Daunomycin-immunoglobulin conjugates, uptake and activity in vitro

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Daunomycin-Immunoglobulin Conjugates Uptake and Activity In Vitro* E. HURWITZ, t R. MARON,I" R. ARNON,I" M. WILCHEK++ and M. SELAt§ ~Department of Chemical Immunology, The Wiezmann Institute of Science, Rehovot, Israel and +Department of Biophysics, The Weizmann Institute of Science, Rehovot, Israel Abstraet--Daunomycin, an anti-tumor drug, the activity of which is known to be exerted mostly in the nucleus, was covalently attached to anti-tumor antibodies or to normal immunoglobulin. The attachment was accomplished either by direct linking or via a dextran bridge. All the conjugates retained a significant amount of their original drug activity. The capacity of these macromolecular conjugates to penetrate the malignant cell and the nucleus was examined. The compounds were found to be able to penetrate the cell membrane in the conjugated form. Daunomycin linked to anti-tumor antibodies entered the cell at a higher rate than daunomycin attached to normal Ig and daunomycin-dextran. In normal cells, no difference in uptake was observed between drug-immunoglobulin or drug-anti-tumor-antibody conjugates. Uptake studies with daunomycin conjugates, radioactively labeled by different methods, suggest that the drug is released from the complex after penetration into the cell but probably enters the nucleus as the free drug.

INTRODUCTION

the antibody was not made directly but through a dextran bridge and thus obtained a high conjugation of the drug to the antibody. We have attempted to clarify the mechanism of action of the conjugates of the drug with antibody or with immunoglobulin by testing the ability of these complexes to enter the cells. Previously it was shown that antibodies directed against the major histocompatibility locus are able to enter the cell and bind to the nucleus [16]. In this study, the uptake of radioactively labeled derivatives of the conjugates by tumor and normal cells and their nuclei was explored in correlation with their activity in vitro.

THE USE of antibodies [1-4] or other specific [5] and non-specific [3, 5, 6-10] macromolecules as carriers of anti-tumor drugs has been investigated by various groups throughout the last few years. The mode of action of these drugs requires their penetration into the cell and mainly into the nucleus, where they both intercalate and adlineate with nuclear DNA [11, 12]. We have used the anthracyclines daunomycin and adriamycin, linked to anti-tumor immunoglobulins or antibodies [13-15] as specific carriers to target cells, in an attempt to minimize the detrimental effect of these toxic drugs on normal cells. The covalently linked drugantibody conjugates were shown to retain an appreciable amount of drug activity in vitro, and, as found recently, also in vivo. We have since synthesized conjugates in which the linking of the anthracyclines to

MATERIALS AND METHODS Daunomycin-HCl (lot 33) was a gift from Farmitalia (Milano, Italy). Tritiated daunomycin (specific" activity 14Ci/mmole) was a gift from New England Nuclear (Mass., U.S.A.). Sodium boro[3H]hydride (specific activity 293 mCi/mmole), and [5-3H]uridine (specific activity 25 Ci/mmole) were purchased from the Radiochemical Centre (Amersham, England). [6-3H]Thymidine (specific activity 4.5 Ci/mmole) was obtained

Accepted 20 February 1978. *The Research described in this publication was supported by a research contract, NCI-No. 1CB 640-49. §Michael Sela is an Established Investigator of the Chief Scientist's Bureau, Ministry of Health, Israel. Abbreviations used: PBS, 0.15M sodium chloride0.01 M sodium phosphate, pH 7.2; Dau, daunomycin; Dex, dextran; NP40, Nonidet P40; Ig, immunoglobulin; TLC, thin layer chromatography. ¢

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E. Hurwitz et al.

from Nuclear Research Center, Dimona, Israel). Sodium borohydride was purchased from Merck (Schuchardt, Germany). Sodium periodate and mannitol were obtained from British Drug House (Poole, Englend). Biogel P-60 was purchased ti~om Bio-Rad (Calif, U.S.A.). Antibodies were prepared in goats against a soluble papain digest of a tumor cell membrane (Yac lymphoma carried out as a line in A/J mice) [17]. The antibodies were purified by the use of glutaraldehydefixed Yac Cells (107 cells/ml with 0.1c}o glutaraldehyde for 90min at room temperature) as an immunoadsorbent and followed by elution with 2 M NaI (Merck, Darmstadt, Germany), by batch elution twice tbr 1 hr at 37°C. The normal goat immunoglobulins were precipitated from goat sera by 33% saturated ammonium sulphate.

