Flow cytometry and Phenotypic/Functional Analysis in Immuno … · 2020-06-10 · Outline • Flow Cytometry Platform Overview • Backround on Adoptive Cell Therapy and T cell Retargeting

Yoav Peretz, Ph.D. Scientific Director

Caprion Biosciences/ImmuneCarta Adjunct Professor McGill University

Flow cytometry and Phenotypic/Functional Analysis in Immuno-Oncology

Adoptive T cell Therapies

Outline

•  Flow Cytometry Platform Overview

•  Backround on Adoptive Cell Therapy and T cell Retargeting Strategies •  Autologous transfer of NY-ESO-1 TCR-transduced T cells

•  Phenotypic and Functional Flow Cytometry Assay Development •  Monitoring NY-ESO-1 T cells (Persistence, Memory, Maturation, Immune Exhaustion markers)

•  Functional profile of NY-ESO-1 specific T cells (ICS)

•  Clinical study •  Flow Cytometry Analysis of the Manufactured Product

•  Immune Monitoring of patient PBMC (Clinical Progression vs Non-Progression)

•  TIL analysis

•  Conclusions

•  Differentiated service offering from 2 proprietary core technologies •  Balanced mix between discovery, pre-clinical and clinical development studies •  GLP operation with rigorous QA/QC

•  Best-in-class, industrialized, and quantitative mass spectrometry technology for unbiased protein biomarker discovery

•  Leading expertise in development and deployment of targeted proteomics using highly multiplexed MRM assays for biomarker development, validation

•  Greater reproducibility, throughput, and multiplexing capabilities than competing technologies

•  Expanded offering of proprietary biomarker assays

•  Differentiated expertise in multiparametric flow cytometry (up to 18 parameters)

•  Provides advanced immune monitoring for pre-clinical and clinical development of vaccines, biologics and immuno- therapeutics

•  Differentiated ability to rapidly develop, validate and deploy customized complex assays for clinical immune monitoring studies

•  Deep expertise in immune-oncology

PROTEOCARTA, is a gel-free, label-free mass spectrometry platform for comprehensive, quantitative and robust measurements of proteins across large sets of samples for biomarker discovery and validation

IMMUNECARTA is a proprietary immune monitoring platform offering multiparametric flow cytometry for comprehensive functional and phenotypical analyses of complex innate and adaptive immune responses

CAPRION’S PROPRIETARY CORE TECHNOLOGIES

Technologies and Applications

ü Enumerate:Discretecellularsubsetsinwholeblood(ex:CD34,TBNKusingTruCount)

ü An,gen-specificresponse:Mul5merdetec5onandiden5fica5onofHLA-restricteds5mulatoryepitopesbyELISPOTandflowcytometry

ü Func,onality:CellSignaling(PhosFlowTM),CytokineSecre,onProfile(ICS),Cellprolifera5on(CFSE),Degranula5on(BAT)

ü Phenotype:CellularDifferen5a5on,Apoptosis,Cellsubset-specificpanels(MDSC,Treg,Tmemory,Bcells,DCs…)

ü Serologicalprofiling:Mul5plexeddetec5onofsolubleinflammatorymediatorsinresponsetoimmunemodula5ngagents(MSD,ELISA)

ü Technology:18-colorMul5parametricSingleCellAnalysis(cellsurface,intra-cytoplasmic,intra-nuclear)

ü ComprehensiveTCellEpitopeMapping:Breadth,Magnitude&SpecificityofHLA-restricteds5mulatoryepitopesbyELISPOT

•  LSR Fortessa equipped with 5 lasers •  Up to 18 unique fluorescent markers can be monitored simultaneously plus with FSC and SSC parameters •  Enables redistribution of fluorescent markers onto other lasers in order to minimize fluorescence spill-over

between channels

Flow Cytometers

Laser Name Wavelength Power Detector

Array Mirror Filter

630 LP 695/40 BP505 LP 530/30 BP

488/10 BP750 LP 780/60 BP690 LP 710/50 BP630 LP 660/20 BP595 LP 610/20 BP505 LP 525/50 BP

450/50 BP735 LP 780/60 BP635 LP 670/30 BP600 LP 610/20 BP

585/15 BP735 LP 780/60 BP710 LP 730/45 BP

670/14 BP690 LP 740/35 BP450 LP 515/30 BP

379/25 BP

Blue 488 nm 50 mW Octagon

Yellow Green 561 nm 50 mW Octagon

Violet 405 nm 50 mW Octagon

UV 355 nm 20 mW Trigon

Red 640 nm 40 mW Trigon

•  Flow panel customization •  Selection of Clone/Fluorochrome

combinations •  Anticipated Antigen Expression Intensity •  Assay conditions (i.e Perm/no perm)

•  Panel setup and qualification is performed before Immune monitoring

•  Titrations •  Instrument settings and compensation •  FMO controls •  Precision on critical readouts

