evidence for antigen-driven tcr𝜷chain convergence …...evidence for antigen-driven tcr 𝜷chain...
TRANSCRIPT
The world leader in serving scienceFor Research Use Only. Not for use in diagnostic procedures.
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
3 For Research Use Only. Not for use in diagnostic procedures.
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.
4 For Research Use Only. Not for use in diagnostic procedures.
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.
5 For Research Use Only. Not for use in diagnostic procedures.
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
6 For Research Use Only. Not for use in diagnostic procedures.
• 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
7 For Research Use Only. Not for use in diagnostic procedures.
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
8 For Research Use Only. Not for use in diagnostic procedures.
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
9 For Research Use Only. Not for use in diagnostic procedures.
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
10 For Research Use Only. Not for use in diagnostic procedures.
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
11 For Research Use Only. Not for use in diagnostic procedures.
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
12 For Research Use Only. Not for use in diagnostic procedures.
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
13 For Research Use Only. Not for use in diagnostic procedures.
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
14 For Research Use Only. Not for use in diagnostic procedures.
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.
15 For Research Use Only. Not for use in diagnostic procedures.
• 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
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