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

[CANCER RESEARCH 35. 1175-1181, May 1975]

Esther Hurwitz, Ronald Levy,1 Ruth MarÃ³n, 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

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 inactivation 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 37Â°.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 (RhÃ´ne-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 37Â°in a humidified atmosphere of 5% CO2 in air. At that chased from the Radiochemical Center (Amersham, Eng time 10 n\ containing 1 /Â¿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 4Â°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 immunoglobulin 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. with free amino groups on the protein, and the resulting The product of the coupling by carbodiimide was substi Schiff base linkages were reduced with sodium borohydride tuted to the extent of 4 moles/mole immunoglobulin and (6). Drug, usually 40 mg/ml in 1 ml PBS, was mixed with a behaved similarly to the periodate oxidation product on a slight molar excess of 0.1 MNaIO4and incubated for 1hrat Bio-Gel P-100 column separation. room temperature in the dark. Glycerol (1 M) was then Pharmacological Activity of Protein-bound Drug. The added to a final concentration of 0.05 Mto consume excess inhibition of [3H]uridine incorporation into test cells (B periodate. The solution of oxidized drug was mixed with 1 leukemia) was used to quantitate drug activity, as described ml of immunoglobulin, 20 to 25 mg/ml in 0.15 Mpotassium in "Materials and Methods." Chart 4 depicts the activity of carbonate buffer (pH 9.5), and incubated for another hr at free daunomycin, daunomycin treated by periodate and room temperature. NaBH4 was then added to a final NaBHÂ«,and 2 different preparations of daunomycin bound concentration of 0.3 mg/ml, and the reaction was allowed to to immunoglobulin by the periodate-borohydride method. proceed for 2 hr at 37Â°. The results demonstrate that a substantial amount of the Method 2. Glutaraldehyde was used for cross-linking drug activity was preserved when it was covalenti}' bound to between the amino group of the drug and the free amino protein by this method. The activity of these conjugates was groups of the protein ( 1). Glutaraldehyde ( 100/Â¿I,0.1%)was less than that of free daunomycin at low concentrations, but added to 1 ml of solution containing 3 mg of immunoglobu bound and free drug produced the same maximal inhibition lin and 0.5 mg drug in PBS and then kept for 15 min at room temperature. Method 3. ECDI was used to bind the drug via its amino (o) ! (b) group to the carboxyl groups of the protein (II). Immuno globulin ( 14 mg) and drug (5 mg) were mixed in 0.75 ml of PBS, and 6.5 mg of ECDI were added in 0.1 ml PBS. The reaction proceeded for 4 hr at room temperature. Free and bound drugs were separated by gel filtration chromatography, using Bio-Gel P-100 or Sepharose 6B. Small traces of free drug were removed from the protein fractions by adsorption chromatography on Porapak Q (24). This material binds free daunomycin and adriamycin very efficiently, allowing protein-bound drugs to pass through the column unretarded. The free drug can then be eluted with acetone or methanol. The degree of substitution Sa was estimated by the drug absorbance at 495 nm, assuming 20 30 IO 20 30 an EÂ¡erÃ³196(2). Protein concentration was measured by the Tube number method of Lowry et al. (19). Chart 2. Separation of free from bound daunomycin. Gel filtration on Bio-Gel P-100 (15 x 1.5 cm), a, periodate-oxidized drug bound to immunoglobulin; b, a mixture ol'drug and immunoglobulin: O, absorbance RESULTS at 280 nm; â€¢¿absorbanceat 495 nm.

