Technology

The technology behind the products and services provided by QVQ is based on the smallest naturally occurring antibody-based molecules, optimized for research and imaging use by directional functionalization.

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

1. 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).

2. VHH sequence analysis

Sequence analyses is a very important step in the characterization of newly selected VHH. Quite often sequences form the basis of patent claims, but is also provide some insight in the maturation process of the VHH. QVQ in collaboration with other groups has determined the (most likely) sequences of the V-genes, whereas Ikbel Achour at al (2008) has determined the D- and J genes of Vecuna alpaca a very close relative to Llama glama. Aligning the newly determined sequences with the germ line V-, D- and J genes and using the large proprietary data base of over 3500 unique sequences of VHH, enables QVQ to determine quite accurate which amino acids of the new sequence are most likely of importance of the functionality and/or the physical and proteolytic stability of the VHH (Dolk et al 2012).  It also enables QVQ to design a rational approach to select family members of related VHH (Koh et al 2010) and to improve by rational design and/or by directed evolution the properties of the selected VHH (Gorlani et al 2012). Finally QVQ has the skills and the contacts to construct reliable 3D structures on basis of amino acid sequences (Calpe et al 2015), the data of more than 20 3D structures of VHH and appropriate programs.

3. VHH production

QVQ offers custom cloning and production of VHH in the yeast Saccharomyces cerevisiae. Plasmids adding different tags at the C-terminus of the VHH are used for cloning of the VHH gene. Characterized yeast strain is used for the production and secretion of the VHH to the culture supernatant. VHH is subsequently purified using affinity and IEX chromatography.

Figure 2: Typical two-face fermentation process to make (e)VHH
Figure 3: QVQ makes all types of variants of VHH in modular forms. Therefore the same VHH is/can be tested from molecule to man.
Figure 3: QVQ makes all types of variants of VHH in modular forms. Therefore the same VHH is/can be tested from molecule to man.
Figure 4: Affinity of random and directionally labeled anti-Her2 VHH 11A4. Random labeling based on NHS-chemistry may inactivate VHH displaying NH2 groups in paratope, while directional labeling away from paratope is not affected by labeling.

4. VHH labeling

Aim of QVQ Research is to deliver VHH that are 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.

The benefit of directional labeling over random labeling is illustrated in Fig. 4, where the affinity of the VHH was to its antigen reduced by 900-fold after random labeling, while the affinity of the same VHH, which was directionally labeled retained nM affinity.

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