Technology

QVQ’s imaging agents are suitable for correlative imaging from nanometer to meter scale. (animation by ScienceTransmitter)

Animals from the camelidae family (i.e. camels, llamas and alpacas) contain a particular class of antibodies that are devoid of light chains. These so-called heavy chain-only antibodies (HcAbs) undergo normal selection and maturation by the animals’ immunse system. For this reason, HcAbs and their the variable domains (VHHs, sdAb or Nanobody), can exhibit high affinities (nM range) and serum stability.

QVQ provides expertise in each step of VHH technology, including immunizations, phage-display selections and screening, production, molecular modeling and genetic optimization. In addition, QVQ offers a considerable number of high quality, off-the-shelf VHHs targeting molecules related to cancer, age related diseases and infectious diseases.

Basic Monoclonal VHH development

Immunization and library construction

QVQ collaborates with Kaneka Eurogentec and Preclinics on the immunization of llamas. The immunizations are conducted according to the applicable animal welfare act at place and all immunizations are reviewed by the internal animal experiment commission of the company before starting the immunization.

QVQ has experience with a large variety of antigen types, ranging from purified natural and recombinant protein up to complex cells and tissue for immunizations and selections. Optimized immunization protocols provide the oppertunity to immunize with relatively low amounts of recombinant protein with high success rates.

At least 2 animals are scheduled for each immunization to ensure diversity of the selected VHH and to avert possible non-responder animals. Four injections with antigen are included in the standard 43-day protocol, which can be extended with additional boost-immunizations for generation of very high specific or affinity VHHs.

After the immunization, titers of antigen-specific antibodies are measured in sera using “in-house made” polyclonal anti-VHH antibodies. In the case of a proper immune response, total RNA is isolated from the white blood cells and this is used to construct phage-display libraries using our proprietary phage-display plasmid. QVQ libraries contain at least >107 (108 on average) unique clones with a correct insert frequency of >90%.

QVQ has experience in using several antigens forms ranging from purified natural and recombinant protein up to complex cells and tissue for immunizations and selections. Optimized immunization protocols allow for immunization with low amounts of recombinant protein with high success rates.

At least 2 animals are scheduled for each immunization to ensure diversity of the selected VHH and to avert possible non-responder animals. Four injections with antigen are included in the standard 43-day protocol, which can be extended with additional boost-immunizations for generation of very high specific or affinity VHH’s.

After the immunization, the titers of antibodies directed against the antigen of interest are measured in sera using “in-house made” polyclonal anti-VHH antibody. In the case of a proper immune response, total RNA is isolated from the white blood cells and this is used to construct phage-display libraries. QVQ libraries contain at least 107 different clones with an correct insert frequency of more than 90%.

Phage diplay selection and screening for binding

Based on extensive experience and available tools, QVQ always proposes a strategy for the selection of target-binding VHHs by phage display-selections. Such strategy is carefully designed to include customer’s requests and wishes. Upon agreement on the selection strategy. QVQ commits to select at least 6 different VHHs belonging to at least 2 different families based on the V-D-J gene combinations.

Importantly, already during phage display-selections,  the  final application of the VHHs and the respective requirements are taken into consideration. These include for example agonistic or antagonistic mode of actions, preferred epitopes, high stability or high specificity.

Production and delivery

VHH are characterized for binding, competition and functionality using the available tools. Moreover, sequences of the different VHHs will be determined. QVQ will combine binding data, sequence data and, if applicable, functional data obtained by the customer in order to select a lead panel of 6 VHHs. These are cloned in a bacterial expression plasmid, produced and purified from the periplasm of Escherichia coli using IMAC.

All the above mentioned steps and all data is collected in the form of a report, which is send to the customer along with purified protein (0,5 mg of each) of the 6 lead clones.

QVQ has ample experience with the production of recombinant VHH from bacterial and yeast hosts for samples up to 500 mg. VHH purification is either by affinity chromatography or IEX-chromatography.

In addition, QVQ has gained experience in translating the production knowledge to perform GMP-approved production of VHHs, making them suitable for diagnostic imaging in human.

Optional Customization of your product

Custom Tags

QVQ can offer numerous tags at the C terminus of the VHHs according to the wishes of the customer, but QVQ has developed a proprietary tag for labelling. Therefore, QVQ products come without tag or with the C-direct tag for directional labeling or coupling, which does not affect the functionality of the VHH.

Custom Labelling

Aim of QVQ is to deliver VHHs that are exclusively labeled directionally. Directional labeling of the VHHs is achieved by using the proprietary C-direct tag that inserts a cysteine residue at the C-terminal end of the VHH. This unpaired cysteine is than used to couple to Maleimide containing molecules via sulfhydryl-maleimide coupling. Because the C-terminal cysteine is located on the opposing end of the VHH, as compared to the eptiope-binding loops., directional labeling of the C-direct tag was proven to not affect the paratope of the VHH. In contrast, common random labeling approaches causes usually inactivation of the paratope.

QVQ offers directional labeling of VHHs at their C-terminus with different fluorescent dyes (FITC, HiLyte, IRDye), biotin, DOTA, NOTA and HRP. On request, other maleimide-labels or dyes can also be used for directional labeling of the VHHs.

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

Optimization

QVQ 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 genes, QVQ can manipulate the VHH sequences to improve their stability and production yields.

Although VHHs are not of human origin, llama antibodies share high homology with those in humans. Based on proprietary and published information, QVQ can modify the VHH sequences to render them even more human-like (humanization).

Fermentation

QVQ has invested in fermentation equipment and can now capitalize on the longstanding knowledge to obtain very high quantities of VHH in a special strain of S. cerevisiae using a proprietary fermentative process.

Two standard protocols are available which are tailored for either well-producing VHH (> 1 g/L) or more difficult to produce VHH (50-800 mg/L).