High affinity humanized antibodies without making hybridomas; immunization paired with mammalian cell display and in vitro somatic hypermutation

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, fuse...

Full description

Saved in:
Bibliographic Details
Published in:PloS one 2012-11, Vol.7 (11), p.e49458
Main Authors: McConnell, Audrey D, Do, Minjee, Neben, Tamlyn Y, Spasojevic, Vladimir, MacLaren, Josh, Chen, Andy P, Altobell, 3rd, Laurence, Macomber, John L, Berkebile, Ashley D, Horlick, Robert A, Bowers, Peter M, King, David J
Format: Article
Language:English
Subjects:
Activation-induced cytidine deaminase
Affinity
Animal tissues
Animals
Antibodies
Antibodies, Monoclonal, Humanized - biosynthesis
Antigens
Arthritis
Biology
Cell surface
Cells (Biology)
Chains
Complement C5 - immunology
Complement component C5
Cytidine deaminase
Cytidine Deaminase - metabolism
Drug Discovery - methods
Flow Cytometry
Fluorescence
Genes
Heavy chains
HEK293 Cells
Humans
Immunization
Immunoglobulin Heavy Chains - genetics
Immunoglobulin Heavy Chains - metabolism
Immunoglobulin Light Chains - genetics
Immunoglobulin Light Chains - metabolism
Immunoglobulins
In vitro methods and tests
Light chains
Lymphatic system
Lymphoid Tissue - immunology
Mammals
Maturation
Mice
Mutagenesis
Proteins
Recombinant Proteins - biosynthesis
Somatic hypermutation
Somatic Hypermutation, Immunoglobulin - genetics
Somatic Hypermutation, Immunoglobulin - immunology
Spleen
Spleen - immunology
Transgenic animals
Citations: Items that this one cites
Items that cite this one
Online Access:Get full text
Tags: Add Tag
No Tags, Be the first to tag this record!
Description
Summary: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.
ISSN:1932-6203
1932-6203
DOI:10.1371/journal.pone.0049458