ISCT Workshop #7 Perspectives in Cell Selection Immunomagnetic Selection

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1 ISCT Workshop #7 Perspectives in Cell Selection Immunomagnetic Selection Carolyn A. Keever-Taylor, PhD Medical College of Wisconsin June 7, 2012

2 History of Available Devices CellPro CEPRATE Avidin/Biotin System European CE mark 1995 FDA Premarket approval 1996 Removed from market 2000 Isolex Available Pre-Approval 1993 European CE mark 1995 FDA Premarket approval 1997 Withdrawn 2010 Available again???? CliniMACS European CE Mark 1997 Available US under IND/IDE 2003 FDA Humanitarian Use Device # Approved indication 2012?

3 Basic principle of Isolex and CliniMACS devices the same. Target cell is retained in the device based on antibody conjugated with a magnetic particle. For CliniMacs it is a paramagnetic particle, for Isolex antibody is on a magnetic bead. Ab on paramagnetic particle Dynal beads on cells Non-retained cells washed through Target cells recovered

4 CD34 Cell Selection- Isolex Apheresis product (8 x 10 9 MNC) Platelet- plasma wash Antibody sensitization Bead rosetting and depletion Isolex Automated Process CD34 Enriched Product

5 Negative Selection + magnetic microbeads conjugated with OKT3 Positive Selection + magnetic microbeads conjugated with anti-cd 34 All other cells CD34+ cells CD 34 neg cells

6 CliniMACS CD34 Reagent System CliniMACS CD34 Reagent CliniMACS Tubing Sets (Standard and Large Scale) CliniMACS plus Instrument Standard: 0.6 x 10 9 CD34 + Cells from 60 x 10 9 Cells Large Scale: x 10 9 CD34 + Cells from x 10 9 Cells CliniMACS PBS/EDTA Buffer

7 Isolex & CliniMacs Compared- MCW Experience Device N CD34 Purity CD34 Recovery CD3 Log Dep Isolex % ± % ± ± 0.3 CliniMacs % ± % ± ± 0.3 Similar CD34 purity but better recovery and more efficient T cell depletion using CliniMACS CliniMacs has more variety of clinical grade antibody conjugates (i.e. CD3, CD25, CD56, CD14, CD4, CD19) for a wider range of engineering.

8 Location of CliniMACS plus Instruments in the USA 84 protocols using CD34 system 162 instruments within 97 institutions in the U.S.

9 Humanitarian Use Device The device is designed to treat or diagnose a disease or condition that affects fewer than 4,000 individuals per year in the U.S. The device is not available otherwise, and there is no comparable device available to treat or diagnose the disease or condition; and The device will not expose patients to unreasonable or significant risk, and the benefits to health from the use outweigh the risks Clinical data must support safety and probable benefit argument Exempt from effectiveness requirement consistent with the HDE requirements

10 HLA-Identical Sibling-Matched, CD34 + Selected, T cell Depleted Peripheral Blood Stem Cells Following Myeloablative Conditioning For First or Second Remission Acute Myeloid Leukemia (AML): Results of Blood and Marrow Transplant Clinical Trials Network (BMT CTN) Protocol 0303 S Devine, S Carter, R Soiffer, M Pasquini, P Hari, A Stein, H Lazarus, C Linker, E Stadtmauer, E Alyea, C Keever-Taylor, and R O'Reilly

11 Secondary Endpoint: CliniMACS CD34 Reagent System Performance Leukapheresis collections from a Matched Related Donor were performed in order to obtain a minimum of 2.0 x 10 6 CD34 + cells/kg Target of 5.0 x 10 6 CD34 + cells/kg and <10 5 CD3 + cells/kg Up to three collections were allowed to achieve the minimum CD34 + cell dose 86 products were processed for 44 patients The CliniMACS Large Scale (LS) Tubing Set or Standard (STND) Tubing Set could be used

12 Donors and Products 47 patients enrolled, 44 proceeded to transplant 86 products collected Total lots (cells from one tubing set) assessed=84 Collections pooled for 2 patients 4 sites processed from 9 to 34 lots 4 sites processed 4 lots

13 CliniMACS CD34 Reagent System Post Processing Performance N=84 % CD34 + Recovery % CD34 + Purity Log 10 TCD % Viability Mean SD ±20.25 ±8.3 ±0.55 ±3.84 Min Max

14 Final Cellular Product Summary Parameter Mean CD34 + dose Median CD34 + dose Mean CD3 + dose Median CD3 + dose Result 8.81 x 10 6 /kg 7.92 x 10 6 /kg 1.51 x 10 4 /kg 0.7 x 10 4 /kg All gram stains/14 day cultures were negative All endotoxin below detection limits No significant infusion related toxicities observed

