CAR T Cell  Development and Commercial Manufacture
MedScope has substantial experience in the transduction of primary T Cells. MedScope is a rapid development organization, and in most instances can initiate value-based discussions within 24 hours. Please contact us at 802-236-8650 or info@medscope.com.

Transduction of Primary T Cells
Transduction of primary cells pose substantial challenges due to biological differences and heterogeneity of primary cell populations. For instance, LVs are commonly pseudotyped with the vesicular stomatitis virus glycoprotein (VSV-G) due to its broad tropism, allowing infection to a broad range of cell types. The low-density lipoprotein receptor (LDLR) is known to be the receptor to VSV-G, which is expressed differentially in T cells depending on their activation state. Stimulation with interleukin-2 (IL-2) is therefore necessary to activate T cells and increase LDL-R expression levels, enabling detectable levels of transduction. T cells are becoming a major target for gene therapy due to increasing use of CAR T cells in cancer immunotherapy. The issues of inefficient mass transport of existing systems combined with such biological barriers highlight the wastes generated in current clinical protocols.

MOI - Lentivector Calculation and Test Methods
Multiplicity of infection (MOI) is a frequently used term in virology which refers to the number of virions that are added per cell during infection. If one million virions are added to one million cells, the MOI is one. Although lentiviral vectors have been widely used for in vitro and in vivo gene therapy researches, there have been few studies systematically examining various conditions that may affect the determination of the number of viable vector particles in a vector preparation and the use of Multiplicity of Infection (MOI) as a parameter for the prediction of gene transfer events. 

Once Lentiviral vectors encoding a marker gene are packaged and supernatants concentrated, the the number of viable vector particles can be determined via in vitro transduction and fluorescent microscopy and FACs analyses. Various factors that may affect the transduction process, such as vector inoculum volume, target cell number and type, vector decay, variable vector – target cell contact,  and adsorption periods were studied. Process conditions and molecular diffusion and transport can also be critcal to maximizing MOI. A MOI assessment on order of 0–32 is  commonly used as an order of magnitude estimate for both existing and new cell line development. 

T Cell Conditioning and Activation
Example: Several vendors provide T Cells, such as AllCells (AllCells, LLC., Alameda, CA, USA). When ready for use, purchased cells are usually first thawed following vendor provided thawing protocol, then  allowed to recover in RPMI 1640 media supplemented with 10% FBS, L-Glutamine, 25 mM HEPES, and 1% Pen/Strep for 24 hours. CD3 and CD28-coated beads (Miltenyi Biotec Inc., San Diego, CA, USA) are then added to the cells at a 1:1 bead:cell ratio with 100 IU/mL human IL-2 (PeproTech, Rocky Hill, NJ, USA) for T cell activation. The cells and beads are then transferred to a 6-well plate at a density of 1-2x106  cells/mL/cm2 . Cells are then ready for transduction after a 24-hour activation period.

T Cell Transduction
Example: Stock of GFP-LV from a  previous cell line transductions can be used for the T cell transduction kinetics experiments. The fVIII LV used to quantify T cell transduction efficiency can be supplied by Expression Therapeutics, LLC (Tucker, GA, USA) and can be prepared by commercial manufacturing organizations for clinical development of a CD34+  fVIII-LV gene therapy product candidate. The fVIII-LV used a CD68 promoter, which is expressed in cells of the myeloid lineage such as granulocytes and monocytes. As such, T cells did not actually express fVIII, so VCN analysis can be used as the primary readout for transduction efficiency. A reduced dose of polybrene at 6µg/mL can be used for all T cell transductions to avoid polybrene-associated toxicity.

T Cell Assessment of Transduction Efficiency
Cells transduced with the GFP-LV can be stained with eFluor 780 viability dye (eBioscience, Inc., San Diego, CA, USA) at least 72 hours post-transduction. T cell purity can then be confirmed with a CD3-V450 (500A2) antibody (BD Biosciences, San Jose, CA, USA). Transduction efficiencies can be determined as the percentage of GFP+ live cells as obtained with a BD LSR II flow cytometer. Genomic DNA can then be harvested from cells using the DNEasy blood and tissue kit (Qiagen, Valencia, CA, USA). Flow cytometry and Real-time quantitative PCR can be used to determine vector copy number(VCN). fVIII-transduced cells received identical processing, without GFP assessment.

Potential Challenges with Transduction of Primary T Cells
There are many biological barriers to T Cell transduction that are not well understood, including mass transport control, diffusion, varying robustness of processed cells, time, temperature, etc. Also eFlour 760 dead cell staining as shown that in many cases cells are not impacted for short transduction times (eg. < 12 hours) but may increase for longer cell transduction times (eg. 24 hours). Also, in some instances, it has been found that cell death can significantly increase for higher MOI, possibly indicating that cell death may be related to total amount vector(as measured by eFLour 780 staining using flow cytometry). To better quantify processing, some developers have defined a Utilization Efficiency of VCN = Estimated Total Vector Copies Integrated/Viral Particles Used In Transduction.

Transducing Primary T Cells Using FVIII-LV 
Primary human T cells can be transduced with product from various vendors. One example is product from Spencer/Doering laboratories, which can provide materials and laboratory documentation for preclinical CD34+ fVIII-LV gene therapy product to transduce primary human T Cells. Utilizing this model, process development can be accomplished to determine CPP, CQA, and as well as optimation DOE process studies. Although in this case fVIII was not produced due to the use of a CD68 myeloid-specific promoter, one can obtain VCN as a readout for successful transduction to assess DOE process parameters. 

How To Reach Us
MedScope is a rapid development organization, and in most instances can initiate and address a wide range of value-based discussions within 24 hours. Our teams and wide technological network in the biopharmaceutical industry enables us to quickly bring substantial knowledge and problem solving skills to help you quickly solve problems. For rapid response, please contact us at 802-236-8650 or info@medscope.com. 




    MedScope - BioProcess Consultants
        QbD/CMC/cGMP Consultants in Gene Therapy, Immunotherapy, Small Molecule, LentiVector Development: Inception to Launch
       802-236-8650   info@medscope.com

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