Potency Assay DevelopmentRegulatory T-cell Function

Smita Ghanekar, Ph.D.BD Biosciences

Types of Treg

Naturally occurring Tregproduced by thymus as a functionally distinct and mature subpopulation of T cells

Inducible or “adaptive”Treg -induced from naïve T cells by antigenic stimulation in the periphery (extrathymic)

Naturally Occurring CD4+CD25+Treg

• Generation is developmentally controlled• Persist in the periphery with stable function• Natural presence in the immune system makes them a

good target for designing ways to treat or prevent immunological diseases

• Congenital deficiency results in impairment of self tolerance and immunoregulation that can lead to severe autoimmunity, immunopathology and allergy in humans

Treg: Potential Clinical Applications

Removal of Treg or blocking of their function to boost immune response using anti-CTLA-4, -CD25, -GITR mAbs

Cancer, Infectious disease

Suppression of Treg Function

Specific suppression of immune responses by transfer of Treg or enhancement of their function; ex vivo generation of regulatory cells using cytokines, pharmacological agents, or modified DCs

Organ Transplantation, Autoimmune disease, Allergy

Enhancement of Treg Function

Potential Therapeutic ApproachTarget Condition

Strategic Manipulation

of Treg

Fehervari and Sakaguchi, 2004

Current Treg Clinical Trials and INDs

Annual growth rate

Treg GVHD: 5-6 trials, 3 INDsTreg T1D: 2 INDsCancer Adoptive Therapy: 2 INDs (Treg depletion)

Indications of potential Treg therapy

Current Technologies to Assess Treg function

Uses animals5-7 daysDifficult to set up

Considered most accurate by some labs

Mouse GHVDModel

Change the paradigmFlow based Multiparametric Subset informationShort term – 7 hoursCan be standardized

Activation Markerse.g. Intracellular Cytokine Staining, CD69, CD154, etc.

Long term – 5 daysDifficult to standardizeIL-2 exhaustionCell deathCFSE labeling difficulties

Flow basedNo radioactivityMultiparametricSubset information

Proliferation with CFSE or VFSE dye dilution

assay

Uses radioactivityLong term - 3 to 5 daysNo subset information

Gold StandardProliferation with3[H]-Thymidine

ConsProsMethod

Our Goals for Treg Functional Assay Development

• To develop an in vitro bioassay that is relevant to a desired in vivo function

• Our assay is a direct measurement of biological activity of the cellular product

• For many cell therapy products, the phenotype of a cell is used as a surrogate

• Challenges

Treg FunctionSuppression of Proliferation

• Conventional suppression assay• Five-day proliferation assay

• Time consuming• Difficult to reproduce• May produce false-positive results

• Depletion of IL-2• Apoptosis of rapidly dividing cells

11.2%85.1%

BD FastImmune Human Regulatory T Cell Function Kit

• Research Use Only GMP Product• Measures suppression of activation markers

expressed by responder T cells• CD154• CD69

• Reduced expression in the presence of Treg• 96-well plate format• Short term: 7-hour activation

BD FastImmune Human Regulatory T Cell Function Kit

• Optimized antibody cocktail for staining• CD4 FITC • CD25 PE• CD3 PerCP-Cy5.5

• Activation markers• CD154 APC• CD69 PE-Cy™7

• Detailed assay and staining procedures• Gating strategy for analysis

BD FastImmune Human Regulatory T Cell Function Kit

• Assay Configuration• Autologous unstimulated PBMCs• Autologous unstimulated PBMCs + Treg

• Different ratios of responders: Treg• CD3/CD28 stimulated PBMCs• CD3/CD28 stimulated PBMCs + Treg

• Different ratios of responders: Treg• Treg alone• Autologous unstimulated PBMCs (for instrument

setup)

Sort CD4+CD25++CD127dim/- CD45RA+ cells

Expand in culture for 13 Days

Co-culture Treg and autologous PBMC at various ratios (e.g. 1 Treg : 1 PBMC)

Activate for 7 hours with stimulus (SEB,CD3/CD28 beads)in presence of CD154

Perform Surface Staining with CD3, CD4, CD69, CD25

Assess reduction in frequency of CD69 and/or CD154 positiveresponder cells in presence of Treg

Treg Suppression AssayHow the Assay Works

Treg-mediated suppression of CD154 and CD69expression in PBMC stimulated with CD3/CD28

CD4- CD4+CD25-

CD4+CD154

CD4+CD69

Unstim PBMC Stim* PBMC Treg+ stim PBMC (0.5:1)

0.6%

MFI=541

1.0%

MFI=1529

20.5%

MFI=6387

28.5%

MFI=4750

8.9%

MFI=3287

12.4%

MFI=3766

*Stimulated with CD3+CD28 beads at 0.25 beads:1 PBMC

% Suppression of activation can be calculated using the following formulas:

1. For the suppression of marker frequency:100-[(%Positive in presence of Treg / %Positive in absence of Treg) x100]

For example, using the numbers from the figures above, for CD4+CD154:100-[(8.9/20.5)x100] = 56.6% suppression

