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.

Keywords: nanobody; phage display; rational design; ribosome display; synthetic library.

Figure 1

(A) Articles per-year based on a “nanobody” search in PubMed. (B) Differences between traditional antibodies and their derivatives with respect to HCAbs. A classical IgG antibody is made up of 12 immunoglobulin domains, distributed in two different pairs of chains: heavy (in blue) and light (in orange). It is functionally divided into the Fc region and the two Fab fragments, where the recognition region is located. On the other hand, both HCAbs and IgNARs are made up of one pair of a single chain type. Different antibody fragments, such as the Fab or a single-chain fragment (scFv) can be derived or constructed. The latter is formed by the heavy and light variable domains, joined by a linker. In contrast, Nbs or VHH are formed solely by the recognition domain of HCAbs or IgNARs.

Figure 2

Strategy and desired physicochemical characteristics for the generation of synthetic libraries. The design and construction of a synthetic library comprise two main factors: the framework and the CDRs. A previously reported universal or Nb-derived framework can be used for a new library. Alternatively, a new framework can be generated from a consensus sequence derived from natural repertoires. Similarly, the design of CDRs can be carried out in a rationalized way, considering the natural diversity in natural repertoires, or be generated in a partially or completely randomized way. While framework selection is more focused on stability, CDR design focuses more on variability. In both cases, the listed characteristics should be considered to obtain a robust and functional synthetic library.