Technology

Technology

Velabs is a spin-out from Europe’s most prestigious life science research and instrumentation institute, the European Molecular Biology Laboratory (EMBL) in Heidelberg, which has been at the forefront of microfluidics-based screening technology development and applications world-wide.

Beside its comprehensive know-how in microfluidics-based screening, Velabs holds a strong IP-Porfolio in the field of microfluidic functional antibody screening.

The droplet-based microfluidic screening technology established at Velabs aims at the fast generation of antibodies against target molecules presented on cellular membrane surfaces or as soluble drug target.

Velabs’ Droplet Microfluidic Technology

  • Velabs uses small aqueous droplets (~20–100µm in diameter) surrounded by oil as miniaturized test tubes
  • Each droplet contains a single pair of one antibody producing cell (B-cells or others from human or mouse) and a target molecule, presented either on the membrane surface of cells or as soluble drug target
  • Detection of functional hits by fluorescence signals from the target/reporter cell
  • Positive droplets are sorted, B-cells are recovered and their antibodies get sequenced and characterized
  • Smallest amounts of starting material (e.g. patient-derived samples) can be reliably analyzed

Velabs’ competitive advantage & know-how

Fully quantitative fluorescence assays

Fluorescent objects such as cells can freely float inside microfluidic droplets, making quantitative measurements very challenging. For example, objects in the focal plane show stronger signals than objects outside of it [A-B]. Velabs is the only company that has established a proprietary 2-color-based normalization procedure of fluorescence signals that can overcome this limitation [C-D], in order to measure modulation of GPCRs and ion channels in a fully quantitative way.

The figure has been reproduced from our article: Shembekar N, Hu H, Eustace D, Merten CA. Single-Cell Droplet Microfluidic Screening for Antibodies Specifically Binding to Target Cells. Cell Rep. 2018 Feb 20;22(8):2206-2215. doi: 10.1016/j.celrep.2018.01.071 which is available under the terms of Creative Commons Attribution License (CC BY)  https://creativecommons.org/licenses/by/4.0/.

To demonstrate the power of this normalization strategy we have compared flow cytometric and droplet microfluidic signals of cells showing identical fluorescence intensities in the red channel (normalization dye), but different intensities in the blue channel (assay channel; e.g. reporter signal). Data from 20,000 events is shown. The results illustrate that only our dual color approach allows identifying all positive events and distinguishing between discrete intensity levels lying on diagonal lines (red lines).

GPCRs

GPCRs are amongst the most important target classes for the development of pharmaceutical agents. These membrane receptors are involved in signaling outside stimuli to the inside of cells, thus being most important triggers and mediators of biological core processes like cellular proliferation, metabolism, development and even programmed cell death.

Up to date, most pharmaceutical activities on GPCRs cover the development of small molecule drug candidates with limited selectivity and often unfavorable side effects, which are especially intolerable in chronic applications. Antibodies are having a much bigger interaction interface with their target proteins and can therefore overcome these selectivity and safety shortcomings.

Velabs is developing a pipeline of proprietary antibodies that will not only bind to, but functionally modulate pharmacologically interesting GPCRs in a wide therapeutic spectrum ranging from antagonizing to agonizing effects. Anti-GPCR functional antibodies developed by Velabs will open novel and un-addressed routes for a safer and more effective treatment of diverse diseases and pathologies associated with GPCR function.

Measurement of calcium flux in target cell after stimulation with agonistic antibody inside droplets
Target cells were probed separately in droplets with an antibody that binds a receptor (left) or an antibody that in addition to binding the receptor also induces a calcium flux in the cells. All cells were labelled with a fluorescent calcium sensitive indicator. Upon agonistic stimulation with a functional antibody, the calcium flux within the target cell (Channel 1) could be detected specifically (~10 fold increased signal) at very high temporal resolution. Data for 10,000 events is shown and activated cell events were gated in the red box along with their percentages in the corner. Velabs’ optimized microfluidic chips enable incubation times ranging from just a few milliseconds to more than 10min and hence allow to exploit these very transient assay signals in the best possible way. The ability to measure hundreds of thousands of cells sequentially, each at the specific sweet spot in terms of incubation time, provides a powerful tool for antibody discovery and clearly sets apart our technology from previous approaches.

Ion Channels

Ion Channels constitute, after GPCRs, the second largest therapeutic target class of modern medicinal drugs. Today, roughly 15% of the most successfully marketed pharmaceutical drugs target ion-channels. Like for GPCRs, most pharma activities are centered in the development of small molecule inhibitors with only limited binding interaction areas towards their ion channel target molecules. Therefore, existing drugs suffer mostly from specificity and selectivity limitations with multiple cross-target reactions and undesirable side effects. This makes it almost impossible to find sound therapeutic combinations between the drug’s target affinity and selectivity and its ability to fine tune channel gating. Velabs develops ion channel antibodies that allow such functional fine tuning of ion channels at high selectivity and superior safety profiles.

Fluorescence-based microfluidic assay for detecting potassium channel agonists

Fluorescent readout was used to measure ion flux following the opening of potassium channels. Control population was compared to cells treated with two different agonists of KV11.1 (hERG) potassium channel (PD-118057 and NS1643). Green fluorescence intensity (channel 1) was measured using Velab’s proprietary microfluidic platform. Data from 10,000 events are shown. Positive (activated) cells are gated in red and percentages are indicated.

Fluorescence-based microfluidic assay for detecting potassium channel antagonists

Fluorescent readout was used to measure blocking of ion flux following forced closing of potassium channels. Control population was compared to cells treated with a highly selective inhibitor of the hERG1 channel – scorpion venom derivate BeKm-1. Green fluorescence intensity (channel 1) was measured using Velab’s proprietary microfluidic platform. Data from 10,000 events are shown. Inhibited cells are gated in red and percentages are indicated. Population of cells with blocked potassium channels can be distinguished based on their lower fluorescence (channel 1) and higher signal in channel 2 due to decreased ion flux.

Procedures and Products