Engineering and characterization of a mouse/human chimeric anti-phencyclidine monoclonal antibody.
Previously, our laboratory produced a high affinity, anti-phencyclidine (PCP) murine monoclonal antibody (mAb6B5) that also binds other PCP-like arylcyclohexylamines. In this project, mAb6B5 is engineered into a mouse/human chimera (ch-mAb6B5) to assess the feasibility of developing it into a medication for PCP and PCP-like drug abuse. To create ch-mAb6B5, the light and heavy chain constant regions of mAb6B5 were replaced with human kappa and IgG(2) constant regions in order to decrease its potential immunogenicity in humans. To be an effective anti-PCP medication, ch-mAb6B5 must retain the critical immunochemical binding properties of mAb6B5. Expression vectors containing ch-mAb6B5 light chain and heavy chain cDNA were constructed and expressed in the murine myeloma cell line P3X63-Ag8.653. Immunoassays confirm that ch-mAb6B5 is indeed a chimera, composed of mAb6B5's PCP-binding variable domains and human kappa and IgG constant regions. Radioimmunoassays show that ch-mAb6B5 has the same drug-binding profile as mAb6B5. Ch-mAb6B5 and mAb6B5 bind PCP with a K(D) of 0.67 nM and 1.17 nM (respectively) and bind PCP-like arylcyclohexylamines 1-[1-(2-thienyl)cyclohexyl]piperidine and N-ethyl-1-phenylcyclohexylamine with similar specificity. Additionally, ch-mAb6B5 and mAb6B5 have the same calculated isoelectric points and molecular weights, critical properties in antigen-antibody interactions. These data demonstrate that mouse/human ch-mAb6B5, a "more human" version of murine mAb6B5, retains mAb6B5's unique drug-binding properties. This work supports our continued efforts to develop ch-mAb6B5 into a medication for PCP and PCP-like drug abuse - introducing the intriguing possibility of using a single therapeutic mAb for treating a class of abused drugs.
Lacy
H Marie
HM
Department of Microbiology and Immunology, College of Medicine, University of Arkansas for Medical Sciences, Little Rock, AR 72205, United States. HMLacy@uams.edu
Gunnell
Melinda G
MG
Laurenzana
Elizabeth M
EM
Owens
S Michael
SM
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D045744:Cell Line, Tumor; D006213:Hallucinogens / Q000276:immunology* / Q000378:metabolism; D006801:Humans; D006825:Hybridomas; D051379:Mice; D008969:Molecular Sequence Data; D010622:Phencyclidine / Q000276:immunology* / Q000378:metabolism; D015202:Protein Engineering / Q000379:methods; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism
D000911:Antibodies, Monoclonal; D006213:Hallucinogens; D011993:Recombinant Fusion Proteins; D010622:Phencyclidine