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Preparation of Highly Purified Regulatory FoxP3+ T Cells Preparation of Highly Purified Regulatory FoxP3+ T Cells
for Clinical Applicationsfor Clinical Applications
FoxP3+ Regulatory T cellsFoxP3+ Regulatory T cells
Application of regulatory T cells in allogeneic BM Application of regulatory T cells in allogeneic BM transplantationtransplantation
Interest in adoptive Treg immunotherapy for autoimmune diseases and Interest in adoptive Treg immunotherapy for autoimmune diseases and GVHDGVHD
T cell depletion decreases incidence and severity of GVHD but increases T cell depletion decreases incidence and severity of GVHD but increases the risk of graft rejection, relapse, and post-transplant infectionthe risk of graft rejection, relapse, and post-transplant infection
Haploidentical donor T cells increase risk of severe GVHDHaploidentical donor T cells increase risk of severe GVHD
Nonablative conditioning regimens for BMT may be unsuitable for Nonablative conditioning regimens for BMT may be unsuitable for patients with high risk diseasepatients with high risk disease
Steroid-refractory acute and chronic GVHD Steroid-refractory acute and chronic GVHD
Graft vs. Host DiseaseGraft vs. Host Disease
Major cause of transplant-related morbidity and mortalityMajor cause of transplant-related morbidity and mortality Chronic GVHD occurs in ~ 30%-40% of BMT recipientsChronic GVHD occurs in ~ 30%-40% of BMT recipients Acute GVHD ~5%-10% occurrenceAcute GVHD ~5%-10% occurrence T-cell proliferation initiated by HLA-mismatched APC interactionT-cell proliferation initiated by HLA-mismatched APC interaction Target organs:Target organs:
Infusion of Treg prior to Tcon enhances GVHD suppressionInfusion of Treg prior to Tcon enhances GVHD suppression
Tumor clearance is not suppressed in Treg recipientsTumor clearance is not suppressed in Treg recipients
From Nguyen et al. Blood, Vol. 109, 2007
Other Ongoing Treg TrialsOther Ongoing Treg Trials
M. Martelli, et al (Perugia)M. Martelli, et al (Perugia)• CD25-enriched Donor Apheresis (haploidentical related BMT)CD25-enriched Donor Apheresis (haploidentical related BMT)
J. Bluestone (UCSF)J. Bluestone (UCSF)• CD24+CD25hiCD127dim sorting + expansion (autologous for Type 1 CD24+CD25hiCD127dim sorting + expansion (autologous for Type 1
Diabetes)Diabetes)
B. Blazar (Minn.) B. Blazar (Minn.) • CD25-enriched with expansion (mismatched unrelated allogeneic BMT)CD25-enriched with expansion (mismatched unrelated allogeneic BMT)
Project goals:Project goals:
• Select Treg to high puritySelect Treg to high purity
• Administer donor regulatory T cells (Treg) to minimize GVHD without Administer donor regulatory T cells (Treg) to minimize GVHD without losing beneficial effects, especially GVLlosing beneficial effects, especially GVL
• Engineer donor grafts to include specific doses of Treg and Tcon Engineer donor grafts to include specific doses of Treg and Tcon without using cell expansionwithout using cell expansion
• Prepare Treg in accordance with regulatory standardsPrepare Treg in accordance with regulatory standards
• Conduct dose escalation study up to 3x10Conduct dose escalation study up to 3x1066 Treg/kg recip. body weight Treg/kg recip. body weight
• Phase 1/2 safety and feasibility trialsPhase 1/2 safety and feasibility trials
Application of Treg in BM TransplantationApplication of Treg in BM Transplantation
Microbial contamination – Microbial contamination – Exposure during “open air” processingExposure during “open air” processing Sterility of reagents and excipientsSterility of reagents and excipients Sterility of sorter fluid pathwaySterility of sorter fluid pathway
Product stability – Product stability – Affect of cryopreservation and thawing on cellAffect of cryopreservation and thawing on cell
Functionality of sorted cells –Functionality of sorted cells – Potency assay predictive of GVHD riskPotency assay predictive of GVHD risk
Unlicensed devices – Unlicensed devices – CliniMACS currently operated under IDE for CliniMACS currently operated under IDE for most applicationsmost applications
Influx sorter not used in a prior clinical trial(s)Influx sorter not used in a prior clinical trial(s)
Regulatory concernsRegulatory concerns
CD25 CliniMACS selection from mobilized peripheral CD25 CliniMACS selection from mobilized peripheral blood enriches for FoxP3blood enriches for FoxP3+ + Treg cells Treg cells
