The Covalent Binding of Daunomycin and Adriamycin to Antibodies, with Retention of Both Drug and Antibody Activities

The Covalent Binding of Daunomycin and Adriamycin to Antibodies, with Retention of Both Drug and Antibody Activities

[CANCER RESEARCH 35. 1175-1181, May 1975] The Covalent Binding of Daunomycin and Adriamycin to Antibodies, with Retention of Both Drug and Antibody Activities Esther Hurwitz, Ronald Levy,1 Ruth Marón, Meir Wilchek, Ruth Arnon, and Michael Sela Departments of Chemical Immunology [E. H., R. L.. R. M.. R. A.. M. S.\ and Biophysics [M. W.] The Weizmann Institute of Science. Rehovol, Israel SUMMARY the two are linked together or, alternatively, as discussed by Isliker et al. ( 14), they might be linked in a manner allowing Daunomycin and adriamycin, two potent cancer chemo- the release of the active agent after reaching the target cell. therapeutic agents, were linked to immunoglobulins, mak Diphtheria toxin has been linked to anti-2,4-dinitrophenyl ing use of various covalent cross-linking methods. The most or anti-mumps virus antibodies, and the resulting conju suitable method for binding of the drugs to the antibodies, gates mediated a selective toxicity towards cells bearing which retained both antibody and drug activity, was perio these determinants on their surface (22, 23). In a different date oxidation of the drug, followed by the linking of the approach, the enzyme glucose oxidase was linked to anti- oxidized drug to the immunoglobulin and subsequent reduc trinitrophenyl antibodies, and these complexes were shown tion of the product with sodium borohydride. The activity of to lead to the toxic iodination of specific target cells in the the drug-antibody conjugates was tested in vitro on tumor presence of lactoperoxidase, glucose, and iodide (26). and normal cell cultures and was found to be similar to that Several reports have appeared in which complexes of of the free drug. A significant amount of antibody activity alkylating drugs with immunoglobulins (4, 7. 9, 10, 18. 30, was retained, as found both with anti-bovine serum albumin 32) and other macromolecules (32) have been studied. For antibodies, assayed by chemically modified bacteriophage, example, chlorambucil has been linked noncovalently to and with anti-mouse tumor antibodies, assayed by C'- antitumor antibodies and has been found to kill the target dependent cytotoxicity. tumor cells more efficiently than either the free drug or the antibody alone, as reported by 4 groups of investigators (4, 7, 9, 30). In 2 of these studies, it was shown that similar INTRODUCTION effects could be obtained by the administration of free drug and antibody separately (4, 30), so it is possible that, when Agents that are effective in killing neoplastic cells usually the drug is administered as a noncovalent complex with also have detrimental effects on normal cells, particularly antibody, it dissociates in vivo and acts separately and the rapidly proliferating ones of the gastrointestinal tract synergistically with the antibody. and bone marrow, and cancer chemotherapy is ultimately Cytotoxic drugs of low molecular weight may retain their limited by its toxicity to these normal tissues. One possible activity after covalent linkage to macromolecules. Thus, approach for increasing the effectiveness of antitumor drugs methotrexate was bound via an azo bond to hamster would be to find methods of altering their distribution in the immunoglobulin (21) as well as to fibrinogen and human body to increase their local concentration at the tumor cell serum albumin (20). The resulting conjugates possessed sites. In this way the selectivity of their toxicity for the significant activity, as did conjugates of a methylhydrazine tumor cells might be enhanced. derivative with fibrinogen and albumin (20) and conjugates Paul Ehrlich (5) was the first to suggest that molecules of several nitrogen mustards with proteins and synthetic with an affinity for certain tissues might be able to serve as polypeptides (31). carriers of cytotoxic agents to concentrate them on the In the present study we have used the antitumor antibiot appropriate target cells in vivo. Various macromolecules ics daunomycin and adriamycin (Chart 1), and we have used have been shown to localize in tumor cells in vivo and were as a model system antibodies against BSA2 or antibodies suggested as possible carriers for cytotoxic drugs (14). With specific for various tumor cells (this paper; Ref. 16). We the development of tumor immunology, many investigators have found conditions whereby the drugs can be covalenti)' have sought to use antibodies to antigenic determinants linked to the antibodies, preserving both the antibody expressed preferentially on tumor cells as carriers of activity and the pharmacological activity of the bound cytotoxic agents. For this approach to succeed, both the drugs. In the accompanying report these preparations of antibody and the toxic agent must retain its activity when antibody-linked drugs will be shown to mediate a cell- specific pharmacological effect in vitro. ' Fellow of the Helen Hay Whitney Foundation. Permanent address: Department of Medical Oncology. Stanford Medical School. Stanford. 