Flow Cytometric Preparation of CD34 + CD90 + Hematopoietic Stem Cells for Transplantation

Transcription

1 Flow Cytometric Preparation of CD34 + CD9 + Hematopoietic Stem Cells for Transplantation B R YA N F OX S TA N F O R D L A B O R AT O R Y F O R C ELL & G ENE M E D I C I N E ISCT Regional Meeting Memphis,TN USA September 3 October 2, 216

2 SCID patients transplanted with purified Blood Stem Cells and Non-Toxic Drugs Chhabra, A., A. M. Ring, K. Weiskopf, P. J. Schnorr, S. Gordon, A. C. Le, H. S. Kwon, et al. "Hematopoietic Stem Cell Transplantation in Immunocompetent Hosts without Radiation or Chemotherapy." Sci Transl Med 8, no. 351 (Aug 1 216): 351ra15. CLINICAL TRIAL APPROVED BY FDA CD34 IN APRIL + CD blood stem cells from patients to correct defective genes FIRST ENROLMENT EXPECTED OCTOBER 216 from healthy donors to replace patients stem cells ADMINISTRATION OF acd117mab TO CREATE SPACE WITHOUT THE TOXICITY OF CHEMOTHERAPY OR RADIATION Irv Weissman & Judy Shizuru 2

3 CD34+ cells are heterogeneous population of stem and progenitor cells CLP HSC MPP T, B & NK Selfrenewing CMP CD34+ CD9+ CD38- Lin- CD34+ CD9- CD38- Lin- CD34+ CD9- CD38+ Lin- Myeloid lineage CD34+CD9+ population contains virtually all long term repopulating cells

4 High Purity HSC by Sequential Process: Immunomagnetic CD34+ Enrichment and CD34+CD9+ Sorting Raw Materials Manufacturing Process Step for Sorted CD34+/CD9+ enrichment In-Process/QC Testing Step 1 Apheresis collection Cell Count Raw Viability Materials Phenotype CliniMACS CD34 Reagent CliniMACS Buffer Human Serum Albumin Step 2 Cell product preparation for CD34 selection CliniMACS LS Column CliniMACS System Step 3 CliniMACS CD34 selection Mouse anti-human CD34-APC Mouse anti-human CD9-RPE. Step 4 CD34/CD9 reagent labeling BD Influx Cell Sorter Step 5 Flow cytometric cell sorting CryoStor CS5 Step 6 Release testing Viability Phenotype CFU assay Step 7 Cryopreservation of Final Product Sterility Endotoxin

5 High Purity HSC by Sequential Process: Immunomagnetic CD34+ Enrichment and CD34+CD9+ Sorting Challenge 1: Adapt and Qualify a RUO Instrument for Therapeutic Cell Sorting Challenge 2: Qualify Sorting and Analysis Reagents Challenge 3: Qualify Sorting Process to Meet Specifications Challenge 4: SCID trial: Establish Absence of Donor Chimerism in Prior Transplant Recipients

6 Challenge 1: Adapt RUO instrument for Therapeutic Cell Sorting First Stanford IND involving sequential Treg enrichment by CliniMACS CD25 selection and cell sorting approved in 28 BD InFlux has been used in three separate Treg Clinical Trials; allows reference for CD34+CD9+ HSC IND submission Treg selection and sort processes neatly translatable to CD34+CD9+ sorting

7 Challenge 1: Adapt and RUO instrument for Therapeutic Cell Sorting Replaceable, g-irradiated fluid pathway includes nozzle assembly, sample line, sheath lines Spike Adapter manufactured In-house to complete closed fluidics path GMP grade, sterile D-PBS in spikable bag as sheath Containment of instrument to maintain aseptic environment HEPA enclosure and BioProtect IV BSC (LCGM)

8 Challenge 2: Qualification of sorting reagents PR3a: a-cd34 Mab, APC-conjugated IgG3 PR13: a-cd9 Mab, PE-conjugated IgG1 CD34 and CD9 hybridomas used for Stanford breast cancer clinical trial in the 199s Hybridomas transferred to CMO for MCB construction, testing, Mab purification, fluorochrome conjugation & fill Fill formulation, stability and performance testing performed at Stanford