CD4 FITC CD25 PE-Cy7
Ungated Ungated CD4 gated (CD4 gated)
FoxP
3 A
lexa
-647
FoxP
3 A
lexa
-647
SS
C
SS
C
CD34 depleted LP CD25 selected cells
32
Most CD25Most CD25++ FoxP3 FoxP3++ cells are CD127 cells are CD127dimdim
Further Treg enrichment requires an additional markersFurther Treg enrichment requires an additional markers
Can use only CD4 and CD127 as sorting markers on CD25-Can use only CD4 and CD127 as sorting markers on CD25-enriched PBMC to obtain high-purity Tregsenriched PBMC to obtain high-purity Tregs
Source of materialsSource of materials Antibodies Antibodies
Sheath fluidSheath fluid Collection/storage medium and additivesCollection/storage medium and additives
Staining preparation for large scale sortStaining preparation for large scale sort Fluorochrome selection/toxicityFluorochrome selection/toxicity
AlignmentAlignment Manual with cells or beadsManual with cells or beads BeadsBeads
Drop delayDrop delay Manual with cells or beadsManual with cells or beads Accudrop beads (Automated)Accudrop beads (Automated)
High speed cell sorting for clinical applicationsHigh speed cell sorting for clinical applications
Influx Cell Sorter -Influx Cell Sorter -
Developed by Cytopeia, Inc (Seattle, WA); manufactured by BD Developed by Cytopeia, Inc (Seattle, WA); manufactured by BD Immunocytometry Systems (San Jose, CA)Immunocytometry Systems (San Jose, CA)
• Efficiency: Efficiency: >> 80% recovery of target cells 80% recovery of target cells
• Abort rate: currently <10% Abort rate: currently <10%
• Cell viability: > 95%Cell viability: > 95%
• Class II certifiable HEPA enclosureClass II certifiable HEPA enclosure
• Containment of instrument to maintain aseptic environmentContainment of instrument to maintain aseptic environment• Replaceable fluid pathway includes nozzle assembly, sample line, sheath linesReplaceable fluid pathway includes nozzle assembly, sample line, sheath lines• IDE-grade Miltenyi CliniMACS PBS as sheathIDE-grade Miltenyi CliniMACS PBS as sheath• Sheath line adapter with spikeSheath line adapter with spike
Preventing contamination of sorted cellsPreventing contamination of sorted cells
Available packaged and Gamma-irradiatedAvailable packaged and Gamma-irradiated DMF availableDMF available Manufacturer DocumentationManufacturer Documentation
Completing the closed fluidics pathCompleting the closed fluidics path
In-house Spike AdapterIn-house Spike Adapter•Tubing and connectors validated Tubing and connectors validated for human usefor human use•Documentation and SOPsDocumentation and SOPs•Integrity testingIntegrity testing•EtO sterilization by ISO 13485-EtO sterilization by ISO 13485-certified CMOcertified CMO•Initial and long-term sterility and Initial and long-term sterility and endotoxin testing programendotoxin testing program
Pouring buffer into tank not good idea for aseptic sortingPouring buffer into tank not good idea for aseptic sorting
How is the Influx set up?How is the Influx set up?
Currently no GMP-grade calibration or drop delay beadsCurrently no GMP-grade calibration or drop delay beads
SOSO
Validated processes and SOPs were created for:Validated processes and SOPs were created for:
Manual stream alignmentManual stream alignment
Manual laser/scatter alignment with pt cellsManual laser/scatter alignment with pt cells
Drop frequency/Piezo/PressureDrop frequency/Piezo/Pressure
Manual drop delay process with pt cellsManual drop delay process with pt cells• Drop deposition onto AO-coated slideDrop deposition onto AO-coated slide
Points to Consider in the Manufacture and Testing of Monoclonal Antibody Points to Consider in the Manufacture and Testing of Monoclonal Antibody Products for Human Use (1997)Products for Human Use (1997)
““As a general guidance, we recommend that each purification protocol include at As a general guidance, we recommend that each purification protocol include at least two orthogonal (i.e. based on different mechanisms) robust virus removal least two orthogonal (i.e. based on different mechanisms) robust virus removal steps (see below). Including these steps would not obviate the need for virus steps (see below). Including these steps would not obviate the need for virus clearance studies, except in the case of products intended for use in feasibility clearance studies, except in the case of products intended for use in feasibility trials in serious or life-threatening conditionstrials in serious or life-threatening conditions”.”.