Drug Binding. Daunomycin or adriamycin were bound to immunoglobulin by the periodate oxidation method. Chart 2a shows the separation of bound from free drug by gel filtration on Bio-Gel P-100. The covalenti}' bound dau nomycin appeared in the exclusion volume of the column. The extent of substitution varied in different preparations and was 2 to 5 moles drug per mole antibody. No binding occurred when drug and protein were simply mixed under the same conditions of concentration, time, and tempera ture (Chart 2b). Rechromatography after storage for 3 weeks at 0Â°,or after treatment of the complex with 1% sodium dodecyl sulfate, showed the drug to remain bound to the protein. However, if the NaBHÂ«reduction step was omitted, the binding was unstable over time. Binding by glutaraldehvde resulted in a higher degree of Chart 3. Separation of free from bound daunomycin. Gel filtration on protein substitution, 7 to 10 moles/mole protein in different Sepharose 6B (15 x 1.5 cm), a. drug cross-linked to immunoglobulin by preparations. However, when the products were separated glutaraldehyde; ft. control, mixture of drug and immunoglobulin; O, on Sepharose 6B, most of the bound drug and half of the absorbance at 280 nm; â€¢¿.absorbanceat 495 nm.

MAY 1975 1177

Table 1 eioo - Time course of drug effect B leukemia cells (IO7) were incubated at 37Â°in the presence of daunomycin, either free or protein bound. 4 ^g/ml. Incorporation of [3H]uridine into cellular material precipitable by trichloroacetic acid was measured and expressed as percent inhibition (100 - % of control cul ^50 ture, containing no drug). % inhibition of [3H]uridine incorporation

daunomycinIncubation Bound

l 23456 time(min)306090120240Freedaunomycin4358616683Daunomycin-anti-BSA2035395376Daunomycin-anti-B leukemia2535485789 Drug concenlrotionl^g/ml) Chart 4. Inhibition of [3H]uridine incorporation (incorp.) by daunomy cin and by daunomycin bound to immunoglobulin (coupled by the periodate-borohydride method). B leukemia test cells. . 2-hr incuba tion; , 4-hr incubation; â€¢¿.freedaunomycin; x. free daunomycin treated with periodate and sodium borohydride (i.e.. the material from the 2nd peak in Chart 2a); A, daunomycin bound to anti-BSA, 6 moles/mole: O. daunomycin bound to anti-B leukemia. Table 2 Non-C'-dependent cytoloxicity of daunomycin and daunomycin-IgG of RNA synthesis at high concentrations. Adriamycin was To 50Mlof 2.10'cells were added 100^1 of IgG (0.7 mg/ml) and/or 1Mg less potent in inhibiting uridine incorporation than was of daunomycin free or bound. The percentage of dead cells was determined daunomycin: 66% as compared to 90% at equivalent by trypan blue uptake. concentrations. The same difference was observed when the bound preparations of the 2 drugs were compared; e.g.. % dead cells at specified incubation time adriamvcin-immunoglobulin gave 52% inhibition of uridine TreatmentNormal incorporation at a concentration at which daunomycin- immunoglobulin inhibited to the extent of 89%. The kinetics IgGAnti-BSAAnti-B of free and bound daunomycin activities are compared in leukemiaFree detail in Table 1, where it can be seen that free drug was more active than the protein-bound drug when assayed at IgGDrug-anti-BSADrug-anti-Bdrug + normal short incubation times, but after longer incubation the same maximal effect was eventually obtained with both the free leukemia2hr1312113620343hr191732Â°4436445hr201627Â°484158 and bound drugs. In Chart 4 and Table 1, one of the ' Counts inaccurate because of the clumping of cells by the antibody. drug-protein conjugates was formed with immunoglobulin- containing antibody directed against the B leukemia test cell, and the other was an immunoglobulin with no specific Table 3 ity toward the test cell. In Chart 4 there is the suggestion Inhibition of[3H]uridine incorporation by daunomycin and daunomycin that drug linked to specific antibody globulin was slightly bound to immunoglobu/in (ftv theperiodate-borohvdride method) on YAC and PCS cells more toxic to the cells than drug linked to the unrelated immunoglobulin, but this difference was not consistently % inhibition of [3H]uridine incorporation observed. Under the conditions of the assay, with no complement present, there was no toxic effect of antibody Bound daunomycin alone on these cells. Another assay of drug effect, namely, direct cell killing (non-C' dependent) as estimated by trypan blue uptake Oig/ml)12461.4369131925Freedaunomycin02666741335526773Daunomycin-anti-BSA0020632237576375Daunomycin-anti-Bleukemia0236668Daunomycin-anti-PC52230576365 (Table 2), confirmed the results of the [3H]uridine incorpo YACcellsPCS ration assay, again demonstrating the activity of protein- bound drug. Other target cells were tested, including other tumor lines such as YAC, a Moloney virus-induced lymphoma in A/J mice, and PCS, a BALB/c plasmacytoma (Table 3), as well as normal mouse and guinea pig spleen cells. Different test cellsDrug cells varied in their sensitivity to the drugs, but in general the relationship between the activity of bound and free drug was similar to that observed in the case of the B leukemia cells. An example of the protein-bound and free drug activities against normal mouse spleen cells is shown in Chart 5.