9 Challenge 2: Qualification of sorting reagents Selection/sorting/release testing antibodies recognize the same markers: Processing Step CD34 Clone CD9 Clone CD34 Enrichment AC11 N/A CD34+/CD9+ Sorting PR3a PR13 8G12 I II III CD34 AC11 PR3a Post-sort Release Analysis 8G12 5E1 CD34 clone 8G12 available in ASR grade Commercially available flow cytometry controls for release assay QC

10 CD34 Selection and Sorting Reagents: Absence of steric hindrance Prestain KG1a CD34 with AC11 Mab Confirm AC11 Binding with 2 o Ab -AC11 +AC11 Sequential PR3a-APC staining isotype +AC11 +PR3a-APC +PR3a-APC Anti-CD34 monoclonal for Selection (AC11) has no detectable impact on PR3a staining of CD34+ cells Compare w/pr3a-apc alone staining

11 Count Count CD34/CD9 Sorting and Analysis Reagents: Absence of Steric Hindrance Isotype Kg1a +PR3a Isotype +8G12 Primary Cells G-CSF Mobilized PBMC CD45+ Gated G-CSF Mobilized PBMC CD34+ Gated PR3a +8G PR3a +8G12 CD34-APC PR3a CD34-PE 8g Isotype +PR13 +5E1 +PR13 Jurkat Isotype +PR13 +5E1 +5E1 PR3a and clone 8G12 sequential staining PR13 and clone 5E1 sequential staining Sorting Antibodies PR3a and PR13 have no impact on post-sort detection using analytical antibodies CD9-PE PR13 CD9BV421 5E1

12 CD9-PE CD9-PE CD9-PE SSC CD9 5E1 pulse width SSC Gating Strategy for Flow Cytometric Cell Sorting of CD34+/CD9+ Cells Products CD34+CD9+ Stained cells ungated CD34+CD9+ Stained cells Singlets gate A B Singlets 96.7% Lymphocytes 81.6% Post Sort Release Analysis CD34+CD9+ Stained cells Lymphocyte FCS gate CD34+CD9+ Stained cells FCS Lymphocyte gate CD45+ Gated %.% 1 3 C Unstained D CD34+ Sort gate 99.3% %.% 1 CD34+CD Stained 1 2 cells CD34+ CD34-APC sort gate 1 4.% 46.51% E CD34+CD9+ Sort Gate % 1 post 1CD34+CD sort Viable CD34-APC cell gated 1 4.5% F % Ungated Post Sort CD34 CD34 8G12 8G12 7-AAD % 53.49% 1 CD34-APC %.82% 1 CD34-APC

13 CD3 CD3 CD3 T-cell Depletion Pre-CD34 Enrichment % 31.88% Post CD34 Enrichment %.1% Post CD34+CD9+ Sort 1 4.9%.4% CD45+ Gated CD3 TCR a/b % 4.88% 1 TCR alpha/beta 99.72%.2% 1 TCR alpha/beta 99.94%.5% 1 TCR alpha/beta

14 Validation Run Cell Testing Assay Type Specifications RHPC-3 RHPC-4 RHPC-5 Viability 7% Viable Cells 99.3% 99.7% 99.3% CFU > 4 CFU/1 cells plated Sterility, Modified USP test Negative for bacteria and Fungus after Negative Negative Negative 14 days Endotoxin-LAL <.5 EU/mL <.5 EU/mL <.5 EU/mL <.5 EU/mL CD34 + CD9 + : 99.99% CD34 + CD9 + : 99.99% CD34 + CD9 + : 99.96% Phenotypic Analysis/Identity > 98% CD34 + CD9 + CD3 + : <.1% CD14 + : <.1% CD19 + : <.1% CD3 + : <.1% CD14 + : <.1% CD19 + : <.1% CD3 + : <.1% CD14 + : <.1% CD19 + : <.1% CD56 + : <.1% CD56 + : <.1% CD56 + : <.1%

