Phage Display for VHH Antibody Discovery
Building on our expertise in library construction and screening, VectorBuilder now offers comprehensive solutions for phage display-based VHH antibody (a.k.a. nanobody) discovery. Leveraging our powerful sequence-guided screening technology with direct access to farmed alpacas, we can identify VHH antibodies with exceptional therapeutic potential for detailed characterization.
Highlights
Wide range of antigen formats for immunization, including soluble protein, short peptide, and more.
In vivo matured VHH antibodies from alpacas, ensuring superb specificity and binding affinity.
Diverse phage panning strategies and proprietary screening technology, enabling high-throughput affinity measurement.
Comprehensive antibody characterization and functional validation assays available.
Service Details and Technical Information
In recent years, phage display technology has become increasingly popular for antibody discovery. Compared to the traditional hybridoma technology, it offers some great advantages, such as higher throughput and the flexibility to accommodate various antibody formats for screening. One phage display library contains millions of antibody candidates and allows them to be tested simultaneously in a single run, making it a highly efficient discovery platform. A typical workflow of phage display-based antibody discovery is shown below:
Antigen Preparation
Animal
Immunization
Immunization
Phage Library
Construction
Construction
Library Screening
and Antibody
Discovery
and Antibody
Discovery
Antibody Characterization
Antibody
Optimization and Engineering
Optimization and Engineering
- Antigen formats: soluble protein, short peptide, DNA, mRNA, whole cell, AAV, and VLPs
- Immunization in alpaca
Choosing the right animal platform and antigen format are both important to successful immunization and downstream candidate identification. Camelids such as alpacas, llamas, and camels are ideal models for nanobody discovery because they naturally possess the heavy-chain antibody, consisting of only two heavy chains. Its variable region, named VHH, allows highly specific antigen binding. Due to VHH’s significantly smaller size and longer CDR3 region, it offers several advantages over conventional antibodies, including better antigen recognition, higher stability and tissue penetration, as well as ease of engineering. With VectorBuilder’s direct access to alpacas, the native B cell repertoire can be expanded through immunization.
We offer multiple antigen formats for immunization, and all antigens prepared by VectorBuilder are subject to strict QC measures. Alternatively, antigens can be prepared and shipped directly by customers. Soluble proteins are usually preferred immunogens because of their easy access and controllable dosage. However, they may not be suitable when the target antigen is a membrane-bound protein or has low immunogenicity. In such cases, other strategies can be considered, including whole cell, DNA, mRNA, VLPs, and virus-based immunization.
- Fully synthetic or native antibody sequences extracted from alpacas
- Library validation by NGS available
Antibody sequences from de novo synthesis, naive animals, and immunized animals can all be used to construct phage display libraries. While synthetic and naive libraries typically offer broader diversity coverage, those based on immunized animals are more relevant to antibodies’ superior affinity. Following collection of sequences, they are cloned into vectors using a phagemid before transformation and assembly. In the constructed libraries, each of the recombinant phages displays a monoclonal antibody on its surface. We further offer NGS to validate the uniformity and overall quality of phage display libraries.
- Diversified panning strategies to enrich functional phage clones
- Ultra high-throughput proprietary sequence-guided screeningRecommend
VectorBuilder has developed a proprietary sequence-guided screening method, offering significantly higher-throughput and unbiased measurement of individual antibody affinity scores. After 3-5 cycles of enrichment, the affinity of top-ranking recombinant phages is further validated by ELISA or flow cytometry.
Leading antibody candidates identified through screening are expressed and purified for comprehensive characterization. Our service portfolio spans:
- Specificity of antigen recognition
- Affinity measurement and ranking by bio-layer interferometry (BLI)
- Antibody sequencing
- Epitope binning
- Cross-reactivity assessment
- In vitro functional analyses
As VHH antibodies discovered through phage display originate from alpacas, they typically require optimization before therapeutic use. At VectorBuilder, we are able to carry your candidates through next stages including:
- Antibody humanization
- Affinity maturation
- Early-stage developability optimization
In addition, we offer antibody engineering services to meet the demands of the fast-evolving therapeutic antibody market, including:
- Fc engineering
- Bispecific engineering
- Bioconjugation
- CAR engineering
- Stable cell line construction
Experimental Validation
Immunization
Figure 1. The alpaca's strong immune responses against AAV immunization. After each round of immunization with a mix of AAV serotypes (1, 2, 4, 5, 8, and 9), the serum was collected from the alpaca and titrated by ELISA. Three (3rd) and four (4th) rounds of immunization significantly increase antibody titers in the serum compared to only two rounds (2nd). NS, negative serum.
Phage display library screening
Figure 2. Five purpose-fitting antibodies discovered through VectorBuilder’s sequence-guided screening. (A) An unbiased affinity score was calculated for each individual phage clone. Our proprietary approach offers a significantly broader dynamic range than the traditional screening method. (B) All antibodies show exceptional binding affinity across multiple AAV serotypes. The affinity of each candidate was validated by ELISA, and pMES4 B4 bacteriophage was used as the negative control (NC).
Antibody characterization
Figure 3. Full characterization of anti-AAV VHH antibodies. Each candidate was re-expressed in the format of VHH-Fc. (A) KD values of the candidates measured by BLI. The results show very high affinity of the two VHH antibodies tested. (B) Binding curves of the candidates to AAV9, which were used to calculate EC50 values. (C) In vitro neutralization assays showing both VHH antibodies effectively block AAV1 from infecting 293T cells. No antibodies were added to the NC group. IMS, immunized mouse serum; NC, negative control.
