recent advances in immune monitoring presentation slides

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Recent Advances in Immune Monitoring | November 5, 2015 1 Copyright © 2015 Covance. All Rights Reserved

WEBINAR: RECENT ADVANCES

IN IMMUNE MONITORING

Sinnathamby Gomathinayagam, PhD

Public

Recent Advances in Immune Monitoring | November 5, 2015 2

Why is Immune Monitoring Necessary?

We monitor the immune system through a

variety of means to:

• Determine the efficacy of therapeutic

approaches, such as the administration of

a vaccine, or to

• Ascertain the safety of an administered

agent such as a biologic that targets the

immune system.

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ELISA SEREX

ELISPOT Western Blot

IHC Cytokine Release Assay

Flow Cytometry PCR

MHC Tetramers TCR Usage

Proliferation Gene Array

CTL Assay 2D Gel Electorphoresis

Limiting Dilution Analysis SELDI

DTH Mass Spectroscopy

Commonly Used Techniques in Immune Profiling

Whelan M et al., Personalized Medicine 2006 3(1), 79-88

SAFETY & EFFICACY ASSESSMENT

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Biologics Targeting The Immune System

Brennan et al., mAbs, 2010:2(3):233-255

Removab®

Orencia®

Stelara®

Tysabri®

Actemra®

Ilaris®

Humira®

Remicade®

Prolia® Soliris®

OKT3®

Enbrel®

Simponi®

Cimzia®

Rituxan®

Mabthera®

Zevalin®

Bexxar®

Simulect®

Amevive®

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Immune Monitoring: “Cytokine Storm”

Cytokine Storm in a Phase 1 Trial of the

Anti-CD28 Monoclonal Antibody TGN1412

Ganesh Suntharalingam, F.R.C.A., Meghan R. Perry, M.R.C.P., Stephen Ward, F.R.C.A., Stephen J. Brett,

M.D., Andrew Castell-Cortes, F.R.C.A., Michael D. Brunner, F.R.C.A., and Nicki Panoskaltsis, M.D., Ph.D.

N Engl J Med 2006; 355:1018-28

The NEW ENGLAND JOURNAL of MEDICINE

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Chemokine Panel Proinflammatory Panel Cytokine Panel

IL-8 IFNy GM-CSF

IP-10 IL-1B IL-12/IL-23

MCP-1 IL-2 IL-5

MCP-4 IL-6 IL-7

MDC IL-8 IL-15

MIP-1α IL-10 IL-16

MIP-1β IL-17A

TNFβ

VEGF

C h e m o k in e P a n e l

0 .0 1 1 1 0 0 1 0 0 0 0 1 0 0 0 0 0 0

1 0 2

1 0 3

1 0 4

1 0 5

1 0 6

1 0 7

IL -8 (H A )

E o ta x in -3

IP -1 0

M C P -1

M C P -4

M D C

M IP -1

M IP -1

T A R C

C o n c [p g /m L ]

EC

L S

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24-plex assay


MULTIPLEX CYTOKINE ASSAYS

© Meso Scale Diagnostics , all rights reserved

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• APRIL / TNFSF13

• BAFF / TNFSF13B

• sCD30 / TNFRSF8

• sCD163

• Chitinase-3-like 1

• gp130 / sIL-6Rβ

• IFN-α2

• IFN-β

• IFN-γ

• IL-2

• sIL-6Rα

• IL-8

• IL-10

• IL-11

• IL-12 (p40)

• IL-12 (p70)

• IL-19

• IL-20

• IL-22

• IL-26

• IL-27 (p28)

• IL-28A / IFN-λ2

• IL-29/IFN-λ1

• IL-32

• IL-34

• IL-35

• LIGHT / TNFSF14

• MMP-1

• MMP-2

• MMP-3

• Osteocalcin

• Osteopontin

• Pentraxin-3

• sTNF-R1

• sTNF-R2

• TSLP

• TWEAK / TNFSF12

Bio-Rad Bio-Plex® Pro Human Inflammation Panel 1, 37-Plex



Source: http://www.bio-rad.com/en-is/sku/171al001m-bio-plex-pro-human-inflammation-panel-1-37-plex

