octet red96 to select high affinity and specific t-cell ... · 2 immunocore is based near oxford in...

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Using Octet RED96 to select high affinity and

specific T-Cell receptors

Dr. Jonathan LowtherSenior Scientist II

22nd April 2015

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Immunocore is based near Oxford in the UK

Immunocore is developing a unique platform of biologics based on soluble T

Cell Receptors (‘monoclonal TCRs’) which target a variety of Class I HLA

epitopes

Anti-Cancer monoclonal TCRs are engineered to produce bi-specific reagents,

‘ImmTACs’ (Immune mobilising monoclonal TCRs Against Cancer)

Lead programme IMCgp100 in phase IIa in UK and USA

In house target validation leading to

an expanding portfolio of >25 validated targets

Strategic partnerships with Genentech, GSK,

MedImmune and Eli Lilly

>140 staff

Immunocore – the world’s leading soluble T cell receptor company

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Antibody based therapeutics can only target cell surface proteins

Cell surface protein

Antibodies make excellent therapeutics…

…but only 10% of targets are cell surface proteins

Cancer cell

CAR

T cell

Antibodies

ADCs

Bi-specifics

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T cell receptors target HLA presented peptides

Intracellular protein

Proteasome

TAP

HLAPeptides

HLA-peptide

Antigen

nearly 100% of targets are

available as HLA-peptides

Cancer cell


CAR

T cell

Antibodies

ADCs

Bispecifics

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T cell receptors target HLA presented peptides

Intracellular protein

Proteasome

TAP

HLAPeptides

HLA-peptide

Antigen

T cellT cell

receptor

T cells scan HLA-peptides

with their T cell receptor

(TCR)

Cancer cell

TCRs have access to a wider choice of targets = access to potentially safer targets


CAR

T cell

Antibodies

ADCs

Bispecifics

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Engineering of ImmTACs

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Engineering Step 1: Stabilising TCRs as soluble proteins

Truncation of trans-membrane domains and relocation of a stabilising

inter-chain disulphide bond

native TCR soluble mTCR

Vα

Cα

Vβ

Cβ

SS

SS

S

SS

SS

S

Vα

Cα

Vβ

Cβ

SS

SS

SS

SS

S

S

Vα

Cα

Vβ

Cβ

SS

SS

S

SS

SS

S

Vα

Cα

Vβ

Cβ

SS

SS

SS

SS

S

S

Cell Surface

Overlay of mTCR and ‘natural’ TCR crystal structures – engineered disulphide bond is highlighted

Boulter et al. (2003) Protein Engineering vol. 16, no. 9 pp. 707-711,

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Engineering Step 1: Stabilizing TCRs as soluble proteins

The two mTCR chains, α and β, are

produced in E.coli as IBs

The α and β chains are refolded in

vitro (chemically controlled reaction)

mTCRs are purified by ion exchange

and gel filtration chromatography

mTCRs are stable at room

temperature for months

Purified, soluble mTCR

disulphide-linked mTCR

β-chainα-chain

Boulter et al. (2003) Protein Engineering vol. 16, no. 9 pp. 707-711,

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α/β TCR

peptide

HLA

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Engineering Step 2: Enhancing TCR affinity

TCR contacts to peptide/HLA is

through six hyper-variable loops

(CDRs)

CDR3s mainly contact the peptide

CDR2s mainly contact the HLA

CDRs

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Engineering Step 3: fusing the high affinity TCR to an effector molecule, an anti-CD3 scFv

ImmTACs (Immune-mobilising mTCR Against Cancer)

are bi-functional biologics

Key points

TCR affinity increased several million fold

Soluble, highly stable protein, low immunogenic format

Anti-CD3 scFv potency optimised

77KDa size provides good tumour penetration

Easy and cheap manufacture (E.coli)

Targeting end:

Soluble high affinity TCR

Effector end:

Anti-CD3 scFv

TCR affinity improved from µM to pM

Liddy et al. Monoclonal TCR-redirected tumour cell killing, Nature Medicine 2012 Jun;18(6):980-7.

doi: 10.1038/nm.2764

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ImmTACs

Mechanism of action

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ImmTAC mechanism of action – T cell redirection

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Cancer cell

T cell

Targeting driven by pM TCR affinity

ImmTAC

therapeutic

engineered TCR specific

for target pHLA with

picomolar affinity

scFv specific for CD3

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1 2

Cancer cellCancer cell

T cell T cell


Low affinity anti-CD3 recruits T cells

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1 2

3


Cancer cell

T cell

T cell T cell



Immune synapse forms

lytic granules

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1 2

3


Cancer cell

T cell

T cell T cell




lytic granules

EM images were taken by Dr. E. Johnson (Dunn School, Oxford)

