INVESTIGATING DUAL-TARGETED DOMAIN ANTIBODY FUSION PROTEINS IN A CANCER MODEL
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Developing efficacious, highly specific therapeutics is an ongoing challenge.
The domain antibody (dAb) fragment platform has shown promise targeting a dAbfused
therapeutic moiety to a specific tissue. This investigation sought to conclude on
the optimum binding affinity combination to maximise tissue-targeting specificity and
localised therapeutic potency by using a panel of dAb fusion proteins with varying
affinity combinations of asialoglycoprotein receptor (ASGPR)-targeting dAb mutants
and human interferon (hIFN) mutants – hIFN-ASGPRdAbs.
The hIFN-ASGPRdAbs were engineered, expressed, 1,4,7-
triazacyclononanetriacetic acid (NOTA)-conjugated and purified with binding affinities
and potencies characterised. Gallium-68 radiolabelling of the NOTA-hIFNASGPRdAbs
was optimised using a high activity fraction in a 10 minute reaction at
ambient temperature in pH4.4 sodium acetate. HepG2 xenograft mouse models
were injected with 68Ga-NOTA-hIFN-dAbs for biodistribution analysis and
PET/CT imaging, followed by mRNA expression analysis of the xenograft tissue.
ASGPR binding affinities of the NOTA-hIFN-ASGPRdAbs ranged from
0.73pM to ~528nM, and their hIFN potency ranged from 6.79pM to ~3.87nM.
Targeting of HepG2 cells was driven by the dAb-mediated ASGPR targeting. Ga-68
radiolabelling efficiencies up to 98.4% and specific activities up to 2.47MBq/μg were
achieved. Xenograft uptake was significantly increased through dAb-mediated
ASGPR targeting compared to the non-ASGPR targeted control 68Ga-NOTA-hIFNCTRLdAb,
achieving 2.42%ID/g vs. 0.68%ID/g, respectively. A trend of increasing
xenograft uptake correlated with increasing ASGPR binding affinity for the panel of
mutants, in spite of very high murine liver uptake. Increased xenograft uptake also
correlated with a more potent hIFN-mediated anti-proliferative mRNA response in
the xenograft tissue. A dAb with a mid-range affinity for ASGPR and maximum
affinity hIFN was concluded to be the optimum combination.
These results showed that the efficacy of a dAb fusion protein can be
influenced by the two intrinsic binding affinities. Hence, dAb-mediated tissue
targeting of a fused therapeutic and engineered affinity synergism strategies may hold
the key to novel, selectively cytotoxic biopharmaceutical drugs.
Authors
Papple, Alex G.Collections
- Theses [4223]