1178 CANCER RESEARCH VOL. 35

100 (O) g 50 Â£ I 40

Â£ÃŽO 50 O Ã¨zo i I 'o

0 0246 (bi Drug concentrtjtion(Â¿Â¿g/ml) 100 Chart 5. Inhibition of [3H]uridine incorporation (incorp.) in normal mouse spleen cells, x, free daunomycin; A, daunomycin anti-BSA: O, daunomycin anti-B leukemia.

The pharmacological activity of daunomycin and 50 adriamycin bound to protein by glutaraldehyde is shown in Chart 6. The drugs lost none of their activity upon binding to protein by this method. In contrast, the EDCI binding method produced complexes with almost complete loss of drug activity (Table 4). Antibody Activity of Drug-substituted Immunoglobulins. D'uq concentration(^j.q/ml) All experiments were done in triplicate. Chart 7 shows the Chart 6. Inhibition of [3H]uridine incorporation (incorp.) by effect of the periodate binding method on the activity of adriamycin (a) and by daunomycin (6). coupled to Â¡mmunoglobulinwith anti-BSA antibody. Approximately 55% of antibody activ glutaraldehyde. B leukemia test cells. O. free drug: A. drug treated by ity was retained, as measured by inactivation of BSA-T4 glutaraldehyde (i.e.. the material from the 3rd peak in Chart 3a); â€¢¿.drug bacteriophage (12), comparing the concentrations of anti anti-B leukemia; x. drug-anti BSA. body necessary to inactivate 50% of the phages. Anti-B leukemia antibody activity measured by complement- Table 4 Drug activity of free daunomycin compared to daunomycin hound to dependent cytotoxicity is described in Chart 8. As can be protein b\- 2 different method* seen, the cytotoxic activity decreased in relation to the B leukemia cells (IO7)were incubated for 2 hr at 37Â°inthe presence of extent of drug substitution. In a drug-antibody conjugate daunomycin. either free of bound to immunoglobulin by I of 2 different with 2 moles drug per mole antibody, 64% of the activity methods. Incorporation of [3H]uridine into cellular material precipitable was retained. In a 2nd preparation with 6 moles/mole, only by trichloroacetic acid was measured and expressed as percent inhibition (100 - % of control culture, containing no drug). 25% of the antibody activity remained. The glutaraldehyde binding method also produced a incorporationDrug % inhibition of [3H]uridine reduction in antibody activity, but when the products were separated into aggregated and nonaggregated fractions (Chart 3a) and tested individually, it was found that the added immunoglobulin immunoglubulin drug44 (periodatebound)38 (ECDIbound)13 aggregated protein had no antibody activity and the nonag (/ig)0.3 gregated protein had full activity compared to the untreated antibody. These results were true for both the BSA-T4 0.6 80 88 II1824 bacteriophage and the complement-dependent cytotoxicity 1 95 94NDDrug- ND"Drug- assays. 15Free " Not done.

