Corporate Tutorial IBA T-CATCH. Cell isolation in pipette tips. ISCT, Paris Lothar Germeroth

Transcription

1 Corporate Tutorial IBA T-CATCH Cell isolation in pipette tips ISCT, Paris 2014 Lothar Germeroth

2 Workshop IBA Introduction of the Streptamer technology Staining with Streptamers off rate assay T-CATCH Cell isolation in pipette tips 2

3 Workshop IBA Introduction of the Streptamer technology Staining with Streptamers off rate assay T-CATCH Cell isolation in pipette tips 3

4 The Streptamer principle reversible staining and isolation MHC - Streptamers Streptag / Streptactin K D =10-6 M fluorochrome D-biotin / Streptactin + D-biotin K D =10-12 M HLA-A*0201/pp Streptamer Knabel, M. et al., 2002, Nat Med CD8 4

5 The Streptamer Complexes MHC-Streptamers Fab-Streptamers Streptactin 5 5

6 basic principle of Fab-Streptamers antigen-binding site antibody recombinant Fab-Streptag monomers affinity modification V L V H C L C H Fc Streptag low binding affinity, fast k off Streptag / Streptactin K D =10-6 M Fab-Streptamers D-biotin / Streptactin K D =10-12 M Knabel et al. 2002, Nat. Med. Stemberger et al. 2012, PloS One

7 basic principle of Fab-Streptamers Fab-multimer Fab-multimer D-biotin Fab-multimer D-biotin ST-PE Fab-multimer D-biotin Fab-multimer CD3 Fab-multimer / Fabmonomer FL1 - FITC

8 Why reversible staining and isolation? Because remaining cell label / sort marker can cause: 1.) changes of the cell population cross linking, activation, receptor blockade, anergy or cell death, depletion 2.) limitations in cell isolation no serial positive enrichment possible negative selection needs complex antibody mixtures 3.) regulatory and clinical issues toxicity, immunogenicity, allergic reactions, classification 4.) economical aspects very difficult process for reagent approval, expensive GMP conform reagents 8

9 Is the dissociation with biotin complete? MHC Neg. control Streptamer Streptamer+bio 100 unstained STPE STPE+bio human: A2 pp iba Gmb H FACS sensitivity: app 100 molecules per cell 9

10 Complete dissociation enables serial selection! marker #1 selection cycle marker #2 selection cycle marker #3 Selection cycle marker1 + fraction marker1 + marker2 + fraction marker1 + marker2 + marker3 + final fraction Streptamers 80nm 2µm Knabel et al. 2002, Nat. Med. Stemberger et al. 2012, PloS One

11 Influence of remaining (sort) marker can the release of bound reagents prevent activation? (early events) activation (intermediate) proliferation (late) + stimulus + ionomycin h 52.8 CD CFSE p-zap70 acd3 (OKT3) CD3- multimer p-erk CD3 multimer + Bio Stim [min] ap-zap70 unstimulated Fab-multimer + D-biotin Fab-multimer no D-biotin mab (OKT3) agapdh

12 Influence of remaining (sort) marker can the release of bound reagents increase effector function? CD25 mab CD4 mab p= CD25 Fab- Streptamer CD4 Fab- Streptamer mab Fab-Streptamer 12 mab Fab- Streptamer page 12 iba Gmb H 12

13 Summary Complete dissociation No stimulation No interference with effector function Serial positive selections possible 13

14 Workshop IBA Introduction of the Streptamer technology Staining with Streptamers off rate assay T-CATCH Cell isolation in pipette tips 14

15 Applications of the Streptamer technology off rate assay 1. antigen-specific staining 2. d-biotin 3. rapid (s) backbone dissociation 4. Dissociation of monomeric MHC I from TCR page 15 page 15

16 Applications of the Streptamer technology Flow cytometry based off rate assay Streptactin PE Biotin directly in FACS tube His-Tag on C L OregonGreen 488 Chromeo488 His adapter technology F. Mohr and C. Stemberger, MIH, unpublished 16

17 Flow cytometry based based k off assay +D-biotin FL1 - FITC FL2 - PE Fab-monomer StrepTactin backbone F. Mohr and C. Stemberger, MIH, unpublished

18 basic principle of Fab-Streptamers original FACS files analysis fit exponential decay t 1/2 CD4 monomer (OregonGreen488) FL1 - FITC time Streptactin backbone (PE) FL2 - PE time

19 Real-time kinetic measurement of MHC I: T-cell dissociation Regression : y = a * e koff * t fluorescence intensity k off = -0,0038; t 1/2 = 182,41 sec k off = -0,0189; t 1/2 = 36,67 sec time [sec] T cell clone A T cell clone B F. Mohr and C. Stemberger, MIH, unpublished page 19