Preparation of daunomyein conjugates The binding of daunomycin to antibodies or to normal Ig was performed as previously reported [13], by oxidation of the sugar moiety of' the daunomycin with periodate, linking it to the amino groups of the protein, followed by reduction of the Schiff bases with sodium borohydride. The only difference was that in the present study mannitol was added to the pure daunomycin in a ratio of 5/1. The oxidation of daunomycin in the absence of mannitol, even when the concentration of sodium borohydride was decreased fi~om 0.1 to 0.02M resulted in loss of solubility and lower yields. The binding of daunomycin to dextran was performed by the use of periodateoxidized dextrans [18]. The Schiff bases formed between the aldehyde groups on the oxidized dextran and the amino group on the sugar moiety of the drug after an overnight incubation at room temperature, were quantitatively reduced by sodium borohydride for stabilization of the binding.

Daunomycin-dextran-antibody or IgG Oxidized dextran to which daunomycin had been linked (ratio of daunomycin to dextran 1:2) was incubated overnight with the immunoglobulin fraction or purified antibody preparation (Ig to dextran ratio 1:1) at 4°C and the resulting complex was reduced by NaBH4 as above. Dextran-bound drug or Ab-dextran bound drug were separated from free drug by gel filtration on Biogel P-60. The conjugates were characterized as follows:

The daunomycin was determined by the absorbance at 495nm, the antibody or Ig by absorbance at 280nm (the absorbance of the drug at 280nm was corrected tbr) and the dextran was quantitated by weight. Each preparation was checked by thin layer chromatography tbr the presence of unbound daunomycin. The various conjugates contained between zero and 3.6~)' ot the drug, indicating that the reduction was incomplete. However, it was impossible to use more drastic conditions for reduction, since this yields a product with damaged daunomycin as indicated by its decoloration. The daunomycin content in the various preparations was determined to be as follows: daunomycin-antibody conjugates; 2-3mole/ mole; daunomycin-dextran conjugates; approximately 1 mole/mole (of dextran T-10, mol.wt 10,000); and in the daunomycindextran-antibody conjugates between 10 and 30 mole/mole antibody.

Determination of drug activity Daunomycin activity was assayed by the inhibition of either uridine or thymidine incorporation. The drug or its conjugates were incubated with the tumor qells for a period of 2hr. Either 3H-uridine or 3Hthymidine were then added for 1 hr at 37°C. The analysis of the incorporated nucleotide was performed as described for 3H-uridine [13]. Overnight incubation (20hr) of the drug or its conjugates in addition to 3H-thymidine gave similar inhibition results to those obtained for the short incubation period.

3H-labeling of daunomycin In addition to tritiated daunomycin (New England Nuclear), daunomycin was labeled either by oxidizing its sugar moiety with periodate and then reducing the resulting aldehyde groups by NaB3H4, or by direct treatment with NaB3H4 without prior oxidation. The labeled preparations were checked by comparison of their chromatographic behaviour in TLC (in chloroform:methanol: water 13:6: 1) to that of unmodified daunomycin. The R r of the oxidized and reduced daunomycin was 0.66, that of the NaBaH4 treated material was 0.68 as compared to R F 0.57 of cold daunomycin. The oxidized and reduced daunomycin lost an appreciable

Daunomycin-hnmunoglobulin Conjugales, Uptake and Activity In Vitro 1

I

r

l

100 ¢..

.9 8 0 =~ 6 0 40

20 L

L

L

L

I

5

I0

15

Drug H.g/ml

Fig. 1. Daunomycin and daunomycin-dextran aclivity determined by inhibition of 3H-uridine (broken line) and 3Hthymidine (continuous line) incorporation. (+) daunornycin; (~ ) daunomycin treated by NaBH4 ([I) daunornycinoxidized by periodate and reduced by NaBH4(AI) daunom3~cin dextran (dextran T40, 50~o) periodate oxidized. part of its drug activity, but the NaBH4 treated compound was as active as the original daunomycin (Fig. 1). The stability of the radioactive label was assessed by incubation of the labeled compounds at pH values 4.7 and 9.5, respectively, for 24hr, after which the solutions were absorbed on a poropak Q column, and both efluent and eluent were monitored for radioactivity. Ahnost no loss in the radioactivity ( ~ 0.3° 5) was observed in acidic or neutral pH. Under basic conditions (pH9.5) a loss of about 9°~} was obtained. These results show that the stability of the label in the daunomycin is quite high and should not interfere with the uptake experiments. This is true both when the label is in the sugar moiety (in the oxidized and reduced preparation) and when it is in the quinone ring or the keto side chain (in the reduced product).

Radioactive labeling of daunomycin-dextran and daunomycin-dextran-antibody conjugates The conjugates were labeled with tritium by different procedures: (1) Tritiated sodium borohydride reduction of the Schiff bases formed during the binding process. (2) Preparation of the conjugates with tritiated daunomycin (catalytic exchange with ~H gas). (3) Combined tritiated daunomycin and reduction by NaB3H4, The stability of the label was determined by dialysis of the compound against PBS for 6 h r and monitoring the radioactivity both inside and outside of the dialysis bag. The loss of

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radioactivity during the dialysis was approximately 4~!~,. When the labeled conjugate was tested by TLC, the anaount of radioactivity retained in the origin was about 70°o of the total.