DISCUSSION The significant finding in this study is that these drugs can be covalently bound to proteins with retention of their toxic In this study we have investigated the method of linking effects on cells. Among the 3 different methods investigated, the toxic agents, daunomycin and adriamycin, to antibodies the drug activity was best preserved with glutaraldehyde so that their activity might be preserved. These drugs are binding and least preserved with ECDI (Chart 6; Table 4). highly active (8, 25) against a wide range of tumors in The mechanism of action of these protein-bound drugs is animals and in man, but their use is ultimately limited by not defined here, but several possibilities exist. One possible their toxicity to normal tissues. The activity of these drugs is mechanism is by simple reversal of the chemical binding and based on their ability to bind to DNA intercalating between the release of free drug. The degrees of reversibility of the the base pairs and inhibiting its template activity for DNA chemical linkages resulting from the 3 binding methods are and RNA polymerase (2, 3, 27). They must therefore enter consistent with this hypothesis. ECDI reacts with the the cell as well as the nucleus to exert their effects. protein carboxyl groups and the amino group of the drug to

MAY 1975 1179

Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1975 American Association for Cancer Research. E. Humit: et al. antibodies can make their way into the nucleus of a living 100- cell (17). Our own preliminary experiments suggest that daunomycin bound to protein by the periodate-borohydride method, but not by the EDCI method, can interact with DNA in solution (unpublished data). Another requirement of the present approach is that the antibodies should retain their activity after binding the drug. The glutaraldehyde binding method, although superior from the point of view of preservation of drug activity, was associated with a variable degree of protein aggregation that completely inactivated the antibody activity. The periodate- o o.i borohydride binding method produced conjugates that Antibody(mq/ml) retained a significant amount of the original antibody Charl 7. The effect of drug binding on anti-BSA antibody activity as activity; however, higher degrees of drug substitution were measured by BSA-TÂ«bacteriophage inactivation. O, anti-BSA; â€¢¿,dauno- associated with less retention of antibody activity (Charts 7 mycin anti-BSA. and 8). 100 In the accompanying paper ( 16) the drug-antibody conju \ gates by the periodate-borohydride method described here have been tested in vitro by a specific cytotoxic method and have been shown to exhibit preferential toxicity for various tumor target cells. â€¢¿o o â€¢¿oW 50 ACKNOWLEDGMENTS

The authors would like to thank Dr. Nechama Haran-Ghera for her gift ..â€¢-* of the B leukemia.

