TOWARD ANTIBODY-DIRECTED ENZYME PRODRUG THERAPY WITH THE T268G MUTANTOF HUMAN CARBOXYPEPTIDASE A1 AND NOVEL IN-VIVO STABLE PRODRUGS OF METHOTREXATE

Antibody-directed enzyme prodrug therapy (ADEPT) has the potential of greatly enhancing antitumor selectivity of cancer therapy by synthesiz ing chemotherapeutic agents selectively at tumor sites, This therapy i s based upon targeting a prodrug-activating enzyme to a tumor by attac hing the enzyme to st tumor-selective antibody and dosing the enzyme-a ntibody conjugate systemically, After the enzyme-antibody conjugate is localized to the tumor the prodrug is then also dosed systemically, a nd the previously targeted enzyme converts it to the active drug selec tively at the tumor, Unfortunately, most enzymes capable of this speci fic, tumor site generation of drugs are foreign to the human body and as such are expected to raise an immune response when injected, which will limit their repeated administration, Ne reasoned that with the po wer of crystallography, molecular modeling and site-directed mutagenes is, this problem could be addressed through the development of a human enzyme that is capable of catalyzing a reaction that is otherwise not carried out in the human body, This would then allow use of prodrugs that are otherwise stable in vivo but that are substrates for a tumor- targeted mutant human enzyme, We report here the first test of this co ncept using the human enzyme carboxypeptidase A1 (hCPA1) and prodrugs of methotrexate (MTX), Based upon a computer model of the human enzyme built from the well known crystal structure of bovine carboxypeptidas e A, we have designed and synthesized novel bulky phenylalanine- and t yrosine-based prodrugs of MTX: that are metabolically stable in vivo a nd are not substrates for wild type human carboxypeptidases A, Two of these analogs are MTX-alpha-3-cyclobutylphenylalanine and MTX-alpha-3- cyclopentyltyrosine, Also based upon the computer model, we have desig ned and produced a mutant of human carboxypeptidase A1, changed at pos ition 268 from the wild type threonine to a glycine (hCPA1-T268G), Thi s novel enzyme is capable of using the in vivo stable prodrugs, which are not substrates for the wild type hCPA1, as efficiently as the wild type hCPA1 uses its best substrates (i.e. MTX-alpha-phenylalanine). T hus, the k(cat)/K-m value for the wild type hCPA1 with MTX-alpha-pheny lalanine is 0.44 mu M-1 s(-1) and k(cat)/K-m values for hCPA1-T286G wi th MTX-alpha-3-cyclobutylphenylalanine and MTX-alpha-3-cyclopentyltyro sine are 1.8 and 0.16 mu M-1 s(-1) respectively, The cytotoxic efficie ncy of hCPA1-268G was tested in an in vitro ADEPT model, For this expe riment, hCPA1-T268G was chemically conjugated to ING-1, an antibody th at binds to the tumor antigen Ep-Cam, or to Campath-1H, an antibody th at binds to the T and B cell antigen CDw52, These conjugates mere then incubated with HT-29 human colon adenocarcinoma cells (which express Ep-Cam but not the Campath. 1H antigen) followed by incubation of the cells with the in vivo stable prodrugs. The results showed that the ta rgeted ING-1:hCPA1-T268G conjugate produced excellent activation of th e MTX prodrugs to kill HT-29 cells as efficiently as MTX itself, By co ntrast, the enzyme-Campath 1H conjugate was without effect, These data strongly support the feasibility of ADEPT using a mutated human enzym e with a single amino acid change.