The cellular uptake of daunomycin and daunomycin conjugates The uptake of daunomycin and its conjugates into cells, and their intracellular distribution, were tested by incubating 5 x 106 tumor or normal cells, with various concentrations of the radioactively labeled cornpounds, at 3 T C and tbr several periods of time mostly up to 4hr. At the termination of the incubation period the cells were washed extensively, and the extracellular unbound drug or drug-conjugates were counted. The cells were then incubated for 30min in 1°/0 Nonidet P40 at 4°C, followed by centrifugation (Sorvall 15,000 rev/min tbr 10min). An aliquot (0.1ml) of the supernatant, which included the fraction rich in cellular membranes and cytoplasmic contents, was counted. The pellets composed of the nuclei rich fraction were dispersed in 0.1~).2ml 1N N a O H and transferred into scintillation vials for counting. The scintillation fluid contained a mixture of toluene, tritone X-100 and 0.1NHC1 at a ratio of 6:3:.t. Testing the stability of daunom~cin conjugates in the presence of cells Yac cells (1 x l07) were incubated for various periods with radioactively labeled daunomycin conjugates. Incubation was tbllowed by centrifugation. Aliquots of the supernatants were applied to columns of biogel P-60. The antibody-bound and the unconjugated drug were detected according to their elution position from the columns and quantitated by radioactivity and by optical density at 495nm. The same tests were performed with the NP-40 supernatants of the postincubated washed tumor cells.

RESULTS

The activity of daunomycin and its various conjugates as measured in vitro is shown in Table 1 and Figs. 1 and 3. The activity was determined by inhibition of either 3Huridine or 3H-thymidine incorporation. When daunomycin was directly linked to anti-Yac antibody or to normal Ig, an appreciable amount of its original activity was retained in the conjugates. Linking of daunomycin

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E. Hurwitz et all Table 1. In vitro activity of daunomycin and daunomycin-conjugates %. inhibition of 3H-uridine incorporation*

Dau cone. #g/ml

Dau

Dau-dex

Dau-~tex-anti-Yac

0.25 0.50 1.00 5.00 15.00 50.00

11 42 60 90 -

0 2 50 80 -

. 8 9 44 63 90

Dau d e x - N I g .

Dau anti-Yac

. 4 17 48 58 92

Dau-NIg

. 22 44 86 -

39 62 90 -

*Yac cells (106) were incubated for 2hr at 37°C in the presence of daunomycin or daunomycinconjugates. Incorporation of [3H]-uridine into cellular material precipitable by trichloroacetic acid was measured and expressed as per cent inhibition (100% of control culture containing no drug).

A

I

B

30 "T:) "0 "0 C)

E

~ZO U

"6

.-e t0

]incubation eime(hrs)

0 DOU

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0

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4

D4u-cles-Ab

0

I

4

Dou-Ab

0

I

4

04u-Nlql

0 ~4

0 2 4

0 24

Oou

Dou-Ab

Oeu-Nl|

Fig. 2. The in vitro uptake of daunomycin and its conjugates by Yac cells (A ) and normal rat lymphocytes (B ), measured as a function of time. Measurements of uptake were made at zero time and after 0.5, 1, 2, 3 and 4 hr following the addition of drug. The total drug accumulation in the cells is denoted by the open bars, whereas the nuclear incorporation by the shaded areas.

to dextran resulted in a reduction of drug activity and when the dextran served as a bridge between the drug and anti-Yac a further decrease in the capacity to inhibit uridine and thymidine incorporation was observed. Daunomycin, in which the amino sugar was periodate-oxidized and reduced, also showed a decrease in activity, whereas the mere treatment by sodium borohydride had no apparent effect on the in vitro activity of the compound, The uptake of daunomycin and its conjugates by Yac or normal cells (rat splenocytes) is depicted in Fig. 2A and B. It is expressed as % uptake of the total radioactivity added to a fixed number of cells. The distribution of daunomycin

or daunomycin-conjugates in the membranes or the cytoplasm and its possible attachment to the nucleus was measured as a function of time of incubation. As can be seen, the free drug controls; daunomycin, oxidized and NaB3H4 reduced, or NaB3H4 treated, penetrated the cells very rapidly. Since the uptake assay was performed by centrifugation and washing at 0°C immediately after the addition of the drug, high intracellular levels of the free drug were observed at the zero point. Optimal levels of uptake was achieved at a drug concentration of 15-30 #g/ml, with a slight drop at 60 #g/ml. Uptake of the free drug reached a peak at l hr incubation, and declined after 3hr. The accumulation of the drug in or on the

