, TX.
Chimeric antigen receptors (CARs) are artificial molecules which can be use to redirect T cell immune response against antigens expressed on the surface of tumor cells. Recent encouraging clinical data from our group and others has shown that T cells engineered with these molecules can produce complete clinical response. Although promising, most current protocols expand engineered T cells nonspecifically (using IL2 and OKT3), which often results in a decrease in transgenic populations over time. Additionally, cell expansion using conventional cultureware is complicated and labor intensive, limiting the broader application of this therapy. With the purpose of optimizing and streamlining CAR-T cell manufacture we assessed whether cell expansion could be improved by; (i) substituting non-specific stimuli with an artificial antigen presenting cell (a-APC) expressing cognate antigen, and (ii) culturing cells in a simple and scalable gas permeable culture device (G-Rex). To expand T cells engineered with a CAR targeting the prostate cancer antigen PSCA we first generated an a-APC cell line using K562 cells engineered to express PSCA antigen and different co-stimulatory molecules including CD80, CD86 and 41BB. When co-cultured with CAR-PSCA T cells in vitro we found that a-APCs co-expressing PSCA, CD80 and 41BB in combination were most effective in inducing T cell expansion, with a 1.9 fold increase in cell numbers when compared with CAR T cells cultured in the presence of IL2 alone. In addition this culture condition enriched for engineered T cells as illustrated by a 2.4±1.2 fold increase in the frequency of transgenic cells after only 7 days of culture. To next assess whether we could simply CAR T cell manufacture we transferred our engineered a-APCs and transgenic T cells to a static G-Rex with a surface area of 600cm2, developed by Wilson Wolf Manufacturing as a closed and GMP-compliant culture system. In this flask O2 and CO2 are exchanged across a silicone membrane at the base, allowing an increased depth of medium above the cells, which provides more nutrients while waste products are diluted. These culture conditions resulted in an increase in the cell output when compared with conventional commercial products such as bags, flasks, and 24-well tissue culture plates, without increasing the number of cell doublings. From an initial seeding density of 1.5E+08 T cells (0.25E+06 cells per cm2) we achieved on average a 110 fold cell expansion in 7 days of culture resulting in the production of 1.51.8E+10 CAR-T cells (25-30E+06 T cells per cm2) using only 6L of culture media (without media change). We found that 10ml media/cm2 (volume:surface area) supported maximal cell output and cell viability was maintained at >95% for 3 days. However, while cell output could not be improved by adding additional media the use of an additional 5ml/cm2 sustained cell viability at >95% for 5 days. This optimized bioprocess is GMP compatible and could be adapted for clinical manufacture of CAR-T cells, decreasing the cost and complexity of this technology and making this therapy more accessible.
576. Exploiting the Mechanism of IntronSplicing in Insect Cells To Produce Viral Vectors Harboring Toxin Genes for Cancer Gene Therapy Haifeng Chen.1 VIROVEK Incorporation, Hayward, CA.
1
Suicide gene therapy has been widely investigated for the treatment of human immunodeficiency virus (HIV) infection, for controlling graft-versus-host disease, and also for the treatment of cancer. While the production of viral vectors carrying suicide genes such as the herpes simplex virus thymidine kinase (HSV-TK) or S223