15 Center to Center Statistical Analysis Sites processing 9 lots compared individually (N=4) Sites processing 4 lots pooled (N=4) Multivariate analysis used a linear mixed effect model to account for repeated measures ( 2 lots for most patients) Pairwise center comparisons were performed with Tukey-Kramer adjustment for multiple comparisons

16 Processing Logistics Products held overnight diluted to 2.0 x 10 8 /ml using concurrent plasma or CliniMACS PBS/EDTA Buffer Controlled 1-8 C overnight Warmed to room temperature next day Platelet and Antibody wash Cell washer if validated (Cobe 2991) Bag method as described in CliniMACS plus User Manual Cell separation steps done per CliniMACS plus User Manual Split products could be pooled for sampling and testing CD34-enriched products infused on day of processing

17 Starting CD3 Starting CD3 % Living Cells % Living Cells X % Living Cells Starting TNC TNC Starting TNC A A Pre-Processing Cell B Counts Starting CD34 x 10 Starting CD34 x Starting CD34 x BB X C Ctr Ctr 1 Ctr Ctr Ctr 1 22Ctr Ctr Ctr 2 33Ctr Ctr 3Ctr 44 Ctrs Ctrs Ctrs p= p= p= C Center Center Ctr Ctr 1 Ctr 1 Ctr 1Ctr 2 Ctr 2 Ctr 2Ctr 3 Ctr 3 Ctr 3Ctr 4 4Ctrs Ctrs Ctrs Center Center p= p= p= D D Center Ctr 1 Ctr Ctr 1 2 Ctr Ctr 2 3 Ctr 3 Ctr 4 Ctr Ctrs 4 Ctrs Ctr 1 Ctr 2 Ctr 3 Center Center Ctr 4 Ctrs 5-8P= P= Center P= Ctr Ctr 1 1 Ctr Ctr 2 2 Ctr Ctr 3 3 Ctr Ctr 4 Ctrs 4 Ctrs Ctr 1 Ctr 2 Ctr 3 p= Center Center Ctr 4 Ctrs 5-8 p= p= Center Differences attributed to mobilization methods

18 % of Starting TNC % of Starting Abs CD34 % TNC and CD34 + Recovery Post Platelet and Antibody Washes A B Ctr 1 Ctr 2 Ctr 3 Ctr 4 Ctrs 5-8 p=0.007 Center 0 Ctr 1 Ctr 2 Ctr 3 Ctr 4 Ctrs 5-8 p=0.002 Center

19 Cobe 2991 Wash vs. Bag Wash Log TCD Bag Cobe 2991 %CD34 Purity %CD34-Enr Rec P=0.04 %CD34 Wash Rec %TNC Wash Rec

20 Log TCD % Living Cells 7-AAD %CD34 Recovery %CD34 Purity Post Processing Outcomes < Ctr 1 Ctr 2 Ctr 3 Ctr 4 Ctrs 5-8 Center P< Ctr 1 Ctr 2 Ctr 3 Ctr 4 Ctrs 5-8 Center P= Ctr 1 Ctr 2 Ctr 3 Ctr 4 Ctrs 5-8 Center P= Ctr 1 Ctr 2 Ctr 3 Ctr 4 Ctrs 5-8 Center P= All centers were able to process grafts that met the study criteria

21 CD34/kg CD34 + Cells x 10 6 /Kg of Patient Weight Infused CD34 + Target Dose 10 6 Ctr 1 Ctr 2 Ctr 3 Ctr 4 Ctrs 5-8 Center P= CD34 + Minimum Dose All patients received the minimum CD34 + dose (>2.0x10 6 cells/kg) 84.1% of patients received > 5 x 10 6 CD34 + cells/kg

22 CD3/kg CD3 + Cells Infused per kg Upper limit of CD3 + dose 10 3 Ctr 1 Ctr 2 Ctr 3 Ctr 4 Ctrs 5-8 Center P=NS No patients received more than 1.0x10 5 /kg CD3 + cells

23 % of CD34-enriched fraction Impurities in Final Cell Collection T Cells B Cells NK Cells Monocytes Contaminating Subset

24 Apheresis per Patient Performed Per Patient to Meet Minimum CD34 + Cell Dose Post Enrichment Target Cell Dose

25 Log TCD Log TCD % Recovery CD34+ Cells % Recovery CD34+ Cells Start Product Factors and Outcome P=NS TNC Loaded (x ) P= CD34+ Cells Loaded (x 10 7 ) P= TNC Loaded (x ) P= CD3+ Cells Loaded (x 10 9 )

26 Cell Processing Conclusions All sites, and all products met and most exceeded study goals for: CD34 + cell infusion dose > 5 x 10 6 /kg CD3 + cell infusion dose < 1 x 10 5 /kg The performance of the CliniMACS CD34 Reagent System was stable and highly reproducible from center to center, resulting in a consistent high degree of CD34 + cell enrichment, TCD and sterility.

