Construction of Synthetic Single Domain Antibody Library
Introduction of Synthetic Library Construction
Synthetic antibody library has been proved to be a highly effective strategy to screen functional antibodies against a wide range of antigens. Moreover, it provides a vast resource of renewable antibody reagents that can rival or exceed those of natural antibodies and can be rapidly isolated through controlled in vitro selections. It is well known that most of the diversity of the antibody molecule is found in the complementarity-determining regions (CDRs). Because of their critical role in the antibody-antigen interaction, these regions are the main targets for randomization in most synthetic library designs.
In contrast to natural immune repertoires, which are limited by the diversity provided by the natural adaptive immune system of the host, synthetic single domain antibody libraries can be designed and constructed with highly diverse and well-defined synthetic repertoires. Each sdAb contains CDRs that correspond to hypervariable sequences, which determine antigen specificity, and conserved framework regions (FRs), which may be crucial in conserving the structural integrity of sdAbs. It is essential to note that the performance and properties of a synthetic library depend on the scaffold chosen as the foundation for the library, the design strategy for diversity, and the overall size of the final library. The use of highly optimized single domain antibody frameworks and the nature and different position of diversity enables the incorporation of defined diversity at positions that are most likely to contribute to antigen recognition. The resulting phage repertoire can achieve 109-10 clones and is primarily populated by correctly folded functional sdAbs.
Fig. 1 Generation of synthetic library.1
Based on naturally occurring VHH or VNAR sequences, synthetic libraries can be constructed by introducing size and sequence variations in the CDRs, using randomized CDR3 primers or error-prone PCR combined with splicing-overlap extension PCR method. These synthetic antibody libraries are constructed through the introduction of synthetic degenerate oligonucleotides precisely into regions most likely to contribute to antigen recognition within a defined framework to finally construct the phage-displayed sdAb library. Synthetic degenerate oligonucleotides are used to perform PCR reactions whereby the CDR regions are randomly combined to assemble into a full sdAb sequence. These products are then cloned into a phage vector and transformed into E. coli cells to create libraries with high diversity.
The Objective of Synthetic Library Construction
These synthetic libraries offer promising new formats because they are very robust to facilitate the processes of isolation, characterization, and optimization, and potent to use for the rapid generation of sdAbs against a variety of targets with high affinities in the nanomolar range. Furthermore, properly designed synthetic sdAbs have excellent biophysical properties to construct second-generation libraries aimed to fine-tune antibody characteristics including affinity, specificity, and manufacturability, making them ideal leads for therapeutic and diagnostic applications.
Advantages of Synthetic Library
The synthetic library has higher CDR3 diversity and utility than the naturally occurring naïve libraries and immune libraries. Natural repertoires lack antibodies against self-antigens, whereas synthetic antibodies are not biased against natural proteins, regardless of source or sequence. Frameworks for synthetic antibodies can be chosen for particular properties, for example, high stability and expression. Besides, experimental conditions can be tightly controlled with relatively easy operation requirements because all steps involved in the screening of antibodies from synthetic libraries are conducted in vitro. Moreover, more complex libraries can be constructed that can produce synthetic antibodies with affinities and specificities beyond the capacity of natural antibodies.
Reference
- Solemani Zadeh, Arghavan, et al. "Efficient construction and effective screening of synthetic domain antibody libraries." Methods and protocols 2.1 (2019): 17. Distributed under Open Access license CC BY 4.0, capture the part A of original Figure 1.
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