working hypothesis is that current large-scale production methods may concentrate proteins that modulate transduction. To address this possibility, we ...
Vector and Cell Engineering/Manufacturing I working hypothesis is that current large-scale production methods may concentrate proteins that modulate transduction. To address this possibility, we characterized the “lentiviral vector proteome” of several different clinical vector lots using an Orbitrap Velos Pro Hybrid Mass Spectrophotometer. Since X-VIVO 10 medium was the final product medium in all LV lots, proteins identified in a sample of unused medium were eliminated from the analysis. We first identified a subset of 24 proteins common to ALL vector lots. Not surprisingly, further analysis showed that 6 of these proteins are both “top 25” exosome markers in the ExoCarta database and known gag-pol interacting proteins listed in the HIV-1 Human Interaction Database. Independent of production size and tangential flow filtration brand, we next identified 57 proteins unique to a low titer LV expressing human beta-globin (BG). We similarly identified 47 proteins unique to a high titer LV expressing adenosine deaminase (ADA). BG lots were enriched in proteasome subunit proteins and ADA lots in ribosomal subunit proteins. Interestingly, the ADA lots were also enriched in several 14-3-3 family proteins that are known to modulate intracellular signaling pathways. It is possible that the mere presence or relative levels of specific proteins, such as 14-3-3 proteins, can affect transduction. We can speculate that the mechanism involves indirect effects of the vector transgene on the producer cells which yields a particle that is more stable and/or has an increased capacity to transduce cells.
458. Development of a Stable Producer Cell Line for Scalable Lentiviral Vector Production for Gene Therapy of Hemoglobinopathie
Sarah Slauson1, Amelia Thomas1, Byoung Ryu2, Melissa Bonner1, Gretchen Lewis1, Gabor Veres1, Janet Chung1, Geoffrey B. Parsons1 1 Bluebird Bio Inc., Cambridge, MA, 2St. Jude Children’s Research Hospital, Memphis, TN Current manufacturing of clinical grade lentiviral vectors (LVVs) for gene therapy applications commonly relies on transient transfection of adherent 293T cells. Improvements in production efficiency and scalability would provide value in meeting the needs for increased amounts of vector required for clinical development. An inducible producer cell line grown in suspension culture represents a potentially more scalable manufacturing process for LVV production which eliminates the need for costly plasmid and transfection reagents. We have engineered a packaging cell line by introducing doxycycline-inducible Gag-Pol, Rev, and VSVG envelope genes into a suspension cell line. Packaging cell clones were isolated by single cell sorting and screened by qPCR for the presence of the delivered genes. Virus production was assessed by transient transfection of a lentiviral vector and doxycycline treatment. A titer of up to 5.0E6 transduction units per milliliter (TU/mL) was observed with packaging cell line stability demonstrated after greater than four months of continuous passage. Next, a self-inactivating lentiviral vector was excised from its plasmid backbone and ligated in vitro to a linear neomycin resistance cassette and used to transfect the lentiviral packaging cell line to generate a panel of producers. Following two weeks of G418 drug selection, adherent colonies were plucked and screened for viral production followed by single cell sorting to isolate individual producer clones. From five different plucked colonies with titers greater than 3.0E6 TU/mL, more than 100 clones were screened and 7 were identified that produced a harvest titer greater than 1.0E7 TU/mL. Four of these clones were successfully re-adapted to suspension and scaled up to 3L culture. LVV generated from individual producer clones was compared for the ability to transduce adult mobilized CD34+ hematopoietic stem cells (HSCs). Interestingly, the vector copy number (VCN) in CD34+ HSCs for the LVV derived from the producer cells varied between S182
clones. Clones that produced the highest VCN have been chosen for further characterization in order to better understand the differences in HSC transduction. These lentiviral producer cell lines represent an important first step toward the creation of a manufacturing process that can better support clinical and commercial development of HSC lentiviral gene therapy.
459. Evaluation of Miltenyi ExpAct and TransAct CD3/28 Beads for CAR-T Cell Manufacturing Xiuyan Wang, Jinrong Qu, Jolanta Stefanski, Oriana BorquezOjeda, Anniesha Hack, Qing He, Teresa Wasielewska, Fang Du, Michel Sadelain, Isabelle Rivière Memorial Sloan Kettering Cancer Center, New York, NY
Adoptive transfer of chimeric antigen receptor (CAR) engineered T cells is a promising emerging strategy to treat cancer patients. Large-scale manufacturing of cGMP-grade CAR T cells using patient T cells selected and activated by CTS™ Dynabeads® CD3/CD28 (Dynabeads) followed by transduction with retroviral vectors is being used in the context of many clinical trials by our laboratory and others. Although we have established a robust protocol using Dynabeads, it is important to explore alternative sources to pre-empt supply chain limitations of this critical reagent. To this end, we evaluated T cell activation with either Miltenyi TransAct CD3/28 (TransAct) beads or Miltenyi ExpAct Treg (ExpAct) beads. In small-scale experiments, PBMCs were directly activated with TransAct or ExpAct beads and compared with our standard T cell selection and activation using Dynabeads. Overall, the transduction efficiency and expansion of T cells were comparable upon activation with all three reagents. The TransAct bead-stimulated cells exhibited comparable effector memory (EM)/central memory (CM) phenotype to that of the Dynabeads stimulated cells. In line with the EM/CM phenotype, CAR T cells stimulated with either TransAct or Dynabeads and tranduced with CD19-targeted CAR demonstrated robust and comparable antitumor activity in a systemic NSG/CD19+ NALM6 tumor mouse model. We further tested the efficacy of TransAct beads using positively or negatively selected T cells in a large-scale cGMP grade CAR-T cell manufacturing setting. Both the transduction efficiency and expansion of selected CD3+ cells activated with TransAct beads and Dynabeads were comparable. CD19-targeted CAR T cells activated by either TransAct or Dynabead were subjected to an in vivo stress test by using decreasing amount of CAR-T cells to treat systemic CD19+NALM6 tumors in NSG mice. In this experimental setting, T cells stimulated with TransAct beads demonstrated equivalent if not better anti-tumor activity than T cells stimulated with Dynabeads. In conclusion,our pre-clinical results suggest that TransAct beads support efficient transduction and expansion of CAR T cells. TransAct activated T cells exhibit antitumor activity equivalent to Dynabeads activated T cells in our NSG/CD19+NAML6 stress test. Therefore, Miltenyi TransAct beads can be used as an alternative to Dynabeads to stimulate T cells in clinical trials aiming at evaluating CAR T cell safety and antitumor activity.
460. Purification of AAV8 Using a Prototype Affinity Resin Compatible with cGMP Manufacturing
Colleen M. Dupuis, Amy A. Medeiros, Samuel C. Wadsworth, K. Reed Clark, Christopher J. Morrison Downstream Process Development, Dimension Therapeutics, Cambridge, MA Affinity interaction based resins have long been the foundation for various purification platforms of biological molecules. For the purification of rAAV, the affinity resin AVB Sepharose HP (AVB) has been used to successfully purify a number of different serotypes. Molecular Therapy Volume 24, Supplement 1, May 2016 Copyright © The American Society of Gene & Cell Therapy
