ISCT Workshop #7 Perspectives in Cell Selection ... · Cell separation steps done per CliniMACS®...
Transcript of ISCT Workshop #7 Perspectives in Cell Selection ... · Cell separation steps done per CliniMACS®...
ISCT Workshop #7
Perspectives in Cell Selection
Immunomagnetic Selection
Carolyn A. Keever-Taylor, PhD
Medical College of Wisconsin
June 7, 2012
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Withdrawn 2010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 #04-0146 2005
Approved indication 2012?
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.
Target cells recovered
Ab on paramagnetic particle
Non-retained cells washed through
Dynal beads on cells
Apheresis product
Platelet- plasma wash
Antibody sensitization
Bead rosetting and depletion
CD34 Enriched Product
(8 x 10 9 MNC)
Isolex
Automated
Process
CD34 Cell Selection- Isolex
+ magnetic microbeads conjugated with anti-CD 34
CD 34 neg cells
Positive Selection
+ magnetic microbeads conjugated with OKT3
All other cells
CD34+ cells
Negative Selection
CliniMACS® CD34 Reagent
CliniMACS® Tubing Sets
(Standard and Large Scale)
Standard:
0.6 x 109 CD34+ Cells
from 60 x 109 Cells
Large Scale:
0.6-1.2 x 109 CD34+ Cells
from 60-120 x 109 Cells
CliniMACS® CD34 Reagent System
CliniMACS®plus Instrument
CliniMACS® PBS/EDTA Buffer
Isolex & CliniMacs Compared- MCW Experience
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.
Device N CD34 Purity CD34 Recovery CD3 Log
Dep
Isolex 110 96.3% ± 4.0 54.8% ± 14.7 4.4 ± 0.3
CliniMacs 16 96.7% ± 8.8 62.3% ± 11.3 5.1 ± 0.3
Location of CliniMACS®plus
Instruments in the USA
162 instruments within 97 institutions in the U.S.
84 protocols using
CD34 system
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
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
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
(BMT CTN) Protocol 0303
Leukapheresis collections from a Matched Related Donor were
performed in order to obtain a minimum of ≥2.0 x 106 CD34+
cells/kg
Target of 5.0 x 106 CD34+ cells/kg and <105 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
Secondary Endpoint: CliniMACS CD34 Reagent System Performance
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
CliniMACS® CD34 Reagent System Post Processing Performance
N=84
% CD34+
Recovery
% CD34+
Purity
Log10 TCD
% Viability
Mean 66.06 93.0 4.78 96.57
SD ±20.25 ±8.3 ±0.55 ±3.84
Min 29.9 61.5 3.2 74.0
Max 125.6 99.8 5.9 100.0
All gram stains/14 day cultures were negative
All endotoxin below detection limits
No significant infusion related toxicities observed
Final Cellular Product Summary
Parameter Result
Mean CD34+ dose 8.81 x 106/kg
Median CD34+ dose 7.92 x 106/kg
Mean CD3+ dose 1.51 x 104/kg
Median CD3+ dose 0.7 x 104/kg
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
Processing Logistics
