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Literature Analysis | Novel VH3 Scaffold Library Boosts Single-Domain Antibody Stability
2025-03-28
On July 31, 2024, in the journal Scientific Reports, Nam Ju Lee et al. published a paper titled A single domain antibody library based on a stability-engineered human VH3 scaffold. The main content of the paper is that natural single-domain antibodies (VHH of camelids) have the characteristics of high stability and small volume. Still, the stability of the variable domain of traditional antibodies is poor. Based on this, the authors conducted phage display screening at high temperatures and identified a stable single-domain antibody variant of human VH3-23 from the framework region 2 randomized human VH library. The synthesized oligonucleotide was randomized into complementary determining regions (CDRs). By increasing the diversity of the library and constructing a human VH single-domain antibody library, the engineered VH3 scaffold retains the human framework. It reduces immunogenicity compared to natural single-domain antibodies. Subsequently, the affinity and other characteristics of the targeted specific binding agent used to identify this library were screened. It was found that the synthetic sdAb library based on stability-engineered human VH scaffolds can be used as a source for high-quality single-domain antibody screening. This human-derived library reduces immunogenicity and increases stability, which is beneficial for improving selection efficiency. In addition, further in vivo validation of immunogenicity and preliminary in vitro experiments (animal models, etc.) are needed. Its discovery provides prospects for antibody development.
Background and Method IntroductionLITERATURE
The affinity and specificity of monoclonal antibodies to targets make them important tools for biological research and treatment. In 1986, the FDA approved the first therapeutic monoclonal antibody, the murine IgG2a CD3-specific transplant rejection drug, OKT3 (also called muromonab). The therapeutic monoclonal antibody drugs supported by the FDA from 2023 to 2024 are presented in the following table (there may be some omissions in data organization):
Tab 1: FDA approved therapeutic monoclonal antibodies
| Antibody | Approval date | Type | Target |
|---|---|---|---|
| Lecanemab | 1/6/2023 | Humanized IgG1ҡ | Amyloid beta protofibrils |
| Rozanolixizumab | 6/26/2023 | Humanized IgG4ҡ | FcRn |
| Pozelimab | 8/18/2023 | Human IgG4ҡ | Complement C5 |
| Mirikizumab | 10/16/2023 | Humanized IgG4ҡ | IL-23p19 |
| Talquetamab | 8/9/2023 | Humanized IgG4ҡ | GPCR5D, CD3 |
| Elranatamab | 8/14/2023 | Humanized IgG2ҡ bispecific | BCMA, CD3 |
| Epcoritamab | 5/19/2023 | Humanized IgG1ҡ/λ bispecific | CD20, CD3 |
| Glofitamab | 6/15/2023 | IgG1ҡ/λ bispecific | CD20, CD3e |
| Retifanlimab | 3/22/2023 | Humanized IgG4ҡ | PD-1 |
| Lebrikizumab | 9/13/2024 | Humanized IgG4ҡ | IL-13 |
| Marstacimab | 10/11/2024 | Fully human | Tissue factor pathway inhibitor |
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Click for inquiryThere are far more therapeutic monoclonal antibodies than the ones mentioned above, so the development of monoclonal antibodies targeting corresponding targets has been ongoing. However, one of the main obstacles to the development of traditional antibody forms is the large molecular weight of monoclonal antibodies, making research on smaller antibody forms a hot topic. Single domain antibodies (VHH), antigen binding fragments (Fab), and single chain antibodies (scFv) The development of antibody libraries is also underway, with smaller antibody fragments allowing for smooth display on phage surfaces. Antibody phage display is a technology that can isolate antibody fragments specific to target antigens in vitro. The functional diversity and specificity of the library (such as folding stability, expression level, solubility, and correct assembly into phage particles) determine the quality of the antibody library, which is crucial for identifying binding agents with the desired target specificity and characteristics. Other obstacles to the development of therapeutic monoclonal antibodies are the easy aggregation of antibody proteins (mainly determined by the variable region) and the formation of dimeric variable forms of scFv (di scFvs, bi scFvs).