27 Conclusions BMT CTN 0303 HCT following myeloablative preparative regimen for patients with AML in CR1 or CR2 can be performed in a multicenter setting using a single TCD method without additional post transplant pharmacologic GVHD prophylaxis All 1 and most 2º endpoints were met, demonstrating: 81.8% Disease Free Survival 6 months post TX No primary graft failure; Consistent neutrophil and platelet engraftment Acute GVHD <23%. No Grade IV agvhd Chronic GVHD <19% at 2 years TRM <20% at 2 years Low risk of relapse, but dependent on remission status The CliniMACS CD34 System consistently produces a graft with > 5 x 10 6 CD34 + cells/kg and <1 x 10 5 C3 + cells/kg

28 Statistical Comparison of 0101 vs Supports Safety of T-Cell Depletion No significant difference between T-Cell Depleted BMT and T-Cell replete BMT for: DFS, OS, TRM, Platelet Engraftment and Relapse Low numbers of CR2 patients in both cohorts make statistical comparisons of relapse inconclusive 0303 patients experienced more infectious episodes/patient (112/44 patients in 0303 versus 162/84 patients in 0101) Did not translate to higher infectious deaths rates (18.2% of deaths in 0101 vs. 20% in 0303)

29 Statistical Comparison of 0101 vs Supports Probable Benefit Argument for T-Cell Depletion Observed advantages of T-Cell depleted allografts include: Significantly reduced incidence of chronic GVHD (p= ) Earlier neutrophil engraftment (p=.00249) Low rates of acute and chronic GVHD in the absence of more traditional pharmacologic prophylaxis

30 CliniMACS CE Marked Reagents 1 Reagent USE Reagent USE CD34 HPC-Enr IFNg Ag-specific Enr CD133 HPC-Enr CD56 All NK Enr CD3/CD19 2 T/B Dplt CD3/CD56 NK T Enr TCR ab biotin 2 abt Dplt spares gdt cells CD19 B Dplt or Enr CD3 T Dplt or Enrich CD14 Monocyte Enr CD4 T helper Dplt or Enr CD1c-biotin Myeloid DC Enr CD8 T effector Dplt or Enr CD304 Plasmacytoid DC Enr CD25 Treg-Enr Anti-Biotin Multiple CD45RA Naïve T Dplt 1 Requires IND or IDE in US 2 NA in US

31 Challenges in Product Analysis Starting product analysis Dual vs Single platform methods Representative sampling Multiple assays as per site procedures Definition of starting product Final product analysis Rare event Background Cells off device vs cells infused Starting and Final product analysis Viability Sampling

32 CD34 assessment on CD34-Enr Products Pre Processing CD34-Enr

33 CD3 Assessment in CD34-Enriched Product Pre Processing CD34-Enr

34 Starting vs CD3/CD19 Enriched Fraction- Effect on Ab for Flow CD3/CD19 Enr Little effect on CD3, marked reduction in CD19 intensity, no effect when using CD20

35 Gating Scheme to Reduce Background Pre vs CD3/CD19 Reduced Fractions Pre Processing CD3/CD19-Red

36 CD3-Reduced CD56-Enriched vs CD3-Reduced Only

37 Single Center Experience vs Published CD34-E CD3-R/CD56-E CD3-R TC NA NA Median 60% -81% (5 studies 991 patients) NA NA CD3-R CD3/CD19-R % CD34 Recovery Median 53% -90% (6 studies 310 patients) CD34-E CD3-R/CD56-E Median 4.6 ->5.0 (5 studies 991 patients) Median (2 studies 25 subjects) CD3-R TC CD3-R CD3/CD19-R (7 studies 319 patients) CD3 Log Depletion

38 Single Center Experience vs Published CD34-E CD3-R/CD56-E 3.2 (2 studies 166 patients) Not reported in 1 study. Median 0.2% in other CD3-R TC CD3-R CD3+CD19 R CD34-E <0.01% CD3-R/CD56-E CD19 Log Depletion (4 studies 272 patients) Not reported, very low Median 49% -53% (2 studies 25 subjects) CD3-R TC CD3-R CD3+CD19 R % CD56 Recovery (2 studies 257 patients)

39 CliniMACS Prodigy Integrated closed system including: CliniMACS Magnetic Separation Unit Centrifugation chamber Optical fractionation control Temperature controlled incubation chamber Observational microscope Multiple input lines Closed fluid path

40

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