Laser, detector and filter combinations for LSR cytometers at Caprion

Adoptive Autologous “T cell” Therapies

ImmTACs (Immune mobilizing monoclonal TCRs against cancer)

Target: Surface protein ex: CD19 in NHL - Ab binding is higher affinity vs TCR - Only approx. 10% of targets are surface bound

DARTs (Dual-Affinity retargeting technology) BITEs (Bi-specific T cell engager)

Target: peptide-HLA - Affinity enhanced TCR ex: NY-ESO-1 in Myeloma - Recognizes endogenously processed Tumor Ag

- Greater number of targets available

TIL

Target: peptide-HLA - Biopsy or resection is required

Target: peptide-HLA - Affinity enhanced and fused to anti-CD3 - No cellular manufacturing

Target: Surface protein ex: CD19 in NHL

•  T cell redirecting Rx •  Can recruit several arms of the immune

system •  Easier/faster to produce •  Less costly

Aleksic, M., et al. (2012). Eur. J. Immunol

Increasing the affinity of Tumor-specific TCRs (NY-ESO-1)

•  All T cells undergo Thymic selection (2040-50 TCR variants)

•  This has evolved to remove autoreactive T cells from an individuals repertoire

•  Therefore the circulating repertoire to self antigens (tumor antigens) are of low affinity

•  MHC down regulation •  Local immunosuppressive mechanisms

•  Are NY-ESO-1 specific T cells Persisting? •  What is the Phenotype of NY-ESO-1 specific T cells with respect to markers of Immune

Dysfunction, Memory and Maturation? •  What is the functionality of NY-ESO-1 specific T cells? •  What is the Phenotype and Function of the Manufactured product prior to infusion?

Adapted from Wherry, J et al. Nature immunology. 2011.

Hierarchical Loss of T Cell Function is Associated with Duration of Antigenic Exposure, Inflammation and Increased Expression of Inhibitory Molecules (PD-1, CD160, 2B4)

Hierarchical Loss of T Cell Function leads to Immune Exhaustion

(Pre-Validation)

Method Development (Pre-validation)

Method Validation Sample Analysis

Identify; Establish Confirm Apply; Monitor

•  Assay Format •  Matrix Selection •  Stability •  LOD/LLOQ •  Precision •  Dilution/Linearity

•  Specificity •  Precision •  Dilution/ Linearity •  LLOQ •  Stability

Assay Phases

Flow Cytometry Panel Setup

Identify; Establish

•  Antibody cocktail •  Titration •  Instrument settings •  Specificity (Isotype) •  Spillover (FMOC)

Method Development of Flow Cytometry Panels

An,bodyPanels(TcellPanel)

Phenotyping

An,body

LiveDeadCD3CD4CD8

PentamerNY-ESO-1157-165(9C)CD45RACCR7

CD45ROCD95CD25CD127LAG-3PD-1TIM-3

Mahnke, Y., et al. (2013). Eur. J. Immunol

Immune inhibition / “exhaustion”

Treg markers

Memory

Lineage

TIM-3PD-1LAG-3

10.0

30.0

0.0

2.0

4.0

6.0

8.0

+++

-++

+-+

--+

++-

-+-

+--

---

Boolean Analysis of “Checkpoint” Marker Expression on T Cell Subsets

CD4

CD8

CD4+ TREG

TIM-3PD-1LAG-3

10.0

30.0

0.0

2.0

4.0

6.0

8.0

+++

-++

+-+

--+

++-

-+-

+--

---

TIM-3PD-1LAG-3

10.020.050.0

0.0

2.0

4.0

6.0

8.0

+++

-++

+-+

--+

++-

-+-

+--

---

Bar Chart LegendSUBJECT_ID: L50SUBJECT_ID: L747SUBJECT_ID: L770

T Cell Panel (Naïve/memory, PD-1, TIM-3, LAG-3)

•  Intra-assay CV below 10% for most

readouts •  This is a function of the number

of events accumulated in each target gate

SEB-stimulated PBMC (72h)

Analysis of Antigen-Specific Responses by Intracellular Cytokine Staining (ICS)

•  Quantification of the frequency of antigen-specific T cells through analysis of cytokine expression combined to Pentamer analysis

•  Assay performed on cryopreserved PBMC •  PBMC are thawed and counted •  PBMC are stimulated and incubated 6hrs prior to staining for Flow

Stimulate cells and treat with protein and transport

inhibitor

Fix and permeabilize cells

Stain cells Flow Cytometry Analysis

1 2

3 4

SEB-stimulated PBMC (6h)

An,bodyPanel

LiveDeadCD3CD4CD8

PentamerNY-ESO-1157-165(9C)CD45RACCR7Ki67

HLA-DRIL-2TNFaIFNg

GranzymeBCD107a

Effector cytokines

Memory

Lineage

Activation/ Proliferation

Analysis of the Distribution of Antigen-Specific CD4 & CD8 T Cell Subsets (Boolean Approach)

