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8

International Congress of Immunology 2016

Abstract Book

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

cellular transplantation.

2716

Editing the genome in cells and mice using CRISPR/Cas9

technology

Herold, M.J.

1,2

, Aubrey, B.J.

1,2,3

, Kelly, G.L.

1,2

, Kueh, A.J.

1,2

, Brennan,

M.S.

1,2

, O’Connor, L.

1,2

, Milla, L.

1,2

, Wilcox, S.

1,2

, Tai, L.

1

, Strasser, A.

1,2

1

The Walter and Eliza Hall Institute or Medical Research,

Melbourne, Australia,

2

Melbourne University, Department

of Medical Biology, Melbourne, Australia,

3

Royal Melbourne

Hospital, Department of Clinical Haematology, Melbourne,

Australia

CRISPR/Cas9 mediated genome engineering provides an

easy and rapid way to edit genes

in vitro

and

in vivo

. Initial

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

in

vitro

and

in vivo

. 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.

336

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.

956

A scalable multiplex assay enabling assessment of TCR

specificity to hundreds of self- and pathogen-derived

antigens

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

cancer.