20 Application of off rate assay Direct determination of TCR affinity for TAA Determination of Fab (scfv) affinities CAR Available upon request 20

21 Workshop IBA Introduction of the Streptamer technology Staining with Streptamers off rate assay T-CATCH Cell isolation in pipette tips 21

22 T-CATCH Cell isolation in pipette tips What is T-CATCH? T-CATCH is an acronym for Tip based Cell AffiniTy Chromatography no magnetic selection process cell selection technology based on the binding of cells to immobilized resins in pipette tips automatization, parallel isolations 22

23 Streptamer selection technologies Nanobeads small ~70nm Microbeads medium ~1.5µm Macrobeads large ~80 µm Non magnetic CATCH Bead Bead cells Fab Fab Fab Bead 23

24 Why non-magnetic selection with T-CATCH? Better yields and purities Faster process times (no centrifugation) Ideally suited for automatisation Enable parallel, serial isolations Clinical selection possible 24

25 T-CATCH magnet free cell isolation Streptactin coated biopolymer matrix > 80µm Fab

26 T-CATCH magnet free cell isolation + D-biotin 100µl resin target cells + D-biotin

27 The CATCH Tips Resin embedded in pipette tips Top screen Resin maintained in discrete area Bottom screen 27

28 T-CATCH magnet free cell isolation Selection tip Removal tip Streptactin coated matrix + Fab Superflow-matrix PBMC or whole blood biotin eluted cell fraction label free target cell population

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30 T-CATCH magnet free cell isolation CD4 enrichment from PBMCs SSC PI 99.1 before 30.3 FSC CD25 negative fraction 2.3 positive fraction CD3 CD % yield 85% recovery

31 T-CATCH magnet free cell isolation CD19 enrichment from whole blood positive fraction CD3 negative fraction SSC PI before FSC FL2 - PE CD19 97% yield 95% recovery

32 n=54 from 18 experiments n=22 from 6 experiments T-CATCH magnet free cell isolation 1ml pipette tip 100µl resin automatization 12 - Speed Catch Streptactin coated selection matrix loading wash positive fraction

33 Serial CATCH tip selection of Tregs (CD4+CD25+) CD4 CD25 PBMC load CD4+ fraction 1*10^7 2,5*10^6 2,5*10^6 Wash 0,09*10^6 0,3*10^6 Biotin elution 2,5*10^6 2,2*10^6 Purity 99% 94% Recovery 97% 82% 33

34 EASY CATCH Isolation system Semi automated system: 8 isolations parallel 3 isolations serial 34

35 SPEED CATCH Isolation System Fully automated system: 12 isolations parallel 3 isolations serial 35

36 Fab Ligands for CATCH Produkt Species Celltype CD4 mouse T CD25 mouse T CD62L mouse T CD3 mouse T CD8 mouse T CD4 Human T CD25 Human Tregs CD62L Human T CD3 Human T CD8 Human T CD45RA Human Naive T CD45RO Human Memory T CD34 Human HSC CD56 Human NK CD154 Human Act. T CD137 Human Act. T CD 105 Human MSC CD73 Human MSC 36

37 T-CATCH Products available end of QII Semi-Automat EASY CATCH Full-Automat SPEED CATCH CDx Tip-S-100 kit CDx Tip-S-1000 kit Tip-R-300 Tip-R , 48, 96 tips per kit; 12,11,10 EUR per tip (capacity 10 million cells per tip) 10 tips per kit 70 per tip (capacity 100 million cells per tip) 12,24, 48, 96 tips per kit (Removal of Fab and biotin for S- 100 kits) Removal of of Fab and biotin for S kits) Fabs MHCs Buffer Buffer set included in the kit price included in the kit price included in the kit price included in the kit price

38 Clinical Streptamer CATCH Selection closed open 38

39 Clinical Streptamer CATCH Selection

40 Clinical Streptamer based Cell Products key advantages No need for GMP-production of Streptamers due to complete dissociation of reagents Not classified as ATMP from EMEA Not classified as medicinal product but as transplant similar to DLI from PEI (directed homologues use) No need for clinical trials for regulatory approval but for reimbursement Optimal regulatory situation for Streptamer-based cell therapeutics 40

41 Summary Streptamer T-CATCH Technology Fully functional effector cells Excellent purities and yields even from whole blood Very uncomplicated and rapid selection process Rapid serial positive selection processes possible Selections from whole blood possible HTS compatible Option for clinical use 41