Tri-virus-specific T cells isolated with the CliniMACS Prodigy CCS

Tri-virus-specific T cells isolated with the
CliniMACS Prodigy CCS-IFN System: Quality
development of an ATMP
Hermann Bohnenkamp, Ph.D.
Miltenyi Biotec GmbH
Agenda
1. Automated generation of trivirus-specific T cells
2. Quality development
3. Summary
2014
Hermann Bohnenkamp
Page 2
Background information
• Indication: Treatment of therapy-refractory infectious and
infection-related diseases
• Viral infections (CMV, AdV, EBV) are a frequent cause of
morbidity and mortality in patients undergoing allogeneic stem
cell transplantations
• Standard antiviral therapy does not cure viral infections
• Only restoration of T-cell immunity cures an infection and can
prevent further infections
• Standard antiviral therapy can be associated with severe side
effects up to necessary discontinuation
Adoptive T cell therapy - using the CCS
Clinical data
Adoptive Transfer of
Adenovirus-specific T cells
•Feuchtinger et al. (2006) Br. J. Haematol.
CMV-specific T cells
•Peggs et al. (2011) Clin. Infect. Dis.
•Feuchtinger et al. (2010) Blood
•Brestrich et al. (2009) Am. J. Transplant.
EBV-specific T cells
•Moosmann et al. (2010) Blood
•Icheva et al. (2012) J. Clin. Oncol.
Existing clinical data
• Efficacy (≥1log decrease in viral load) in treatment-refractory
patients:
• 62.5%: EBV-positive PTLD
• 83%: CMV infections
• 50%: AdV infections
• Safety: four reports of acute GvHD (Grade ≥2) induction post Tcell transfer in 66 patients
Enrichment of Ag-specific T cells
Workflow scheme of CCS
isolation
Enrichment of IFN-gamma secreting cells
CliniMACS Cytokine Capture System (CCS)
Pro
• Fast
• not HLA-restricted
• CD4+ and CD8+ T cells
• CE-approved*
• Multi-antigen-specific T cells possible
Con
• In vitro stimulation necessary
* In the USA the CliniMACS CCS is available for use under IND application or IDE
PepTivator (overlapping peptide pools)
Configuration
• Mixtures of 15-mer peptides
• 11 aa peptide overlap, spanning over the whole sequence
of a protein
• Covers CD4 epitopes (12-15 aa) as well as CD8 epitopes
(8-10 aa)
15 aa
11 aa
Kiecker et al., 2004
Tr-virus-specific T cells
5 Peptide pools are used for the re-stimulation of T cells:
•CMV: pp65
•EBV: EBNA-1, BZLF1, LMP2a
•AdV: AdV5 Hexon
15 aa
11 aa
Process scheme
Automated generation of virus-specific T cells
Tubing set,
buffer, media
Preparation
Priming
Cell sample, reagents,
programming
0.5 h
Preparation
Sample Preparation
2h
Antigen Incubation
4h
Wash and Labeling with CatchMatrix
Secretion Phase
1h
1h
Wash and Labeling with Enrichment Reagent
Wash and Separation Process
1h
2.5 h
Automated steps
Manual steps
Final Handling
Enriched IFN-g+ cell fraction
CliniMACS Prodigy CCS system
CliniMACS plus vs Prodigy
Tri-virus-specific T cells
Analysis of mixed or separated stimulation
Tri-virus-specific T cells
Analysis of mixed or separated stimulation
stimulation with 1 antigen
w/o
pp65
IE-1
EBNA1
hexon
stimulation with 4 antigens
simultaneous
separate
loading
0.22%*
0.06%*
0.03%*
0.04%*
0.35%*
0.28%*
<0.01%*
1.59%*
0.33%*
0.27%*
0.09%*
2.24%*
1.99%*
CD8
CD4
<0.01%*
secreted IFN-
Specificity for each antigen sustained within
CD4+ T cells
Specificity for each antigen sustained within
CD8+ T cells
Agenda
1. Automated generation of trivirus-specific T cells
2. Quality development
3. Summary
2014
Hermann Bohnenkamp
Page 16
Workflow
Process for tri-virus-specific T cells
Donor with a T-cell response to the
viral PepTivator antigen(s) of the
virus causing the infection
1
Confirmation of donor
eligibility
(donor pre-test)
Automated CliniMACS Prodigy IFNCCS process
2
Enrichment of antigenspecific T cells
Quality control of enriched antigenspecific T cells:
Assessments of identity, purity and
potency by flow cytometry
3
Characterisation and
composition of the
final cellular product
May 12, 2014
Page 17
Considerations for flow-cytometric analysis
Development of a test system that ensures operator
independent results
•Pre-mixed panel for staining of T cells (Kits)
•Single platform analysis (MACSQuant Analyzer):
•Automated measurement
•Predefined experiment settings as well as acquisition and analysis
templates
•Application of automated gating strategy to data files
May 12, 2014
Hermann Bohnenkamp // Clinical Marketing // CliniMACS Prodigy CCS-IFN System
Page 18
Workflow
Process for tri-virus-specific T cells
1
2
3
Confirmation of donor
eligibility (donor pre-test)
Enrichment of antigenspecific T cells
Characterisation and
composition of the final
cellular product
Confirmation of donor eligibility – workflow
(donor pre-test)
PepTivator HTA
Rapid Cytokine Inspector
(CD4/CD8 T Cell) Kit,
human
•96-well format
•CMV pp65
•AdV5 Hexon
•EBV (LMP2A, BZLF1,
EBNA-1)
•5-colour-flow panel
May 12, 2014
MACSQuant
Express Mode
Page 20
Conformation of donor eligibility
Panel design
Excitation
Emission
Filter
405nm
450nm
VioBlue
CD3
488nm
525nm
FITC
CD8
488nm
585nm
PE
IFN-
488nm
655-730nm
PerCP
PI/CD14/CD20
638nm
655-730nm
APC
CD4
May 12, 2014
CD4/CD8
T
Cell
Fluorochrome
Detection Cocktail
Hermann Bohnenkamp // Clinical Marketing // CliniMACS Prodigy CCS-IFN System
Page 21
Rapid Cytokine Inspector
Express mode and gating strategy
May 12, 2014
Hermann Bohnenkamp // Clinical Marketing // CliniMACS Prodigy CCS-IFN System
Page 22
Workflow
Process for tri-virus-specific T cells
1
2
3
Confirmation of donor
eligibility (donor pre-test)
Enrichment of antigenspecific T cells
Characterisation and
composition of the final
cellular product
Characterisation and composition of the final
cellular product
Quality control of enriched antigen-specific T cells:
Assessments of:
•Identity
•Purity
•Potency
Flow-cytometric analysis of the final product
Potency
•A relevant biological function of antigen-specific T cells is their ability to
secrete IFN-gamma upon stimulation.