Problem: No GMP-grade Mab reagents available at start of initial Treg trialProblem: No GMP-grade Mab reagents available at start of initial Treg trial
Solution: Solution: CD4 and CD127 Mab engineering run material made CD4 and CD127 Mab engineering run material made available from vendoravailable from vendorRepurify with FDA approvalRepurify with FDA approvalUse until GMP Mabs availableUse until GMP Mabs available
Monoclonal antibody reagents for cell sortingMonoclonal antibody reagents for cell sorting
Monoclonal antibody in PBS by manufacturer
Ig purification by Protein A chromatography
Low pH elution with 0.1M Citric Acid, pH 2.5
pH adjustment to 3.5 with sterile Na-Citrate, 1 hour hold
Neutralization with sterile Na-Citrate
Viral load reduction by passage through Viresolve NFP capsule filter
Succinimidyl ester mediated conjugation of Ig with fluorochrome
Separation of labeled Ig from unbound fluorochrome by Sephadex chromatography
Concentration of conjugated antibodies by UFDF to 200 micrograms/ml
Sterile filtration (0.22 μm), aliquot and freeze (-86°C)
Purity testing:- Western Blot analysis ((≥ 95% IgG)- Protein A concentration (≥ 0.2 mg/ml)
Sterility testing compliant with USP <71>
Specificity and fluorescence intensity assessment by flow cytometry
Ultrafiltration/diafiltration with sterile PBS
Endotoxin by LAL (< 5 EU/ml)
Monoclonal reagent repurification processMonoclonal reagent repurification process
In Process TestingIn Process Testing
• Purity: SDS-PAGE on purified antibodyPurity: SDS-PAGE on purified antibody Residual Protein A testingResidual Protein A testing
• Viral Clearance testingViral Clearance testing
1)1) A Amplification on mplification on Mus dunniMus dunni cells; supernatant co-culture cells; supernatant co-culture withwith PG4 S+L- cellsPG4 S+L- cells 2) Co-incubation with cell lines MRC-5, VERO, NIH 3T3; 2) Co-incubation with cell lines MRC-5, VERO, NIH 3T3; assess hemadsorption, hemagglutination and cytopathic changes assess hemadsorption, hemagglutination and cytopathic changes
Antibodies for Selection, Sorting and Identity Assessment Antibodies for Selection, Sorting and Identity Assessment
General parameters to consider for antibodies General parameters to consider for antibodies • GMP compliant preparative antibodies GMP compliant preparative antibodies • Antibodies recognizing unique epitopes from preparative absAntibodies recognizing unique epitopes from preparative abs
• Specificity in detecting target antigens Specificity in detecting target antigens • Compatibility with process and other reagents (antigen loss Compatibility with process and other reagents (antigen loss
during fixation, spectral overlap, sensitivity) during fixation, spectral overlap, sensitivity) • ASR (analyte specific reagents) antibodies when available for ASR (analyte specific reagents) antibodies when available for
release analysis. Validate RUO abs for analysis. Commercial release analysis. Validate RUO abs for analysis. Commercial FC controls where appropriateFC controls where appropriate
Examples:Examples:• Use of an independent marker to establish purity (FoxP3 in Treg)Use of an independent marker to establish purity (FoxP3 in Treg)• Use of antibodies recognizing non-cross inhibiting epitopes to Use of antibodies recognizing non-cross inhibiting epitopes to
CD4 for sorting and analysis CD4 for sorting and analysis
FoxP3+ Post-Sort Cell Analysis for Treg PurityFoxP3+ Post-Sort Cell Analysis for Treg Purity
Validated independent Validated independent measurement of CD4 and measurement of CD4 and
FoxP3FoxP3
*CD4 clone L200 for sorting & SK3 for analysis**CD4 clone L200 for sorting & SK3 for analysis**CD25 ab staining blocked by selection reagent**CD25 ab staining blocked by selection reagent*
Infusion Release Criteria:Infusion Release Criteria:EndotoxinEndotoxinFoxP3%FoxP3%ViabilityViabilitySterility by Sterility by Gram stainGram stainCell CountCell CountDoseDose
Reportable Data:Reportable Data:14-day sterility14-day sterilityAncillary phenotyping by flowAncillary phenotyping by flowMLR inhibitionMLR inhibition
Thawed Treg Thawed Treg InfusionInfusion
Sorting of CD4+/CD127Sorting of CD4+/CD127lowlow cells improves purity of Treg cells improves purity of Treg compared to CD25+ enrichmentcompared to CD25+ enrichment
Treg (>1X10Treg (>1X1066/kg to 3X10/kg to 3X1066/kg)/kg) Validated reagents for independent verification of Treg Validated reagents for independent verification of Treg
content for product releasecontent for product release Vendor initiated internal GMP Mab quality processVendor initiated internal GMP Mab quality process
AcknowlegementsAcknowlegements
Our Stanford BMT Patients & Their FamiliesOur Stanford BMT Patients & Their Families
Stanford HealthcareStanford HealthcareDavid DiGiusto, PhD. - Executive Director of Stem Cell and David DiGiusto, PhD. - Executive Director of Stem Cell and Cellular Therapy OperationsCellular Therapy Operations