Hybribody, our optimization services

Good is nice but better is great. You deserve more! If you want to increase the affinity of your nanobody for particularly challenging application. We provide you a unique optimization platform using the yeast to meet your needs.

Applications

Increase the affinity of your current nanobody form the lama or a synthetic library
Make it work in living cells (intrabodization)

Key benefits

Cost-effective and quick optimization methods
choose the library construction method which is the most adapted to your project
no drift effect like in phage display
possibility to have apparent kD measurement.

How does it work?

Extracellular optimization

Hybrigenics services can perform optimization using yeast display. Here, we construct a library of mutant VHHs (nanobodies), and we express it at the surface of the yeast cells using various systems. Different possibilities are available for the mutagenesis (random or CDR targeted).

Your antigen will be labeled with a fluorophore.

Several rounds of selection using a cell sorter, with decreasing concentration of the antigen will allow to select the nanobodies with the highest affinities. The DNA of the positive clones is then sequenced and analyzed. Contrary to the phage display, yeast display allows to control in the same time antigen/antibody equilibrium and display.

Intracellular optimization or Intrabodization

If your antigen is a soluble protein, we can use the Yeast Two-Hybrid technology to optimize your VHH (nanobody) or to make it work intracellularly (intrabodization). Hybrigenics will build a library from your VHH (nanobody) by mutagenesis and clone it into our Y2H prey vector by gap repair. This vector allows the fusion of the Activation Domain (AD) of a transcription factor to the single-domain antibody sequence. This Y2H Antibody library (made of VHH nanobodies) is then transformed into a genetically modified yeast strain lacking the HIS3 gene necessary for the synthesis of histidine. The antigen of interest is fused to the DNA Binding Domain (DBD) of a transcription factor in a Y2H bait vector and then transformed into a second yeast strain of opposite mating type compared to the prey yeast strain.

Put together, the two yeast strains mate and form diploids expressing both the antigen and a VHH nanobody from the library. If the antibody recognizes the antigen, it allows for the reconstitution of the functional transcription factor. It activates the transcription of the HIS3 reporter gene and allows yeast cells to grow on a selective medium lacking histidine. By increasing the selection pressure with 3-aminotriazol (a competitive inhibitor of the Histidine reporter), we can force the selection towards the VHH with the highest affinities. The DNA of the positive clones is then sequenced and analyzed.

Material required

For intracellular optimization or intrabodization

the sequence of your nanobody (form the lama or a synthetic library)
5 µg of the cDNA of your antigen

For extracellular optimization

the sequence of your nanobody (form the lama or a synthetic library)
100 µg of your purified antigen

Deliverables

Full report containing the description of the selection process
Sequences and cDNA clones of up to 5 VHH, in the expression vector of your choice for E. coli production or mammalian production (minibody)
For yeast display optimization, apparent Kd measurement using the method published by Hunter S.A. and J.R. Cochran (Methods Enzymol., 2016).

References

Please see testimonial from Dr Olivier Christophe (INSERM U1176, Le Kremlin Bicêtre, France)

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