Antibody–drug conjugates (ADCs) are bifunctional
molecules
combining the targeting potential of monoclonal antibodies with the
cancer-killing ability of cytotoxic drugs. This simple yet intelligently
designed system directly addresses the lack of specificity encountered
with conventional anti-cancer treatment regimes. However, despite
their initial success, the generation of clinically sustainable and
effective ADCs has been plagued by poor tumor penetration, undefined
chemical linkages, unpredictable pharmacokinetic profiles, and heterogeneous
mixtures of products. To this end, we generated a SNAP-tag-based fusion
protein targeting the epidermal growth factor receptor (EGFR)a
biomarker of aggressive and drug-resistant cancers. Here, we demonstrate
the use of a novel click coupling strategy to engineer a benzylguanine
(BG)–linker–auristatin F (AuriF) piece that can be covalently
tethered to the EGFR-targeting SNAP-tag-based fusion protein in an
irreversible 1:1 stoichiometric reaction to form a homogeneous product.
Furthermore, using these recombinant ADCs to target EGFR-overexpressing
tumor cells, we provide a proof-of-principle for generating biologically
active antimitotic therapeutic proteins capable of inducing cell death
in a dose-dependent manner, thus alleviating some of the challenges
of early ADC development.
Current treatments for asthma help to alleviate clinical symptoms but do not cure the disease. In this study, we explored a novel therapeutic approach for the treatment of house dust mite allergen Der p 1induced asthma by aiming to eliminate specific population of B-cells involved in memory IgE response to Der p 1. To achieve this aim, we developed and evaluated two different proDer p 1-based fusion proteins; an allergen-toxin (proDer p 1-ETA) and an allergen drug conjugate (proDer p 1-SNAP-AURIF) against Der p 1 reactive hybridomas as an in vitro model for Der p 1 reactive human B-cells. The strategy involved the use of proDer p 1 allergen as a cell-specific ligand to selectively deliver the bacterial protein toxin Pseudomonas exotoxin A (ETA) or the synthetic small molecule toxin Auristatin F (AURIF) into the cytosol of Der p 1-reactive cells for highly efficient cell killing. As such, we demonstrated recombinant proDer p 1-fusion proteins were selectively bound by Der p 1-reactive hybridomas as well as primary IgG1 + B cells from HDM sensitized mice. The therapeutic potential of proDer p 1-ETA` and proDer p 1-SNAP-AURIF was confirmed by their selective cytotoxic activities on Der p 1 reactive hybridoma cells. The allergen-toxin demonstrated superior cytotoxic activity, with IC50 values in the single digit nanomolar value, compared to the allergen drug conjugate. Altogether, the proof-of-concept experiments in this study provide a promising approach for the treatment of patients with house dust mite-driven allergic asthma.
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