Humanization and characterization of an anti-ricin neutralization monoclonal antibody.
Ricin is regarded as a high terrorist risk for the public due to its high toxicity and ease of production. Currently, there is no therapeutic or vaccine available against ricin. D9, a murine monoclonal antibody developed previously in our laboratory, can strongly neutralize ricin and is therefore a good candidate for humanization. Humanization of D9 variable regions was achieved by a complementarity-determining region grafting approach. The humanized D9 (hD9) variable regions were further grafted onto human heavy and light chain constant regions to assemble the complete antibody gene. A foot-and-mouth-disease virus-derived 2A self-processing sequence was introduced between heavy and light chain DNA sequences to cleave the recombinant protein into a functional full-length antibody molecule from a single open reading frame driven by a single promoter in an adenoviral vector. After expression in mammalian cells and purification, the hD9 was demonstrated to have equimolar expression of the full-length antibody heavy and light chains. More importantly, the hD9 exhibited high affinity to ricin with K(D) of 1.63 nM, comparable to its parental murine D9 (2.55 nM). In a mouse model, intraperitoneal (i.p.) administration of hD9, at a low dose of 5 µg per mouse, 4 hours after the i.p. challenge with 5×LD50 ricin was found to rescue 100% of the mice. In addition, administered 6 hours post-challenge, hD9 could still rescue 50% of the mice. The hD9 has the potential to be used for prophylactic or therapeutic purposes against ricin poisoning.
Defence Research and Development Canada - Suffield, Medicine Hat, Alberta, Canada. wei-gang.hu@drdc-rddc.gc.ca
Wei-Gang
WG
Hu
Junfei
J
Yin
Damon
D
Chau
Laurel M
LM
Negrych
John W
JW
Cherwonogrodzky
D000256:Adenoviridae / Q000235:genetics; D000595:Amino Acid Sequence; D000818:Animals; D061067:Antibodies, Monoclonal, Humanized / Q000235:genetics* / Q000276:immunology / Q000493:pharmacokinetics / Q000627:therapeutic use; D000915:Antibody Affinity; D000992:Antitoxins / Q000235:genetics* / Q000276:immunology / Q000627:therapeutic use; D002619:Chemical Warfare Agents / Q000506:poisoning*; D022801:Complementarity Determining Regions / Q000235:genetics / Q000276:immunology; D004279:DNA, Viral / Q000235:genetics* / Q000378:metabolism; D005260:Female; D029544:Foot-and-Mouth Disease Virus / Q000235:genetics; D005822:Genetic Vectors; D006207:Half-Life; D006801:Humans; D007143:Immunoglobulin Heavy Chains / Q000235:genetics / Q000276:immunology; D007147:Immunoglobulin Light Chains / Q000235:genetics / Q000276:immunology; D007274:Injections, Intraperitoneal; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010939:Plant Poisoning / Q000276:immunology / Q000401:mortality / Q000517:prevention & control*; D015202:Protein Engineering; D012276:Ricin / Q000506:poisoning*; D015996:Survival Rate
D061067:Antibodies, Monoclonal, Humanized; D000992:Antitoxins; D002619:Chemical Warfare Agents; D022801:Complementarity Determining Regions; D004279:DNA, Viral; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D012276:Ricin