High Affinity Humanized Antibodies without Making Hybridomas; Immunization Paired with Mammalian Cell Display and In Vitro Somatic Hypermutation

Abstract

A method has been developed for the rapid generation of high-affinity humanized antibodies from immunized animals without the need to make conventional hybridomas. Rearranged IgH D(J) regions were amplified from the spleen and lymph tissue of mice immunized with the human complement protein C5, fused with a limited repertoire of human germline heavy chain V-genes to form intact humanized heavy chains, and paired with a human light chain library. Completed heavy and light chains were assembled for mammalian cell surface display and transfected into HEK 293 cells co-expressing activation-induced cytidine deaminase (AID). Numerous clones were isolated by fluorescence-activated cell sorting, and affinity maturation, initiated by AID, resulted in the rapid evolution of high affinity, functional antibodies. This approach enables the efficient sampling of an immune repertoire and the direct selection and maturation of high-affinity, humanized IgGs.

A general approach to antibody thermostabilization

“…C5-C345C was similarly produced in E. coli as described. 29 bNGF and IL-17A with N-terminal his 6 tags were expressed transiently in HEK293 c-18 cells and purified using standard Nichelate affinity purification. TNF, RANKL, HER2, and EGFR were purchased from R&D Systems and IgE was purchased from Abcam.…”

Humanization of Antibodies Using Heavy Chain Complementarity-determining Region 3 Grafting Coupled with in Vitro Somatic Hypermutation

“…In addition, because this method minimizes the number of mutations needed to humanize and affinity-mature an antibody, the APE896 HC possessed greater sequence identity to its parental V gene segment (IGHV3-23) (89%) than did the original hybridoma rat V region sequence to either its originating rat germ line segment (86%) or to the closest human V gene IGHV4 -59 (60%). The adaptive nature of the methodology and its ability to mature antibodies to high potency facilitates the use of human V regions with distant homology to the originating murine sequence, providing more flexibility in gene selection based on additional criteria such as manufacturability and known expression characteristics (30).…”

High Affinity Maturated Human Antibodies from Naïve and Synthetic Antibody Repertoires

“…The human antibody repertoire represents a diverse collection of immunoglobulin gene segments that encodes for heavy (V H ) and light chain (V L ) domains [34], forming an unique set of antigen-binding sites [35,36]. The heavy chain (HC) locus is located at chromosome 14, comprises of V H , D, J H and C H gene segments.…”

“…AID-mediated mutagenesis serves as a useful method to enhance antibody affinities through sequence diversity by introducing point mutations, such as single amino acid substitutions or indels (insertions, deletions) specifically on the antibody CDR sequences. Nucleotide transversions and duplications are among the most complicated to design into a library but possible with the AID-mediated mutagenesis approach [34]. The AID enzyme is capable of generating indels which are localized in CDR regions, while affinity maturation through somatic mutation further improves the antibody binding and specificity [100].…”