evidence for antigen-driven tcr𝜷chain convergence …...evidence for antigen-driven tcr 𝜷chain...

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Geoffrey M. Lowman, PhDSenior Staff Scientist EACR - 02 July 2018

Evidence for antigen-driven TCR𝜷 chain convergence in the tumor infiltrating T cell repertoire

2 For Research Use Only. Not for use in diagnostic procedures.

Conflicts

The authors are full-time employees of ThermoFisher Scientific


Need for Biomarkers in Immuno-Oncology

• Biomarkers for prediction of adverse events during immunotherapy

• Biomarkers for prediction of response/non-response

Adapted from Sharma et al. Cell, 2015;161(2):205 with permission from Elsevier

Combination immunecheckpoint therapy

Combination Immunotherapy

PD-1 or CTLA-4

TumorDendritic

Cell

Macrophage

T cell

Checkpoint blockade may

target PD-1 and CTLA-4

pathways to upregulate

T cell cytotoxic response

Adapted from Ott, et al. Clin Cancer Res. 2013;19(19):5300

with permission from AACR.


Constant Leader FR1 FR2

Diversity (D) Joining (J)Variable (V)

CDR3

FR3

CDR1 CDR2

Sequencing Beta

Chain of T Cell

Receptors to

Characterize

Immune Status

AmpliSeq Primers ~330-340 bp

Long Read NGS assay capturing all 3

CDRs (1,2 &3). Uniquely captures

germline TRBV polymorphism from

blood/tissue to investigate

predisposition to adverse events.

Flexible input/sequencing depth

requirements covering samples with

low, medium, and high clonal

diversity.

Superior informatics for accurate

clonality and β chain sequence

assessment without interference from

primer bias.

10ng-

1mg

Ion Torrent Oncomine™ TCR Beta – LR Assay

Allelic variants may alter interaction of CDR1 and 2 with HLA

V-gene polymorphism Clonality / TCR convergence

TCRComplex.png used under creative commons license

https://creativecommons.org/licenses/by-sa/3.0

https://creativecommons.org/licenses/by-sa/3.0


• Tumor neoantigens may stimulate the

proliferation of T cells possessing a stereotyped

TCRβ amino acid sequence.

• Due to the degeneracy of the amino acid code, T cell

clones having the same amino acid sequence may

have different nucleotide sequences.

• Measuring the features of convergent TCRs,

rather than all detected TCRs, allows one to

eliminate noise from TCRs that are not involved

in tumor antigen specific responses.

Detecting Tumor Antigen Driven T Cell Expansion

Proliferation of

convergent T cells

(small fraction of

total T cells)

Tumor antigen stimulates T cells with

stereotyped TCRβ amino acid features

Extraneous TCRs in gray

Venturi, et al. PNAS 2006


TCR Convergence May Be a Hallmark of Chronic Antigen Stimulation

Priming and

proliferation of tumor

antigen specific T cells.

T cells do not destroy

tumor.

Tumor antigen continues

to prime naive T cells with

shared antigen specificity.

Over time, convergent

TCR groups become

detectable in peripheral

blood.

Tumor Ag


Example of a Convergent TCR Group in an Individual With Melanoma

• This sample contains three TCR sequences that are identical in amino acid space but have distinct

CDR3 NT junctions owing to differences in non-templated bases at the V-D-J junction.

Variable Joining CDR3 AA CDR3 NT Frequency

TRBV7-8 TRBJ2-7 ASSLGQAYEQY GCCAGCAGCTTAGGTCAGGCATACGAGCAGTAC 1.8E-3

TRBV7-8 TRBJ2-7 ASSLGQAYEQY GCCAGCAGCTTGGGACAGGCCTACGAGCAGTAC 4.8E-4

TRBV7-8 TRBJ2-7 ASSLGQAYEQY GCCAGCAGCTTAGGGCAGGCCTACGAGCAGTAC 9.9E-05


Example of a Convergent TCR Group in an Individual With Melanoma

• This sample contains three TCR sequences that are identical in amino acid space but have distinct

CDR3 NT junctions owing to differences in non-templated bases at the V-D-J junction.

• Note: Base substitution PCR and sequencing errors can create artifacts that resemble convergent

TCRs. The Ion Torrent sequencing platform has very low rates of base substitution errors.