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1 2

3


Cancer cell

T cell

T cell T cell




lytic granules

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T cell

Lytic granules migrate and release – killing target by apoptosis

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1 2


T cell T cell



4

T cell

Lytic granules migrate and release – killing target by apoptosis

EM images were taken by Dr. E. Johnson (Dunn School, Oxford)

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µM TCR (Wild Type)

CDR1α

CDR2α

CDR3α

CDR1β

CDR2β

CDR3β

nM

nM

nM

nM

nM

nM

pM TCR

individualCDR libraries

CDR* combinations

phage TCR soluble TCR

Affinity maturation: phage display selection and CDR* combination

Yi et al. (2005) Nature Biotechnology vol. 23, no. 3 pp. 349-354

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Molecular Evolution to increase mTCR affinity

Selection

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BIAcore binding analysis of TCR mutant combinations

-100

-40

20

80

140

200

260

320

380

440

500

560

620

680

740

800

-500 -200 100 400 700 1000 1300 1600 1900 2200 2500

Time sec

Re

sp

on

se

RU

3 CDRs mutated combined:t ½ = 8hours KD in the pM range

WT TCR: t ½ = 6.4sec

KD in the µµµµM range

2 CDRs mutated combined:t ½ = 15 minKD in the low nM range

1CDR mutated:t ½ = 10.5 minKD in the high nM range

Affinity enhanced TCRs have vastly extended antigen residence times

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Typical SPR experiments in Biochemistry, Immunocore

Experiment Time (Biacore) No. TCRs (Biacore)

Single concentration low – medium affinity 0.5 day multiple

Single concentration - high affinity 1 day ≤ 4

Equilibration titration - low affinity 1 day ≤ 4

Equilibration titration - high affinity (single

cycle kinetics)1 day ≤ 2

Alanine scanning and X-scanning 2 – 3 days 1

Cross-reactivity screen - low affinity 1 day multiple

ILT2 titration for pHLA validation - multiple

CD3 assay for ImmTAC scFv validation 1 day ≤ 4

Biacore is reliable but limited throughput particularly for high affinity TCRs/ImmTACs

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Low Affinity HLA1 TCR1 Interaction in 1x Kinetics Buffer with Double Referencing

Kinetics Analysis: 1:1 global fit Equilibrium Titration Analysis

KD (M) KD Error kon (1/Ms) kon Error kdis (1/s) kdis Error

4.81E-06 2.52E-07 6.44E+04 3.24E+03 3.10E-01 4.50E-03

KD 4.30E-06 ±1.5E-07M

Good agreement between kinetics and equilibrium titration data

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Medium Affinity HLA1 TCR2 Interaction in 1x Kinetics Buffer with Double Referencing

Kinetics Analysis: 1:1 global fit Equilibrium Titration Analysis

KD 2.70E-07 ±1.8E-08M



2.69E-07 1.14E-08 1.04E+05 4.25E+03 2.79E-02 3.28E-04

Good agreement also with KD from SPR

KD (M) kon (1/Ms) kdis (1/s)

2.39E-07 2.38E+05 6.86E-02SPR Data

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High Affinity HLA1 TCR3 Interaction in 1x Kinetics Buffer with Double Referencing

Kinetics Analysis: 1:1 global fit


1.35E-10 <1.0E-12 1.56E+05 6.39E+02 2.10E-05 5.60E-08

SPR KD Data:KD (M) kon (1/Ms) kdis (1/s)

7.90E-11 1.73E+05 1.37E-05

Good agreement (within 2 fold) with SPR data

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Octet: ranking high affinity ImmTACs

Sample ID kon (1/Ms) kdis (1/s) t1/2 (hr) KD (M)

ImmTAC 21 1.26E+05 1.73E-05 11.1 1.38E-10

ImmTAC 22 6.18E+04 3.31E-05 5.8 5.36E-10

ImmTAC 23 6.14E+04 1.95E-05 9.9 3.18E-10

ImmTAC 24 4.07E+04 2.97E-04 0.6 7.29E-09

ImmTAC 25 7.49E+04 4.87E-05 4.0 6.50E-10

ImmTAC 26 6.53E+04 1.75E-04 1.1 2.68E-09

*ImmTAC 10 5.62E+04 2.23E-05 8.6 3.96E-10

Ranking high affinity ImmTACs at a single concentration

Experimental set-up [ImmTAC] = 50nM, 15 min on, 60 min off, with double referencing

Affinities compared to current best ImmTAC kept as an internal standard, in this case ImmTAC 10

From the panel below ImmTAC 21 was selected for testing in cellular assays

*internal standard

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Octet: investigating TCR specificity

Wild-type peptide containing L-Tryp at P5

TCR-07 WT TCR-01 WT

Substituted peptide containing 1-Nal at P5

TCR-07 WT TCR-01 WT

Incorporating unnatural amino acid 1-Nal abolished binding by TCR-07 WT

but did not affect binding by TCR-01 WT

pHLA KD(TCR-01 WT) (µM) KD(TCR-07 WT) (µM)

