Nanobodies and Their Applications | Nanobody Series (Part 1)

Since the advent of monoclonal antibody technology, it has been widely applied in scientific research, clinical diagnosis, treatment and other fields. Particularly after the launch of CTLA antibodies in 2011 and two PD-1 monoclonal antibody drugs in 2014, the research and development of antibody drugs has boomed worldwide, accompanied by an exponential expansion of the market scale. Traditional antibodies feature a typical Y-shaped structure, consisting of four peptide chains: paired heavy chains and light chains. In the 1990s, scientists discovered heavy-chain antibodies in camelids and cartilaginous fish. Ablynx named such heavy-chain antibodies nanobodies, which have diversified antibody structural forms and expanded the application scope of antibodies.

Structural Forms of Nanobodies

Nanobodies possess a unique structural configuration. During antibody formation, aberrant recombination occurs in the constant region of the heavy chain, resulting in the loss of the CH1 domain found in conventional antibodies. Consequently, the constant region of the light chain (CL) can no longer pair with the constant region of the heavy chain. Instead, the variable region of the heavy chain is directly linked to the hinge region, CH2 and CH3 domains. Such molecules are termed heavy-chain antibodies or single-domain antibodies. Composed solely of the variable region of the heavy chain, nanobodies have a molecular weight of approximately 15 kDa and a nanometer-scale size, hence the name coined by Ablynx, a pioneer in this field.

Structural Characteristics and Application Scenarios of Nanobodies

Compared with traditional antibodies, nanobodies exhibit distinct physicochemical properties and are applicable to diverse scenarios:

With low molecular weight and small size, nanobodies feature strong tissue penetration capacity, making them ideal for drug delivery.

The CDR3 region of nanobodies contains longer amino acid sequences and presents a distinctive conformation relative to that of traditional antibodies. Equipped with unique antigen recognition sites, nanobodies can specifically recognize the pocket and tunnel structures formed by membrane proteins and ion channel proteins.

The domains of nanobodies have excellent hydrophilicity. In the FR2 framework region, the hydrophobic amino acids that form the interaction interface between conventional antibodies and light chains are largely replaced by highly hydrophilic amino acids in nanobody VHH domains.

Nanobodies are easy to express, with favorable thermostability and higher resistance to proteases compared with traditional antibodies.

Nanobodies have a short serum half-life and can be readily cleared from the body, which renders them suitable for clinical diagnosis.

The germline sequences of nanobodies share high similarity with those of human antibodies, simplifying subsequent humanization and engineering modification. Combined with their small molecular weight, nanobodies induce weaker immunogenicity.

Nanobodies demonstrate affinity comparable to or even superior to that of traditional antibodies.

Preparation Process of Nanobodies

Taking alpaca nanobodies as an example, the preparation procedures are briefly described as follows:

After alpaca immunization, whole blood is collected and peripheral blood mononuclear cells (PBMCs) are isolated to construct immune libraries or natural libraries.

Total RNA is extracted and purified from PBMCs. Using mRNA as the template, reverse transcription is performed to synthesize cDNA. The obtained cDNA is then subjected to two rounds of PCR amplification to acquire VHH sequences, followed by the construction of screening libraries (bacterial, yeast or mammalian cell libraries).

Taking phage display technology as an example, helper phages are used to infect the bacterial library to prepare the phage library.

Solid-phase or liquid-phase screening is carried out based on the specific binding between antigens and antibodies.

Monoclonal strains enriched via screening are sequenced according to the results of phage ELISA.

Expression vectors are constructed (for expression in bacteria, yeast or mammalian cells) to produce nanobodies from positive clones.

Verification is performed on the recombinantly expressed nanobodies.

Generally, 3 to 5 consecutive rounds of panning (Steps 3 to 6) enable effective enrichment and screening of nanobodies targeting specific antigens. After sequence acquisition via sequencing analysis and recombinant expression, preliminary evaluation of nanobodies can be conducted in vitro by means of ELISA, FACS and functional verification assays.

Prospect

With in-depth research on nanobodies, their applications in scientific research, clinical diagnosis and clinical treatment will become increasingly extensive. For instance, they can streamline the construction of bispecific antibodies; nanobody-based antibody-drug conjugates (ADCs) gain enhanced tissue penetration; nanobodies help build more stable and efficient delivery systems for nucleic acid drugs. In fundamental research, nanobodies are conducive to stabilizing protein structures for protein structure analysis. Given their multiple superior properties, nanobodies are bound to embrace broader development prospects.