Anti-Cocaine Catalytic Antibodies:  A Synthetic Approach to Improved Antibody Diversity

Abstract

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 × 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.

RETRACTED ARTICLE: Recent advances for the treatment of cocaine abuse: Central nervous system immunopharmacotherapy

“…A related antibody-based approach to cocaine addiction treatment used catalytic antibodies specifi c for cocaine and the cleavage of its benzoyl ester ( Figure 1 ). [31][32][33][34][35][36] The effi ciency of catalytic antibodies has been demonstrated in rodent models of cocaine overdose and reinforcement, but kinetic constants for all reported antibody catalysts are marginal and thus improved rates …”

Subunit Stabilization and Polyethylene Glycolation of Cocaine Esterase Improves In Vivo Residence Time

“…The most effective vaccines to date have been against bovine serum albumin-or cholera toxin B-conjugated norcocaine (Fox et al, 1996;Kantak et al, 2000), and their success in eliciting good antibody titers has resulted in the initiation of clinical trials in humans (Kosten et al, 2002;Kinsey et al, 2010). A variant of passive immunization using catalytic monoclonal antibodies, which can not only sequester but also hydrolyze cocaine, was unfortunately not therapeutically active for more than 72 h after administration (Landry et al, 1993;Yang et al, 1996;Matsushita et al, 2001). Other than the vaccines against bovine serum albumin-or cholera toxin B-conjugated norcocaine, none of the other immunotherapeutic-based biologics has shown promise.…”

CART peptides as modulators of dopamine and psychostimulants and interactions with the mesolimbic dopaminergic system

“…Moreover, injection of D3 dopamine receptor agonists lowers levels of CART mRNA in the accumbens [24], and DA receptors are found on CART neurons [25,26]. Injection of CART 55-102 into the ventricles causes an increased turnover of DA [27,28]. CART-containing neurons in the nucleus accumbens core project to the substantia nigra zona compacta, while projections from the shell seem to be located more medially in the ventral tegmental area [29].…”