Cytokine Capture System for Production of Virus Specific T Cells

May 18, 2021

Susan Berrigan MLTCellular Therapy Laboratory Technologist IIIAlberta Precision LaboratoriesAlberta Blood & Marrow Transplant Program& Alberta Cellular Therapy Program

CliniMACS® Prodigy CCS System

In the allogeneic transplant setting opportunistic pathogens frequently reactivate from latency during prolonged immunosuppression

Often patients who fail to respond to conventional therapy for these infection have few options left for treatment until now...

The CCS System is a fully automated labeling and enrichment procedure for the selection of antigen specific Interferon gamma (IFN-γ) secreting CD4 and CD8 T Cells

The specificity of the enriched T Cells is determined by the virus or viruses that you want to targetThis done by using the MiltenyiBiotec MACS® GMP PeptivatorPeptide Pools

Currently Miltenyi has peptivators for the following Viruses for GMP Manufacturing:

AdV- AdenovirusBkV- Bk VirusHCMV- CMV VirusEBV- Epstein Barr VirusHPV16- Human Papilloma virusThese are the viruses that can be treated using the CCS system.

treated using the CCS system.

Prior to starting the CCS IFN-γenrichment process, donor samples must be tested for reactivity to the virus specific antigen(s)

This is done by performing intracellular staining using IFN-gamma Rapid Cytokine Inspector with RCI Anti IFN gamma

Once the donor is deemed appropriate then a MNC, Apheresis product is collected

Make note that for the CCS system you can only process a total WBC of 1 X 10^9. ( not very many)

Sample Preparation

The MNC, Apheresis product is enumerated for CD3, CD4, CD8, CD45 (WBC), and viability.Calculate how much volume of the product that is to be removed to ensure that you are processing no more than 1 X 10^9 total WBC

Any excess is cryopreserved.

Reagent Preparation

Prepare reagents 0.5% HSA, elution buffer, required peptivors, CCS system reagents

The fully automated processes is divided into the following steps:

1. Initial washing and antigen incubation and stimulation2. Cell labelling with the Catch Matrix reagent3. Secretion phase4. Cell labelling with the Enrichment reagent5. Washing6. Magnetic Enrichment7. Elution and Separation

The pre process steps include the loading of the tubing, column, buffers and reagent onto the prodigy

May 18, 2021A L B E R T A P R E C I S I O N L A B O R A T O R I E S14

Definition of process end

Once the tubing is primed and the sample and reagents loaded, the automated process takes 12 hours but must be completed in 36 hours. You can pause the process for operational purposes and have a delay between cell washing and antigen stimulation.The QC sample to be removed must be taken 70 minutes prior to the end of processing. We set the time for finishing for 9:00 am the next morning with removal of the QC sample at 7:55 am. The QC sample is equivalent to the Precolumn sample.

Step 1: Loading, Washing and Stimulation:The CTP is washed and then stimulated with the virus specific antigen (s). It is between these two steps that the delay in processing takes place.

Step 2 Cell Labelling I

CD45 cells are labelled with a bispecific antibody(CatchMatrixreagent) One end of the antibody binds onto the CD45 cell surface the other end is specific for the IFN gamma which will be secreted by the stimulated CD4 and CD8 cells

Step 3: Secretion PeriodDuring the secretion period the bispecific antibody binds the secreted IFN-gamma to the surface of the cytokine releasing cells.

Step 4: Cell Labelling IIThe IFN gamma that is bound to the surface of the secreting cells are then labelled with Anti IFN gamma antibody that is conjugated to the MACS microbeads

Step 5 :Washing

After the two step labelling process, the excess antibodies are removed by washing of the cells

QC sampleThe QC sample is removed at the predetermined time and is taken prior to the Enrichment step of the run. It is enumerated for CD3,CD4, CD8, CD45, and viability

Step 6: Enrichment

Labelled IFN gamma secreting cells that are bound with the MACS microbeads are retained within the column that is surrounded by a magnetic field.

Any cells that are “unbound” will pass through to the “Non Target Cell bag

Step 7: Elution and Separation

Once the column washes are complete, the magnetic field surrounding the column is removed and the IFN-gamma secreting cells will be eluted into the Target Cell bag.

The final volume for infusion is between 6 to 8 mls

Efficacy and QC testing

The Target cell bag is tested for the following:CD3/CD4 CD3/CD8CD3/CD4 IFN-γ %CD3/CD8 IFN-γ %70 to 80% CD4/CD8 IFN-γ % is seen in patient products that are good responders to the virus specific peptivator.< 70% CD4/CD8 IFN-γ is seen in patient products who are not great responders to the virus specific peptivator.

Patient response to the virus specific T Cells is usually seen within two weeks after infusion with a decrease in their viral load