TIMEPOINT: Day 1 TIMEPOINT: Day 2 TIMEPOINT: Day 3

Pie Chart Arc LegendIFN gamma +

IL-2 +

TNF alpha +

IL-17 +

Pie SliceIL-17

TNF alphaIL-2

IFN gamma

10.0

15.0

0.0

2.0

4.0

6.0

8.0

++++

+++-

++-+

+-++

-+++

++--

+-+-

+--+

-++-

-+-+

--++

+---

-+--

--+-

---+

----

Bar Chart LegendTIMEPOINT: Day 1

TIMEPOINT: Day 2

TIMEPOINT: Day 3

Pie SliceIL-2

0.0

5.0

10.0

15.0

+

Freq

uenc

y of

CD

8 (%

)

Deconvolute

Total IL-2 secretion

4 3 2 1 0 # of Functions

Polyfunctional Monofunctional



IL-2 +

TNF alpha +

IL-17 +

Pie SliceIL-17

TNF alphaIL-2

IFN gamma

10.0

15.0

0.0

2.0

4.0

6.0

8.0

++++

+++-

++-+

+-++

-+++

++--

+-+-

+--+

-++-

-+-+

--++

+---

-+--

--+-

---+

----


TIMEPOINT: Day 2

TIMEPOINT: Day 3



IL-2 +

TNF alpha +

IL-17 +

Pie SliceIL-17

TNF alphaIL-2

IFN gamma

10.0

15.0

0.0

2.0

4.0

6.0

8.0

++++

+++-

++-+

+-++

-+++

++--

+-+-

+--+

-++-

-+-+

--++

+---

-+--

--+-

---+

----


TIMEPOINT: Day 2

TIMEPOINT: Day 3

D1 D2 D3

Flow cytometry Panel Qualification

FMOC No CD45RA FMOC No CCR7 Full Cocktail

Ungated

0 50K 100K 150K 200K 250KFSC-A

0

50K

100K

150K

200K

250K

FSC-H

95.7

Singlets

0 50K 100K 150K 200K 250KFSC-A

0

50K

100K

150K

200K

250K

SSC-A

84.9

0 103 104 105

<V450-A>: CD3

0

102

103

104

105

<Aqu

a-A>

: Viab

ility

92.6

72.7

Lymphocytes Viables CD3

0 103 104 105

<Qdot 605-A>: CD4

0

103

104

105

<Qdo

t 655

-A>:

CD8

64.4

29

0 103 104 105

<APC-Cy7-A>: CD45RA

0

103

104

105

<PE-

Cy7-

A>: C

CR7

48.3 0

0.010751.7

0 103 104 105

<APC-Cy7-A>: CD45RA

0

103

104

105

<PE-

Cy7-

A>: C

CR7

0 0.116

47.152.8

0 103 104 105

<APC-Cy7-A>: CD45RA

0

103

104

105

<PE-

Cy7-

A>: C

CR7

31.4 21.8

22.923.9

0 103 104 105

<APC-Cy7-A>: CD45RA

0

103

104

105

<PE-

Cy7-

A>: C

CR7

6.45 6.74e-3

0.06493.5

0 103 104 105

<APC-Cy7-A>: CD45RA

0

103

104

105

<PE-

Cy7-

A>: C

CR7

0.0116 5.81e-3

3.7296.3

0 103 104 105

<APC-Cy7-A>: CD45RA

0

103

104

105

<PE-

Cy7-

A>: C

CR7

5.15 0.38

3.3691.1

L205 (SEB)

WAVE (PMA-IONO)

Gated on CD8

0 50K 100K 150K 200K 250KFSC-A

0

50K

100K

150K

200K

250K

FSC-

H

90

0 50K 100K 150K 200K 250KFSC-A

0

50K

100K

150K

200K

250K

SSC-

A

72.2

Ungated Singlets Lymphocytes Viables CD3

CD4 CD8 CD8-Pentamer

Viables CD3 Viables CD3

0 103 104 105

<V450-A>: CD3

0

102

103

104

105

<Aqu

a-A>

: Viab

ility

64.2

63.1

0 103 104 105

<Qdot 605-A>: CD4

0102

103

104

105

<PE-

A>: P

entam

er

0.481

0 103 104 105

<Qdot 605-A>: CD4

0

103

104

105

<Qdo

t 655

-A>:

CD8

27.5

60.1

0 103 104 105

<Qdot 655-A>: CD8

0102

103

104

105

<PE-

A>: P

entam

er

5.1

0 103 104 105

<APC-Cy7-A>: CD45RA

0

103

104

105

<PE-

Cy7-

A>: C

CR7

5.46 0.213

0.29894

0 103 104 105

<APC-Cy7-A>: CD45RA

0

103

104

105

<PE-

Cy7-

A>: C

CR7

4.52 0.529

2.4792.5

0 103 104 105

<APC-Cy7-A>: CD45RA

0

103

104

105

<PE-

Cy7-

A>: C

CR7

4.33 0.718

3.2491.7

•  Fluorescence minus One Control •  Specificity/Sensitivity •  Spill Over •  Signal detection threshold

