Programmed death-ligand 1

(PD-L1)

anti-PD-L1

Rabbit mAb E1L3N

Goat anti-Rabbit

Cleaved

VeraTag

Capillary Electrophoresis

PD-L1 VeraTag® Assay

Slide based FFPE

DTT

VeraTag units = RF/mm2 = Relative Fluorescence per mm2

tumor

Conclusions

AACR 2015

#LB-281

Development of a sensitive and quantitative PD-L1 immunoassay superior to IHC with

application in human FFPE tissue samples Jerry Wallweber, Ahmed Chenna, Roy Ravanera, David Stathas, Weidong Huang and Christos Petropoulos

Introduction: Cancer immunotherapy approaches and targets are rapidly expanding for the

treatment of many different types of cancer. The programmed cell death-1 receptor (PD-1)

and its ligand PD-L1 have garnered a great deal of interest lately, partially due to current

therapeutic agents demonstrating a long and durable clinical response with low toxicities in

several cancer types. Binding of the PD-1 receptor on activated T-cells to PD-L1 expressing

tumor cells reduces T-cell activation, thereby evading an immune response against tumor

progression. PD-L1 expression by immunohistochemistry (IHC) has been shown to be a

prognostic and potential predictive biomarker for response to both anti-PD-L1 and anti-PD-1

therapy, however, currently the IHC assay is not standardized and the definitions used for

positivity are variable and subjective due to a visual scoring system. A lack of sensitivity of the

IHC assay may partially explain the observed response to therapy in patients whose tumors

were identified as IHC=0/PD-L1 negative. In an attempt to offer a more sensitive and

quantitative assay, we developed a PD-L1 protein expression assay using the VeraTag®

technology. The PD-L1 VeraTag assay utilizes the release of a unique fluorescent reporter

(VeraTag), which is measured with high sensitivity via capillary electrophoresis to accurately

and objectively quantify the amount of PD-L1 protein expression in FFPE samples.

Methods: The anti-PD-L1 rabbit monoclonal antibody E1L3N (Cell Signaling Technologies)

was utilized in the VeraTag assay together with a goat anti-rabbit secondary antibody

conjugated to the VeraTag reporter. FFPE cancer cell line lines were used to optimize antigen

retrieval, primary antibody concentration and signal/background ratio, with emphasis on the

lower end of the dynamic range.

Results: VeraTag measurements of PD-L1 protein expression correlated to both IHC and

PD-L1 gene expression (CCLE, R squared=0.7212) in FFPE cancer cell lines. The PD-L1

protein expression by VeraTag and IHC was compared across a group of FFPE squamous

cell carcinoma of the head and neck (SCCHN) and HER2- and HER2+ breast samples.

VeraTag assays for the measurement of the HER-family of receptors (HER1, HER2 and

HER3 total, HER1-HER1 homodimer, HER2-HER3 heterodimer, phospho-HER3 and HER3-

PI3 kinase complex) were evaluated for correlation to PD-L1 protein expression in these two

cancer types. There was good agreement between the VeraTag and IHC measurements of

PD-L1 protein expression, with the added advantage of the VeraTag assay providing an ~5-

fold range of PD-L1 expression within the IHC=0 category.

Conclusions: We have developed a sensitive and quantitative measurement of PD-L1

protein expression in FFPE human SCCHN and breast cancer samples utilizing the VeraTag

technology. Measurement of PD-L1 expression from clinical samples with the VeraTag assay

is warranted.

Abstract

Monogram Biosciences/LabCorp Inc., South San Francisco, CA

Quantitative FFPE VeraTag® Assays

PD-L1 Protein and mRNA Distributions

VeraTag® Assay Workflow

PD-L1 IHC compared to VeraTag®

PD-L1 vs EGFR-family of Biomarkers

Correlations with significant p-values (p<0.05) are shown in red.

Spearman r HER1/EGFR HER2 HER3 HER1-HER1 Homodimer HER2-HER3 Heterodimer phospho-HER3 HER3-PI3 kinase Complex

Breast n/a 0.4583 0.2848 n/a 0.3893 0.4410 0.4796SCCHN -0.0580 0.1100 -0.0569 -0.2786 0.1092 0.2151 0.4840

Spearman correlation coefficients from the relationship between PD-L1 and a

panel of EGFR-family of biomarkers as measured by VeraTag.

Wednesday, April 22nd, 2015, 8am – 12pm

0.2

5

0.5 1 2 4 8

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0.25

0.5

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4

8

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PD-L1 VeraTagOperator #1

PD

-L1

Ve

raT

ag

Op

era

tor

#2

Reproducibility -

19 FFPE SCCHN tumors

18 of the 19 tumors (94.7%)

are within 2-fold.

PD-L1 VeraTag® Assay Characteristics

Background in FFPE cell lines and tumors

PD-L1 VeraTag

Isotype control

MB

453

MB

468

H1650

MB

231

H441

100643A

2

120635A

1

7291841

120149B

2

98910B

1

0.125

0.25

0.5

1

2

4

8

16

32

64

Ve

raT

ag

Cell lines Tumors

PD-L1 VeraTag® and mRNA Correlation Screening a panel of 14 FFPE cell lines.

