Lead discovery projects.
A full project including immunization, library construction, panning and delivery of clones can be performed within a few months.
The tailor-made single-domain antibody-discovery projects on inquiry can include the following building blocks:
- Design and implementation of an immunization protocol in llamas
- Determination of immune responses in the sera
- Cloning of VHH-repertoires in phage-display libraries
- Panning for the desired epitope and specificity
- Picking of masterplates with monoclonal single-domain antibodies
- Production in 96-wells format
- Screening for binding in ELISA
- Sequence analysis and annotation of clusters
- Delivery of different single-domain antibodies (sequence and >0.2 mg protein).
The building blocks of the services of QVQ.
Immunization
Immunizations of llamas are done in collaboration with Kaneka Eurogentec, Preclinics, and Diaclone. All immunizations are conducted according to the applicable animal welfare act at place and reviewed by the animal experiment commission of the company. A standard project includes the immunization of 2 animals in a 43-day program. We can combine different targets and different antigen formats including recombinant proteins, cells, and other complex antigens. After immunization, we always confirm immune responses of the animals by serum titration ELISA.
Library construction
Immune libraries are generated by cloning VHH genes from the llamas’ PBMCs into our proprietary phagemid vectors. QVQ’s immune libraries contain on average 108 transformants with ~100% correct insert frequency.
QVQ also offers different ready-to-go libraries, such as pooled immune libraries, as well as synthetic, combinatorial, or variant libraries.
Phage-display panning
QVQ proposes a phage-display panning strategy that, based on own experience and available tools, should result in the selection of the desired target binders. Such a strategy is carefully designed to include customer’s requests and wishes. Importantly, the prospective final application of the single-domain antibodies is taken into consideration while determining the phage-display selection strategy.
The expertise of the QVQ team is used to select for single-domain antibodies with specific characteristics, such as antagonism, targeting preferred epitopes, high stability, or high specificity.
Screening panning outputs
Outputs from phage-display selections are screened for binders in different ways:
1) by picking single clones into 96-wells plates (masterplates), producing those in small-scale and screening the crude periplasmic extracts in ELISA. In addition, VHH sequences and sequence cluster information are delivered.
2) by next-generation sequencing (NGS) of the polyclonal outputs, generating >50.000 reads per sample. The NGS reads are annotated for VHH sequences and analyzed for sequence clusters.
Both approaches generate a lead panel of up to 12 sdAbs with unique sequences.
Production and delivery
QVQ selects a lead panel ~12 different single-domain antibodies based on a combination of binding data, sequence data and, if available, functional data. These lead candidates are produced and purified from the periplasm of Escherichia coli using IMAC. Standard quality control includes SDS-PAGE to confirm protein integrity and ELISA for determining antigen binding and affinity. QVQ delivers the purified single-domain antibody protein of the lead clones (0.2 mg of each).
Optional Follow-up Services
- Pharmacological characterization (e.g. target engagement, epitope binning, SPR, flow cytometry, cell-based functional assays)
- Larger scale production and purification
- Custom functionalization (labeling, fusion proteins)
- Engineering, optimization and humanization
Report and Deliverables
QVQ provides a detailed report describing all the above-mentioned steps and data, including all VHH sequences determined in the project (for immune libraries: lead clones, the full masterplates, and all NGS data. For synthetic libraries only sequence of the hits are shared). After production and purification, at least 0.2 mg of protein per lead clone will be delivered.
All intellectual property corresponding to the lead discovery project or the production, purification, and characterization of single-domain antibodies is transferred to the customer and is fully owned by the customer to allow protection and further development of the molecules.