i 0 0.5 1.0 1.5 2.0 REFERENCES Antibody(mg/ml) Chart 8. The effect of drug binding on anti-B leukemia antibody I. Avrameas, S. Coupling of Enzymes to Proteins with Glutaraldehyde. activity as measured by C' cvtotoxicity against B leukemia cells. â€¢¿.anti-B Use of the Conjugates for the Detection of Antigens and Antibodies. leukemia; O. duunomycin anti-B leukemia. 2 moles/mole; A. daunomycin- Immunochemistry. 6: 43 52. 1969. anti-B leukemia. 6 mules/mole. 2. Bernard. J.. Paul. R., Boiron. M., Jacquillet. C., and Marci, R. Rubidomycin. p. 12. Berlin: Springer-Verlag, 1969. 3. Calendi. E.. Di Marco. A.. Reggiani. M.. Scarpinato. B.. and give an irreversible bond. This conjugate was almost entirely Valentini. L. On Physico-chemical Interactions between Daunomycin inactive (Table 4). The periodate-oxidized drug forms and Nucleic Acids. Biochim. Biophys. Acta. 103: 25 49. 1965. reversible Schiff base linkages with the protein amino 4. Davies, D. A. L.. and O'Neil. G. J. In vivo and in vitro Effects of groups, which could dissociate if they were incompletely Tumor Specific Antibodies with Chlorambucil. Brit. J. Cancer. reduced by the sodium borohydride. This conjugate was -'Â«(Suppl.IK 285 298. 1973. moderately active (Charts 4 and 5; Tables 1 and 4). 5. Ehrlich. P. Collected Studies on Immunity. Vol. 2, pp. 442 447. New Glutaraldehyde cross-links the drug and the protein via their York: John Wiley and Sons. Inc.. 1906. amino groups yielding potentially reversible Schiff base 6. Erlanger. B. F.. and Beiser. S. M. Antibodies Specific for Ribonucleo- linkages, and these conjugates were fully active compared to sides and Nucleotides and Their Reaction with DNA. Proc. Nati. the free drug (Chart 6). However, simple chemical dissocia Acad. Sei. U. S., 52: 68 74. 1964. 7. Flechner. I. The Cure and Concomitant Immunization of Mice tion does not fit with the experimental data. Complete Bearing Ehrlich Ascites Tumors by Treatment with an Antibody- dissociation of the glutaraldehyde-linked material would alkylating Agent Complex. European J. Cancer, 9: 741 745, 1973. have had to occur to explain the data of Chart 6. With 8. Frei, E. Prospectus for Cancer Chemotherapy. Cancer. 30: 1656 regard to the periodate-borohydride-linked drug, it is un 1661. 1972. likely that the drug would extensively dissociate on exposure 9. Ghose, T., and Nigam. S. P. Antibody as Carrier of Chlorambucil. to cells when it did not dissociate on prolonged storage or Cancer, 29: 1398 1400, 1972. after exposure to sodium dodecyl sulfate. 10. Ghose. T.. Norvell. S. T.. Guclu, A., Cameron. D.. Bodurtha. A., and Another possibility is that the drug-protein conjugates Macdonald. A. S. Immunochemotherapy of Cancer with Chloram- enter the cells by pinocytosis and are digested intracellularly bucil-carrying Antibody. Brit. Med. J.. 3: 495 499, 1972. 11. Goodfriend, T. L., Levine. L., and Fasman. G. D. Antibodies to to liberate either free drug or small drug-peptide conjugates. Bradykinin and Angiotensin: A Use ol'Carbodiimides in Immunology. This mechanism has been proposed to explain the activity of Science. 144: 1344 1346. 1964. complexes of daunomycin with DNA (33). A 3rd, more 12. Haimovich, J., Hurwitz, E.. Novik. N.. and Sela. M. Preparation of remote, possibility is that protein-drug conjugates can enter Protein-Bacteriophage Conjugates and Their Use in Detection of the nucleus and interact with the DNA without needing to Anti-protein Antibodies. Biochim. Biophys. Acta, 207: 115 124. 1970. be digested. There is at least 1 report that suggests that 13. Haran-Ghera. N.. and Peled. A. Thvmus and Bone Marrow Derived