•The isolation process itself is a suitable form of potency assay.
May 12, 2014
Hermann Bohnenkamp // Clinical Marketing // CliniMACS Prodigy CCS-IFN System
Page 24
Identity
1. The product is characterized by flow cytometry
• Number of leukocytes
• T cells, IFN-g+/CD4+ and /CD8+ T cells
2. Subsequent expansion of an aliquot (14 days) for
determination of single specificities (not release relevant)
• Very low cell number directly after isolation
• Determination of % of CMV-, AdV- and EBV-specific T cells
21-Nov-2013
EMA/HTA advice
Confidential
Flow-cytometric analysis of the final product
Panel design
Excitation
Emission
Filter
Fluorochrome
Specificity
405nm
450nm
VioBlue
CD45
488nm
525nm
FITC
CD3
488nm
585nm
PE
IFN-
488nm
655-730nm
PerCP
PI/CD14/CD20
638nm
655-730nm
APC
CD4
638nm
750nm
APC-Vio770
CD8
May 12, 2014
Hermann Bohnenkamp // Clinical Marketing // CliniMACS Prodigy CCS-IFN System
Page 26
Flow-cytometric analysis of the final product
Gating strategy – number of leukocytes
1: time-gate
2: single cells
3: CD45+ cells
4: leukocytes (debris excluded)
4a: viable leukocytes
5: lymphocytes
5a: viable lymphocytes
May 12, 2014
Hermann Bohnenkamp // Clinical Marketing // CliniMACS Prodigy CCS-IFN System
Page 27
Flow-cytometric analysis of the final product
Gating protocol – CD3+ T cell frequency
1: time-gate
2: single cells
3: CD45+ cells
4: cells (debris excluded)
5: viable cells
6: CD3+ cells
May 12, 2014
Hermann Bohnenkamp // Clinical Marketing // CliniMACS Prodigy CCS-IFN System
Page 28
Flow-cytometric analysis of the final product
Gating protocol – IFN-+ T cell frequency
1: time-gate
2: single cells
3: CD45+ cells
4: viable CD14- CD20- cells
5: lymphocytes
6: CD3+ cells
7: CD4+ cells
7a: CD4+ IFN-γ+ cells
8: CD8+ cells
8a: CD8+ IFN-γ+ cells
May 12, 2014
Hermann Bohnenkamp // Clinical Marketing // CliniMACS Prodigy CCS-IFN System
Page 29
Composition of final cell product
Final product (n=8)
Median
Min
Max
Total number of viable leukocytes
3.13×105
9.89×104
2.45×106
% CD3+ T cells
56.34
45.14
77.83
Total number of viable T cells
1.90×105
4.73×104
1.12×106
Total number of IFN-g+/CD4+ T cells
4.63×104
2.06×103
2.91×105
Total number of IFN-g+/CD8+ T cells
1.20×104
2.81×103
6.23×105
May 12, 2014
Hermann Bohnenkamp // Clinical Marketing // CliniMACS Prodigy CCS-IFN System
Page 30
Purity of the starting material and the final
product
• Depletion of all unwanted cells, including potentially
alloreactive T-cells by at least 99.9%
• Safer, more specific treatment than unselected DLIs
May 12, 2014
Hermann Bohnenkamp // Clinical Marketing // CliniMACS Prodigy CCS-IFN System
Page 31
Characterisation and composition of the final
cellular product
Quality control of enriched antigen-specific T cells:
Further assessments of:
•Sterilty
•Alloreactivity
•Shelf life
•Phenotype/Genotype
May 12, 2014
Hermann Bohnenkamp // Clinical Marketing // CliniMACS Prodigy CCS-IFN System
Page 32
Summary
Tri-virus-specific T cells
• Automated enrichment of tri-virus-specific T cells using the
CliniMACS CCS (IFN-gamma)
• For the quality development a robust flow cytomteric
platform has been developed:
• Pre-mixed panels (Kits)
• Single platform analysis with automated measurment and
predefined settings and aquisition templates
• Application of automated gating strategy to data files
Tri-virus-specific T cells isolated with the
CliniMACS Prodigy CCS-IFN System: Quality
development of an ATMP
Thank you!

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