Products held overnight diluted to ≤2.0 x 108/mL using
concurrent plasma or CliniMACS® PBS/EDTA Buffer
Controlled storage @ 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
Pre-Processing Cell Counts
Ctr 1 Ctr 2 Ctr 3 Ctr 4 Ctrs 5-80
5
10
15
20
p=0.0005
0.0022
0.0038
Center
Sta
rtin
g T
NC
Ctr 1 Ctr 2 Ctr 3 Ctr 4 Ctrs 5-80
50
100
150
200
2500.0252
0.0138
p=0.0150Center
Sta
rtin
g C
D34 x
10
7
Ctr 1 Ctr 2 Ctr 3 Ctr 4 Ctrs 5-80
10
20
30
40
P=0.0463
0.0271
Center
Sta
rtin
g C
D3
Ctr 1 Ctr 2 Ctr 3 Ctr 4 Ctrs 5-880
85
90
95
100
1050.0164
0.01750.0411
p=0.0026Center
% L
ivin
g C
ells
A B
C D
Ctr 1 Ctr 2 Ctr 3 Ctr 4 Ctrs 5-80
5
10
15
20
p=0.0005
0.0022
0.0038
Center
Sta
rtin
g T
NC
Ctr 1 Ctr 2 Ctr 3 Ctr 4 Ctrs 5-80
50
100
150
200
2500.0252
0.0138
p=0.0150Center
Sta
rtin
g C
D34 x
10
7
Ctr 1 Ctr 2 Ctr 3 Ctr 4 Ctrs 5-80
10
20
30
40
P=0.0463
0.0271
Center
Sta
rtin
g C
D3
Ctr 1 Ctr 2 Ctr 3 Ctr 4 Ctrs 5-880
85
90
95
100
1050.0164
0.01750.0411
p=0.0026Center
% L
ivin
g C
ells
A B
C D
X 1
010
Ctr 1 Ctr 2 Ctr 3 Ctr 4 Ctrs 5-80
5
10
15
20
p=0.0005
0.0022
0.0038
Center
Sta
rtin
g T
NC
Ctr 1 Ctr 2 Ctr 3 Ctr 4 Ctrs 5-80
50
100
150
200
2500.0252
0.0138
p=0.0150Center
Sta
rtin
g C
D34 x
10
7
Ctr 1 Ctr 2 Ctr 3 Ctr 4 Ctrs 5-80
10
20
30
40
P=0.0463
0.0271
Center
Sta
rtin
g C
D3
Ctr 1 Ctr 2 Ctr 3 Ctr 4 Ctrs 5-880
85
90
95
100
1050.0164
0.01750.0411
p=0.0026Center
% L
ivin
g C
ells
A B
C D
Differences attributed to mobilization methods
X 1
09
% TNC and CD34+ Recovery Post Platelet and Antibody Washes
Ctr 1 Ctr 2 Ctr 3 Ctr 4 Ctrs 5-80
25
50
75
100
1250.010
0.005
0.019
p=0.007Center
% o
f S
tart
ing
TN
C
Ctr 1 Ctr 2 Ctr 3 Ctr 4 Ctrs 5-80
50
100
150
0.048
p=0.002
0.007
Center
% o
f S
tart
ing
A
bs C
D34
A B
Cobe 2991 Wash vs. Bag Wash
0 2 4 6 60 80 100
%TNC Wash Rec
%CD34 Wash Rec
%CD34-Enr Rec
%CD34 Purity
Log TCD
P=0.04
Cobe 2991
Bag
Post Processing Outcomes
Ctr 1 Ctr 2 Ctr 3 Ctr 4 Ctrs 5-850
60
70
80
90
100
110
P=0.0439
0.024
Center
%C
D34 P
uri
ty
Ctr 1 Ctr 2 Ctr 3 Ctr 4 Ctrs 5-870
80
90
100
110 0.0232
0.0196
P=0.0032Center
% L
ivin
g C
ells
7
-AA
D
Ctr 1 Ctr 2 Ctr 3 Ctr 4 Ctrs 5-80
50
100
150
P<0.0000
0.024
<0.001
0.002
Center
%C
D34 R
eco
very
Ctr 1 Ctr 2 Ctr 3 Ctr 4 Ctrs 5-83
4
5
6 0.046
P=0.0207Center
Lo
g T
CD
All centers were able to process grafts that met the study criteria
CD34+ Cells x 106/Kg of Patient Weight Infused
Ctr 1 Ctr 2 Ctr 3 Ctr 4 Ctrs 5-8106
107
108
0.029
P=0.0403Center
CD
34/k
g CD34+
Target
Dose
All patients received the minimum CD34+ dose (>2.0x106 cells/kg)
84.1% of patients received > 5 x 106 CD34+ cells/kg
CD34+
Minimum
Dose
Ctr 1 Ctr 2 Ctr 3 Ctr 4 Ctrs 5-8103
104
105
P=NSCenter
CD
3/k
g
CD3+ Cells Infused per kg
Upper limit
of CD3+
dose
No patients received more than 1.0x105/kg CD3+ cells
Impurities in Final Cell Collection
T Cells B Cells NK Cells Monocytes0
2
4
6
8
1020
22
24%