Variable Joining CDR3 AA CDR3 NT Frequency

TRBV7-8 TRBJ2-7 ASSLGQAYEQY GCCAGCAGCTTAGGTCAGGCATACGAGCAGTAC 1.8E-3

TRBV7-8 TRBJ2-7 ASSLGQAYEQY GCCAGCAGCTTGGGACAGGCCTACGAGCAGTAC 4.8E-4

TRBV7-8 TRBJ2-7 ASSLGQAYEQY GCCAGCAGCTTAGGGCAGGCCTACGAGCAGTAC 9.9E-05


TCRB sequencing of melanoma tumor biopsies

(N=63) revealed the presence of expanded T

cell clones having convergent TCRB chains.

TCR convergence appears elevated within

melanoma biopsies compared to healthy PBL

(N=4).

TCR Convergence Defines the Melanoma Infiltrating T Cell Repertoire

Convergent TCR Frequency in PBL

and Melanoma Biopsy

Adapted from Looney et al, AACR 2018

p<1E-4


TCRB sequencing of melanoma tumor biopsies

(N=63) revealed the presence of expanded T

cell clones having convergent TCRB chains.

TCR convergence appears elevated within

melanoma biopsies compared to healthy PBL

(N=4).

Is TCR convergence also elevated in PBL

from individuals with cancer?

TCR Convergence Defines the Melanoma Infiltrating T Cell Repertoire

Convergent TCR Frequency in PBL

and Melanoma Biopsy

Adapted from Looney et al, AACR 2018

p<1E-4


Tumor Antigen Specific T Cells May Be Detected in Peripheral Blood

Tumor antigen

specific T cells enter

peripheral blood

Source: http://www.sciencedirect.com/science/article/pii/S1074761313002963

License: https://creativecommons.org/licenses/by/4.0/

Labels were removed.

T cell infiltration

of tumor

T cells are

primed with

tumor antigen

Cancer Immunity Cycle

http://www.sciencedirect.com/science/article/pii/S1074761313002963

https://creativecommons.org/licenses/by/4.0/


TCRB Convergence to Predict Response to Immunotherapy from Pre-Treatment Peripheral Blood

Note: Research sample set includes cancers of varying stages treated with three checkpoint blockade

agents. Furthermore, checkpoint blockade agents were utilized at low dose (1 mg/kg). Increasing dose

may convert some non-responding individuals having higher TCR convergence into responders.

Convergent TCR Frequency vs

Response for Adenocarcinoma

Research Subjects

No Objective

Clinical Response

Objective

Clinical Response

0.005

0.010

0.015

0.020

Convergent TCR Frequency in PBL and

Response for Adenocarcinoma Patients (p=.027)

Con

ve

rgen

t T

CR

fre

qu

en

cy

No Objective

Clinical Response

N=3

Objective Clinical

Response

N=8

Converg

ent T

CR

Fre

quency

p=.027

Converg

ent T

CR

Fre

quency

Convergent TCR Frequency vs

Response for Melanoma

Research Subjects

p=.177

No Objective

Clinical Response

N=8

Objective Clinical

Response

N=9

ROC for individuals having

melanoma or adenocarcinoma

Specificity

Sen

sitiv

ity

1.0 0.8 0.6 0.4 0.2 0.0

0.0

0.2

0.4

0.6

0.8

1.0

Adenocarcinoma, AUC=.92

Melanoma, AUC=.69

ROC for Prediction of Response


TCR Profiling May Provide the Most Direct Measurement of Tumor Immunogenicity

Tumor acquires non-

synonymous mutation

Non-synonymous

mutation is translated

Translated protein is

broken into peptides

Peptide is presented

by HLA

T cells recognize

peptide and proliferate

Anti-tumor T cell responses

Tumor mutation burden

profiling (TMB)

HLA typing

TCRβ sequencing of

peripheral blood

Sequence of Events to Elicit

Anti-Tumor T Cell Response

• Convergent TCRs are the result of T cell responses to

antigen, and thus may serve as a metric for

quantifying tumor immunogenicity.

• We propose that TCR convergence is a T cell

repertoire feature which preferentially arises following

chronic antigen stimulation rather than the acute but

transient antigen challenges elicited by infectious

disease.

• Given that tumor antigens may prime T cells over

months or years time, measurements of TCR

convergence may provide an accurate pre-treatment

estimate of tumor immunogenicity.


• Tim Looney

• Elizabeth Linch

• Lauren Miller

• Alex Pankov

• Denise Topacio-Hall

• Alice Zheng

• Gauri Ganpule

• Jim Godsey

• Mark Andersen

• Fiona Hyland

• Simon Cawley

• Rob Bennett

Acknowledgements

© 2018 Thermo Fisher Scientific Inc. All rights reserved. All (other)

trademarks are the property of Thermo Fisher Scientific and its subsidiaries.

evidence for antigen-driven tcr𝜷chain convergence …...evidence for antigen-driven tcr 𝜷chain...

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