Human ovarian cancers specifically bind daunorubicin-OC-125 conjugate: an immunofluorescence study.

This study was designed to test the specific binding to human ovarian serous adenocarcinomas of a drug-antibody conjugate [daunorubicin (DNR-OC-125], made from a new analog (PIPP-DNR) of daunorubicin that chemically links the drug to monoclonal antibodies. We recently reported that the DNR-OC-125 conjugate is selectively toxic in vitro to dividing cell populations of the human ovarian cancer cell lines SK-OV-3 and OVCAR-3 that express the CA-125 antigen [F. Sweet, L. O. Rosik, G. M. Sommers, and J. L. Collins, Gynecol. Oncol. 34, 305-311 (1989)]. In the present study, immunofluorescence data show that the DNR-OC-125 conjugate has high affinity and specificity for proliferating malignant cells from human ovarian tumors. The results demonstrate that the DNR-OC-125 conjugate retains the specific binding to CA-125 antigenic sites characteristic of the OC-125 monoclonal antibody moiety. The DNR-OC-125 conjugate selectively binds to CA-125 antigen-positive ovarian cancerous tissue in both cryostat and paraffin-embedded tissue sections. This is consistent with the earlier in vitro data from dividing populations of two human ovarian cancer cell lines that revealed retention by the DNR-OC-125 conjugate of both the specificity due to OC-125 and the cytotoxicity of daunorubicin. The present immunofluorescence studies in the DNR-OC-125 conjugate is tested on human ovarian serous tumors indicate that the OC-125 monoclonal antibody can indeed serve as a cancer-targeting carrier for daunorubicin and its analogs.

Electrophilic analogues of daunorubicin and doxorubicin.

Daunorubicin (DNR) or doxorubicin (DOX) was modified with one of four "linker reagents" to produce electrophilic drug analogues for synthesis of bioconjugates. Synthesis and characterization of two new reagents [p-isothiocyanatobenzoyl chloride and 3-(p-isothiocyanatophenyl) propionyl chloride] are described here for the first time. Adding one of the new reagents, bromoacetyl bromide, or p-(fluorosulfonyl)-benzoyl chloride in chloroform to an alkaline aqueous solution of DNR (or DOX) provided excellent yields of the corresponding, electrophilic 3'-N-amide analogue. The DNR and DOX analogues were characterized by thin-layer chromatography, nuclear magnetic resonance spectroscopy, and infrared spectroscopy. Bioconjugates were produced with the electrophilic DNR or DOX analogues by mixing them with bovine serum albumin (BSA), mouse IgG, or a monoclonal antibody (OC125, which specifically binds to the CA125 antigen from human ovarian carcinoma). The relative reactivity of the 3'-N-substituents toward protein is p-(fluorosulfonyl)benzoyl greater than phenylisothiocyanato greater than bromoacetyl. Overall, the new phenyl isothiocyanate acid chlorides are superior to p-(fluorosulfonyl)benzoyl chloride or bromoacetyl bromide as reagents with which to produce electrophilic DNR or DOX analogues for conjugation with monoclonal antibodies. The bioconjugates DNR-OC125 and DOX-OC125 are selectively toxic to two human ovarian cancer cell lines in vitro (1) and bind with high specificity to human ovarian tumor sections (2) that express the CA125 antigen.

Affinity alkylation of human placental 3 beta-hydroxy-5-ene-steroid dehydrogenase and steroid 5----4-ene-isomerase by 2 alpha-bromoacetoxyprogesterone: evidence for separate dehydrogenase and isomerase sites on one protein.

We have copurified human placental 3 beta-hydroxy-5-ene-steroid dehydrogenase and steroid 5----4-ene-isomerase, which synthesize progesterone from pregnenolone and androstenedione from fetal dehydroepiandrosterone sulfate, from microsomes as a homogeneous protein based on electrophoretic and NH2-terminal sequencing data. The affinity alkylator, 2 alpha-bromoacetoxyprogesterone, simultaneously inactivates the pregnene and androstene dehydrogenase activities as well as the C21 and C19 isomerase activities in a time-dependent, irreversible manner following first order kinetics. At four concentrations (50/1-20/1 steroid/enzyme M ratios), the alkylator inactivates the dehydrogenase activity (t1/2 = 1.5-3.7 min) 2-fold faster than the isomerase activity. Pregnenolone and dehydroepiandrosterone protect the dehydrogenase activity, while 5-pregnene-3,20-dione, progesterone, and androstenedione protect isomerase activity from inactivation. The protection studies and competitive kinetics of inhibition demonstrate that the affinity alkylator is active site-directed. Kitz and Wilson analyses show that 2 alpha-bromoacetoxyprogesterone inactivates the dehydrogenase activity by a bimolecular mechanism (k3' = 160.9 l/mol.s), while the alkylator inactivates isomerase by a unimolecular mechanism (Ki = 0.14 mM, k3 = 0.013 s-1). Pregnenolone completely protects the dehydrogenase activity but does not slow the rate of isomerase inactivation by 2 alpha-bromoacetoxyprogesterone at all. NADH completely protects both activities from inactivation by the alkylator, while NAD+ protects neither. From Dixon analysis, NADH competitively inhibits NAD+ reduction by dehydrogenase activity. Mixed cofactor studies show that isomerase binds NAD+ and NADH at a common site. Therefore, NADH must not protect either activity by simply binding at the cofactor site. We postulate that NADH binding as an allosteric activator of isomerase protects both the dehydrogenase and isomerase activities from affinity alkylation by inducing a conformational change in the enzyme protein. The human placental enzyme appears to express the pregnene and androstene dehydrogenase activities at one site and the C21 and C19 isomerase activities at a second site on the same protein.