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MULTI-ARRAY ®

Meso Scale Diagnostics,

LLC (Electro-

chemiluminescence)

Bio-Plex

Bio-Rad (Luminex)

A2® Beckman Coulter

(Bead based

chemiluminescence)

FAST Quant Whatman

Scleicher & Schuell

BioScience ( Slide

array system)

IL-12p70

Manufacturer’s calibrator interval, ng/Lb 2500–0.6 4411–0.3 4800–6.5 50 000–12.2

Quantifiable interval, ng/Lc 2500–0.6 4411–0.27 577–7.1 625–2.4

Signal intervald ≅2 700 000–200 ≅23 000–30 ≅1000–25 ≅16 000–100

Mean CV within quantifiable intervale 9.60% 2.80% 8.70% 3.40%

IL-2

Manufacturer’s calibrator interval, ng/L 2500–0.6 2161–0.1 6630–9.1 10 000–2.4

Quantifiable interval, ng/L 2500–2.4 540–2.1 245–9 10 000–2.4

Signal interval ≅600 000–200 ≅18 000–100 ≅4000–100 ≅2000–30

Mean CV within quantifiable interval 5.00% 5.90% 10.00% 3.20%

IL-6


Quantifiable interval, ng/L 2500–0.6 138–2.1 577–7.1 625–2.4

Signal interval ≅1 300 000–400 ≅6000–80 ≅1000–25 ≅16 000–100


IL-10


Quantifiable interval, ng/L 2500–0.6 269–1.05 175–6.5 50 000–195

Signal interval ≅500 000–300 ≅70 000–20 ≅3000–70 ≅1500–300

Mean CV within quantifiable interval 5% 8% 6% 4%

IL-1β


Quantifiable interval, ng/L 2500–0.6 59–0.2 538–6.6 625–2.4

Signal interval ≅1 300 000–500 ≅9000–50 ≅3000–50 ≅20 000–400


Calibrators

Manufacturer’s recommended standard dilution factorf 1/4 Dilution 1/4 Dilution 1/3 Dilution 1/4 Dilution

Calibration curve 7 Concentrations 8 Concentrations 7 Concentrations 7 Concentrations

Comparison of the performance of 4

multiplex immunoassay platforms

The MULTI-ARRAY and Bio-Plex assays had the best performance with the lowest limits of detection, and the MULTI-ARRAY

system had the most linear signal output over the widest concentration range (105 to 106).



Clin Chem. Author manuscript; available in

PMC 2010 July 19.

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Flow Cytometry in Immune Monitoring

CHALLENGES FACED

Limited amount of blood and other tissue

material available from patients

Need for standardized procedures across

clinical trial sites

PBMC isolation and cryopreservation

Viability of samples upon thawing

Experience of staff

Whole blood vs. PBMC

Phenotyping vs. Functional

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Antigen-specific stimulation of T cells results in cytokine expression. Intracellular staining with IFNγ

FITC/CD69 PE/CD8 PerCPCy5.5/CD3 APC by Ficoll or CPT-processed PBMC from one representative HIV+ donor.


Functional Assays Intracellular Cytokine Analysis

Ruitenberg et al. BMC Immunology

2006 7:11 doi:10.1186/1471-2172-7-11

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Intracellular Cytokine Analysis

BioMed Research International Volume 2013, Article ID

726239, 11 pages http://dx.doi.org/10.1155/2013/726239

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Hickling et al., 1998

Tetramer Analysis

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Tetramer Analysis

Immunology, 146, 11-22

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Discovery of novel epitopes and

validation Immune Monitoring

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Analysis of T cell activation

ELISPOT analysis

Source: Covance Translation and

Biomarker Solutions

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Cells 2015, 4, 84-95; doi:10.3390/cells4010084


COMPARATIVE MULTI-DONOR STUDY OF IFNγ SECRETION

AND EXPRESSION BY HUMAN PBMCS USING ELISPOT

SIDE-BY-SIDE WITH ELISA AND FLOW CYTOMETRY ASSAYS


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Cells 2015, 4, 84-95; doi:10.3390/cells4010084


