Vitols et al.: M e t h o t r e x a t e p r o d r u g s a c t i v a t e d b y e n z y m e / a n t i b o d y c o n j u g a t e s 125
P t e r i d i n e s
Vol. 3, p p . 1 2 5 - 1 2 6
Short Communication
Activation of Methotrexate Prodrugs by Enzyme/Monoclonal Antibody
Conjugates
K . S. Vitols, E. Haenseler1', Y. M o n t e j a n o , T. Baer2) and F. M. H u e n n e k e n s
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 m a j o r goals in cancer chemotherapy is the development of procedures for the selective delivery of drugs to t u m o r cells. This strategy, when perfected, will allow drugs to be used at the high concentrations necessary for virtually complete (i. e., > 9 9 % ) eradi-cation of the t u m o r cells without concomitant destruc-tion of normal cells. M o n o c l o n a l antibodies targeted to t u m o r 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 f r o m 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 a m o u n t s of the active drugs f r o m inert p r o d r u g s at the t u m o r 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 a m i n o acid) are relatively non-toxic, but these derivatives can be hydrolyzed by
carboxypep-" R e c i p i e n t of an E O R T C F e l l o w s h i p . P r e s e n t a d d r e s s : Insti-t u Insti-t e of Clinical C h e m i s Insti-t r y , U n i v e r s i Insti-t y H o s p i Insti-t a l , Z u r i c h , SwiInsti-tz- Switz-e r l a n d . 2) P r e s e n t A d d r e s s : Byk G u l d e n P h a r m a z e u t i k a , K o n s t a n z , G e r m a n y . P t e r i d i n e s / V o l . 3 / N o . 1/2 C o p y r i g h t © 1992 Walter de G r u y t e r - Berlin - N e w Y o r k
tidases to yield the active parent drug. Alanine, phen-ylalanine, aspartate and arginine derivatives of M T X (Fig. 1) have been synthesized in good yield by con-ventional pathways (2, 3). It Should be noted, how-ever, t h a t 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 carboxypeptidasemediated cleavage of the M T X -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 M T X oc-peptides by various carboxypeptidases, and particularly M T X -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 M T X - A l a / C P - A combi-nation was demonstrated in a model system in which immobilized CP-Α was embedded in a semi-solid me-dium containing the p r o d r u g and LI210 cells (4). The crucial question, however, was whether cytotoxic combinations of M T X could be produced by the a m o u n t 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.\ M e t h o t r e x a t e p r o d r u g s activated by e n z y m e / a n t i b o d y c o n j u g a t e s MTX α-Peptides o II r, r\ O C-NH-CH-COOH
// \
11 1 1CHaN —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 M e t h o t r e x a t e α-peptides.
Table 1. Cytotoxicity of M e t h o t r e x a t e α P h e n y l a l a n i n e ( M T X -Phe) f o r U C L A - P 3 Cells Treated with C a r b o x y p e p t i d a s e - A ( C P - A ) / M o n o c l o n a l A n t i b o d y KS1/4 C o n j u g a t e D r u g Enzyme- % or P r o d r u g M o n o c l o n a l Inhibition C o n j u g a t e M T X 100 M T X - P h e C P - A / K S l / 4 94 M T X - P h e — 23 — C P - A / K S l / 4 3
In vitro cytotoxicity against U C L A P 3 h u m a n lung a d e n o c a r -c i n o m a -cells (104 cells/well) was m e a s u r e d in 96-well plates as
described previously (5). In samples treated with enzyme-anti-b o d y c o n j u g a t e , excess of the latter was r e m o v e d enzyme-anti-by extensive washing. G r o w t h was stopped a f t e r 72 h. I n h i b i t i o n is expressed relative to t h a t observed with M T X (designated as 100%). C o n c e n t r a t i o n s of M T X a n d M T X - P h e , 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 U C L A - P 3 human lung adenocarcinoma cells (2 χ IO4 cells/well) in
96-well plates demonstrated that the amount of free ΟΡ-Α required to produce ID5 0 values for M T X prodrugs
equal to that of M T X (ca. 5 χ 10 8 m) was 10 m U /
well for MTX-Ala, and 1 m U 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 m U 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 ID5 0 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: ID5 0 in the
presence of conjugate approached that of M T X (5.2 χ Ι Ο- 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 M T X 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 M T X peptide prodrugs.
This work was supported by an Outstanding Inves-tigator Grant (CA39836) from the National Cancer Institute and a Grant (CH-31) f r o m 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) F A S E B J o u r n a l 4, 1 8 8 - 1 9 3 .
2. K u e f n e r , U., L o h r m a n n , U., M o n t e j a n o , Y. D . , Vitols, K . S. & H u e n n e k e n s , F. M . (1989) Biochemistry 28, 2 2 8 8 - 2 2 9 7 . 3. Kuefner, U., Esswein, Α., L o h r m a n n , U., M o n t e j a n o , Y.,
Vitols, Κ . S. & H u e n n e k e n s , F. M . (1990) Biochemistry 29, 1 0 5 4 0 - 1 0 5 4 5 .
4. Vitols, K . S., M o n t e j a n o , Y. D . , K u e f n e r , U. & H u e n n e k e n s , F. M . (1989) Pteridines 1, 6 5 - 7 0 .
5. Haenseler, E., Esswein, Α., Vitols, K . S., M o n t e j a n o , Y., Mueller, Β. M . , Reisfeld, R . A . & H u e n n e k e n s , F. M. (1992) Biochemistry, 31, 8 9 1 - 8 9 7 .
