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.

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We have combined gene cloning with an assay for prohormone biosynthesis and processing in Xcnopus oocytes to identify the genes that encode m prohormone processing enzymes. The coinjection of RNA encoding murine prohormone convertase 1 (mPC1), a m endoprotease, along with prooppiomlanocortin RNA into an oocyte results in the appropriate cleavage after paired basic residues in the proopiomelanocortin polyprotein necery to generate corticotropin.

In this study, we characterize the sequences required for the cleavage of prohormones in Xenopus oocytes. We demonstrate that the yeast a-factor and the Aplysia egglaying hormone (ELH) precursors are not cleaved in oocytes following simple pairs of basic residues, such as Lys-Arg, but that the ELH precursor is cleaved following the consensus sequence Arg-Xaa-(Lys/Arg)-Arg. This motif is conserved among precursors that are cleaved in virtually all mammalian cell types. Mutations that generate this sequence in the a-factor prohormone also result in efficient processing within oocytes. Cleavage at this consensus sequence may be due to the action of the Xenopus homologues of mammalian furin.