title: Characterization of neutralizing mouse-human chimeric and shuffling antibodies against botulinum neurotoxin A.
abstract: Mouse-human chimeric monoclonal antibodies that could neutralize botulinum neurotoxins were developed and an attempt was made to establish mouse hybridoma cell clones that produced monoclonal antibodies that neutralized botulinum neurotoxin serotype A (BoNT/A). Four clones (2-4, 2-5, 9-4 and B1) were selected for chimerization on the basis of their neutralizing activity against BoNT/A and the cDNA of the variable regions of their heavy (V(H)) and light chains (V(L)) were fused with the upstream regions of the constant counterparts of human kappa light and gamma 1 heavy chain genes, respectively. CHO-DG44 cells were transfected with these plasmids and mouse-human chimeric antibodies (AC24, AC25, AC94 and ACB1) purified to examine their binding and neutralizing activities. Each chimeric antibody exhibited almost the same capability as each parent mouse mAb to bind and neutralize activities against BoNT/A. From the chimeric antibodies against BoNT/A, shuffling chimeric antibodies designed with replacement of their V(H) or V(L) domains were constructed. A shuffling antibody (AC2494) that derived its V(H) and V(L) domains from chimeric antibodies AC24 and AC94, respectively, showed much higher neutralizing activity than did other shuffling antibodies and parent counterparts. This result indicates that it is possible to build high-potency neutralizing chimeric antibodies by selecting and shuffling V(H) and V(L) domains from a variety of repertoires. A shuffling chimeric antibody might be the best candidate for replacing horse antitoxin for inducing passive immunotherapy against botulism.
authors.affiliation: Department of Veterinary Science, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, 1-58, Rinku Orai-Kita, Izumisano, Osaka 598-8531, Japan. mukamoto@vet.osakafu-u.ac.jp
authors.forename: Masafumi
authors.initials: M
authors.lastname: Mukamoto
authors.forename: Hiroaki
authors.initials: H
authors.lastname: Maeda
authors.forename: Tomoko
authors.initials: T
authors.lastname: Kohda
authors.forename: Chikateru
authors.initials: C
authors.lastname: Nozaki
authors.forename: Motohide
authors.initials: M
authors.lastname: Takahashi
authors.forename: Shunji
authors.initials: S
authors.lastname: Kozaki
mesh_terms: D000818:Animals; D000907:Antibodies, Bacterial / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification; D000911:Antibodies, Monoclonal / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification; D000992:Antitoxins / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification; D019274:Botulinum Toxins, Type A / Q000037:antagonists & inhibitors*; D016466:CHO Cells; D018929:Cell Culture Techniques; D006224:Cricetinae; D006801:Humans; D007143:Immunoglobulin Heavy Chains / Q000235:genetics / Q000276:immunology; D007135:Immunoglobulin Variable Region / Q000235:genetics / Q000276:immunology; D051379:Mice; D008807:Mice, Inbred BALB C; D009500:Neutralization Tests; D010957:Plasmids; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology
chemical_list: D000907:Antibodies, Bacterial; D000911:Antibodies, Monoclonal; D000992:Antitoxins; D007143:Immunoglobulin Heavy Chains; D007135:Immunoglobulin Variable Region; D011994:Recombinant Proteins; D019274:Botulinum Toxins, Type A