The folate receptor (FR) is an important biomarker for many cancers, and its overexpression on tumours can be exploited for targeted therapy, diagnosis and imaging.

Paper-based lateral flow immunoassays (LFIAs) are one of the most widely used point-of-care (PoC) devices, however, their application in early disease diagnostics is often limited due to insufficient sensitivity for the requisite sample sizes and the short timeframes of PoC testing. To address this, we developed a serum-stable, nanoparticle catalyst-labelled LFIA with a sensitivity surpassing that of both current commercial and published sensitivities for paper-based detection of p24, one of the earliest and most conserved biomarkers of HIV. We report the synthesis and characterization of porous platinum core-shell nanocatalysts (PtNCs), which show high catalytic activity when exposed to complex human blood serum samples. We explored the application of antibody functionalized PtNCs with strategically and orthogonally modified nanobodies with high affinity and specificity toward p24 and establish the key larger nanoparticle size regimes needed for efficient amplification and performance in LFIA. Harnessing the catalytic amplification of PtNCs enabled naked-eye detection of p24 spiked into sera in the low femtomolar range (ca. 0.8 pg•mL-1), and the detection of acute phase HIV in clinical human plasma samples in under 20 minutes. This provides a versatile absorbance-based and rapid LFIA with sensitivity capable of significantly reducing the HIV acute phase detection window. This diagnostic may be readily adapted for detection of other biomolecules; as an ultrasensitive screening tool for infectious and non-communicable diseases, and can be capitalized upon in PoC settings for early disease detection.

Targeting protein degradation with Proteolysis-Targeting Chimeras (PROTACs) is an area of great current interest in drug discovery. Nevertheless, although the high effectiveness of PROTACs against a wide variety of targets has been established, most degraders reported to date display limited intrinsic tissue selectivity, and do not discriminate between cells of different types. Here we describe a strategy for selec-tive protein degradation in a specific cell type. We report the design and synthesis of a trastuzumab-PROTAC conjugate (Ab PROTAC 3) in which E3 ligase-directed degrader activity is caged with an antibody linker which can be hydrolyzed following antibody-PROTAC internal-ization, releasing the active PROTAC and inducing catalytic protein degradation. We show that 3 selectively targets bromodomain-containing protein 4 (BRD4) for degradation only in HER2 positive breast cancer cell lines, whilst sparing HER2 negative cells. Using live cell confocal microscopy, we show internalization and lysosomal trafficking of the conjugate specifically in HER2 positive cells, leading to release of active PROTAC in quantities sufficient to induce potent BRD4 degradation. These studies demonstrate proof-of-concept for tissue-specific BRD4 degradation, overcoming limitations of PROTAC selectivity, with significant potential for application to novel targets.

Recent advances in nanomedicine have shown that dramatic improvements in nanoparticle therapeutics and diagnostics can be achieved through the use of disease specific targeting ligands.

Although recent methods for the engineering of antibody–drug conjugates (ADCs) have gone some way to addressing the challenging issues of ADC construction, significant hurdles still remain. There is clear demand for the construction of novel ADC platforms that offer greater stability, homogeneity and flexibility. Here we describe a significant step towards a platform for next-generation antibody-based therapeutics by providing constructs that combine site-specific modification, exceptional versatility and high stability, with retention of antibody binding and structure post-modification. The relevance of the work in a biological context is also demonstrated in a cytotoxicity assay and a cell internalization study with HER2-positive and -negative breast cancer cell lines.