Structural Insights into the Design of Synthetic Nanobody Libraries

Abstract

Single domain antibodies from camelids, or nanobodies, are a unique class of antibody fragments with several advantageous characteristics: small monomeric size, high stability and solubility and easy tailoring for multiple applications. Nanobodies are gaining increasing acceptance as diagnostic tools and promising therapeutic agents in cancer and other diseases. While most nanobodies are obtained from immunized animals of the camelid family, a few synthetic nanobody libraries constructed in recent years have shown the capability of generating high quality nanobodies in terms of affinity and stability. Since this synthetic approach has important advantages over the use of animals, the recent advances are indeed encouraging. Here we review over a dozen synthetic nanobody libraries reported so far and discuss the different approaches followed in their construction and validation, with an emphasis on framework and hypervariable loop design as critical issues defining their potential as high-class nanobody sources.

Therapeutic Potential of Intrabodies for Cancer Immunotherapy: Current Status and Future Directions

“…The identification of cancer mutations which are uniquely expressed in tumor cells has increased enormously in the last ten years [ 54 ] and some intrabodies already exist against some neoantigens [ 28 , 29 , 30 , 31 , 32 , 33 , 34 ]. Now intrabodies against almost every TAA or neoantigen can be selected by phage display or ribosomal display using antibody repertoires comprising scFvs, nanobodies, shark antibodies, human VHs and VLs or DARPins [ 8 , 16 , 17 , 19 , 148 , 149 ] and evaluated in appropriate xenograft tumor mouse models. Conferring to tumor mouse models it would be very useful to analyze the effect of intrabodies in immunodeficient or humanized mice transplanted with human tumor tissue.…”

“…Intracellular TAAs and neoantigens are targeted by single domain antibodies selected from camels, sharks or human VH or VL antibody repertoires by phage display or ribosomal display [ 16 , 17 , 19 ]. Recently a new technique was established for the selection of small molecules, peptide aptamers and cytoplasmic intrabodies: localization-based interaction screening (SOLIS) [ 82 ].…”

“…In addition, intracellular neoantigens are important targets for intrabodies. After recombinant expression of the neoantigen, purification, biochemical characterization and analysis of its function, intrabodies can be selected by phage display [ 16 , 17 , 18 , 19 ] ( Figure 1 ).…”

“…The other group are single domain antibodies (sdAbs) comprising only the variable domain of the heavy chain VHH from camels (nanobodies) or sharks or human VH and VLs and are stable in the cytoplasm or nucleus [ 19 ]. They can be selected by phage display or ribosomal display from immune, naïve or synthetic single domain antibody repertoires [ 16 , 17 , 18 , 19 ] and inactivate their targets by altering their conformation or interfere with the binding of the target protein to its corresponding binding partner. sdAbs were isolated against intracellular TAAs [ 19 ], including the recent examples of hypoxia-inducible factor-1 (HIF-1) [ 24 ], serine/threonine protein kinase AKT2 [ 25 ], p53 C-terminal region involved in the interaction with Twist1 [ 26 ] and chemokine receptor US28 [ 27 ].…”

“…In addition, intracellular neoantigens are important targets for intrabodies. After recombinant expression of the neoantigen, purification, biochemical characterization and analysis of its function, intrabodies can be selected by phage display [16][17][18][19] (Figure 1). To select an appropriate neoepitope for adoptive T-cell therapy and cancer vaccines, several additional evaluation steps using bioinformatics and immunological screening assays are necessary [3,[20][21][22].…”