[Lingkang Times CDMO] Overview of Minimal Functional Antigen-Binding Fragments-Nanobodies

1. Nanobody structure

IgG antibodies are Y-shaped structures composed of light and heavy chains. Each heavy chain contains 3 constant regions (CH1/CH2/CH3) and 1 variable region (VH). Each light chain contains 1 constant region (CL) and 1 variable region (CL). The molecular mass is generally around 150 kDa. In 1993, Belgian scientists first reported nanobodies in the journal Nature, a heavy chain antibody variable region derived from camelids that naturally lacks light chains. The molecular mass is only 15 kDa. The length of the protein crystal structure is 4 nm and the diameter is 2.5 nm. It is the smallest naturally occurring functional antigen-binding fragment discovered by humans so far.

Figure 1 Schematic structural diagram of traditional antibodies, heavy chain antibodies, and nanobodies

The VH domain of conventional monoclonal antibodies is usually combined with the VL domain and requires a total of six CDRs to show complete antigen-binding ability, while VHH only has three CDRs to show antigen-binding ability. Although the lack of the VL domain results in a smaller paratope, the range of antigens that VHH may bind is limited, but the longer complementarity determining region CDR1 not only increases the size of the antigen-binding site, but can also form a wider loop structure that is not found in the VH domain. In addition, the CDR3 region of VHH is longer. The average length of the CDR3 region of human and mouse antibody VH is 9-12 amino acids, and the CDR3 region of VHH is 16-18 amino acids, which can form a special loop. This loop can bind to clefts and cavities in the spatial conformation of proteins, thereby identifying cavities and cryptic concave epitopes such as enzyme active sites and cryptic viral epitopes, helping to predict new potential biological targets and discover new pharmacological targets. It can also be used as an enzyme inhibitor, receptor agonist or antagonist. The extended CDR3 region also allows the nanobody to exhibit a convex alignment, as opposed to the concave or planar alignment typically exhibited by the VH-VL domains of monoclonal antibodies.

In addition, in the FR2 region of conventional monoclonal antibody VH, there are four amino acids involved in the interaction with VL. These four amino acids are V37, G44, L45, and W47. In the nanobody VHH, these four amino acids have been mutated, namely F(Y)37, E44, R45, and G47. These four positions have changed from hydrophobic amino acid residues to hydrophilic amino acid residues, making VHH more soluble. It should also be noted that substitution of the amino acid residue of L11 for S11 in the FR1 region is commonly observed, and this phenomenon is more common in dromedary VHH sequences than in llama VHH sequences.

Human VH3 and camel VHH germline genes are highly homologous. Therefore, only a few changes to the VHH gene are needed to humanize the antibody. Nanobodies with high affinity, high specificity, and high stability can be obtained through genetic engineering technology.

Figure 2 Comparison between VH and VHH

2. Advantages, disadvantages and applications of nanobodies

Advantages:

· Small size (increases tissue and blood-brain barrier penetration)

· Unique antigen binding epitope

· High affinity (KD at nM/pM level)

· High solubility

· Thermal stability

· Tolerate a wider pH range

· Protease resistance

· Low immunogenicity

· Highly combinable (bivalent/multivalent/fusion antibodies)

· Can be produced using a variety of expression systems

Defects:

· Unit price forms often require additional steps to modify

· Small size results in low serum persistence or rapid renal clearance

· Inability to exert effects related to the Fc region

Disease treatment applications:

· Research has found that nanobodies can be used as an antidote to neutralize the venom of snakes, scorpions, spiders, etc. or as a better inhibitor of myotoxicity;

· Nanobodies can bind to the surface antigens of pathogenic bacteria with high specificity, antagonize the adhesion of bacteria to host cells, and can treat severe infections caused by the emergence of drug-resistant bacteria;

· Nanobodies can serve as efficient neutralizing agents. Nanobodies targeting the receptor-binding domain (RBD) of the viral spike protein have been developed to competitively inhibit the interaction between RBD and human angiotensin-converting enzyme 2 (ACE2) and neutralize the virus;

· Examples of targeted treatments for tumors and autoimmune diseases.

The nanobody CAR-T cell candidate (named Ciltacabtagene Autoleucel) was approved by the FDA for the treatment of multiple myeloma in February 2022. Its treatment approach is to develop chimeric antigen receptor (CAR) T cells that express nanobodies specific to tumor antigens, extract T cells from the patient, and genetically modify them to express tumor antigen-specific nanobodies, and then infuse them back into the patient.

Nanobodies against tumor antigens can be combined with radiopharmaceuticals for specific delivery to tumors. An I-131-conjugated anti-HER2 nanobody for the treatment of breast and gastric cancer is currently in Phase 2 clinical trials; in 2018, caplacizumab was approved by the EU for the treatment of acquired thrombosis. Thrombocytopenic purpura; ALX-0061, which targets the interleukin-6 receptor, is used to treat rheumatoid arthritis and systemic lupus erythematosus, and is currently in phase 2 clinical trials; in September 2022, ozoralizumab, which targets tumor necrosis factor-α, was approved for marketing in Japan for the treatment of rheumatoid arthritis.