FMOC IFNγ (Gated on CD8)

FMOC No IFNγ

Full Cocktail

Full Cocktail

CD3

0.674 5.29 02.01 5.01 35.4 8.48

IL-2 TNFa IFNg Ki-67 HLA-DR Granzyme B CD107a

CD3

0.741 5.47 10.21.84 7.64 33.6 9


CD3

5.1 74.69.35e-4

94.1 62.5 96.9 45.2


CD3

5.78 7781.7

95.8 71.1 98.6 42.9


L205

(S

EB

) W

AVE

(P

MA

-ION

O)

Viables CD3

0 103 104 105

<Qdot 605-A>: CD4

0

103

104

105

<Qdo

t 655

-A>:

CD8

64.4

29

FMOC No IFNγ

Protocols for Pentamer Staining combined with ICS

Protocol 1: Pentamer staining BEFORE stimulation

•  Plate cells (1.5X106 cells per well) •  Add pentamer and Incubate for 10 min at RT •  Wash •  Add stimulators and CD107a (or DMSO in unstimulated

samples) and Golgi Plug/Stop, and incubate for 6h at 37°C

•  Wash •  Add surface cocktail and incubate 30 min at 4°C •  Wash •  Fix •  Permeabilize •  Add intracellular cocktail and incubate 30 min at 4°C •  Wash •  Acquire data on flow cytometer

Protocol 2: Pentamer staining AFTER stimulation

•  Plate cells (1.5X106 cells per well) •  Add stimulators and CD107a (or DMSO in unstimulated

samples) and Golgi Plug/Stop, and incubate for 6h at 37°C

•  Wash •  Add pentamer and incubate for 10 min at RT •  Wash •  Add surface cocktail and incubate 30 min at 4°C •  Wash •  Fix •  Permeabilize •  Add intracellular cocktail and incubate 30 min at 4°C •  Wash •  Acquire data on flow cytometer

•  We observed that the frequency of NY-ESO-1 TCR detected and backround levels observed by ICS varied depending on the protocol used

•  Following TCR triggering, the complex gets downregulated •  Pentamer staining triggers the TCR

Protocol Comparison (L205, Unstimulated)

0 103 104 105

<Qdot 655-A>: CD8

0102

103

104

105

<PE-

A>: P

entam

er

0.632

0 103 104 105

<Qdot 655-A>: CD8

0102

103

104

105

<PE-

A>: P

entam

er

0.964

CD3


0 11.8 22.53.34 9.91 94.7 15.7

CD3


0 0.17 0.7952.1 6.81 72.4 0.738

0 103 104 105

<Qdot 605-A>: CD4

0

103

104

105

<Qdo

t 655

-A>:

CD8

59.2

33.4

CD3

6.54e-3 0.305 1.021.97 7.13 37.3 0.953


0 103 104 105

<Qdot 605-A>: CD4

0

103

104

105

<Qdo

t 655

-A>:

CD8

58.2

33.6

CD3

3.25e-3 0.0537 0.3561.95 6.49 37 0.491


Gated on CD8

Gated on CD8-Pentamer

Gated on CD8


Protocol 1

Protocol 2

STAIN BEFORE STIM

STAIN AFTER STIM

•  Better pentamer separation and detection frequency

•  Lower backround levels

•  Staining before stimulation induces backround

11.8 22.5

0.17 0.78

Protocol Comparison (L205, CEF)

0 103 104 105

<Qdot 655-A>: CD8

0102

103

104

105

<PE-

A>: P

entam

er

0.535

CD3


0 67.4 78.33.6 7.59 93.2 41.1

0 103 104 105

<Qdot 655-A>: CD8

0102

103

104

105

<PE-

A>: P

entam

er

0.65

CD3


0.0667 30.4 31.52.07 6.53 47.5 21.6

0 103 104 105

<Qdot 605-A>: CD4

0

103

104

105

<Qdo

t 655

-A>:

CD8

59.7

33

CD3

3.24e-3 0.942 1.652.12 6.84 37 1.55


0 103 104 105

<Qdot 605-A>: CD4

0

103

104

105

<Qdo

t 655

-A>:

CD8

58.3

33.7

CD3

9.01e-3 0.524 0.9962.15 6.13 35.8 1.1


Protocol 1

Protocol 2

•  CMV-specific responses are detected (TNFα and IFNγ)

