The foundation of any successful bispecific project is the quality of the initial building blocks. This process almost always begins with the generation of a high-diversity library followed by VHH phage display. Unlike traditional hybridoma technology, phage display allows for the direct selection of binders with specific biochemical properties.

A typical workflow involves immunizing a camelid, such as a llama or alpaca, with the target antigen over a six-to-eight-week period. Following the final boost, peripheral blood mononuclear cells (PBMCs) are isolated for RNA extraction. The VHH repertoire is then amplified via RT-PCR and cloned into a phagemid vector. It is crucial to maintain a library size of at least 108 to 109 transformants to ensure sufficient diversity for the VHH phage display process.

During selection, the stringency of the wash steps is a key parameter. We often use increasing concentrations of Tween-20 (from 0.05% to 0.1%) to eliminate low-affinity binders. Competitive elution with the natural ligand of the target can also be used to steer the selection toward specific epitopes. This ensures that the chosen VHH Antibody will have the desired functional activity when later incorporated into a bispecific format.

After three to four rounds of VHH phage display, individual clones are expressed in the periplasm of E. coli (typically TG1 or HB2151 strains). ELISA is the standard bench-level method for identifying positive binders. However, for bispecific design, we also look at the melting temperature (T_m) at this stage, as only the most stable VHHs will survive the fusion process.

Expression yields usually decrease during the move to bispecifics. A single VHH might yield 50 mg/L in a lab setting. The bispecific yield often drops by 40% or even 60%. Monitoring these change is essential before we scale up the process.

E. coli is used for single VHH units. Yields are often between 10 to 100 mg/L. Bispecifics are usually expressed in yeast or mammalian cells. HEK293 or CHO systems are preferred for complex formats. If an Fc-fusion is needed, mammalian expression is required. It extend the half-life from 4 hours to over 7 days. During the expression phase, we monitor the culture supernatant via SDS-PAGE to check for proteolytic cleavage of the linker, which is a frequent failure mode for tandem VHHs.

Purification is typically achieved through Immobilized Metal Affinity Chromatography (IMAC) if a His-tag is present, followed by Size Exclusion Chromatography (SEC). SEC is the most critical step for characterizing a VHH Antibody because it allows us to quantify the monomeric fraction. We look for a clean, symmetrical peak. Any leading shoulders or broad peaks usually indicate the presence of dimers or higher-order aggregates, which are unacceptable for clinical candidates.

In our experience, the thermal stability of a bispecific molecule is generally dictated by the weakest link—the domain with the lowest Tm. Differential Scanning Fluorimetry (DSF) is used to determine the unfolding profile. If a single VHH Antibody shows a Tm of 65ºC but the fusion shows a Tm of 52ºC, this suggests that the linker or the presence of the second domain is destabilizing the fold.

Working at the bench often reveals issues that are not covered in theoretical papers. For instance, the choice of the signal sequence for periplasmic expression in E. coli can greatly impact the folding of VHH Antibodies. The PelB leader sequence is generally effective, but we have found that OmpA sometimes yields better results for more hydrophobic sequences.

Another common issue is the behavior of certain clones. Some VHHs tend to bind non-specifically to plastic surfaces or chromatography resins. Non-specific interactions causes misleading results in ELISA or SPR. Small amounts of surfactant are added and salt concentration in the running buffer is increased to 300 mM NaCl. This mitigate the issues during VHH characterization.

VHH domains are building blocks for bispecific therapeutics and phage display libraries have 10⁹ variants. Researchers identify modules for various formats. The transition to a bispecific construct require careful work. Linkers of 15 to 20 amino acids are common and pI matching is necessary. The pI of domains should match within 1.0 unit. Physical characterization is rigorous. Through systematic testing of orientations and expression parameters, it is possible to create stable and highly potent molecules. The modularity of VHHs ensures that as the demand for more sophisticated multi-specifics grows, the toolkit for antibody engineering will remain flexible and efficient.

Alpha Lifetech provides a comprehensive and fully integrated Antibody Discovery Platform to support your custom bispecific antibody development. Utilizing advanced Phage Display Technology and Yeast Display Technology, our platform is designed for the discovery and engineering of high-affinity antibodies across multiple formats, including VHH, Fab, and scFv.

[1] Mullin M, McClory J, Haynes W, et al. Applications and challenges in designing VHH-based bispecific antibodies: leveraging machine learning solutions. MAbs. 2024 Jan-Dec;16(1):2341443. doi: 10.1080/19420862.2024.2341443. Epub 2024 Apr 26. PMID: 38666503; PMCID: PMC11057648.
[2] Labrijn AF, Janmaat ML, Reichert JM, et al. Bispecific antibodies: a mechanistic review of the pipeline. Nat Rev Drug Discov. 2019 Aug;18(8):585-608. doi: 10.1038/s41573-019-0028-1. PMID: 31175342.
[3] Muyldermans S. Nanobodies: natural single-domain antibodies. Annu Rev Biochem. 2013;82:775-97. doi: 10.1146/annurev-biochem-063011-092449. Epub 2013 Mar 13. PMID: 23495938.
[4] Barron N, Dickgiesser S, Fleischer M, et al. A Generic Approach for Miniaturized Unbiased High-Throughput Screens of Bispecific Antibodies and Biparatopic Antibody-Drug Conjugates. Int J Mol Sci. 2024 Feb 8;25(4):2097. doi: 10.3390/ijms25042097. PMID: 38396776; PMCID: PMC10889805.