Notes:

a. -1, -2 and -3 represent different FFPE preparations of the same cell line.

b. PD-L1 mRNA (log2) available from CCLE at http://www.broadinstitute.org/ccle/home

H441

MB231

MB468

MB453

H1650

3 4 5 6 7 8 9 10

0.25

0.5

1

2

4

8

16

32

PD-L1 mRNA (log2)

PD

-L1

Ve

raT

ag

Pearson R2 = 0.7881

Reproducibility -

5 replicates of 5 FFPE cell lines

Pearson R2 = 0.7210

H441

MB231 H1650

MB468

MB453

3 4 5 6 7 8 9 10

0.25

0.5

1

2

4

8

16

32

PD-L1 mRNA (log2)

PD

-L1

Ve

raT

ag

FFPE Cell Linea VeraTag mRNA (log2)

b

BT474 1.21 4.23

MB453-1 0.87 4.50

MB453-2 1.54 4.50

MB453-3 1.61 4.50

MCF7 1.11 4.57

MB468 1.12 5.07

SKBR3 0.89 5.08

TE4 1.82 5.60

SNU869 1.91 5.66

H1650 3.06 5.84

TE6 2.57 6.12

BXPC3-1 3.18 6.65

BXPC3-2 3.39 6.65

CAL27-1 3.56 6.97

CAL27-2 3.65 6.97

MB231 6.77 8.16

H441-1 17.73 8.96

H441-2 19.56 8.96

PD-L1

79 FFPE tumors

38 Breast cancer

27 SCCHN

14 NSCLC

PD-L1 protein expression by VeraTag and mRNA expression from CCLE both

show an increase in NSCLC and SCCHN over Breast cancer.

p<0.0001

Bre

ast

Lung-N

SC

SC

CH

N

0.25

0.5

1

2

4

8

16

PD

-L1

Ve

raT

ag

p<0.0001

FFPE tumors

from Asterand Bioscience Cancer cell lines from CCLE

Mann-Whitney test, p<0.0001

p = 0.0002

All

Bre

ast

Colo

recta

l

Endom

etr

ium

Kid

ney

Liv

er

Lung

Lung-S

C

Lung-N

SC

Oesophagus

Ovary

Pancre

as

Skin

Sto

mach

Head&

Neck

4

5

6

7

8

9

10

11

12

PD

-L1 m

RN

A (

log

2)

1.Pathology Review and

Macro-dissection

Template

2. Macro-dissection

3.Deparaffinization/

Rehydration

4.Antigen Retrieval 5.Antibody Incubation

& Wash

6.Photoactivated

VeraTag Release

7.VeraTag Separation

& Detection on C.E.

V

8.VeraTag Identification

& Quantification by

VeraTag Informer

9.H&E Tumor

Identification and

Area Analysis

10.Final Sample and Batch

Normalized VeraTag Result

h

V

1O2

Slide based FFPE

Cell line %CV

MB453 9%

MB468 11%

H1650 15%

MB231 5%

H441 9%

0.25 0.5 1 2 4 8 16

0.25

0.5

1

2

4

8

Breast cancer

PD-L1 VeraTag

H3P

I3K

Vera

Ta

g

Spearman r = 0.4796

p-value = 0.0027

0.25 0.5 1 2 4 8 16

0.25

0.5

1

2

4

8

SCCHN cancer

PD-L1 VeraTag

H3P

I3K

Vera

Ta

g

Spearman r = 0.4840

p-value = 0.0165

PD-L1 correlated with HER3-PI3 kinase Complex

Preclinical models support a role for the oncogenic PI3K pathway

in the regulation of PD-L1 protein expression.

Activation by:

PTEN loss or

PIK3CA mutation

Inhibition by:

PI3K, AKT or mTOR inhibitors

PI3K pathway PD-L1 protein

Consistent with this model, VeraTag measurements of PD-L1 protein expression

significantly and positively correlated with HER3-PI3 kinase complex.

•We have developed a quantitative and reproducible PD-L1 assay using the VeraTag technology to

measure PD-L1 protein expression in FFPE samples.

•The PD-L1 VeraTag assay correlated significantly to mRNA expression in a panel FFPE cell lines

over an ~50-fold dynamic range.

•The PD-L1 VeraTag assay had good reproducibility within FFPE cell lines (5 – 15% CV) and

between operators with SCCHN tumors (94.7% of the samples within 2-fold).

•The elevated distributions of PD-L1 protein as measured by VeraTag in NSCLC and SCCHN

tumors over Breast cancer tumors paralleled the relative distributions of PD-L1 mRNA in caner cell

lines derived from the same tissue types.

•The PD-L1 VeraTag assay provided a range of expression within the IHC 1+ and 2+ categories.

•VeraTag measurements of PD-L1 protein expression correlated with HER3-PI3 kinase complex in

both Breast and SCCHN tumors, supportive of a role for the PI3K pathway in the regulation of PD-

L1 protein expression.

•Clinical evaluation of PD-L1 protein expression by an objective, quantitative and reproducible

VeraTag assay may help stratify patients for anti-PD-1 or anti-PD-L1 therapies.

PD-L1 IHC utilizing the same primary antibody (E1L3N) as the VeraTag assay.

PD-L1 VeraTag separated

IHC 0 and 1+ by 7.9-fold

Cell line IHC VeraTag

MB453 0 0.39

H1650 1+ 3.08

MB231 3+ 7.90

H441 3+ 23.27

PD-L1

PD-L1 VeraTag provided a wide range of

expression in both IHC 1+ and 2+.

HER2 Receptor

anti-HER2

anti-HER2 Cleaved

VeraTag

Capillary

Electrophoresis

HERmark® Assay

Slide based FFPE

VeraTag units = RF/mm2 = Relative Fluorescence per mm2

tumor

1O2

SCCHN

NSCLC

Breast cancer

Cell Lines

0 1+ 2+ 3+0.25

0.5

1

2

4

8

16

32

PD-L1 VeraTag vs IHC

IHC

PD

-L1

Ve

raT

ag