Hybridoma populations enriched for affinity-matured human IgGs yield high-affinity antibodies specific for botulinum neurotoxins.
The affinity-matured human antibody repertoire may be ideal as a source for antibody therapeutics against infectious diseases and bioterror agents. Hybridoma methods for cloning these antibodies have many potential advantages, including convenience, high-yield antibody expression, and the ability to capture the antibodies in their native configurations. However, they have been hindered by hybridoma instability and limited accessibility of antigen-specific, class-switched human B-cells. Here, we describe an efficient, three-step method that uses human peripheral blood B-cells to produce stable hybridoma populations that are highly-enriched for affinity-matured human IgG antibodies. Peripheral blood mononuclear cells (PBMCs) are (a) selected for expression of CD27, a marker of post-germinal center B-cells, (b) cultured in vitro to promote B-cell proliferation and class-switching, and (c) fused to a genetically modified myeloma cell line. Using this strategy, we cloned 5 IgG antibodies that bind botulinum neurotoxins (BoNT), the causes of the food-borne paralytic illness, botulism, and Category A Select Bioterror agents. Two of these antibodies bind BoNT with low picomolar affinities. One (30B) is the first high-affinity human antibody to bind serotype B BoNT, and another (6A) is able to neutralize a lethal dose of serotype A BoNT in vivo in pre- and post-exposure models. This optimized hybridoma method will broadly enable access to the native human antibody repertoire.
Adekar
Sharad P
SP
Cardeza Foundation for Hematologic Research and Kimmel Cancer Center, Thomas Jefferson University, 1015 Walnut Street, Philadelphia, PA 19107, United States.
Jones
R Mark
RM
Elias
M D
MD
Al-Saleem
Fetweh H
FH
Root
Michael J
MJ
Simpson
Lance L
LL
Dessain
Scott K
SK
D000818:Animals; D000907:Antibodies, Bacterial / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D000918:Antibody Specificity; D001402:B-Lymphocytes / Q000276:immunology; D001905:Botulinum Toxins / Q000235:genetics / Q000276:immunology*; D019274:Botulinum Toxins, Type A / Q000235:genetics / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D006825:Hybridomas / Q000276:immunology*; D007074:Immunoglobulin G / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D007700:Kinetics; D016014:Linear Models; D051379:Mice; D009500:Neutralization Tests; D012329:RNA, Bacterial / Q000737:chemistry / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D020349:Surface Plasmon Resonance; D018127:Tumor Necrosis Factor Receptor Superfamily, Member 7 / Q000276:immunology
D000907:Antibodies, Bacterial; D007074:Immunoglobulin G; D012329:RNA, Bacterial; D018127:Tumor Necrosis Factor Receptor Superfamily, Member 7; C096323:rimabotulinumtoxinB; D001905:Botulinum Toxins; D019274:Botulinum Toxins, Type A