Drew C Deniger

James C Yang

Jared J Gartner

Katarina Trebska-McGowan

Ken-ichi Hanada

Mini Bharathan

Pasetto Anna

Paul F Robbins

Steven A Rosenberg

Journal for ImmunoTherapy of Cancer

Springer - Publisher Connector

POSTER PRESENTATION Open Access
Tetramer based approach for efficient identification
and isolation of neo-antigen specific CD8 T cells
from peripheral blood (PBL) of patients with
metastatic cancers
Mini Bharathan1, Katarina Trebska-McGowan2, Pasetto Anna2, Drew C Deniger1, Ken-ichi Hanada2, Jared J Gartner2,
James C Yang3, Steven A Rosenberg1, Paul F Robbins3*
From 30th Annual Meeting and Associated Programs of the Society for Immunotherapy of Cancer (SITC 2015)
National Harbor, MD, USA. 4-8 November 2015
Background
Adoptive cell therapy with T cells bearing mutation speci-
fic T cell receptors (TCR) can be an effective method for
treating metastatic cancers. The objective of this study was
to identify mutation reactive T cells in the circulation of
patients with different types of metastatic cancer.
Methods
The strategy utilized whole exome sequencing data to
identify somatic non-synonymous mutations and then in-
silico algorithms to predict minimal epitopes encoding
mutated amino acids for each patient specific HLA-allele.
CD8-enriched PBL from each patient were stained with
tetramers generated in house by a UV-exchangeable tech-
nique as previously described for A*02:01, A*03:01,
A*11:01, B*07:02, and a commercial tetramer was acquired
for B*57:01. Based on the initial staining frequency (+tetra-
mer+ T cells recognizing 7 unique neo-antigens from the
PBL of 4 patients (ranging from 1 to 4 per patient). We
enriched the frequencies of CD8+tetramer+ cells from 0.5
to >85%, 0.3 to >65% and 0.01 to 3% from the PBL of
patients with colorectal (3971-A*02:01), lung (4014-
B*57:01), and ovarian (4067-B*07:02) cancers respectively,
using individual tetramers. Populations reactive with three
HLA-A*11:0-restricted and one HLA-A*03:01-restricted
neoantigens were also isolated from the PBL of lung can-
cer patients 4014 and 4037, respectively, using a pooled
tetramer approach.
Results
Overall, the isolated T cells recognized mutated epitopes
when co-cultured with autologous CD14+ monocytes
pulsed with mutated peptides in the context of appropriate
MHC-I alleles including HLA-A*02:01, HLA-A*03:01,
HLA-A*11:01, HLA-B*07:02, and HLA-B*57:01, with reac-
tivity detected using IFN-g ELISA. Using single cell PCR,
we could clone the TCRs reactive with an HLA-*02:01-
presented colon cancer neoantigen and an HLA-B*57:01-
presented lung cancer neoantigen. Evaluation of PBL
retrovirally-transduced with these TCRs demonstrated
that they bound to tetramers and secreted IFN-g when co-
cultured with CD14+ monocytes pulsed with appropriate
mutated peptides.
Conclusions
To conclude, tetramers offer a sensitive, fast and reliable
methodology to isolate mutation specific tumor reactive
T cells from PBL of cancer patients. Furthermore, this
method facilitates the identification, and cloning of muta-
tion reactive TCR with which to construct receptor-
engineered T cells for adoptive T cell therapy.
Authors’ details
1NIH/NCI, Bethesda, MD, USA. 2NIH/NCI Surgery Branch, Bethesda, MD, USA.
3National Institutes of Health, Bethesda, MD, USA.
3National Institutes of Health, Bethesda, MD, USA
Full list of author information is available at the end of the article
Bharathan et al. Journal for ImmunoTherapy of Cancer 2015, 3(Suppl 2):P47
http://www.immunotherapyofcancer.org/content/3/S2/P47
© 2015 Bharathan et al. This is an Open Access article distributed under the terms of the Creative Commons Attribution License
(http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided
the original work is properly cited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/
publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
Published: 4 November 2015
doi:10.1186/2051-1426-3-S2-P47
Cite this article as: Bharathan et al.: Tetramer based approach for
efficient identification and isolation of neo-antigen specific CD8 T cells
from peripheral blood (PBL) of patients with metastatic cancers. Journal for
ImmunoTherapy of Cancer 2015 3(Suppl 2):P47.
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Bharathan et al. Journal for ImmunoTherapy of Cancer 2015, 3(Suppl 2):P47
http://www.immunotherapyofcancer.org/content/3/S2/P47
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Tetramer based approach for efficient identification and isolation of neo-antigen specific CD8 T cells from peripheral blood (PBL) of patients with metastatic cancers

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Tetramer based approach for efficient identification and isolation of neo-antigen specific CD8 T cells from peripheral blood (PBL) of patients with metastatic cancers

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