•  Better pentamer separation and detected frequency

Gated on CD8

Gated on CD8



•  Stimulation with CEF induces TCR downregulation

STAIN BEFORE STIM

STAIN AFTER STIM

67.4 78.3

30.4 31.5

CohortsandStudyDesign:PhaseI/IIasinglearmtrialthatenrolled20pa5entswhoreceivedAutologousStemCellTransplantfollowedbygene-modifiedTcells(NY-ESO-1C259)InclusioncriteriaPa5entscreeningforHLA*0201,LAGE-1orNY-ESO-1posi5vemyeloma(70screened/50excluded)ClinicalSites:2clinicalsitesObjec,vesPrimary:Determinethesafetyandtolerabilityofautologousgene5callymodifiedTcells.Secondary:Evaluatecorrelatesoftreatmentefficacybymeasuringimmuneparameterssuchasthepersistenceandfunc5onalityofNY-ESO-1transducedTcells.Hypothesis:Adop5vetransferofNY-ESO-1enhancedTCR’swouldimprovethedura5onofASCTinadvancedMM

Rapoport, A.P. et al. July 2015. Nature Medicine

Clinical Study Design

Investigational Drug Manufacturing Process and Screening

Patient Screening (NY-ESO-1/LAGE-1 mRNA

and HLA-A*0201)

Blood Cell Collection (Leukapheresis)

Enrichment/ Selection of CD3+ T cells

Activation/Lentiviral Transduction (NY-ESO-1

TCR)

T cell Expansion

Harvesting/Bead Removal & Formulation

T cell Product and Release Testing

T cell Infusion Mean: 2 x 109 / donor

Clinical sites

Central Manufacturing Site

Deplete Monocytes and CD25+ cells

Bead-based activation/expansion (CD3/CD28) >20% transduction efficiency

1 day

1 day

8-12 days

8-10 days

Patient conditioning and lymphodepleting chemotherapy

Clinical Blood Sample Processing & Storage Capabilities

SST No additive

Standard K2/3EDTA

Na-Heparin

Whole blood samples

CytoCHEX K3EDTA

Leukapheresis ACD

CPT Na-Heparin Na-Citrate

Frozen serum

aliquots PBMC

isolation

FICOLL

Frozen plasma aliquots

PBMC Cryovials

Sample assayed fresh (within a stability

window)

SPIN

PBMC isolation

FICOLL

Samples assayed in batches

All procedures are conducted using approved SOP

Smart Tube

Fixative

ThawingSta,s,csoftheCohort

0 10 20 30 40 50 60 70 80 90

100 110

0 25 50 75 100 125 150 175 200

Viab

ility

Recovery

N = 206 Viability = 95% (median) Recovery = 61% (median)

PhenotypingAssay-HierarchicalGa,ng

•  Noted differences in CD4/CD8 ratios and frequencies of CD4 between both donors for both the MP and the baseline timepoint

•  CD4 frequencies were approx. 3 times higher in donor 201 MP and at baseline

L205

WAVE049B

01411-253_MP

01411-253_Day-50

01411-253_Day21

01411-253_Day42

01411-253_Day1000

20

40

60

80

100

120

Freq

uenc

y of

tota

l

Singlets

L205

WAVE049B

01411-253_MP

01411-253_Day-50

01411-253_Day21

01411-253_Day42

01411-253_Day1000

20

40

60

80

100

Freq

uenc

y of

via

ble

CD3

CD4+ lymphocytes

L205

WAVE049B

01411-253_MP

01411-253_Day-50

01411-253_Day21

01411-253_Day42

01411-253_Day1000

20

40

60

80

100

120

Freq

uenc

y of

sin

glet

Lymphocytes

L205

WAVE049B

01411-253_MP

01411-253_Day-50

01411-253_Day21

01411-253_Day42

01411-253_Day1000

20

40

60

80

100

Freq

uenc

y of

via

ble

CD3

CD8+ lymphocytes

L205

WAVE049B

01411-253_MP

01411-253_Day-50

01411-253_Day21

01411-253_Day42

01411-253_Day1000

20

40

60

80

100

120

Freq

uenc

y of

lym

phoc

ytes

Viable CD3+ lymphocytes

L205

WAVE049B

01411-253_MP

01411-253_Day-50

01411-253_Day21

01411-253_Day42

01411-253_Day1000.0

0.5

1.0

1.5

2.0

2.5

CD4/

CD8

Ratio

CD4/CD8 Ratio

253 (Clinical Progressor)

