TY - JOUR
T1 - ALTHEA Gold Libraries™
T2 - antibody libraries for therapeutic antibody discovery
AU - Valadon, Philippe
AU - Pérez-Tapia, Sonia M.
AU - Nelson, Renae S.
AU - Guzmán-Bringas, Omar U.
AU - Arrieta-Oliva, Hugo I.
AU - Gómez-Castellano, Keyla M.
AU - Pohl, Mary Ann
AU - Almagro, Juan C.
N1 - Publisher Copyright:
© 2019, © 2019 The Author(s). Published with license by Taylor & Francis Group, LLC.
PY - 2019/4/3
Y1 - 2019/4/3
N2 - 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.
AB - 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.
KW - Phage display
KW - Protein A filtration
KW - human albumin
KW - lysozyme
KW - next-generation sequencing
KW - semisynthetic libraries
KW - tumor necrosis factor
UR - http://www.scopus.com/inward/record.url?scp=85062374314&partnerID=8YFLogxK
U2 - 10.1080/19420862.2019.1571879
DO - 10.1080/19420862.2019.1571879
M3 - Artículo
C2 - 30663541
AN - SCOPUS:85062374314
SN - 1942-0862
VL - 11
SP - 516
EP - 531
JO - mAbs
JF - mAbs
IS - 3
ER -