Wild-type (P5=W) 3.6 8

Mimic (P5=1Nal) 5 No binding

L-Tryptophan 1-Naphthylalanine

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Specificity: X-scanning TCRs

Each residue along the peptide sequence is replaced by X

X = all other amino acids except for wild-type and cysteine

� e.g. TAX (HLA-A2)

� LLFGYPVYV WT

� XLFGYPVYV pHLA1

� LXFGYPVYV pHLA2

� LLXGYPVYV pHLA3

� LLFXYPVYV pHLA4

� LLFGXPVYV pHLA5

� LLFGYXVYV pHLA6

� LLFGYPXYV pHLA7

� LLFGYPVXV pHLA8

� LLFGYPVYX pHLA9

Biotinylated pHLA prepared

for each X-substituted peptide

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Octet: X-scan experimental set-up

Sensors loaded in diagonal format to ensure equal loading

Limited TCR for sample plate so 3 experiments required for full scan (1) X1, X2, X3, irrelevant

(2) X4, X5, X6, irrelevant (3) X7, X8, X9, irrelevant

Sample plate

Sensor plate

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Octet: X-scan plots

KD values estimated from steady state plots and normalised to KD for

wild-type peptide

X1 X2 X3

X4 X5 X6

X7 X8 X9

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X-scan profile generated on Octet

X-scan profile gives useful information on peptide specificity

We will be able to generate these profiles even quicker on RED384

0.00

0.20

0.40

0.60

0.80

1.00

1.20

1.40

1.60

1.80

2.00

WT X1 X2 X3 X4 X5 X6 X7 X8 X9

KD

(X)/K

D(W

T)

µM Affinity TCR

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Octet: Titration of non-biotinylated pHLA-T2R8 against immobilised biotinylated TCR

Loading Sample ID Sample ID Conc. (nM) kobs (1/s) kdis (1/s) KD (M)

bio-a22b31 T2R8 non bio 15.7 1.55E-02 1.12E-02 4.02E-08



bio-a22b31 T2R8 non bio 125 4.15E-02 1.45E-02 6.69E-08



Chi^2/DoF 0.000131702

R^2 0.997383463

RMax 0.746242978

KD 8.60E-08 ±4.6E-09M


Good agreement (within 2 fold) with SPR data (111nM)

This experimental set-up may be useful for screening large numbers of pHLA

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Octet timeline at Immunocore so far…….

Contacted ForteBioJanuary 2014

Demo RED96March 2014

Installation RED96September 2014

Installation RED384March 2015

Follow-up meetingJan 2015

Site-licence installedMar 2015

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Octet (RED96) performance

ExperimentTime

(Biacore)No. TCRs (Biacore)

Time (RED96 Octet)

No. TCRs (RED96 Octet)

Single concentration low affinity 0.5 day multiple 0.5 day multiple

Single concentration - high affinity 1 day ≤ 4 1 day ≤ 14

Equilibration titration - low affinity 1 day ≤ 4 1 day ≤ 20

Equilibration titration - high affinity

(single cycle kinetics)1 day ≤ 2 1 day ≤ 14

Alanine scanning and X-scanning 2 – 3 days 1 1 day ≤ 3

Cross-reactivity screen - low

affinity1 day multiple Not done yet Not done yet

ILT2 titration for pHLA validation - multiple Not done yet Not done yet

CD3 assay for ImmTAC scFv

validation1 day ≤ 4 Not done yet Not done yet

Octet has higher throughput particularly for high affinity TCRs/ImmTACs

Octet much faster for Ala/X-scan specificity experiments

SPR still favoured for some experiments particularly validation assays

because response is proportional to molecular weight

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Octet: challenges

Current challenges

Maximising instrument time while producing reliable data

• Optimising experimental set-up for each experiment type

Octet and Biacore

• Immunocore has 3 x Biacore3000 and 1 x Biacore T200

• Often the systems agree very well but sometimes can get large discrepancy between the 2

systems for same TCR

• Some possible solutions: (1) use one system only per TCR series (2) always have an

internal standard for each experiment

We have already overcome some challenges

Initially some difficulties with data fitting - double-referencing is important, partial

versus full fit, PBS + 0.005% Tween sample buffer or 1 x KB buffer

Follow-up meetings and ForteBio support have been very helpful for Octet users

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Acknowledgements

Pall ForteBio

� David Pennington, Phil Buckle, Lisette Deddens, Martin Feasey, Tom,

Yixin + support group

Immunocore

� All Protein Science; data from Viren Patel, Katrin Weiderhold, Marine

Raman, Emma Baston

� Steve Megit

octet red96 to select high affinity and specific t-cell ... · 2 immunocore is based near oxford in...

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