Immunoassays support research by coping with a difficult matrix
A number of serious eye diseases, such as neovascular age-related macular degeneration (AMD) and diabetic macular edema, involve increase in the leakage of blood vessels in the eye. This is caused by vascular endothelial growth factor (VEGF) and while such diseases can be treated with protein drugs that neutralize VEGF, the drugs must be administered frequently. A research group has developed an elegant approach that significantly extends the half-life of the drugs. This exciting work involved handling a challenge that is common to much of eye research –
running immunoassays on precious samples in difficult matrices.
A peptide add-on that extends drug half-life
Protein drugs such as ranibizumab, a recombinant, fully humanized monoclonal anti-VEGF-A antibody, can be used to rescue the vision of patients suffering from retinal vascular disease by neutralizing VEGF-dependent leakage. But there is a drawback – treatment with these drugs involves intraocular injection as often as once a month. In an effort to reduce the stress and risk associated with such frequent injections, a research team at Novartis Institutes for BioMedical Research in Cambridge, Massachusetts, USA has developed a novel approach to extend the half-life of the drug treatment.
The team at Novartis reasoned that modifying a drug so that it could bind to hyaluronan, a major macromolecular component of the eye’s vitreous humor, could extend its half-life in the eye. They fused a 97-amino acid peptide (HABP) that binds to hyaluronan with a number of protein drugs and tested a number of HABP-Fab fusion proteins that were based on the Fab version of brolucizumab, an anti-VEGF protein in human clinical trials. Experiments in rabbits showed that one hybrid Fab, NVS24, not only retained its ability to neutralize hVEGF and reduced vascular leakage but was also active at least four weeks after a single dose, in contrast to other drugs that rapidly lost their potency. They called this construct LAVA1 (Long-Acting Anti-VEGF Antibody 1).
Boosting drug activity
Building on this and other discoveries, the team used a rabbit model to compare the efficacy of ranibizumab in its free form and as a HABP fusion protein. The results were impressive, showing that protein drugs fused with the HABP could be administered at doses as much as 50-fold lower compared with unmodified drugs and still have comparable or superior activity compared to the unmodified drug.
Immunoassays for challenging matrices
Researchers into eye research frequently face the challenge of measuring analytes in the presence of complex matrices and in samples that are available in very small amounts. The team at Novartis used a traditional plate-based ELISA to measure protein drug levels in rabbit samples but decided to use Gyrolab xP workstation to measure free LAVA1 and ranibizumab in samples from cynomolgus monkeys. By doing so they could exploit the matrix tolerance of Gyrolab assays that enabled a final dilution of 1:2 in Rexxip buffer to maximize sensitivity. The results confirmed the power of HABP-fusion in extending the duration of drug potency.
A Gyrolab assay was used to measure levels of free drug in cynomolgus monkey eyes after injection of equimolar amounts of LAVA1 and ranibizumab. Levels of free LAVA1 in the vitreous, retina and RPE-choroid were consistently higher than free ranibizumab. Reproduced from Figure 4, Ghosh et al, 2017.
Fusing protein drugs with hyaluronan-binding peptides promises to reduce the frequency of treating AMD and other eye diseases to no more than four times a year. Approaches like this could revolutionize the treatment of diseases in other hyaluronan-rich environments, such as synovial fluid-filled joints. Measuring protein analytes in these environments demands immunoassays that can cope with complex matrices and can minimize the consumption of precious samples.
You can find out more about how Gyrolab systems handle small sample volumes and difficult matrices here: http://www.gyros.com/why-gyros/technology/
and in the blog article ‘The small, the complex, and the precious’