Fig 1: scFv structure and other formats
Single-domain antibodies (sdAbs) are derived from alpacas, cougars, and sharks. They are currently the smallest and most binding antibodies, with a size of only 12-15 kDa, while full-length antibodies range from 150-160 kDa, Fab is approximately 50 kDa, and scFv is approximately 25 kDa. Due to the lack of CH1 domains in traditional antibody light and heavy chains, single-domain antibodies are more likely to penetrate the blood-brain barrier than other forms of antibodies. In addition, single-domain antibodies have stable structures, can be stored for a long time under high-temperature conditions, are not easily degraded by proteases, and have the advantage of acid and alkali resistance, which is conducive to improving their bioavailability. Although single-domain antibodies lack light chains, they can still tightly bind to antigens like traditional antibodies due to their long CDR3. In addition, the CDR3 of single-domain antibodies can form a concave-convex structure, which has the advantage of easy recognition of antigen-hidden epitopes. Single-domain antibodies have a simple structure with only one heavy chain variable region and no post-translational modifications, which makes them easy to transform into different forms and can be expressed on a large scale in prokaryotic and eukaryotic expression systems, greatly reducing experimental costs. However, the main difference between traditional variable regions and natural single-domain antibodies lies in the presence of frame 2 (FR2), which can lead to poor solubility and stability of non-camelid VH domains. Synthetic VH sdAb libraries based on human variable domain scaffolds (many containing camelid-like FR2 sequences) can be an attractive alternative for camelid animal immunity.
Fig 2: Single domain antibody (VHH) structure
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Method, Results, and ConclusionLITERATURE
By constructing an FR2 randomized VH library, diverse CDRs were introduced into the VH scaffold, reassembled into the pComb3X vector, and electroporated into TG1 E. coli to construct the final single-domain antibody library. Remove the constructed library for rescue, followed by phage display screening of protein A superantigen. Use typical phage display screening methods to screen for 3 rounds. Heat the precipitated bacteriophages at 70°C for 4-6 rounds, cool them on ice to bind to the antigen, and heat them at 80°C for 7-8 rounds, cool them on ice to bind to the antigen. In addition to the target protein A superantigen, the other four test antigens were also screened. Single colonies were selected from the final round of screening for ELISA screening to identify specific binding, followed by Ni NTA/SEC purification, SPR binding assay, and other analyses. The following results were obtained:
Construct a randomized library of FR2 with a size of 1.1 × 10^7, establish stable variants of VH3 FR2, select appropriate variants for the final construction of the single domain antibody library, and then perform selection through a set of test antigens to validate the constructed sdAb library. By comparing the binding activity before and after heat treatment, as shown in Figure 3, comparing the binding activity before and after heat treatment with that without heat treatment, the heat-treated bacteriophages showed significant effects on CARS1 #1, CARS1 #3、CARS #8. The relative binding activities of ovalbumin #7, ovalbumin #8, and HSA #23 were 92%, 102%, 97%, 80%, 85%, and 48%, respectively. The sdAb retained approximately 50% or more of its binding activity with homologous antigens, indicating high thermal stability and/or refolding ability.
Fig 3: Binding activities of sdAbs before and after heat treatment
As shown in Figure 4, an ELISA assay was performed to evaluate its binding activity against SARS-CoV-2 RBD Fc, human ACE2 HEL, CARS1, and When testing a set of antigens including ovalbumin and HSA, all six clones only significantly bound to their target antigens and did not significantly bind to nontarget antigens, confirming the high specificity of the screened antigens.
Fig 4: Evaluation of binding specificity by ELISA
The final author identified an optimized non-camelid animal FR2 sequence based on human VH3 sdAb, which has the characteristics of high purification yield, high thermal stability, and high monomer content. CDR diversity was introduced into the VH scaffold, and the constructed sdAb library was able to screen for target-specific conjugates with ideal properties.
ReferenceLITERATURE
Lee, N.J., Jung, M., Yang, H.Y. et al. A single-domain antibody library based on a stability-engineered human VH3 scaffold. Sci Rep 14, 17747 (2024). https://doi.org/10.1038/s41598-024-68680-5