15 Validation Run Cell Yields and Dosage Estimates RHPCA3 Fraction TNC CD3 Count CD3/Kg CD3 Log Depletion (max) (2Kg patient) HPC-A 42X1 9 12X1 9 4X1 6 CD X X1 5 2.X CD34 + /CD9 + 58X RHPCA4 Fraction TNC CD3 Count CD3/Kg CD3 Log Depletion HPC-A 22X1 9 13X1 9 42X1 6 CD X X CD34 + /CD9 + 11X RHPCA5 Fraction TNC CD3 Count CD3/Kg CD3 Log Depletion HPC-A 33X X1 9 29X1 6 CD X1 6 2.X1 5 1.X CD34 + /CD9 + 35X >5X1 5 /kg CD34+CD9+ HSC target dose 3X1 4 /kg max. CD3 dose Achieved 6-7 logs of CD3 depletion ~2 logs CD3 depletion beyond CD34 enrichment alone

16 Challenge 4: Assessment of Donor Chimerism for SCID Trial Inclusion Prior transplant recipient inclusion: Donor chimerism must be undetectable Status of donor chimerism based on genomic DNA, Short Tandem Repeat (STR) analysis of recipient peripheral white blood cells (1% detection limit) Lymphocytes can persist for years whereas granulocytes are turned over rapidly. Analysis of granulocytes closer to real time status of donor chimerism. Isolate CD15+ granulocytes containing <1% contaminating white blood cells from other lineages by flow cytometric cell sorting

17 Generation of Donor Chimerism A STR Analysis A (Recipient) B (Donor) B STR Analysis Spike to 25% B CD3+ RBC Deplete 1 ml aliquot CD15+ sort RBC Deplete 1 ml aliquot CD15+ sort RBC Deplete 2 ml aliquot 495, Sorted CD15+/CD45+/ SSC hi cells 5, Sorted CD15+/CD45+/ SSC hi cells Sort CD3+ Sort CD15+/ CD45+/SSC hi CD3+ purity CD15+ purity AB15 STR analysis AB3 STR analysis Add 1% CD15+ B (5, cells)

18 SSC SSC SSC pulse width SSC 7AAD SSC Sort Gating Strategy of SSC hi CD15+ Granulocytes CD SSChiCD Viable CD Singlets FCS *CD45-V45 1 CD45-V45 *CD15-APC SSC hi CD15+ sort %.% % 99.7% CD3+ Lymphs *CD3-FITC SSC lo CD3+ sort %.1% *CD3-FITC %.% *CD15-APC

19 Percent Donor Chimerism Determined by STR Analysis Post Sort Donor Chimerism Recipient:Donor Pair 25% CD3 Spike 1% CD15 Spike AB % % CD % 1% EF % 1% GH % 2% IJ % 1%

20 In Conclusion Robust CD34+CD9+ Cell Sorting Process for Approved SCID Trial Adapted and Qualified BD Influx for Therapeutic Cell Sorting Qualified Preparative and Analytical Reagents Qualified Sorting Process Demonstrating the Ability to Meet and Exceed Specifications Preparative Flow Cytometry to Establish Chimerism Status of Prior Transplant Recipients Additional Applications Tumor cell reduction in breast cancer and multiple myeloma Potential gene therapy applications such as genome editing in sickle cell anemia

21 Acknowledgements S TA N F O R D B M T C E L L U L A R T H E R A P Y FA C I L I T Y R AN D A L L A R M S T R O N G K E R I TAT E, P HD C Y N T H I A T U D I S C O K E V I N S H E E H AN, P HD S TA N F O R D B L O O D & MA R R O W T R A N S P L A N TAT I O N P R O G R A M J U D I T H S H I Z U R U, M D, P HD H YE- S OOK K W O N, P HD S TA N F O R D U N I V E R S I T Y S C H O O L OF M E D I C I N E D AV I D D IG I U S T O, P HD - E X E C U T I V E D I R E C T O R OF S T E M C E L L A N D C E L L U L A R T H E R A P Y O P E R AT I O N S I R V I N G W E I S S M AN, M D - D I R E C T O R, IN S T I T U T E OF S T E M C E L L B I O L O G Y A N D R E G E N E R AT I V E M E D I C I N E