Figure 3 Nanobodies and related therapeutic product information (from reference 1)

3. Nanobody preparation (immune library)

4. Phage display nanobody library construction

Obtain the antigen gene sequence, construct it into an expression vector through homologous recombination, express the antigen and affinity purify it. Domesticated camelid animals were immunized by subcutaneous injection. After the immunization, peripheral blood was collected using vacuum blood collection tubes. Peripheral blood mononuclear cells (PBMC) were centrifuged and RNA was extracted and further reverse transcribed to generate cDNA. The VHH fragment was amplified through two rounds of PCR. After purification and recovery, the VHH fragment was digested with restriction enzymes and cloned into a phagemid vector. The ligation product was then electroporated into E. coli to complete the construction of a specific phage library. Finally, the library quality was evaluated through detection of library library capacity and correct insertion rate of VHH fragments.

2. Phage display nanobody library screening

After obtaining a high-quality phage library, phage display technology is used for library screening. The screening process includes four stages: "adsorption binding - non-specific washing - specific elution - enrichment amplification". At the beginning of each round of screening, the library needs to be adsorbed and combined with solid-phase adsorbed antigens. After completing the washing steps to remove non-specific antigen-binding and low-affinity phages, the specific phages are dissociated and eluted and infected with E. coli for amplification and enrichment for the next round of screening. The library is enriched through screening.

3. Specific Nanobody Screening

The phage is transfected again into new E. coli cells, using antibiotics as selectable markers, to express the antibodies. The obtained antibody with high cell-binding activity was subjected to an enzyme-linked immunosorbent assay (ELISA) to detect the binding ability of the antibody and the antigen, and the affinity constant simulation curve was measured. Typically, equilibrium dissociation constants in the nanomolar/picomolar range are ideal.

The selected Nanobodies are then subjected to sequence analysis.

Figure 4 Schematic diagram of a conventional method for generating antigen-specific Nanobodies using phage display libraries.

4. Expression of specific Nanobodies

E. coli: Currently, periplasmic expression is the most common method for producing VHH. The periplasmic space of E. coli can provide an oxidative environment, promote the formation of disulfide bonds, and facilitate the correct folding of foreign proteins. Commonly used signal peptides to mediate the entry of recombinant proteins into the periplasm include PelB, OmpA, PhoA, etc. After the signal peptide is cleaved by signal peptidase, the target protein can be obtained. The expression level of Nanobodies in the periplasm is usually low. In order to improve the expression level of Nanobodies in the periplasm, measures such as fusion expression of Nanobodies with tag proteins and co-expression with molecular chaperones can be adopted. In recent years, commercial E. coli host cells, including BL21 (DE3) strain, Rosetta-gami B (DE3) pLysS strain, Origami 2 (DE 3) strain and Shuffle T7 expression, have been frequently used for VHH expression.

Yeast: Pichia pastoris is a methanotrophic yeast that can utilize methanol as its sole carbon source. Foreign proteins are expressed intracellularly or secreted extracellularly in Pichia pastoris. During secretory expression, Pichia pastoris secretes only a small amount of endogenous proteins, and exogenous proteins are the main components in the culture medium. The degree of glycosylation of foreign proteins in Pichia pastoris is the same as that of mammalian cells, which reduces the immunogenicity of foreign proteins. Compared with Saccharomyces cerevisiae, Pichia pastoris is more suitable for high-density culture, and the expression amount of foreign proteins is more than 10 times that of Saccharomyces cerevisiae, which is beneficial to industrial production. Some studies have found that Pichia pastoris mutant strains are more suitable for protein expression. Currently, Pichia pastoris expresses nanobodies, and the output can reach the g/L level.

Mammalian cells: Mammalian cells are best suited for the production of therapeutic antibodies, and the antibodies produced by them are minimally immunogenic. The yield of VHH-Fc in batch culture in CHO can reach 100 mg/L. The fusion expression of Fc and Nanobodies can extend the half-life of Nanobodies, activate other immune responses, and enhance the antiviral ability of Nanobodies. However, Fc does not improve the ability of Nanobodies to cross the blood-brain barrier.

Others: There are studies on the expression of anti-NDOM-VHH (fermenter) in Brevibacillus spp., and the secretion of nanobodies is as high as 3g/L; there are successful expression studies on filamentous fungi such as Aspergillus oryzae, insect expression systems and plant expression systems with high yields.

4. Summary

Since the discovery of nanobodies, due to their advantages that conventional antibodies and antibody fragments do not have, the research on nanobodies has developed rapidly and involves many fields such as diagnostic tools and therapeutic methods, attracting the attention of the medical market. Of course, the application of nanobodies still requires a lot of research and development, but they will definitely play an important role in the future development of the medical market.

References

1. A Review of Generation, Diagnostics and Therapeutics. Int. J.Mol. Sci. 2023, 24, 5994.

2. Expression of single-domain antibody in different systems. Appl Microbiol Biotechnol 102, 539–551 (2018).

3. Screening and identification of endocytic EpCAM nanobodies[J]. Acta Pharmaceutical Sinica, 2020, 55(10):9.DOI:10.16438/j.0513-4870.2020-1359.

4. Camelid immunoglobulins and nanobody technology. Vet Immunol Immunopathol. 2009 Mar 15;128(1-3):178-83.doi:10.1016/j.vetimm.2008.10.299.