2. For the suppression of marker expression level:100-[(MFI in presence of Treg / MFI in absence of Treg) x100]

For example, using the numbers from the figures above, for CD4+CD154:100-[(3287/6387)x100] = 48.5% suppression

Calculating Percent Suppression

7h Activation Marker vs 5d Proliferation Suppression Assay

Proliferation (VFSE assay) Suppression - 5 DaysCD3+CD4- gated

0.0

10.0

20.0

30.0

40.0

50.0

60.0

70.0

80.0

90.0

100.0

0:1 0.125:1 0.25:1 0.5:1

Treg:PBMC%

Sup

pres

sion

S39

S41

S43

S44

Activation marker (CD69) Suppression - 7 HoursCD3+CD4- gated

0.0

10.0

20.0

30.0

40.0

50.0

60.0

70.0

80.0

90.0

100.0

0:1 0.125:1 0.25:1 0.5:1

Treg:PBMC

% S

uppr

essi

on(C

D69

)

S39

S41

S43

S44

Challenges

• Cellular product manufacturing

• Factors/sources that influence assay variability

• Well-defined reagents

• Instrumentation calibration

• Following detailed SOP

Suppression Assay ReproducibilityEffect of Treg Expansion Conditions on

Suppressive Function

35.6221.37% CD154 Sup. @ 0.25:1

36.2521.20% CD69 Sup. @ 0.25:1

66.8068.10%FoxP3

50ml18mlVolume

126.0x10650.5x106Total Cell Number

Harvest: (Day 14)

50ml in 2xT75 flasks*18.0ml in 6 well of 6*Day 12

16.8ml in T75 flask6.0ml in 2 well of 6*Day 9 (restimulated)

9.0ml to 9 well of 24*2.0 ml in 2 well of 24*Day 7 (rapamycin removed)

1.5ml to 3 well of 24*350 μl in 2 well of 96*Day 5

add 300 μl media+ rIL2add 50 μl media + rIL2Day 2

450 μl in 1 well of 24200 μl in 1 well of 96Day 0

CD45RA+CD45RA+Sorted as

All cells @ 2.5x105/ml

Condition 2Condition 1Sort #32

Activation Marker Expression ReproducibilityInter-assay and Intra-assay

donor1

donor2

donor3

donor4

donor5

0

25

50

75

100

% C

V(C

D69

+/C

D4+

res

pons

e)

donor1

donor2

donor3

donor4

donor5

0

25

50

75

100

% C

D69

+/C

D4+

res

pons

e

0

25

50

75

100Assay 1Assay 2Assay 3Assay 4Assay 5

1 2 3 4 5Donor #

% C

D69

+/ C

D4+

res

pons

eCryopreserved PBMC from 5 donorsActivation assay performed 5 times3 replicates per assay

Beta Test SitesSummary

•All test sites seemed very happy with the assay once they got it to work.

•Why the assay didn’t initially work at some test sites – Did not follow the protocol!

•What was done differently-Different ActivatorsDifferent platesDifferent activation timesDifferent cell numbersDifferent gating strategyDifferent mediaDifferent staining cocktailsQuality of cryopreserved PBMC post-thaw

Optimal Activation Conditions

Activators tested:• soluble CD3 + CD28/49d costim.• CD2/CD2R• SEB+CD3/CD28 beads• CD3/CD28 beads

Looking for good CD69 and CD154 expression without loss of CD3 resolution

Loss of CD3 Resolution with Activation

Unstim CD2/CD2RSEB+CD3/28

CD3/28 0.25:1CD3/28 0.5:1CD3/28 1:1

Activation with CD3/CD28 beads

0.010.020.030.040.050.060.070.080.090.0

100.0

Unstim 1:1 0.5:1 0.25:1

Bead:PBMC

%C

D69

Expr

essi

on

D1D2D3

0.010.020.030.040.050.060.070.080.090.0

100.0

Unstim 1:1 0.5:1 0.25:1

Bead:PBMC%

CD

154

Expr

essi

on

D1D2D3

CD69 CD154

Suppression of CD154 and CD69CD3/CD28 Bead Activation

05

1015202530354045505560

0:1 0.5:1 0.25:1

Treg:PBMC

%C

D69

Expr

essi

on

0.5:10.25:1

05

1015202530354045505560

0:1 0.5:1 0.25:1

Treg:PBMC

%C

D15

4Ex

pres

sion

0.5:10.25:1

CD69 CD154

28%

41%

15%

30%

Assay Plate ComparisonExpression of CD69 and CD154

0

10

20

30

40

50

CD154

7hr V Bot t om 1

7hr U Bot t om 1

7hr V Bot t om 2

7hr U Bot t om 2

7hr V Bot t om 3

7hr U Bot t om 3

% Expression of CD154

0

5

1015

20

25

3035

40

45

CD69

% P

ositi

ve

7hr V Bottom 17hr U Bottom 1

7hr V Bottom 2

7hr U Bottom 2

7hr V Bottom 37hr U Bottom 3

% Expression of CD69

Polypropylene V-Bottom vsPolypropylene U-Bottom

CD154 SuppressionCD4+ T cellsDonor 2 (S34)