2The abbreviations used are: BSA. bovine serum albumin; ECDI. Calif. l-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride; PBS. 0.15 Received November 4, 1974; accepted January 20, 1975. vi NaCl:0.01 M phosphate. pH 7.2. MAY 1975 1175 Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1975 American Association for Cancer Research. E. Hurwit: et al. O OH cytoma (PCS) in BALB/c mice (28) were similarly main tained by serial passage in their respective inbred strains. Antisera H3CO O OH Antiserum to BSA was produced in rabbits by weekly s.c. injections of 2 mg BSA emulsified in complete Freund's adjuvant. Antibody activity was measured by the inactiva- tion of BSA-coated bacteriophage (12). Rabbit antisera to the B leukemia cells were prepared by 4 to 5 i.v. injections of IO8leukemia cells at 5-day intervals. Antibody activity was measured by complement-dependent cytotoxicity. Five x IO6 target cells were incubated with dilutions of the antisera for 15 min at 37°.Agarose- absorbed guinea pig complement, at a 1:5dilution, was then added, and after a further 30 min incubation the reaction was stopped by the addition of 0.25 volume of 0.01 M EDTA. Viable cells were counted by trypan blue exclusion, R = , adriamycin and the titer of the antiserum was taken as the dilution that gave 50% cytotoxicity. Titers of 1:100 to 1:200 were R = >daunomycin obtained against the B leukemia cells. The immunoglobulin fractions of the antisera were prepared by precipitation with Chart I. The structure of daunomycin and adriamycin. ammonium sulfate at 33% saturation. MATERIALS AND METHODS Drug Activity Drugs The pharmacological activity of daunomycin and Daunomycin hydrochloride was obtained as the product adriamycin was measured primarily by their inhibition of Cerubidine (Rhône-Poulenc, Paris, France) or as the pure cellular RNA synthesis. The assays were carried out in compound, a gift of the same manufacturer. Adriamycin microtiter plates (Dynatech Laboratories, Sussex, England) hydrochloride was supplied by the Drug Development in Eagle's minimal essential medium containing penicillin Branch, National Cancer Institute (Bethesda, Md.), in vials and streptomycin (Microbiological Associates, Jerusalem, containing 10 mg drug mixed with 50 mg lactose, or as the Israel). Cells were suspended in medium at a concentra pure compound, a gift of the Farmitalia Company (Milan, tion of 2 x 1C7cells/ml and dispensed into the wells of the Italy). plates in 50-^1 aliquots. Drugs were diluted in PBS and then added to the cells in 50-^1 amounts. The plates were Chemicals and Reagents incubated for 2 hr (unless otherwise stated in the text) at [5-3H]LJridine (specific activity, 25 Ci/mmole) was pur 37°in a humidified atmosphere of 5% CO2 in air. At that chased from the Radiochemical Center (Amersham, Eng time 10 n\ containing 1 /¿Ciof [5-3H]uridine were added land). Sodium periodate and sodium borohydride were to each well and, after another 1 to 2 hr of incubation, 25 purchased from British Drug House (Poole, England). //I of 25% trichloroacetic acid were added and the plates Glutaraldehyde was obtained from Ladd Research In were placed at 4°overnight. Trichloroacetic acid precipi dustries (Jerusalem, Israel). ECDI was purchased from tates were washed, solubilized in NaOH, and transferred Ott Chemical Co. (Muskegon, Mich.). Porapak Q, 50 to vials for counting as previously described (29). The to 80 mesh, was purchased from Waters Associates (Bos scintillation mixture consisted of toluene-based scintilla ton, Mass.). Bio-Gel P-100 was obtained from Bio-Rad tion solution:Triton X-100:0.1 N HC1 (6:3:1). The HC1 was Laboratories (Los Angeles, Calif.), and Sepharose 6B included to counteract chemiluminescence. Assays were was purchased from Pharmacia (Uppsala, Sweden). performed in triplicate, which generally had less than 10% Guinea pig complement was obtained from Grand Island variation. Biological Co. (New York, N. Y.). Another assay of drug activity used was the measurement of cytotoxicity as judged by trypan blue dye uptake. Tumor Cells A dimethylbenzanthracene-induced leukemia in SJL/J Binding of Daunomycin and Adriamycin mice (13) was maintained by s.c. passage in the syngeneic to Immunoglobulin mouse strain. The cells of this leukemia have immunoglobu- lin on their surface (13) and are thus referred to as B Three different methods of covalent binding were used, leukemia cells. A Moloney virus-induced lymphoma each taking advantage of the amino sugar moiety of the (YAC) in A/J mice (15) and a mineral oil-induced plasma- drugs (Chart 1). 1176 CANCER RESEARCH VOL. 35 Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1975 American Association for Cancer Research. Binding of Daunomycin and Adriamycin to Antibodies Method 1. Periodate oxidation of the drugs was per protein appeared in an aggregated form (Chart 3), probably formed to cleave the bond between C-3 and C-4 of the as a result of protein-protein cross-linking by glutaralde- amino sugar, producing carbonyl groups capable of reacting hyde.

View Full Text

Details

  • File Type
    pdf
  • Upload Time
    -
  • Content Languages
    English
  • Upload User
    Anonymous/Not logged-in
  • File Pages
    8 Page
  • File Size
    -

Download

Channel Download Status
Express Download Enable

Copyright

We respect the copyrights and intellectual property rights of all users. All uploaded documents are either original works of the uploader or authorized works of the rightful owners.

  • Not to be reproduced or distributed without explicit permission.
  • Not used for commercial purposes outside of approved use cases.
  • Not used to infringe on the rights of the original creators.
  • If you believe any content infringes your copyright, please contact us immediately.

Support

For help with questions, suggestions, or problems, please contact us