Objective To screen the anti-CD22-specific nanobodies to provide a basis for immunotherapy agents. Methods The naive phage nanobody library was constructed and its diversity was analyzed. Three rounds of biotinylated streptavidin liquid phase screening were performed by using biotinylated CD22 antigen as the target, and the sequence of nanobodies against CD22 were identified by ELISA and gene sequencing. Results The capacity of the constructed naive phage nanobody library was 3.89×10⁹ CFU/mL, and the insertion of effective fragments was higher than 85%. Based on this library, seven anti-human CD22 nanobodies were screened, and the amino acid sequence comparison results showed that the overall similarity was 70.34%, and all of them were hydrophilic proteins. The results of protein-protein complex docking prediction showed that the mimetic proteins of the five nanobody sequences could be paired and linked to CD22, and the main forces were hydrophobic interaction and hydrogen bonding. Conclusion This study provided a basis for the study of chimeric antigen receptor T cells targeting CD22, successfully constructed the natural phage nanobody library and obtaining five anti-CD22-specific nanobodies.
Camelus/immunology* ; Single-Domain Antibodies/chemistry* ; Peptide Library ; Humans ; Animals ; Sialic Acid Binding Ig-like Lectin 2/genetics* ; Amino Acid Sequence ; Molecular Docking Simulation

Nanobodies (Nbs), the unique single-domain antibodies discovered in the species of Camelidae and sharks, are also known as the variable domain of the heavy chain of heavy-chain antibody (VHH). They offer strong antigen targeting and binding capabilities and overcome the drawbacks such as large size, low stability, high immunogenicity, and slow clearance of conventional antibodies. Nbs can be boosted by bioconjugation with toxins, enzymes, radioactive nucleotides, fluorophores, and other functional groups, demonstrating potential applications in the diagnosis and treatment of human and animal diseases. This article introduces the structures and characteristics of Nbs, the construction and screening of Nb libraries, and the strategies for affinity maturation and then reviews the current applications of Nbs in diagnosis and treatment, providing a reference for the development of diagnostic reagents and clinical therapies for infectious diseases.
Single-Domain Antibodies/chemistry* ; Animals ; Humans ; Camelidae/immunology*

Nanobodies are derived from the variable domain of the heavy-chain antibodies (HCAbs) that occur naturally in the serum of Camelidae. They are the smallest antibody fragments capable to bind antigens. With the characteristics of their increased solubility, increased domain stabilities, nanomolar affinities, easy crossing the blood-brain barrier, easy generation, engineering, optimization and tailoring, easy humanization, nanobodies have extensive application prospects in diagnosis and detection. Although nanobody has demonstrated tremendous success, a number of practical challenges limit its broader applications in disease diagnosis and detection, including construction of a phage library and selection of nanobody fragments with high affinity and immunogold labeling technique. Here, we review several recent findings on the use of nanobodies in molecular diagnostics and suggest some practical strategies in resolving the current challenges in this attractive research area, particularly to optimize the affinity, solubility, humanization of nanobodies.
Humans ; Immunoglobulin Heavy Chains ; chemistry ; Single-Domain Antibodies ; chemistry ; drug effects

Small molecule antibodies are naturally existed and well functioned but not structurally related to the conventional antibodies. They are only composed of heavy protein chains or light chains, much smaller than common antibody. The first small molecule antibody, called Nanobody was engineered from heavy-chain antibodies found in camelids. Cartilaginous fishes also have heavy-chain antibodies (IgNAR, "immunoglobulin new antigen receptor"), from which single-domain antibodies called Vnar fragments can be obtained. In addition, free light chain (FLC) antibodies in human bodies are being developed as therapeutic and diagnostic agents. Comparing to intact antibodies, common advantages of small molecule antibodies are with better solubility, tissue penetration, stability towards heat and enzymes, and comparatively low production costs. This article reviews the structural characteristics and mechanism of action of the Nanobody, IgNAR and FLC.
Animals ; Camelids, New World ; immunology ; Humans ; Immunoglobulin Light Chains ; chemistry ; immunology ; Receptors, Antigen ; immunology ; Sharks ; immunology ; Single-Chain Antibodies ; chemistry ; immunology ; therapeutic use ; Single-Domain Antibodies ; chemistry ; immunology ; therapeutic use