L205

WAVE049B

01411-201_MP

01411-201_Day-50

01411-201_Day7

01411-201_Day21

01411-201_Day42

01411-201_Day100

01411-201_Day180

01411-201_Day270

01411-201_Day3600

20

40

60

80

100

120

Freq

uenc

y of

tota

l

Singlets

L205

WAVE049B

01411-201_MP

01411-201_Day-50

01411-201_Day7

01411-201_Day21

01411-201_Day42

01411-201_Day100

01411-201_Day180

01411-201_Day270

01411-201_Day3600

20

40

60

80

100

Freq

uenc

y of

via

ble

CD3

CD4+ lymphocytes

L205

WAVE049B

01411-201_MP

01411-201_Day-50

01411-201_Day7

01411-201_Day21

01411-201_Day42

01411-201_Day100

01411-201_Day180

01411-201_Day270

01411-201_Day3600

20

40

60

80

100

120

Freq

uenc

y of

sin

glet

Lymphocytes

L205

WAVE049B

01411-201_MP

01411-201_Day-50

01411-201_Day7

01411-201_Day21

01411-201_Day42

01411-201_Day100

01411-201_Day180

01411-201_Day270

01411-201_Day3600

20

40

60

80

100

Freq

uenc

y of

via

ble

CD3

CD8+ lymphocytes

L205

WAVE049B

01411-201_MP

01411-201_Day-50

01411-201_Day7

01411-201_Day21

01411-201_Day42

01411-201_Day100

01411-201_Day180

01411-201_Day270

01411-201_Day3600

20

40

60

80

100

120

Freq

uenc

y of

lym

phoc

ytes

Viable CD3+ lymphocytes

L205

WAVE049B

01411-201_MP

01411-201_Day-50

01411-201_Day7

01411-201_Day21

01411-201_Day42

01411-201_Day100

01411-201_Day180

01411-201_Day270

01411-201_Day3600.0

0.5

1.0

1.5

2.0

2.5

CD4/

CD8

Ratio

CD4/CD8 Ratio

201 (Clinical Responder)

L205

WAVE049B

01411-201_MP

01411-201_Day-50

01411-201_Day7

01411-201_Day21

01411-201_Day42

01411-201_Day100

01411-201_Day180

01411-201_Day270

01411-201_Day3600

2

4

6

8

10

12

Freq

uenc

y of v

iable

CD3

Pentamer-CD8

L205

WAVE049B

01411-201_MP

01411-201_Day-50

01411-201_Day7

01411-201_Day21

01411-201_Day42

01411-201_Day100

01411-201_Day180

01411-201_Day270

01411-201_Day3600

2

4

6

8

10

12Fr

eque

ncy o

f viab

le CD

3 Pentamer-CD4

L205

WAVE049B

01411-201_MP

01411-201_Day-50

01411-201_Day7

01411-201_Day21

01411-201_Day42

01411-201_Day100

01411-201_Day180

01411-201_Day270

01411-201_Day3600

20

40

60

80

100

Freq

uenc

y of P

enta

mer

-CD4

TReg (CD127-CD25+) in Pentamer-CD4

201 (Clinical Response)

•  Loss of NY-ESO-1 T cell persistence is associated with clinical progression

NY-ESO-1TCellPersistence

253 (Clinical Progression)

L205

WAVE049B

01411-253_MP

01411-253_Day-50

01411-253_Day21

01411-253_Day42

01411-253_Day1000.0

2.0

4.0

6.0

8.0

Freq

uenc

y of v

iable

CD3

Pentamer-CD8

L205

WAVE049B

01411-253_MP

01411-253_Day-50

01411-253_Day21

01411-253_Day42

01411-253_Day1000.0

0.5

1.0

1.5

Freq

uenc

y of v

iable

CD3

Pentamer-CD4

L205

WAVE049B

01411-253_MP

01411-253_Day-50

01411-253_Day21

01411-253_Day42

01411-253_Day1000.0

2.0

4.0

6.0

8.0

10.025.050.075.0

100.0

Freq

uenc

y of P

enta

mer

-CD4

TReg (CD127-CD25+) in Pentamer-CD4

0.56% 0.22%

MemoryProfileofNY-ESO-1Pentamer-CD4Freq

uencieso

fparen

t(%

ofP

entamer-CD4

)

•  Distinct distribution of memory/maturation profiles within NY-ESO-1 CD4 T cells (MP and Patient sample) •  Progression associated with Effector phenotypes contrary to TCR persistence associated with a Central memory phenotype

TIMEPOINT: MP TIMEPOINT: Day21 TIMEPOINT: Day42 TIMEPOINT: Day100

Pie SliceCD45ROCD45RA

CCR7Subset

SUBJECT_ID0,0

10,0

20,0

30,0

40,0

50,0

+++

Pentamer-CD401411-253

-++


+-+


--+


++-


-+-


+--


---


Bar Chart LegendTIMEPOINT: MP

TIMEPOINT: Day21

TIMEPOINT: Day42

TIMEPOINT: Day100



CCR7Subset

SUBJECT_ID0,0

20,0

40,0

60,0

+++


-++


+-+


--+


++-


-+-


+--


---



TIMEPOINT: Day21

TIMEPOINT: Day42

TIMEPOINT: Day100


TIMEPOINT: MP TIMEPOINT: Day7 TIMEPOINT: Day21 TIMEPOINT: Day42 TIMEPOINT: Day100 TIMEPOINT: Day180 TIMEPOINT: Day270 TIMEPOINT: Day360