1180 CANCER RESEARCH VOL. 35

Lymphatic Leukemia in Mice. Nature, 241: 396-398, 1973. Antigens on the Cells. Science, 169: 68 70, 1970. 14. Isliker, H., Cerottinin. J. C., Jaton, J.-C., and Magnenat, G. Specific 24. Niederwieser, A. Adsorption on Neutral Polystyrene Resin. A Simple and Non-specific Fixation of Plasma Proteins in Tumors. In: Method for Extraction of 2,4-Dinitrophenyl Derivatives from Aqueous Chemotherapy of Cancer, pp. 278-288. Amsterdam: Elsevier Publish Solution and for Decoloration of Protein Hydrolysates. J. ing Co., 1969. Chromatog.. 54: 215 223, 1971. 15. Klein, E., and Klein, G. Antigenic Properties of Lymphomas Induced 25. O'Bryan. R. M., Luce. J. K., Talky, R. W., Gottlieb, J. K.. Baker. L. by Moloney Agent. J. Nati. Cancer Inst., 32: 547-568, 1964. H., and Bonadonna, G. Phase II Evaluation of Adriamycin in Human 16. Levy, R., Hurwitz, E., MarÃ³n, R., Arnon, R., and Sela. M., The Neoplasia. Cancer. 32: I 8. 1973. Specific Cytotoxic Effects of Daunomycin Conjugated to Antitumor 26. Philpott, G. W.. Shearer. W. T.. Bower. R. J., and Parker, C. W. Antibodies. Cancer Res., 35: 1182-1186, 1975. Selective Cytotoxicity of Hapten-substituted Cells with an Antibody- 17. Lewis, G. M., Pegrum, G. D., and Evans, C. A. Intracellular Location Enzyme Conjugate. J. Immunol., ///: 921 929, 1973. of Specific Antibodies Reacting with Human Lymphocytes. Nature, 27. Pigram. W. J., Fuller, W., and Hamilton, L. D. Stereochemistry of 247: 463 465, 1974. Interaction of Daunomycin with DNA. Nature. 17: 235 236, 1972. 18. Linford, J. H., Froese, G.. Berczi, I., and Israels, L. G. An Alkvlating 28. Potter, M., and Robertson. C. L. Development of Plasma Cell Agent-Globulin Conjugate with Both Alkylating and Antibody Activ Neoplasms in BALB/c Mice after Intraperitoneal Injection of Paraf ity. J. Nati. Cancer Inst., 52: 1665-1667, 1974. fin-Oil Adjuvant, Heat-killed Staphylococcus Mixtures. J. Nati. 19. Lowry, O. H., Rosebrough. N. J.. Farr, A. L., and Randall. R. J. Cancer Inst., 25: 847-861, 1960. Protein Measurement with the Folin Phenol Reagent. J. Biol. Chem., 29. Rosenberg. S. A., Levy, R.. Schlechter, B., Ficker, S., and Terry, W. 193: 265-275, 1951. D. A Rapid Microassay of Cellular Immunity in the Guinea Pig and 20. Magnenat, R., Schindler. R., and Isliker, H. Transport d'Agents Mouse. Transplantation, 13: 541 545, 1972. Cytostatiques par les ProtÃ©inesPlasmatiques. European J. Cancer, 5: 30. Rubens. R. D., and Dulbecco, R. Augmentation of Cytotoxic Drug 33-40, 1969. Action by Antibodies Directed at Cell Surface. Nature, 248: 81 82. 21. Mathe, G., Tran, B. L., and Bernard, J. Effet sur la LeucÃ©mie1210de 1974. la Souris d'une Combinaison par Diazolation d'AmÃ©thopterineet de 31. Szekerke. M., Wade. R., and Whisson, M. E. Some Serum Protein a-Globulines de Hamsters Porteurs de Cette LeucÃ©mieparHÃ©tÃ©ro- Nitrogen Mustard Complexes with High Chemotherapeutic Selectiv greffe. Compi. Rend., 246: 1626-1628, 1958. ity. Nature. 215: 1303 1304. 1967. 22. Moolten. F. L.. Capparell. N. J.. and Cooperband, S. R. Antitumor 32. Szekerke, M., Wade, R., and Whisson. M. E. The Use of the Mac- Effects of Antibody-Diphtheria Toxin Conjugates: Use of Hapten- romolecules as Carriers of Cytotoxic Groups (Part II) Nitrogen coated Tumor Cells as an Antigenic Target. J. Nati. Cancer Inst.. 49: Mustard-Protein Complexes. Neoplasma. 19: 211 215. 1972. 1057-1062, 1972. 33. Trouet, A., Deprez-de Compeneere, D., and De Duve, C. Chemother 23. Moolten, F. L., and Cooperband, S. R. Selective Destruction of Target apy through Lysozymes with a DNA-Daunorubicin Complex. Nature. Cells by Diphtheria Toxin Conjugated to Antibody Directed against 239: 110 112. 1972.

MAY 1975 1181

Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1975 American Association for Cancer Research. The Covalent Binding of Daunomycin and Adriamycin to Antibodies, with Retention of Both Drug and Antibody Activities

Esther Hurwitz, Ronald Levy, Ruth Maron, et al.

Cancer Res 1975;35:1175-1181.

Updated version Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/35/5/1175

E-mail alerts Sign up to receive free email-alerts related to this article or journal.

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Subscriptions Department at [email protected].

Permissions To request permission to re-use all or part of this article, use this link http://cancerres.aacrjournals.org/content/35/5/1175. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.