COMPARATIVE MULTI-DONOR STUDY OF IFNγ SECRETION AND

EXPRESSION BY HUMAN PBMCS USING ELISPOT SIDE-BY-SIDE

WITH ELISA AND FLOW CYTOMETRY ASSAYS


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Bendall, S.C.et al., 2012; Trends in Immunology

CyTOF® is a registered trademark of Fluidigm Canada Inc

Time of Flight

Antibodies labeled with elemental isotopes

Nebulizer

Analysis

Cell 1

Cell 2

Cell 3

.FCS

Quadrupole

Integrate per cell

Heavy (>100Da)

Reporter atomic ions

Light (<100Da)

Overly abundant ions


MASS CYTOMETRY (CyTOF®)

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Weighing the Pros and Cons of CyTOF®

Pros

1. Traditional labelling

techniques can be used

2. No autofluorescence; No

spectral overlaps

3. Minimal to no color

compensation

4. Designing panels is easier

Cons

1. Slow acquisition of samples

(>1000 events per second)

2. Much cleaner sample needed

3. Limited set of commercial

antibodies

4. Complex data analysis

5. Cells cannot be recovered

CyTOF® is a registered trademark of Fluidigm Canada Inc

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Maecker and Harari

Journal for ImmunoTherapy of Cancer

(2015) 3:44 DOI 10.1186/s40425-015-0085-x


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MHC II MHC I

CD4+ T CD8+ T

Endosomal Cytosolic

Antigen Processing and Presentation

© 2009 QIAGEN, all rights reserved

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T-cell Mediated Anti-Tumor Immunity

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Tumorigenesis

Isolation and identification of tumor specific epitopes

Potential use as peptide vaccine

Epitope Identification

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Tumor cells express

HLA-class I/peptide

complexes on their surface

Immunoprecipitation

of HLA-class I/peptide

complexes

Isolation of

HLA-class I

bound peptides

Identification of tumor

specific epitopes

Generation and validation

of epitope specific CTLs

Approach to Identify Tumor Epitopes

Presented by HLA Class I

Mass-spectrometry

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Isolation

Of “buffy coat”

Stimulate

with peptide

Three cycles of

restimulation

with peptide

Test effector function

of CTLs by ELISpot assay

(IFNg or Granzyme B)

Generation of Peptide Specific CTLs in vitro

Culture

“buffy coat”

derived cells

Public


18-33 0C 37 0C

Can the Identified Peptides with a Putative

HLA-A2 Binding Motif be Bound by HLA-A2?

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T2

T2+p13

IgG FITC W6/32 BB7.2

T2+Flu

T2+L1

10e0 10e1 10e2 10e3 10e4Green Fluorescence (GRN-HLog)

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BB7.2

Peptide pulsed T2

MF

I

Stabilization of Surface HLA Class I by

Synthetic Peptides

Shetty, V., Sinnathamby, G., Nickens, Z., Shah, P., Hafner et al., J of

Proteomics 74(5):728-43

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Michael A. Morse et al. Clin Cancer Res 2011;17:3408-3419

Immune Monitoring Using ELISPOT analysis

CLINICAL TRIALS

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https://en.wikipedia.org/wiki/Antibody-dependent_cell-

mediated_cytotoxicity#/media/File:Antibody-dependent_Cellular_Cytotoxicity.svg

Gated on CD3-CD56+ lymphs

Antibody Dependent Cellular Cytotoxicity

(ADCC)

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The Essential Role of Immune Monitoring

► Immune monitoring is a crucial component of

clinical trials

► A variety of techniques can be employed to

assess soluble mediators as well as cell

associated biomarkers

► Multiplex immunoassays to determine the levels

of soluble mediators and flow cytometry-based

assays to determine the levels of activation

markers on the surface of immune cells have

played a major role in immune monitoring

► Employing the right technique at the right time is

critical to determine the safety and efficacy of a

therapeutic intervention

ASSESSING SAFETY AND EFFICACY

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Sinnathamby Gomathinayagam, PhD

[email protected]

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Covance Inc., headquartered in Princeton, NJ, is the drug

development business of Laboratory Corporation of

America® Holdings (LabCorp®). Covance is the marketing

name for Covance Inc. and its subsidiaries

around the world.