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Withdrawn 2010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


50

100

150

200

2500.0252

0.0138

p=0.0150Center

Sta

rtin

g C

D34 x

10

7


10

20

30

40

P=0.0463

0.0271

Center

Sta

rtin

g C

D3


85

90

95

100

1050.0164

0.01750.0411

p=0.0026Center

% L

ivin

g C

ells

A B

C D


5

10

15

20

p=0.0005

0.0022

0.0038

Center

Sta

rtin

g T

NC


50

100

150

200

2500.0252

0.0138

p=0.0150Center

Sta

rtin

g C

D34 x

10

7


10

20

30

40

P=0.0463

0.0271

Center

Sta

rtin

g C

D3


85

90

95

100

1050.0164

0.01750.0411

p=0.0026Center

% L

ivin

g C

ells

A B

C D

X 1

010


5

10

15

20

p=0.0005

0.0022

0.0038

Center

Sta

rtin

g T

NC


50

100

150

200

2500.0252

0.0138

p=0.0150Center

Sta

rtin

g C

D34 x

10

7


10

20

30

40

P=0.0463

0.0271

Center

Sta

rtin

g C

D3


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


25

50

75

100

1250.010

0.005

0.019

p=0.007Center

% o

f S

tart

ing

TN

C


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


60

70

80

90

100

110

P=0.0439

0.024

Center

%C

D34 P

uri

ty


80

90

100

110 0.0232

0.0196

P=0.0032Center

% L

ivin

g C

ells

7

-AA

D


50

100

150

P<0.0000

0.024

<0.001

0.002

Center

%C

D34 R

eco

very


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


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


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

CD34 assessment on CD34-Enr Products

CD34-Enr

Pre Processing

CD3 Assessment in CD34-Enriched Product

CD34-Enr

Pre Processing

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

Gating Scheme to Reduce Background Pre vs CD3/CD19 Reduced Fractions

CD3/CD19-Red

Pre Processing

CD3-Reduced CD56-Enriched vs CD3-Reduced Only

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:

isct workshop #7 perspectives in cell selection ... · cell separation steps done per clinimacs®...

Documents