0102030405060708090

100

0.5:1 0.25:1 0.125:1

7hr V Bottom 7hr U Bottom

CD69 SuppressionCD4+ T cellsDonor 2 (S34)

0102030405060708090

100

0.5:1 0.25:1 0.125:1

Treg to PBMC%

Sup

pres

sion

7hr V Bottom 7hr U Bottom

% S

uppr

essi

on

Assay Kinetics for CD154 Suppression7 hour vs 20 hour

0

10

20

30

40

50

CD154

% P

ositi

ve

7hr V Bottom 120hr V Bottom 1

7hr V Bottom 220hr V Bottom 2

7hr V Bottom 320hr V Bottom 3

0102030405060708090

100

0.5:1 0.25:1 0.125:1

Treg to PBMC

% S

uppr

essi

on

7hr V Bottom 3

20hr V Bottom 3

0102030405060708090

100

0.5:1 0.25:1 0.125:1

Treg to PBMC

% S

uppr

essi

on

7hr V Bottom 2

20hr V Bottom 2

0102030405060708090

100

0.5:1 0.25:1 0.125:1

Treg to PBMC

% S

uppr

essi

on

7hr V Bottom 1

20hr V Bottom 1

% Expression of CD154

% Suppression of CD154

Assay Kinetics for CD69 Suppression 7 hour vs 20 hour

0

5

10

15

20

25

30

35

40

45

CD69

% P

ositi

ve

7hr V Bottom 1

20hr V Bottom 17hr V Bottom 2

20hr V Bottom 2

7hr V Bottom 3

20hr V Bottom 3

0102030405060708090

100

0.5:1 0.25:1 0.125:1

Treg to PBMC

% S

uppr

essi

on

7hr V Bottom 1

20hr V Bottom 1

0102030405060708090

100

0.5:1 0.25:1 0.125:1

Treg to PBMC

% S

uppr

essi

on

7hr V Bottom 2

20hr V Bottom 2

0102030405060708090

100

0.5:1 0.25:1 0.125:1

Treg to PBMC

% S

uppr

essi

on

7hr V Bottom 3

20hr V Bottom 3

% Expression of CD69

% Suppression of CD69

Different Responder Cell Numbers in Activation Plate

CD154 CD69

Donor 1CD4+ CD154

0.0010.0020.0030.0040.0050.0060.0070.0080.0090.00

100.00

0:1 0.125:1 0.25:1 0.5:1

Treg:PBMC

% S

uppr

essi

on

5x105 PBMC2.5x105 PBMC

CD4+ CD69

0.0010.0020.0030.0040.0050.0060.0070.0080.0090.00

100.00

0:1 0.125:1 0.25:1 0.5:1

Treg:PBMC

% S

uppr

essi

on

5x105 PBMC2.5x105 PBMC

CD4+CD154

0.0010.0020.0030.0040.0050.0060.0070.0080.0090.00

100.00

0:1 0.125:1 0.25:1 0.5:1

Treg:PBMC

% S

uppr

essi

on

5x105 PBMC2.5X105 PBMC

CD4+CD69

0.0010.0020.0030.0040.0050.0060.0070.0080.0090.00

100.00

0:1 0.125:1 0.25:1 0.5:1

Treg:PBMC

% S

uppr

essi

on5x105 PBMC2.5X105 PBMC

Donor 2

Excluding Treg from Suppression Analysis

Treg:PBMC = 0.5:1, n=19

Narrow Wide0

102030405060708090

100

CD25 gate

% S

uppr

essi

on

CD3+/CD4+/CD154 CD3+/CD4+/CD69

Narrow Wide0

102030405060708090

100

CD25 gate

% S

uppr

essi

on

P=0.0226 P=0.0414

46.5% 35.7% 36.6% 26.8%

Unstim/Unstained Unstim/Stained Stim/StainedA. C.B.PBMC Treg

narrow wide

9.3%

Conclusions

•Different stimulation times may result in variable suppression outcomes – still recommend 7 hours but, if decide to go longer, should pick one time and always use that time throughout a study.

•Different plates result in different suppression activity – highly recommended use polypropylene V-bottom plates. Polystyrene doesn’t work!

•Different responder cell numbers in the assay may result in different suppression amounts – appears to vary by donor. Should try to always use the same number of cells throughout a study.

•Different CD25 gating methods may result in different suppression outcomes - recommend the narrow CD25- gating strategy .

•Need to Follow the Protocol!!!

CD154 expression estimates liver graft rejection risk

Ashokkumar et al., 2009: Allospecific CD154+ T Cells Associate with Rejection Risk After Pediatric Liver Transplantation. Am J Transplantation 9:179

CD154 expression estimates liver graft rejection risk

Rejector Non-Rejector

Ashokkumar et al., 2009: Allospecific CD154+ T Cells Associate with Rejection Risk After Pediatric Liver Transplantation. Am J Transplantation 9:179

Threshold IR > 1.13

Acknowledgements

• Joyce Ruitenberg• Angelica Igano• Christopher Boyce• Ravi Hingorani• Tim Fong• Jurg Rohrer• Skip Maino

Smita_Ghanekar@bd.com