| Summary: | <p>PET and SPECT imaging of
intranuclear epitopes using antibodies (IgG) tagged to cell
penetrating peptides (CPPs), such as TAT (GRKKRRQRRRPPQGYG) has great potential
given the versatility, specificity and sensitivity typical of antibodies. However, this process is
technically challenging because of the location of the target. Previous research has
demonstrated a variety of intranuclear epitopes that can be targeted with TAT-IgG-based
radioimmunoconjugates (RICs). In this thesis, I set out to determine the technical limitations
of intranuclear PET/SPECT using IgG-TAT-based RICs, notably the lower target abundance
detection threshold.</p>
<p>I stably transfected the lung adenocarcinoma cell line H1299 with an enhanced green
fluorescent protein (EGFP)-tagged histone protein (H2B-EGFP) and generated four cell lines
expressing increasing levels H2B-EGFP. EGFP levels were quantified using western blot, flow
cytometry, and ELISA (0 - 1 Mcopies/cell, including H1299 WT cells). In parallel, a murine
anti-GFP monoclonal antibody was produced, purified and validated for selective binding to
H2B-EGFP.</p>
<p>Having established a model system for interrogating the limits IgG-TAT RICs, I developed a
novel conjugation strategy to label antibodies with azide modified derivatives of the CCP TAT
(TAT-N<sub>3</sub>) and chelator DTPA (N<sub>3</sub>-Bn-DTPA) using strain-promoted alkyne-azide
cycloaddition (SPAAC) click chemistry. This method of conjugation, referred to as the DBCON3
method, was optimised and followed by radiolabeling with <sup>111</sup>In (<sup>111</sup>In-DTPA-GFP-G1-
TAT) prior to in vitro comparison with the conventional method of IgG-TAT RIC conjugation
(EDC-NHS crosslinking), using the H2B-EGFP / GFP-G1 model system.</p>
<p>With this new conjugation strategy in place, the cellular uptake of <sup>111</sup>In -DTPA-GFP-G1-TAT
across the H2B-EGFP transfected clones was evaluated to determine the lower abundance detection threshold in vitro. Using the same model, tumour uptake in xenograft-bearing mice
was quantified to determine the smallest amount of target epitope that could be detected using
<sup>111</sup>In-DTPA-GFP-G1-TAT in vivo.</p>
<p>Finally, I attempted to apply IgG-TAT RICs to a practical application by imaging the DNA
damage response (DDR) by targeting the serine/threonine protein kinase ataxia telangiectasia
mutated (ATM), an apical regulator of the cellular response to DNA double strand breaks
(DSBs).</p>
<p>Here, I present a proof-of-concept demonstration of what is currently possible with RIgG-TAT
based RICs whilst simultaneously providing a platform for further exploration and optimisation
of this technology, potentially yielding a new generation of more potent whole-body immunoimaging
devices.</p>
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