International Congress of Immunology 2016
on immortalized mouse antigen-presenting cells (RAW264.7
macrophages) expressing high levels of surface MHC (H2-Kd).
Genomic exchange at the H2-K locus was achieved via co-
transfection of cells with a plasmid carrying Cas9 and guide
RNA targeting the H2-Kd locus and a homology donor repair
template encoding an alternate MHC allele (H2-Kb). Following
homology directed repair, modified cells were isolated by flow
cytometry based on expression of new MHC. Engineered cells
expressing the alternate H2-Kb allele were able to present a
model antigen peptide (OVA-SINFEKLL) and activate a cognate
T cell hybridoma line, demonstrating they were active for
downstream immune functions. We envision this approach
could be used in the future to improve MHC/HLA matching in
Editing the genome in cells and mice using CRISPR/Cas9
, Aubrey, B.J.
, Kelly, G.L.
, Kueh, A.J.
, O’Connor, L.
, Milla, L.
, Wilcox, S.
, Tai, L.
, Strasser, A.
The Walter and Eliza Hall Institute or Medical Research,
Melbourne University, Department
of Medical Biology, Melbourne, Australia,
Hospital, Department of Clinical Haematology, Melbourne,
CRISPR/Cas9 mediated genome engineering provides an
easy and rapid way to edit genes
experimental strategies utilised proved to have a low efficiency
and were not broadly applicable to all cell types. In order to
overcome this hurdle and to allow for efficient modification of
genes in the haematopoietic system, we have developed a novel
drug-inducible lentiviral system to deliver the CRISPR/Cas9
platform to cells permitting efficient genome engineering
. Additionallywe have recently also implemented
the CRISPR/Cas9 technology for producing genetically modified
mice and to use it as a screening tool for the identification of
novel cell death regulators.
Cancer-specific T cell receptor isolation by single cell PCR
for cancer immunotherapy
Hibbert, L., Teng, M., Ryan, R., Baker, D., Clark, V., Wiiliams, L.,
Conlon, L., Hale, K., Davis, S., Weigand, L., Paston, S., Molloy, P.,
Vuidepot, A., Hassan, N., Jakobsen, B.
Immunocore Ltd, Abingdon, United Kingdom
Malignant cells may be recognised by T cells binding cell surface
Class I HLA (Human Leukocyte Antigen)-peptide complexes
presenting disease-associated epitopes. Many cancer patients
have been shown to generate CD8 cytotoxic T cell responses to
tumour-associated antigens. However this is often insufficient
for the immune system to clear tumours, resulting in progression
of cancer. This is in part due to the low avidity of these T cells as
well as the ability of cancer cells to develop escape mechanisms
to avoid destruction by T cells. To overcome these issues, we
have engineered novel, bi-functional protein therapeutics
termed ImmTACs (Immune mobilising monoclonal TCR
Against Cancer) which re-direct the immune system to target
and destroy tumour cells with a high degree of potency and
specificity. An ImmTAC comprises a high affinity ‘monoclonal’ T
cell Receptor (mTCR) targeting a cancer-associated HLA-peptide
complex, fused to an anti-CD3 scFv domain which activates an
anti-tumour T cell response.
In order to produce ImmTACs, we have developed an integrated
in-house process leading to the isolation of TCRs specific for
validated cancer epitopes. High affinity ImmTACs are then
generated through affinity maturation by phage display. We
have developed a method of rapidly identifying TCR chains
from T cell clones using targeted amplification from single cells.
We describe the steps leading to cloning of wild type TCRs
with single cell PCR. We present data to illustrate the successful
isolation of TCRs as a result of this procedure.
A scalable multiplex assay enabling assessment of TCR
specificity to hundreds of self- and pathogen-derived
Klinger, M., Taniguchi, R., Hu, J., Hayes, T., Wittkop, T., Asbury, T.,
Moorhead, M., Emerson, R., Sherwood, A., Robins, H., Faham, M.
Adaptive Biotechnologies Corp, South San Francisco, United States
Monitoring antigen-specific T-cells is critical for the study
of immune responses and development of biomarkers and
immunotherapeutics. We developed and validated a novel
multiplex assay (MIRA, or Multiplexed Identification of TCR
Antigen specificity) that combines conventional immune
monitoring techniques and TCR repertoire sequencing to assess
T-cell specificity to large numbers of query antigens. MIRA is
a sensitive assay enabling detection of antigen-specific TCR
clonotypes well below the limit of detection of conventional
immune monitoring assays including flow cytometry and
ELISPOT. Here we report the results from a scaled-up version of
the assay using 270 different query peptide antigens (159 self-
and 111 pathogen-derived). We identified >500 TCR clonotypes
at frequencies as low as 1 per million T-cells that were specific to
41 query antigens from 6 healthy HLA-A*02-positive individuals.
Most of the antigen-specific TCRs identified recognized one
of 27 different peptides derived from a variety of pathogens
including CMV, EBV, Flu, Rotavirus, HSV, mTB, WNV and HIV. A
subset of antigen-specific TCRs recognized one of 14 different
peptides derived from self including MART1, RCC, BCL-2, MAGE,
STEAP1, KLK4, CAMEL and MOG. These data support the notion
that escape and survival of self antigen-specific T-cells occurs
without causing overt autoimmunity in healthy individuals. We
show MIRA can be used to assess TCR specificities to hundreds
of query antigens simultaneously. The assay is highly scalable
and easily modified to accommodate thousands of additional
query antigens. This technology may be used to monitor T-cell
specificity to antigens relevant to infection, autoimmunity and