CCR7Subset

SUBJECT_ID0.0

20.0

40.0

60.0

80.0

+++


-++


+-+


--+


++-


-+-


+--


---



TIMEPOINT: Day7

TIMEPOINT: Day21

TIMEPOINT: Day42

TIMEPOINT: Day100

TIMEPOINT: Day180

TIMEPOINT: Day270

TIMEPOINT: Day360



CCR7Subset

SUBJECT_ID0.0

20.0

40.0

60.0

80.0

+++


-++


+-+


--+


++-


-+-


+--


---



TIMEPOINT: Day7

TIMEPOINT: Day21

TIMEPOINT: Day42

TIMEPOINT: Day100

TIMEPOINT: Day180

TIMEPOINT: Day270

TIMEPOINT: Day360201

(Clinical Response)

MemoryProfileofNY-ESO-1Pentamer-CD8Freq

uencieso

fparen

t(%

ofP

entamer-CD8

)TIMEPOINT: MP TIMEPOINT: Day21 TIMEPOINT: Day42 TIMEPOINT: Day100


CCR7Subset

SUBJECT_ID0,0

20,0

40,0

60,0

+++


-++


+-+


--+


++-


-+-


+--


---



TIMEPOINT: Day21

TIMEPOINT: Day42

TIMEPOINT: Day100



CCR7Subset

SUBJECT_ID0,0

20,0

40,0

60,0

+++


-++


+-+


--+


++-


-+-


+--


---



TIMEPOINT: Day21

TIMEPOINT: Day42

TIMEPOINT: Day100



CCR7Subset

SUBJECT_ID0.0

20.0

40.0

60.0

80.0

+++


-++


+-+


--+


++-


-+-


+--


---



TIMEPOINT: Day7

TIMEPOINT: Day21

TIMEPOINT: Day42

TIMEPOINT: Day100

TIMEPOINT: Day180

TIMEPOINT: Day270

TIMEPOINT: Day360



CCR7Subset

SUBJECT_ID0.0

20.0

40.0

60.0

80.0

+++


-++


+-+


--+


++-


-+-


+--


---



TIMEPOINT: Day7

TIMEPOINT: Day21

TIMEPOINT: Day42

TIMEPOINT: Day100

TIMEPOINT: Day180

TIMEPOINT: Day270

TIMEPOINT: Day360



•  Distinct distribution of memory/maturation profiles within NY-ESO-1 CD8 T cells (MP and Patient sample) •  Progression associated with Effector phenotypes contrary to TCR persistence associated with a Central memory phenotype

CheckpointMoleculeExpression(NY-ESO-1Pentamer-CD8)

TIM-3PD-1LAG-3Subset

SUBJECT_ID

40.080.0

0.0

5.0

10.0

15.0

20.0

+++


++-


+-+


-++


+--


-+-


--+


---



TIMEPOINT: Day7

TIMEPOINT: Day21

TIMEPOINT: Day42

TIMEPOINT: Day100

TIMEPOINT: Day180

TIMEPOINT: Day270

TIMEPOINT: Day360TIM-3PD-1LAG-3Subset

SUBJECT_ID

40,080,0

0,0

5,0

10,0

15,0

20,0

+++


++-


+-+


-++


+--


-+-


--+


---



TIMEPOINT: Day21

TIMEPOINT: Day42

TIMEPOINT: Day100

Freq

uencieso

fparen

t(%

ofP

entamer-CD8

)

•  No accumulation of checkpoints on NY-ESO-1 CD8 T cells in both donors •  Switch in phenotype during progression (TIM-3 to PD-1 switch)



ICSAssay-IFNγ(NY-ESO-1Pentamer-CD8)

IFNgSubset

STIMULATIONSUBJECT_ID

0.0

20.0

40.0

60.0

80.0

+Pentamer-CD8

T201411-201

+Pentamer-CD8T2-NY-ESO01411-201

+Pentamer-CD8PMA-IONO01411-201


TIMEPOINT: Day7

TIMEPOINT: Day21

TIMEPOINT: Day42

TIMEPOINT: Day100

TIMEPOINT: Day180

TIMEPOINT: Day270

TIMEPOINT: Day360

IFNgSubset

STIMULATIONSUBJECT_ID

0,0

20,0

40,0

60,0

80,0

+Pentamer-CD8

T201411-253

+Pentamer-CD8T2-NY-ESO01411-253

+Pentamer-CD8PMA-IONO01411-253

Freq

uencieso

fparen

t(%

ofP

entamer-CD8

)Bar Chart Legend

TIMEPOINT: MP

TIMEPOINT: Day21

TIMEPOINT: Day42

TIMEPOINT: Day100



•  Presence of NY-ESO-1 specific IFNγ secretion in both patients

ICSAssay-IFNγ, TNFα, andIL-2 �(NY-ESO-1Pentamer-CD8)

