Over the last decades, the development of therapeutic antibodies and the resulting products have become a constantly growing factor in markets and clinical setups. Typical application fields are cancer and inflammation. Here, mostly strong binders are in use that either serve as immunogenic flag on malignant cells for further immune-modulated destruction or that block certain dysregulated and pathological signal cascades by strongly binding to the involved receptor epitopes or corresponding ligands.
In addition to pure binding, our technology also allows to select modulating antibodies suitable for the regulation of somehow mistuned signal cascades that need additional enhancement or damping. Up to date, small molecules constitute the biggest class of modulatory drugs.
However, their specificities and side effect profiles are often very poor, resulting in undesired off-target effects with significant toxicity, especially in chronic applications. Modulating functional antibodies have the potential to overcome these limitations due to their unmet target specificity and biological nature. Therefore, the development of therapeutic and functional modulatory antibodies on certain pharmacological interesting target classes like GPCRs is becoming a major therapeutic focus of pharma companies developing biologicals.
Due to the limitations of current screening technologies, finding of functional modulatory antibody candidates needs to be split in two different steps. First, a pool of strongest binders is identified and second, the top binders of that pool are checked for their function.
As binding strength does not directly correlate with function – only very few binding mAbs have good function – this two-step approach often fails or does not yield the desired functional candidates. Numerous re-iterations and time-consuming tedious repetitions are needed to identify best candidates. In many cases, this process may take several years or results in project termination!
VERAXA’s one-step technology detects simultaneously binding and function and quantitatively sorts out all of the best functional hits at unmatched speed. Identifying mAB drug candidates can thus be reduced from years to days and developmental risks will be significantly reduced.
VERAXA has established fluorescence-based assays and procedures for ultra-fast detection and sorting of functional antibodies with a modulatory action on complex membrane receptors like GPCRs. The amount of the intracellular fluorescence signals we detect is a direct proxy for receptor modulation strength and the correlated downstream cellular signaling effects and changes. We can measure the normalized agonizing or antagonizing strength of the antibodies in real time and use these signals as prime filter pass for the sorting of hits. Screening is performed on our workstations on proprietary capillary chip structures that host tiny little droplets (10 to 150 µm in diameter) containing both a single antibody secreting B-cell clone and a reporter cell expressing the target receptor. Co-encapsulation of both entities or fusion of the entities containing droplets enables direct physical contact of B-cell secreted antibodies and reporter cells and results in a miniaturized true assay compartment.
Typically, we screen up to several hundred of B-cell clones per second. It is important to note, that after droplet fusion, all B-cell clones under investigation are passing the detection unit on chip at the exact same time point after signal induction, enabling fine tuning to obtain maximal S/N ratios. This time resolution can be precisely adjusted between milliseconds to seconds or minutes and affects in a strictly synchronous manner all screened B-cell clones at unpredendented temporal resolution.
The typical workflow for the assessment of antibody agonizing functions on GPCRs can be summarized as follows:
- Immunization of mice (or other appropriate animals) with cells overexpressing the human target
- Droplet generation and loading with B-cells and reporter cells.
- After fusion of droplets with B-cells and reporter cells, each droplet contains a single pair of one antibody producing cell (B-cells or others from humans or mice) and a reporter cell expressing the target receptor on its surface.
- Only functional B-cell secreting mAbs that induce GPCR modulation (agonistic or antagonistic) induce measurable fluorescent signals which are fully quantitatively analyzed and used as a trigger for the sorting process.
- After sorting of positive droplets, encapsulated B-cells are recovered and sequenced and the expressed mAb further characterized.
By using our technology and assays, VERAXA can as well screen antibodies with antagonizing functions. An antibody-mediated antagonism of receptor signaling (normally induced by its activating natural ligand) is possible in two different ways:
- by binding to the membrane receptor and blocking the ligand binding site on the receptor or
- by binding to the natural ligand and inhibiting ligand-receptor interactions. The relevant fluorescent readout of receptor mediated intracellular signal cascades is analyzed in droplets housing a competition mix of B-cell, receptor cell and natural ligand.
The relevant fluorescent readout of receptor mediated intracellular signal cascades will be analyzed in droplets housing a competition mix of B-cell, receptor cell and natural ligand.