Expression and Purification of Nanobody

Introduction of Nanobody

Nanobody (Nb) comprises only two heavy chains, and its variable domain of heavy chain is called VHH(heavy chain of heavy-chain antibody). The molecular weight of VHH prepared by recombinant expression in vitro is only 15kDa, about one-tenth of the traditional antibody and one-half of the antigen-binding fragment (scFV, VH-VL).

Since there are no light chains, the nanobody has only three heavy chain antigen recognition regions (CDR regions). To compensate for the lack of biological activity of light chain CDRs, nanobody increase the amino acid length in the CDR3 part (16-18 amino acid residues, corresponding to the traditional antibody CDR3 has 8-15 amino acids) to increase the diversity and specificity of CDR.

Figure 1: Structure diagram of the Nanobody（KMD Bioscience）

Expression and Purification Strategies of Nanobody

Unlike the classical hybridoma technique for monoclonal antibody preparation, the whole process of Nanobody development mainly includes alpaca immunization, phage library construction, antibody screening, expression purification, and verification. After immunizing the alpaca, B lymphocytes were isolated from the peripheral blood of the alpaca, total RNA was extracted and reverse-transcribed into cDNA, which was used as a template for PCR amplification to obtain a variety of nanobody gene fragments, and then connected to the vector to construct phage library. Then, the antigen-specific nanobodies were obtained by multiple rounds of panning, and sequenced, expressed, and verified.

Figure 2: Production process of Nanobody（KMD Bioscience）

KMD Bioscience has established a complete mammalian expression system for antibody expression, including but not limited to cell lines such as FreeStyle 293-F cell line and Expi CHO-S. Recombinant antibodies were expressed and prepared in conjunction with a high-expression vector designed by KMD Bioscience (containing a full-length CMV promoter and an optimized secreted signaling peptide sequence). After the expression was completed, the nanobody was purified by a nickel column. Then the purified nanobody was tested by Biacore affinity.

Figure 3: Picture of expression result of Fc-VHH nanobody（KMD Bioscience）

Advantages of Nanobody

The biophysical and biochemical properties of nanobodies are particularly stable, including solubility, heat resistance, and proteolytic resistance.

②Large-scale production of nanobody is easier to achieve. Because of the long amino acid sequence and complex protein structure, the production of traditional antibodies requires complex instruments and can only be realized in eukaryotic systems. In addition, in the use of clinical antibody drugs, to achieve therapeutic effects, usually give large doses of drugs. These two aspects lead to the production of traditional antibodies requiring the use of a large number of mammalian cells and lengthy screening and purification steps, and the production cost is very expensive. Nanobodies can be expressed in a large number of microbial systems (such as bacteria, yeast, and fungi), and can be quickly screened in display libraries, making nanobodies have lower production costs.

③ The immunogenicity of nanobodies is low. The structure of the nanobody is very similar to that of the heavy chain variable region VH of a human antibody, including three hypervariable regions hypervariable region (HVR) and four framework regions (FR) on both sides and its gene homology is 90%. This means that the immunogenicity of nanobodies is relatively low, and so far, there have been no reports of adverse reactions caused by the immunogenicity of nanobodies.

④ Nanobody molecular weight is small, tissue penetration ability is strong, and blood clearance is faster. The passive diffusion rate of drugs between cells in tissues is inversely proportional to the size of drug molecules. Therefore, compared with the traditional antibody (150), the monovalent nanobody with the size of 15 kDa has better tissue penetration and can reach the focal tissues that the traditional antibody cannot reach, such as solid tumors and other unstable tissues.

Application of Nanobody

Because of its small molecular weight, a nanobody is a single gene code, easy to genetic engineering modification, and can be polymerized by short linking sequences to form a polyvalent or multi-specific antibody structure. Nanobodies are easy to form new fusion molecules with other structures (such as BSA, IgG-Fc, etc.). In the new fusion molecule, the nanobody is bound to its target antigen, and the part fused with the nanobody can play the corresponding function, so it can be used in combination with other drugs, or applied to diagnosis and as an experimental research tool in a variety of fields, and the application scenario is broad.

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