TNFaIL-2IFNg

SubsetSTIMULATIONSUBJECT_ID

40.0

45.0

0.0

10.0

20.0

30.0

+++

Pentamer-CD8T2-NY-ESO01411-201

++-


+-+


-++


+--


-+-


--+


---



TIMEPOINT: Day7

TIMEPOINT: Day21

TIMEPOINT: Day42

TIMEPOINT: Day100

TIMEPOINT: Day180

TIMEPOINT: Day270

TIMEPOINT: Day360

TNFaIL-2IFNg

SubsetSTIMULATIONSUBJECT_ID

25,0

30,0

0,0

5,0

10,0

15,0

20,0

+++


++-


+-+


-++


+--


-+-


--+


---



TIMEPOINT: Day21

TIMEPOINT: Day42

TIMEPOINT: Day100

Freq

uencieso

fparen

t(%

ofP

entamer-CD8

)

•  Persistence and functional diversification of responses are apparent only in patient 201 •  Presence of IL-2 secretion capacity is also present only for patient 201



•  Clinical sample procurement •  Clinical data and pathology report available

ELISpot Multiparametric Flow Cytometry

•  Network of clinical and academic collaborators •  Drafting protocol and consent forms for

submission to the Research Ethics Board

•  Sample preparation •  Mechanical disruption of tumor sample +/-

enzymatic digestion into a single cell suspension

•  Sample shipment in temperature-controlled containers for immune monitoring and/or proteomics analysis

•  Immune monitoring and/or proteomic analysis •  Phenotypic and functional single cell profiling

Clinical Immune Monitoring of Tumor Infiltrating Lymphocytes (TIL)

Tumor Microenvironment and Frequency Regulatory T cells

FoxP

3

CD25

Healthy donor PBMC control

Inverted CD4/CD8 ratio in situ versus PBMC

Increased frequency of intra-tumoral TREG as compared to PBMC samples

CD

45

CD

4

CD

45

CD

4

CD8

TIL from NSCLC tumor

samples

CD8

FoxP

3

CD25

Phenotypic Characterization of Lymphocytes from Tumor and Adjacent Tissues (n=10)

% o

f CD

4 or

CD

8 T

cells

ex

pres

sing

m

arke

r of i

nter

est

GITRCD0.0

1.0

2.0

3.0

4.0

5.0

+4

+#

+8

+

137CD0.0

1.0

2.0

3.0

4.0

+4

+#

+8

+#

PD1CD0.0

20.0

40.0

60.0

+4

+#

+8

+#

TIM3CD0.0

3.0

6.0

9.0

12.0

+4

+#

+8

+#

Bar Chart LegendSAMPLE_TYPE: Adjacent Tissue

SAMPLE_TYPE: Tumor

Increased expression of several “checkpoint”

markers on TIL as compared to

lymphocytes from healthy adjacent tissue

TIM3PD1137GITRCD

20.0

40.0

60.0

0.01.02.03.04.0

++++8

-+++8

+-++8

--++8

++-+8

-+-+8

+--+8

---+8

+++-8

-++-8

+-+-8

--+-8

++--8

-+--8

+---8

----8

% o

f CD

8 T

cells

ex

pres

sing

m

arke

r of i

nter

est Coexpression of PD1

and TIM-3 on TIL as compared to lymphocyte

from healthy adjacent tissue

Assessment of the Functionality of Tumor Infiltrating Lymphocytes

Healthy Donor PBMC control NSCLC Tumor Sample

Significant decrease in the frequency of IFNγ-producing CD8 and CD4 TILs compared to PBMC following ex vivo anti-CD3/anti-CD28 stimulation

CD

4

CD8

CD

3

IFNγ

Conclusion

•  Flow cytometry is a unique technology that gathers phenotypic and functional data on single cells from heterogeneous mixtures such as blood and tissues.

•  Relative distribution of phenotypic and functional subsets

•  Predictive and/or correlative value with markers of clinical disease progression

•  Several factors must be considered when developing a Flow Cytometry panel •  Sample Matrix, Integrity and Stability

•  Clone/Fluorophore Combinations

•  Anticipated Antigen Expression Levels

•  Signal Specificity (Isotype and FMO Controls)

•  Defining optimal duration of stimulation and staining procedures

•  Trending Control

For additional information, please visit

our website:

www.caprion.com

or contact us:

[email protected]

Booth 535 Poster presentation by Laetitia Cortes Title: Metaproteomic analysis of the infant fecal microbiome

Documents

Flow cytometry and Phenotypic/Functional Analysis in Immuno … · 2020-06-10 · Outline • Flow Cytometry Platform Overview • Backround on Adoptive Cell Therapy and T cell Retargeting