of C
D34-e
nri
ch
ed
fra
ctio
n
Contaminating Subset
Apheresis per Patient Performed Per Patient to Meet Minimum CD34+ Cell Dose Post Enrichment
Target
Cell Dose
Start Product Factors and Outcome
0 5 10 150
50
100
150
P=NS
TNC Loaded (x 1010)
% R
eco
very
CD
34+
Cells
0 5 10 153
4
5
6
7
P=0.06
TNC Loaded (x 1010)
Lo
g T
CD
0 50 100 150 2000
50
100
150
P=0.02
CD34+ Cells Loaded (x 107)
% R
eco
very
CD
34+
Cells
0 10 20 30 403
4
5
6
7
P=0.05
CD3+ Cells Loaded (x 109)
Lo
g T
CD
Cell Processing Conclusions
All sites, and all products met and most exceeded
study goals for:
CD34+ cell infusion dose > 5 x 106/kg
CD3+ cell infusion dose < 1 x 105/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.
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 106 CD34+ cells/kg and <1 x 105 C3+ cells/kg
Conclusions BMT CTN 0303
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)
Statistical Comparison of 0101 vs. 0303 Supports Safety of T-Cell Depletion
Observed advantages of T-Cell depleted allografts
include:
Significantly reduced incidence of chronic GVHD
(p=.000434)
Earlier neutrophil engraftment (p=.00249)
Low rates of acute and chronic GVHD in the
absence of more traditional pharmacologic
prophylaxis
Statistical Comparison of 0101 vs. 0303 Supports “Probable Benefit” Argument
for T-Cell Depletion
Reagent USE Reagent USE
CD34 HPC-Enr IFNg Ag-specific Enr
CD133 HPC-Enr CD56 All NK Enr
CD3/CD192 T/B Dplt CD3/CD56 NK T Enr
TCR ab biotin2 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
CliniMACS CE Marked Reagents1
1 Requires IND or IDE in US
2 NA in US
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
Challenges in Product Analysis
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
Single Center Experience vs Published
Median 53% -90% (6 studies 310 patients)
2.8 -4.4 (7 studies 319 patients)
Median 4.6 ->5.0 (5 studies 991 patients)
Median 60% -81% (5 studies 991 patients)
NA
NA
Median 3.03 -5.3 (2 studies 25 subjects)
40 60 80 100
CD3/CD19-R
CD3-R
CD3-R TC
CD3-R/CD56-E
CD34-E
NA
NA
% CD34 Recovery
2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0
CD3/CD19-R
CD3-R
CD3-R TC
CD3-R/CD56-E
CD34-E
CD3 Log Depletion
Single Center Experience vs Published
2.2-4.4 (4 studies 272 patients)
36-68 (2 studies 257 patients)
3.2 (2 studies 166 patients)
Not reported, very low
Median 49% -53% (2 studies 25 subjects)
Not reported in 1 study. Median 0.2% in other
0.0 1.0 2.0 3.0 4.0 5.0
CD3+CD19 R
CD3-R
CD3-R TC
CD3-R/CD56-E
CD34-E
CD19 Log Depletion
20 40 60 80 100
CD3+CD19 R
CD3-R
CD3-R TC
CD3-R/CD56-E
CD34-E <0.01%
% CD56 Recovery
CliniMACS® Prodigy
CliniMACS Magnetic Separation Unit
Centrifugation chamber
Optical fractionation control
Temperature controlled incubation chamber
Observational microscope
Multiple input lines
Closed fluid path
Integrated closed system including: