Baer, T.

Haenseler, E.

Huennekens, F. M.

Montejano, Y.

Vitols, K. S.

RERO DOC Digital Library

Vitols et al.: Me tho t rexa te p rod rugs act ivated by enzyme/an t ibody conjuga tes 125 Pteridines Vol. 3, pp . 1 2 5 - 1 2 6 Short Communication Activation of Methotrexate Prodrugs by Enzyme/Monoclonal Antibody Conjugates K. S. Vitols, E. Haenseler1 ', Y. Montejano, T. Baer2) and F. M. Huennekens Division of Biochemistry, Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, California 92037, U. S. A. (Received M a r c h 1992) One of the major goals in cancer chemotherapy is the development of procedures for the selective delivery of drugs to tumor cells. This strategy, when perfected, will allow drugs to be used at the high concentrations necessary for virtually complete (i. e., > 99%) eradi-cation of the tumor cells without concomitant destruc-tion of normal cells. Monoclonal antibodies targeted to tumor antigens offer considerable promise for ef-fecting the desired selectivity. To date, however, only limited success has been achieved in clinical trials with monoclonals as vehicles for carrying drugs. Two lim-itations are evident in the use of drug-monoclonal conjugates: (a) Relatively few drug molecules can be linked to each monoclonal without impairing binding activity; and (b) A mechanism is needed to release covalently-bound drug from the antibody. In an effort to overcome these problems, attention has focused recently on the attachment of enzymes rather than drugs to monoclonals, thereby taking advantage of the catalytic power of the enzyme to generate large amounts of the active drugs f rom inert prodrugs at the tumor site [reviewed by Senter (1)]. Methotrexate (MTX), employed extensively in cancer chemotherapy, is well-suited to the prodrug/enzyme-monoclonal conjugate strategy. α-Peptides of the drug (i.e., derivatives in which the a - C O O H has been linked to an amino acid) are relatively non-toxic, but these derivatives can be hydrolyzed by carboxypep-" Recipient of an E O R T C Fellowship. Present address: Insti-tu te of Clinical Chemistry , Universi ty Hospi ta l , Zur ich, Switz-er land. 2) Present Address : Byk Gu lden Pha rmazeu t ika , Kons tanz , Ge rmany . Pter idines / Vol .3 / No . 1/2 Copyr igh t © 1992 Walter de Gruyte r - Berlin - New York tidases to yield the active parent drug. Alanine, phen-ylalanine, aspartate and arginine derivatives of MTX (Fig. 1) have been synthesized in good yield by con-ventional pathways (2, 3). It Should be noted, how-ever, that direct coupling of the amino acid to M T X results in considerable racemization of the L-gluta-mate moiety in the drug which, in turn, limits the carboxypeptidase-mediated cleavage of the MTX-amino acid linkage. This can be avoided by coupling the Glu-amino acid dipeptide to 4-amino-4-deoxy-10-methylpteroic acid. Hydrolysis of the MTX oc-peptides by various carboxypeptidases, and particularly MTX-Ala by carboxypeptidase-A (CP-Α), has been exam-ined in detail (2). These reactions, although slow relative to the rates with standard substrates for the enzymes (e.g.. hippurylphenylalanine for CP-Α), are still sufficient to generate toxic quantities of the drug ( 2 ) . Regional selectivity of the MTX-Ala/CP-A combi-nation was demonstrated in a model system in which immobilized CP-Α was embedded in a semi-solid me-dium containing the prodrug and LI210 cells (4). The crucial question, however, was whether cytotoxic combinations of MTX could be produced by the amount of CP-Α that could be attached to target cells via a monoclonal antibody. Accordingly, CP-Α deri-vatized with succinimidyl 4-(N-maleimidome-thyl)cyclohexane-l-carboxylate and a monoclonal an-tibody (KS 1/4, directed toward a surface antigen on carcinoma cells) derivatized with N-succinimidyl 3-(2-pyridyldithio)propionate were reacted to produce a thioether-linked, enzyme-antibody conjugate prep-aration (5). The latter, purified by sequential H P L C size-exclusion and D E A E chromatography, contained 126 Vitols et al.