148562-39-4Relevant academic research and scientific papers
Anti-cocaine catalytic antibodies: A synthetic approach to improved antibody diversity
Yang,Chun,Arakawa-Uramoto,Wang,Gawinowicz,Zhao,Landry
, p. 5881 - 5890 (2007/10/03)
Catalytic antibodies are potential therapeutic agents for drug overdose and addiction, and we previously reported the first such artificial enzymes to degrade cocaine. However, as described herein, these catalytic monoclonal antibodies (Mab's) were found to have nearly identical complementarity-determining regions (CDR's). Such limited diversity among catalytic antibodies of similar specificity has been reported previously and poses a problem since the capacity of any single group of homologous catalytic antibodies to yield one of high activity, whether through repetitive screening of hybridomas or through antibody mutagenesis, is unpredictable. One strategy to increase the diversity of the immune response to an analog would be to vary the tether site of the immunogenic conjugate thereby exposing unique epitopes for immunorecognition. We now report the syntheses of three immunogenic conjugates of a transition-state analog (TSA) of cocaine benzoyl ester hydrolysis which have identical phosphonate monoester core structures but varying tether sites for attachment to carrier protein: TSA 1 at the methyl ester, TSA 2 at the 4'-phenyl position, and TSA 3 at the tropane nitrogen. Mixed phosphonate diester precursors were obtained from phosphonic dichlorides and ecgonine alkyl esters through our 1H-tetrazole catalysis method. We found that all three analogs provided catalytic antibodies that hydrolyze cocaine at the benzoyl ester; the most active catalytic antibody, Mab 15A10, displayed a rate acceleration (k(cat)/k(uncat) = 2.3 x 104) sufficient to commence preclinical studies. On competitive ELISA, all nine catalytic antibodies, regardless of the eliciting antigen, bound TSA 1 with high affinity but four bound TSA 3 poorly and five failed to bind TSA 2 despite the inhibition of all antibodies by free TSA (TSA 4). A comparison of heavy and of light chain CDR's showed four discrete groups with TSA 1 and 3 each yielding two non-overlapping families of catalytic antibodies; TSA 2 yielded one antibody with CDR's nearly identical to those of the largest group of catalytic antibodies elicited by TSA 1. The failure of TSA 2 and TSA 3 to bind to catalytic antibodies derived from alternative immunogenic conjugates demonstrates that the tether site does limit the catalytic antibodies produced and supports the general strategy of varying the attachment to carrier protein.
Generation of polyclonal catalytic antibodies against cocaine using transition state analogs of cocaine conjugated to diphtheria toxoid
Basmadjian,Singh,Sastrodjojo,Smith,Avor,Chang,Mills,Seale
, p. 1902 - 1911 (2007/10/03)
Six novel transition state analogs (TSAs) of cocaine (10-14 and 17) and one non-cocaine, p-aminophenyl-phosphonyl ester of cyclohexanol (19), were synthesized and characterized by 1H- and 13C-NMR and FAB-MS. (1R)-ecgonine methyl ester or cyclohexanol were subjected to phenylphosphonylation in the presence of dicyclohexyl carbodiimde (DCC) and 4-N,N-dimethyl aminopyridine (4-DMAP). TSA-IV (10), however, was synthesized from norcocaine which was protected with dibromoethane to yield 4 before acid hydrolysis, esterification and phenylphosphonylation were carried out. TSA-III (11) TSA- I (12) and (19), using various length spacer arms, were coupled with the immunogenic protein, diphtheria toxoid (DT). The TSAs coupled with DT were used to immunize mice and after appropriate boosts their sera were tested for the presence and tiler of anti-TSA polyclonal antibodies using ELISA. Preliminary results show that the mice immunized with these TSAs produced high titers of polyclonal catalytic antibodies, except for (19), with the ability to hydrolyze the substrate 125I-4'-iodococaine in an in vitro assay, even in the presence of noncatalytic anti-TSA antibodies.
