QVQ research offers two types of services: Research Services Agreement (RSA) and Research Collaboration Agreement (RCA). While all generated libraries and sequences in the project are ownership to the customer in case of RSA, libraries and VHH can be exploited by QVQ for research and imaging purposes in the case of RCA. The price of the RCA is therefore reduced.

Camelids express next to conventional tetrameric antibodies consisting of the light and heavy chains, functional dimeric antibodies consisting of heavy chains only (HCAb) (Fig. 1). For many applications, minimizing the multi-domains monoclonal antibodies is highly appreciated.
Dissection of the antigen binding domain from conventional monoclonal antibodies; mAbs (150 kDa) yields a single chain variable fragment (sFv, ~30 kDa), or Fab fragment (~60 kDa), which are still large and complex proteins. Dissection of the antigen binding domain of a HCAb results in a small, single polypeptide called VHH or Nanobody (~15 kDa). Next to the small size, VHH are highly soluble, show the ability to refold to their functional structure after denaturation and are able to target alternative epitopes, which are not targeted by conventional mAbs and their minimized versions. Therefore, VHH are the preferred tool for tumor targeting and diagnostics. Moreover, VHH are predicted to have a brilliant future as therapeutics.

Figure 1: Schematic representation of conventional mAb and HCAb mAb. (Hamers-Casterman 1993)

QVQ Research scientists lean on more than 40 years of experience in llama antibody technology. Fields of expertise include, but are not limited to, immunization, phage display libraries, selection and characterization of VHH, production and purification of the selected VHH and modification of the VHH such as directional labeling, coupling etc… In addition to the valuable experiences, QVQ Research disposes of many tools such as plasmids and strains to implement all fields of expertise set above, independently and to the best expectations. Moreover, QVQ Research did prove to be a reliable partner for development of llama antibodies for any desired application since 2010.
QVQ Research scientists have track records of > 90% success rate in different application of llama VHH. Founder of QVQ (Professor Verrips) was involved in the llama antibody technology from the pioneer work in the 90ties of the previous century, when Professor Hamers discovered the occurrence of HCAb in the blood of Camelides. Since then Professor Verrips and his group was involved in studying different aspects of llama antibodies, such as application as therapeutics in different diseases and as ligands for affinity chromatography, next to ways of producing VHH and determining their structures.
QVQ Research has established a track record of delivering custom-tailored services in developing llama antibody projects since its foundation in 2010. Services include llama immunization, construction of phage-display libraries, custom-tailored selection procedures, production of VHH equipped with different tags in bacteria and Yeast.

Immunization and library construction

QVQ Research outsources immunizations of llamas a company specialized in the immunizations and production of polyclonal antibodies. The immunizations are conducted according to the applicable animal welfare act at place. All immunizations are reviewed by the internal animal experiment commission of the company before starting the immunization.
QVQ Research has experience of using several antigens forms ranging from purified natural and recombinant protein up to complex cells and tissue for immunization. Immunization can also be done by locally expression of antigen through immunization with plasmid DNA and purified proteins are reconstituted into liposomes or virus-like particles.
At least 2 animals are scheduled for each immunization to ensure diversity of the selected VHH and to avert possible non-responder animals.
During immunization, single domain antibody titers directed against the antigen of interest is measured using “in-house made” polyclonal antibody directed against isolated VHH. In concordance with the client, the immunization schedule is either extended or ended at the usual 43 day after immunization, after which lymphocytes are isolated from the peripheral blood and their RNA is purified.
QVQ Research disposes of different sets of primers to amplify the sequence of the antigen binding domain of the heavy chain antibodies. Usually, VHH of VH3 family are amplified and cloned into phage display plasmids, however on customer’s request, VHH of VH4 family can be included in the library or cloned to form a VH4-dedicated library. (De Schacht et al. 2010)

QVQ Research disposes of different phagemid plasmids for cloning of the VHH libraries. The parental plasmid is pHEN was modified to code for different tags or no tag at all at the C terminus.
QVQ Research considers only libraries with size exceeding 107 different clones and insert frequency of more than 90% qualified to be used for selection of VHH binding to desired antigen.
Next to immune VHH libraries, QVQ Research dispose of many libraries constructed from PBLs of llamas immunized with complex antigen (such as cells and tissues), which have the potency to contain VHH directed against individual antigens. Moreover, different libraries can be pooled to construct a high diversity non-immunized VHH library.

Phage display and VHH selection

QVQ Research always proposes a strategy to achieve selection of VHH binders by phage display based on own experience and available tools. Such strategy is carefully designed to include customer’s requests and wishes and after agreement on the selection strategy, QVQ Research commits to select at least 6 VHH belonging to at least 2 different families based on the V-D-J gene combinations. VHH are characterized using the tools available for binding, competition and functionality either as preliminary samples or as purified VHH proteins. Moreover, sequence of the different VHH is determined. VHH are subsequently, cloned in an expression plasmid devoid of the filamentous phage gene3 and purified using IMAC from the periplasm of Escherichia coli. QVQ Research disposes of expression plasmids displaying different tags at the C terminus.
Data describing all the above mentioned steps are collected in the form of a report, which is send to the customer along with purified VHH protein (400-500 μg).

VHH sequence analysis

QVQ research disposes of a large database of VHH sequences, which are used for analysis of VHH production, stability and protease resistance. Moreover, due to wealth of information from mutagenesis of VHH, QVQ Research is able to manipulate the sequence of the selected VHH to generate more stable VHH and VHH produced at large yield.

Since VHH are not of human origin, and although llama origin of VHH was never an impediment for its application, QVQ research can modify the VHH sequence to render it more human-like sequence, based on the information disclosed in the paper of Vincke et all (2012) and other proprietary information.

Fermentative production of (e)VHH using a special strain of S. cerevisiae and a proprietary process.

VHH production

QVQ Research has ample experience with the production of recombinant VHH from bacterial and yeast hosts for samples up to 100 mg. Moreover, QVQ research disposes of plasmids for the expression of VHH with a myriad tags at the C terminus. VHH purification is either by affinity chromatography or IEX-chromatography.

Currently, QVQ is gaining experience on production under GMP regime of VHH that will be applied for diagnostic imaging in human.

VHH labeling

Aim of QVQ Research is to deliver VHH that are exclusively labeled directionally. This is achieved by extending the VHH protein with a tag containing a cysteine residue at the C terminus. This unpaired cysteine is than used to couple to Maleimide containing molecules at a site that was proven to not affect the paratope of the VHH, while random labeling causes usually inactivation of the paratope.

QVQ delivers VHH that are labeled directionally at the C terminus with different fluorescent dyes (FITC, Dylight, Hilyte, IRDye), biotin, DOTA, NOTA and HRP). Moreover, QVQ also label the VHH on request with the preferred maleimide label or dye.