The VIB Nanobody Core has experience in a wide range of immunization protocols with a broad range of antigens. Recombinant protein is often available and is the most straightforward option for immunization. However, for more difficult target such as multispan membrane proteins other options need to be employed like DNA, cell, liposome, nano-disc or VLP immunizations. Proteins from different species can be combined to aim for a multi-species specific antibody response. Adapting the immunization protocol in terms of number of injections and how these are spread in time can also have a profound effect on the outcome of the immunization. When the immunization has run it's course blood samples are collected, allowing to generate immune antibody libraries. Pre-immunization blood samples can also be taken upon request.

After immunization, RNA and subsequently cDNA is prepared from the animal's immune cells. Using this material we are able to generate Nanobody immune libraries for phage display using different types of vectors with variations in tags, cloning procedures, etc. The resulting libraries are subjected to a thorough QC which also gives the size of the library and the percentage of clones with a Nanobody insert. The finished library can then be screened in a variety of ways.

An immune (phage) library can be used to find specific Nb binders to the antigen used for immunization, a process called panning. Many different can be offered and customized panning strategies: recombinant antigens coated on a plate, beads or in solution, on cells expressing the antigen, using liposomes, nanodiscs, VLPs, ... On top of that competition to parts of the antigen or similar antigens can be added to fine-tune the outcome further. The phages binding to the antigen are recovered,amplified and used for one or two more rounds of panning. During the panning process bacterial colonies are generated containing a single Nb clone each.

After a round of panning large number of single clone colonies are screened for reactivity to the antigen in question. This screening process can use any of the modalities: recombinant antigen in ELISA, flow cytometry screening on antigen expressing cells, etc. After the screening process all the antigen binding clones are DNA sequenced. The whole package of positive clones is offered to our clients as both DNA sequences and as E.coli frozen slants. The number of clones are not limited or CDR3 groups delivered.

At first the Nanobody sequences are grouped in CDR3 groups. Nanobodies belonging to the same CDR3 group (same B-cell lineage) are very similar and their amino acid sequences suggest that they are from clonally-related B-cells resulting from somatic hypermutation or from the same B-cell but diversified due to RT and/or PCR error during library construction. Nanobodies belonging to the same CDR3 group recognize the same epitope but their other characteristics (e.g. affinity, potency, stability, expression yield, etc.) can be different. The sequencing analysis is provided at no cost when Nanobodies are generated.

The sequences can be analyzed in depth to identify uncommon framework residues, VH like Nanobodies or sequence liabilities such as unpaired cysteins, methionins, ...

When a large collection of Nbs is obtained after the screening procedure, it is useful to filter the clones by performing off-rate ranking. Using crude periplasmic extract the clones are gauged which CDR3 group has a higher relative off-rate compared to the other members of the group and compared to other groups. With this information the number of Nanobodies that need to be produced and purified before further characterization can be reduced. The off-rate values obtained are not yet the exact off-rates for the Nanobodies, but already represent a good estimate of how long an interaction is maintained.

In an epitope binning experiment a criss-cross competition experiment is used with a set of Nanobody clones selected from different CDR3 groups (clones within a group already share an epitope) to gauge if these clones interfere with each others binding to the antigen. Clones falling in the same bin recognize the same or overlapping epitopes. Independent bins (not blocked in any direction by another bins Nbs) have distinct and independent epitopes. When the binding of one Nb only partially interferes with the binding of the second Nb (or in just one direction of the assay), the epitope of the second Nb can either partially overlap/obstruct the epitope of the first Nb or the binding of one of the Nbs induces a conformational shift in the antigen that distorts or makes inaccessible the epitope of the other Nb. In that case no definite statements can be made about the independence of the epitopes in question. If the clones gave good clear signals during the off-rate ranking, which is performed before the binning, this experiment can also be run using periplasmic extracts without prior purification of the Nanobodies.

Although producing and purifying Nanobodies is not the mainstay of our activities, we do offer this service in order to obtain µg to mg quantities of Nanobody protein which can be used for the further characterization of Nanobody clones either in our or our client's facility. We express the Nanobodies in an E. coli expression strain, by default with a His-tag. However other tags like HA, FLAG or BAD are also possible. After making a periplasmic extract the default purification procedure consists of a IMAC combined with a size exclusion step. This combination leads to Nanobodies that are, in most cases, >90% pure. Higher quantity productions and productions in other hosts or strains are available through our sister facilty, the Protein Service Core.

When a purified Nanobody (or/and antibody) is available,the full range of binding parameters can be determined: kon, koff, KA,KD. We have both Octet and Biacore machines present and use either one depending on the use case: number of samples, nature and sizes of antigens and analytes, ... Additionally the same setup used for affinity determination can be used for competition studies between different anti/nano-bodies or different antigen/ligand combinations.

Even the Nanobody with the best functional characteristics will not be developed further if it turns out to be unstable or when it easily aggregates. Consequently we offer a number of procedures to determine how stable a Nanobody remains when experiencing heatstress (Tm determination) or an oxidative buffer shock. The stability in different buffers can also be easily determined using our Uncle machine. Additionally, we can determine freeze/thaw stability and check how easily the protein precipitates with PEG.

If the sequence of a Nanobody with desirable functional characteristics holds some liabilities for it's further development, we can correct some of these problematic features. This includes "repairing" framework abnormalities or preventing pyroglutamate formation or oxidizable methionins, etc. Changing the Nanobody structure always goes hand-in-hand with affinity determinations and stability experiments to gauge if the affinity and/or stability of the Nanobody was not decreased during the process. Especially for high-risk interventions such as loop-grafting or affinity maturation follow-up experiments are critical.

Another intervention which is possible (partial) humanization of Nanobodies. The Nanobody sequence cannot be made fully equal to a human VH due to the stability problems this would cause. However, several amino-acids in the framework regions can normally be adapted to a more human sequence without compromising the Nanobody's affinity.

The small size of Nanobodies allows us to use them as building blocks for all kinds of manifold constructs.

• The apparent affinity, or avidity, can be tweaked by making bi- or trivalent constructs. This can either improve the affinity on a single target molecule if it possesses multiple epitopes, or it can bring several target molecules together which can be functionally relevant. Another way of increasing the avidity for a molecule is to use a bi-paratopic construct consisting of two Nanobodies against two different epitopes on the same molecule. The epitope binning service can shed light on which couples that would function best together in that regard.
• Multi-specific constructs can be used to bring molecules or cells together, like two (or more) components of a heterodimeric receptor complex or two cells for an anti-cancer immunotherapy.
• Increasing the serum half life of a Nanobody through addition of a half-life extension module. This prolongs the serum half-life of a Nanobody construct up to the half-life of IgG sized constructs. The Nanobody Core has a multi-species albumin specific Nanobody that was shown to improve the half life of a bi-specific Nanobody. This anti-SA module is available for licensing.

In some cases Nanobodies exibit a functional effect on their own like blocking or activating receptors. However, in many case a Nanobody needs to functionalized by coupled to another molecule to it. We can fuse a range of proteins and fluorophores to your Nanobody. A Nanobody coupled to GFP or a FITC-like dye can be used as a probe in many biological studies. Another popular fusion is to an antibody Fc domain, making Nano-antibodies. These molecules can, depending on the chosen Fc domain, activate immune cells through their Fc-receptors.