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How Much Do You Know About Nanobodies?
Release time:
2024-12-26
About Antibodies
Antibodies are protective proteins produced by the body in response to antigen stimulation. They are Y-shaped proteins composed of four linked polypeptide chains.Two of these chains, with relatively larger molecular weights, are called heavy chains, while the other two are light chains. Each chain has two ends: the C-terminus and the N-terminus. Analysis of the sequences of the four chains reveals that approximately 110 amino acids near the N-terminus exhibit significant variability, known as the variable region. In contrast, other regions of the sequence remain relatively consistent, referred to as the constant region.
The variable region is the part of the antibody that binds to the antigen. The variable regions of both the heavy and light chains contain three highly variable areas composed of amino acids, known as complementarity-determining regions (CDRs), specifically CDR1, CDR2, and CDR3. Among these, CDR3 exhibits the highest degree of variability.
About VHH
Not all antibodies have the structure described above. In 1993, a type of antibody consisting only of the variable region of the heavy chain was discovered in camelids. This discovery was published in Nature. Later, heavy-chain antibodies were also found in sharks, but to date, camelids remain the primary species used for VHH antibody research.
Nanobodies, or heavy-chain single-domain antibodies (VHH-variable domain of heavy chain antibody), are antibodies that lack light chains and have a heavy chain constant region missing the CH1 domain. The variable region of the heavy chain, VHH, can be cloned and expressed individually, retaining antigen-binding activity comparable to that of full heavy-chain antibodies. Due to their crystal width of just 2.5 nm, length of 4.8 nm, and a molecular weight of about 15 kDa, VHHs are the smallest known units capable of binding antigens, and are therefore also known as nanobodies.
Significance of VHH Antibody Research
Immunogenicity is positively correlated with the molecular weight of an antibody. Because nanobodies have a small molecular weight, they exhibit lower immunogenicity and possess stronger tissue penetration capabilities. They can cross the blood-brain barrier and penetrate dense tissues such as tumors.
Nanobodies are highly soluble, structurally simple, and suitable for expression in both prokaryotic systems (e.g., Escherichia coli) and eukaryotic systems (e.g., yeast).
VHH Library Construction
Animal-Derived Nanobody Libraries
Most nanobodies currently come from camelids. However, the process of obtaining animal-derived nanobodies through immunization of camelids is lengthy and expensive, requiring large quantities of purified proteins and extensive livestock and veterinary facilities. Additionally, due to immune tolerance to self-antigens, animal-derived antibodies often fail to bind to conserved regions, which are typically responsible for driving key protein functions.
Artificially Synthesized Nanobody Libraries
Due to the complex process of obtaining nanobodies from camelids, there have been many challenges in nanobody research. As a result, an artificially synthesized nanobody library has been developed, and a yeast library has been constructed. Based on the camelid antibody genes and considering the characteristics of nanobodies, variants are designed in the complementarity-determining regions (CDRs). For specific positions within the CDRs, random sequences are introduced to reflect the high diversity at these locations. In particular, highly diversified sequences are introduced at 4 positions in CDR1, 1 position in CDR2, and at 7, 11, and 15 consecutive positions in CDR3, which corresponds to the highest degree of variability observed in the CDR3 region of nanobodies under natural conditions.
After evaluation, the artificially synthesized nanobodies were found to be comparable to those obtained through animal immunization. Through yeast surface display and high-throughput sequencing, the library contains over 10^8 full-length nanoclones and is capable of expression and display on the yeast surface.
VHH Antibody Screening
Currently, the most commonly used methods for VHH antibody display screening are phage display and yeast display. Compared to the phage expression system, yeast, as a eukaryotic expression system, can perform post-translational modifications to express and fold complex eukaryotic proteins. Therefore, it overcomes the limitations of phage display technology, particularly in the identification of conformationally selective nanobodies.
Phage Display
Phage display technology uses a phage vector to clone the gene encoding a protein into an appropriate position within the gene for the phage coat protein. This allows the foreign protein to be expressed as a fusion with the coat protein, displaying it on the surface of the phage.
Yeast Display
The variable region of the antibody sequence is fused with the lectin Aga2p for expression. The Aga2p subunit binds to the Aga1p subunit, which is fixed on the yeast cell wall via two disulfide bonds. Flow sorting technology is then used to select the antibodies that specifically target the antigen.
Yeast Two-Hybrid (Y2H) Screening
The antigen gene is expressed in vivo by fusing it with the GAL4 DNA-binding domain (DNA-BD/bait). The VHH clone is inserted into the DNA-activation domain (DNA-AD/prey) plasmid, and through Y2H screening, VHH antibodies specific to the antigen are identified.
Experimental Process and Deliverables
Library Construction
Nanjing Pronet Biotech used the nanobody scaffold sequence IGHV1S1-IGHV1S1S5 and aligned it with nanobody sequences from the PDB database. Based on this alignment, a VHH synthetic sequence was designed with random point mutations in the CDR1-3 regions. The design includes both conserved sites and diversity at random sites with variations of 2, 4, or 18 amino acids. The CDR3 region was further designed with length polymorphism, offering three lengths: 12, 16, and 20 amino acids. The VHH synthetic gene sequence was cloned into a DNA-AD prey plasmid and transformed into yeast, resulting in a yeast library containing over 10^8 full-length nanoclones.
Library Screening
By fusing the antigen gene with the GAL4 DNA-binding domain (DNA-BD/bait) for in vivo expression, and screening with the VHH yeast prey library, VHH antibodies specific to the antigen were obtained through Y2H screening.
Self-activation was verified by confirming that pGBKT7-C04 did not exhibit any self-activation activity.
The AH109 yeast strain containing the pGBKT7-C04 bait plasmid was prepared as a competent host. The library plasmid pGADT7-VHH was transformed into the strain, and the cells were plated on SD-TLH selective plates. A total of 192 clones were selected after two rounds of screening.
A total of 192 positive yeast clones were PCR amplified, sequenced, and analyzed using Seqman and BLAST. Ultimately, 26 gene sequences were obtained. The 26 gene sequences were subjected to reverse validation, and the results showed that all 26 positive clones could grow normally on SD-TL deficient plates. The 26 positive clones could also grow on SD-TLH deficient plates, and on SD-TLHA and SDTLHA+X-α-gal plates. Additionally, 23 clones were able to turn blue on the SD-TLHA+X-α-gal deficient plates.
Delivered screening result sequences
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