Fetal lamb 3 beta, 20 alpha-hydroxysteroid oxidoreductase: dual activity at the same active site examined by affinity labeling with 16 alpha-(bromo[2'-14C]acetoxy)progesterone.

3 beta,20 alpha-Hydroxysteroid oxidoreductase was purified to homogeneity from fetal lamb erythrocytes. The Mr 35,000 enzyme utilizes NADPH and reduces progesterone to 4-pregnen-20 alpha-ol-3-one [Km = 30.8 microM and Vmax = 0.7 nmol min-1 (nmol of enzyme)-1] and 5 alpha-dihydrotestosterone to 5 alpha-androstane-3 beta, 17 beta-diol [Km = 74 microM and Vmax = 1.3 nmol min-1 (nmol of enzyme)-1]. 5 alpha-Dihydrotestosterone competitively inhibits (Ki = 102 microM) 20 alpha-reductase activity, suggesting that both substrates may be reduced at the same active site. 16 alpha-(Bromoacetoxy)progesterone competitively inhibits 3 beta- and 20 alpha-reductase activities and also causes time-dependent and irreversible losses of both 3 beta-reductase and 20 alpha-reductase activities with the same pseudo-first order kinetic t1/2 value of 75 min. Progesterone and 5 alpha-dihydrotestosterone protect the enzyme against loss of the two reductase activities presumably by competing with the affinity alkylating steroid for the active site of 3 beta,20 alpha-hydroxysteroid oxidoreductase. 16 alpha-(Bromo[2'-14C]acetoxy) progesterone radiolabels the active site of 3 beta,20 alpha-hydroxysteroid oxidoreductase wherein 1 mol of steroid completely inactivates 1 mol of enzyme with complete loss of both reductase activities. Hydrolysis of the 14C-labeled enzyme with 6 N HCl at 110 degrees C and analysis of the amino acid hydrolysate identified predominantly N pi-(carboxy[2'-14C]methyl)histidine [His(pi-CM)].(ABSTRACT TRUNCATED AT 250 WORDS)

Daunorubicin conjugated to a monoclonal anti-CA125 antibody selectively kills human ovarian cancer cells.

The present study was designed to test the in vitro efficacy for human ovarian cancer cells of daunorubicin (DNR) conjugated to a monoclonal antibody (OC125). The OC125 antibody specifically binds to the antigenic protein CA125 from human ovarian carcinoma. New analogs of DNR containing various linker groups were conjugated to mouse monoclonal anti-CA125 antibody (DNR-OC125); nonspecific murine IgG1 (DNR-IgG1); or bovine serum albumin (DNR-BSA). The DNR-protein conjugates were all stable for several days in neutral solutions at room temperature. The DNR-OC125 conjugates selectively killed dividing cell populations but not nondividing cell populations of two human ovarian cancer cell lines (SK-OV-3 or OVCAR-3) that express the CA125 antigen. Equivalent concentrations of DNR-IgG1 or DNR-BSA conjugates were neither toxic to the dividing nor the nondividing populations of SK-OV-3 or OVCAR-3 cells. Only those DNR-protein conjugates linked to OC125 were cytotoxic to dividing cell populations of both cell lines. This indicates that cytotoxicity is dependent on OC125 antibody-CA125 antigen binding which concentrates DNR on the ovarian cancer cells. We advance the hypothesis that following antibody-antigen binding, DNR is released from the conjugate and it intercalates in DNA by a mechanism similar to that of the unmodified DNR. The new DNR-OC125 conjugate may be useful for delivering DNR to ovarian tumors that express the CA125 antigen because the drug-antibody conjugates (1) retain the cytotoxic characteristics of the unmodified drug: (2) specifically kill the human ovarian cancer cells that express the CA125 antigen; and (3) are completely stable for days in neutral solutions at room temperature.