\ Methotrexate prodrugs activated by enzyme/ant ibody conjugates MTX α-Peptides o II r, r\ O C-NH-CH-COOH // \ 11 1 1 CHaN —U y— C-NH-CH R CH, \ = / (CH,), COOH MTX-Ala R = -CH, MTX-Phe R = - C H , - ^ MTX-Asp R = -CH,COOH / NH, MTX-Arg R = -CH,CH,CH,NHC ζ" ^ NH, © Figure 1. Structures of Methotrexate α-peptides. Table 1. Cytotoxicity of Methotrexate α-Phenylalanine (MTX-Phe) for U C L A - P 3 Cells Treated with Carboxypept idase-A (CP-A)/Monoclonal Ant ibody KS1/4 Conjugate Drug Enzyme- % or Prodrug Monoclonal Inhibition Conjugate M T X 100 MTX-Phe C P - A / K S l / 4 94 MTX-Phe — 23 — C P - A / K S l / 4 3 In vitro cytotoxicity against UCLA-P3 human lung adenocar-cinoma cells (104 cells/well) was measured in 96-well plates as described previously (5). In samples treated with enzyme-anti-body conjugate, excess of the latter was removed by extensive washing. Growth was stopped af ter 72 h. Inhibit ion is expressed relative to tha t observed with M T X (designated as 100%). Concentrat ions of M T X and MTX-Phe , 1 χ ΙΟ"6 M. NH, approximately equal amounts of 1 : 1 and 2 : 1 (en-zyme : antibody) conjugates. Binding activity of the conjugate (1.8 χ IO5 molecules/cell) was similar to that of unreacted antibody. In vitro cytotoxicity studies using UCLA-P3 human lung adenocarcinoma cells (2 χ IO4 cells/well) in 96-well plates demonstrated that the amount of free ΟΡ-Α required to produce ID50 values for MTX prodrugs equal to that of MTX (ca. 5 χ 10 8 m) was 10 mU/ well for MTX-Ala, and 1 mU for MTX-Phe. How-ever, calculations based upon the number of antigen binding sites per cell and the CP-Α activity of the KS1/4 conjugate indicated that only about 0.03 mU of enzyme would be present on cells saturated with conjugate. It was a pleasant surprise to find that, when the lung tumor cells were treated with the con-jugate, and excess unbound conjugate was removed by extensive washing prior to addition of the prodrug, the ID50 for MTX-Ala improved from 8.9 χ 10 6 M (no CP-A or conjugate) to 1.5 χ 10~6 m for cells containing bound conjugate. With MTX-Phe as a prodrug, the results were even better: ID50 in the presence of conjugate approached that of MTX (5.2 χ ΙΟ - 8 m). The conjugate-mediated toxicity of MTX-Phe is illustrated by an experiment (Table 1 ) in which identical concentrations of the drug and prodrug are compared for their ability to inhibit cell replication. Increased potentiation of prodrug cytotoxicity by an-tibody-bound CP-Α over that afforded by free enzyme is attributed to enhanced effectiveness of MTX gen-erated adjacent to folate transporters at the cell sur-face. These results clearly demonstrate the in vitro chemotherapeutic potential of carboxypeptidase-monoclonal antibody conjugates used in conjunction with MTX peptide prodrugs. This work was supported by an Outstanding Inves-tigator Grant (CA39836) from the National Cancer Institute and a Grant (CH-31) from the American Cancer Society. Studies with monoclonal antibody KS 1/4 were performed in collaboration with Drs. Ralph Reisfeld and Barbara Mueller. The authors thank Ms. Susan Burke for expert assistance in prep-aration of the manuscript. References 1. Senter, P. D. (1990) FASEB Journal 4, 1 8 8 - 1 9 3 . 2. Kuefner, U., Lohrmann , U., Monte jano , Y. D. , Vitols, K. S. & Huennekens, F. M. (1989) Biochemistry 28, 2 2 8 8 - 2 2 9 7 . 3. Kuefner, U., Esswein, Α., Lohrmann , U., Monte jano , Y., Vitols, Κ. S. & Huennekens, F. M. (1990) Biochemistry 29, 10540-10545 . 4. Vitols, K. S., Monte jano , Y. D. , Kuefner, U. & Huennekens, F. M. (1989) Pteridines 1, 6 5 - 7 0 . 5. Haenseler, E., Esswein, Α., Vitols, K. S., Monte jano , Y., Mueller, Β. M. , Reisfeld, R. A. & Huennekens, F. M. (1992) Biochemistry, 31, 8 9 1 - 8 9 7 . Pteridines / Vol. 3 / No. 1 β 

Activation of Methotrexate Prodrugs by Enzyme/Monoclonal Antibody Conjugates

Abstract

Similar works

Full text

Available Versions

RERO DOC Digital Library