ALTHEA Gold Libraries™: antibody libraries for therapeutic antibody discovery

Abstract

We describe here the design, construction and validation of ALTHEA Gold Libraries™. These single-chain variable fragment (scFv), semisynthetic libraries are built on synthetic human well-known IGHV and IGKV germline genes combined with natural human complementarity-determining region (CDR)-H3/J H (H3J) fragments. One IGHV gene provided a universal V H scaffold and was paired with two IGKV scaffolds to furnish different topographies for binding distinct epitopes. The scaffolds were diversified at positions identified as in contact with antigens in the known antigen-antibody complex structures. The diversification regime consisted of high-usage amino acids found at those positions in human antibody sequences. Functionality, stability and diversity of the libraries were improved throughout a three-step construction process. In a first step, fully synthetic primary libraries were generated by combining the diversified scaffolds with a set of synthetic neutral H3J germline gene fragments. The second step consisted of selecting the primary libraries for enhanced thermostability based on the natural capacity of Protein A to bind the universal V H scaffold. In the third and final step, the resultant stable synthetic antibody fragments were combined with natural H3J fragments obtained from peripheral blood mononuclear cells of a large pool of 200 donors. Validation of ALTHEA Gold Libraries™ with seven targets yielded specific antibodies in all the cases. Further characterization of the isolated antibodies indicated K D values as human IgG1 molecules in the single-digit and sub-nM range. The thermal stability (Tm) of all the antigen-binding fragments was 75°C-80°C, demonstrating that ALTHEA Gold Libraries™ are a valuable source of specific, high affinity and highly stable antibodies. ARTICLE HISTORY

Isolation and characterization of high affinity and highly stable anti-Chikungunya virus antibodies using ALTHEA Gold Libraries™

“…In the previous sections we reported the characterization of a panel of anti-CHIKV antibodies obtained using UV-inactivated CHIKV particles as selectors and ALTHEA Gold Libraries™ [21,22] as source of antibodies. Among other applications, these antibodies can be used as substrate for developing serological assays in clinical research and/or in surveillance and control of CHIKV outbreaks.…”

“…In addition, throughout a three-step construction process the functionality and thermal stability of the libraries was improved. This process led to a repertoire of a highly diverse source of functional and developable human antibody fragments as shown with four therapeutic targets in three laboratories [21].…”

“…Panning with viral particles and screening for CHIKV speci c clones Having quali ed CHIKV-033 isolate, we used UV-inactivated puri ed particles as selectors to isolate human antibodies from ALTHEA Gold Libraries™ [21]. The panning was performed in solid phase, e.g., ELISA plates.…”

“…The second transition is close or above 80 o C. In contrast, the control antibodies have a single transition at 61 o C or 65 o C for 4N12 and 4J21, respectively. The reason for such a high stability is that ALTHEA Gold Libraries™ were built to generate highly stable antibodies [21], whereas, the control antibodies were isolated from a CHIKV convalescent patient and, while the control antibodies are highly potent, they did not evolve to be stable at high temperatures.…”

“…Once quali ed, UV-inactivated CHIKV particles were used as selectors to discover high a nity and highly stable antibodies from ALTHEA Gold Libraries™ [21,22]. The speci city of the antibodies was assessed by ELISA using viral particles of Venezuelan equine encephalitis virus (VEEV), which is similar to CHIKV, both belonging to the Alphavirus genus.…”