How VLPs are unlocking the next generation of antibody therapeutics for challenging transmembrane proteins
Transmembrane proteins play crucial roles in cell signaling transduction, immune response regulation, and ion/small molecule transportation. Their involvement in diseases like cancer, autoimmune disorders, and infectious diseases makes them the prime targets for therapeutic antibodies. However, these proteins remain among the most challenging targets for antibody drug development due to the insolubility of membrane protein and difficulty of presenting in native confirmations under normal assay conditions. Fortunately, the Virus-like particles (VLPs), are now reshaping this landscape by providing a natural bilipid layer to display transmembrane proteins in a soluble format with native target protein conformation. Additionally, the viral capsid structure of VLPs enhances the immunogenicity of membrane proteins with low abundance, making it a robust tool for antibody discovery. Here, we explore applications of VLP-displayed antigens in antibody discovery and screening including immunization, yeast display screening, and phage display screening.
Immunization
Immunization is the first and also the most critical step in the early stages of antibody discovery campaigns. VLPs are particularly suitable for immunization campaigns, as they mimic the repetitive structure of viral surfaces, which enhances the immune response to the target antigen. This leads to boosted production of strong antibodies for otherwise poorly immunogenic proteins. Additionally, VLPs have a high display density of target protein compared to cell lines with overexpressed proteins, further improving the outcome of immunization..
We have validated the strength of VLP-displayed antigens for immunization in an in-house immunization campaign using CXCR4 VLP. Antiserum generated using CXCR4 VLP for immunization produced polyclonal antibodies against CXCR4, demonstrating that our VLPs are a robust way to boost immunization and obtain antibodies for the protein of interest. Furthermore, the VLPs produced sufficient anti-CXCR4 antibodies even with a small dosage of VLP (0.5µg), lowering raw material requirements for immunization.
Yeast Display Screening
Once antibodies are generated through immunization, the screening and optimization process begins. Yeast display is one of the most powerful techniques used to isolate and refine antibodies with high affinity and specificity. In this mid-stage of antibody discovery, VLP-displayed antigens are used to present complex transmembrane proteins in their native state to yeast-displayed antibody libraries. This allows researchers to identify lead antibodies that bind specifically to the functional, conformational epitopes of the target antigen.
By integrating VLP-displayed antigens into yeast display campaigns, researchers can more accurately screen for antibodies that recognize the target protein's native structure, leading to better candidates for therapeutic development. Our catalog of fluorescent VLPs can streamline this process by enabling easy cell sorting without the need for secondary labeling. This is useful for identifying target-antigen-binding monoclonal antibodies (mAb) in lead screening. Our FITC-equivalent GPRC5D VLP was successfully used in yeast display to identify GPRC5D mAbs. See the full case study here.
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Phage Display Screening
Phage display, another critical screening technique, is often employed alongside yeast display or as a standalone method, especially in the later stages of an antibody campaign. It allows for the high-throughput screening of antibody libraries against VLP-displayed antigens, enabling researchers to discover high-affinity antibodies with therapeutic potential.
VLPs can be biotinylated and immobilized on streptavidin-coated plates or beads, which allows for the effective selection of phage-displayed antibodies with high specificity for the target protein. Phage display screening can be iterative, requiring multiple rounds to enrich for the best candidates, and is often used to refine and validate antibodies discovered through immunization or yeast display. Our Biotinylated VLP-displayed antigens can bind to a streptavidin-coated plate or streptavidin beads to identify high-affinity phages, which are then purified, amplified, and sequenced.
Figure 3. Biotinylated GPRC5D VLP ELISA assay shows the VLP-displayed antigen can generate a strong enough signal for use in phage panning.
Limitations: Phage display screening is time-consuming and may require multiple rounds of selection to isolate high-affinity binders. It’s important to note that phage display is most effective when used with immunized libraries, as naive libraries can yield low-affinity antibodies. By using immunized animals from earlier campaigns, researchers can generate a robust pool of antibodies for further screening.
Conclusion
VLP-displayed antigens offer a powerful and versatile platform for various steps in antibody drug development including immunization and screening. However, considerations of VLP size and complexity, as well as assay limitations, must be carefully considered to maximize the effectiveness of VLPs in antibody drug development. Understanding both the strengths and weaknesses of VLP-displayed antigens allows researchers to leverage this technology effectively.
KACTUS has been developing and validating VLP-displayed antigens since 2019, applying them in a range of techniques such as immunization, yeast display, ELISA, SPR, and BLI. Over the years, we’ve built a robust expertise across these applications.
Our efforts have culminated in an off-the-shelf catalog of high-performing VLP-displayed antigens, featuring various multi-pass transmembrane proteins and other difficult-to-express targets including GPRC5D, Claudin 18.2 Claudin 6, CCR2b, GPC3, and others. If you’d like to learn more about our products and services, please contact our team at support@kactusbio.us.