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Daunomycin-Immunoglobulin Conjugates, L'ptake and Activity In Vitro

nuclei (shaded areas in the chart) paralled its uptake by the intact cell and amounted roughly to two thirds of the total cellular content. 3H-daunomycin, prepared by catalytic exchange, behaved somewhat differently; i.e. most of the drug added (90% or more) penetrated the nucleus rapidly. In addition, no reduction in its uptake was noted after 3hr and no decline in its uptake was observed at a concentration of 60 #g/ml. Daunomycin-antibody conjugates labeled by NaBaH4 reduction, whether directly bound or via a dextran bridge, penetrated into the Yac cells and attached to their nuclei, although at a much lower rate than that of the free drug (Fig. 2A). As depicted by the results, daunomycin bound directly to antibodies entered the cells at higher levels than the drug bond, through dextran, to normal Ig. The optimal concentrations were similar to those obtained for free drug without an observed dependence on the amount of antibody in the preparation (the range of concentration tested with 1-3 mg/ml). Drug bound to antibody via the dextran bridge was taken up somewhat more readily at higher concentrations (60-80 /lg/ml). The uptake of the bound substances proceeded at a slower rate than that of free drug, peaked at 2 - 3 h r and slightly decreased after 4hr. Except for the decline after 4hr, this corresponds well with the activities of the bound and free drug (Fig. 3). In assays of drug activity maximal levels are reached by the free drug after approximately 90min, while longer incubation periods (up to 4 h r ) are needed for daunomycin conjugate with either dextran or with antibodies via dextran for reaching their maximal potency. As is also depicted in Fig. 2, the accumulation of drug on the nuclei paralleled uptake by whole cells, comprising half to two thirds its value. In normal non-related cells (rat splenocytes) the uptake of free drug was slightly lower than that by the tumor cells. The daunomycin conjugates with either anti-Yac antibodies or normal Ig (linked directly), were taken up at low rates (Fig. 2B) and did not differ from each other. This was expected, since these cells do not bind goat anti-Yac. Daunomycin-dextran and d a u n o m y c i n dextran-antibody conjugates were tested for their stability in their intracellular and extracellular form. As can be seen from 'Fable 2 both compounds preserve the conjugated

form in the presence of cells up to 24hr. After cell penetration an appreciable amount of free drug remains bound to the antibody dextran (70-75%) and to the dextran alone (43-44%) in the detergent supernatant. In each case the antibody in the conjugate adds to the stability of the complex. The uptake of conjugates labeled at different positions in the complex is depicted in Fig. 4. In c o n j u g a t e s , whereby only the drug is radioactive, the label accumulates mostly (90°/'0) in the nucleus. On the other hand, in conjugates labeled at the attachment I

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I 3

1 4

30 E

e~

o

20

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Incubation period (hrs)

Fig. 3. The relationship between daunomycin or its conjugates uptake (A) and activity (B) as a function of the incubation period with Yac cells. ( 0 ) daunomycin; (x) daunomycin-antiYac; (+) daunomycin-dextran-anti-Yac. Concentration (f daunomycinfree or in the conjugates is 5 #g/ml. Table 2. Stability of daunomycin conjugates in the presence of Yac cells and after their penetration into the cells

Conjugated drug, %, of total* Cellsupernatant~ NP40supernatant++ Incubation (hr) 3 24 3H-dau~tex 3H-dau-dex-Ab

65 95

52 89

Incubation (hr) 3 24 43 70

49 75

*The results reported are an average of two experiments. tYac tumor cells were incubated for 3 and 24hr with the conjugates and the supernatants were applied to biogel P-60 columns and analyzed. ++Theabove cells were extensivelywashed and incubated for 30min in 0.5ml 1% NP40. Aliquots of these supernatants were applied to biogel P-60, columns and analyzed.

E. H u r w i t z et al.

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DO

®

ir-

g EQ . o

50

1

(a)

(b)

Dau

(a)

(b) (c)

Dau-dex-Ab

Fig. 4. Daunomycin and daunomycin-conjugates in or on the nucleus after cell penetration. The percent of cpm on the nucleus

out of the total penetration cpm was measured with differently labeled compounds. Dau: (a) NaB3H4 treated (b) Tritiated. Daunomycin dextran anti-Yac (Ab): Reduced by NaB3H4. (b) Conjugated with tritiated daunomycin. (e) Both conjugated with tritiated daunomycin and reduced by NaB3H4. Incubation ./or 1 hr; shaded area,./br 3 hr; open area.

point between the drug and the macromolecules total radioactivity is divided between the cytoplasmic contents and the nucleus (only about half to two thirds in the latter). Similarly, when tritiated daunomycin was bound to dextran or to dextranantibody, and the reduction was performed with tritiated sodium borohydride, radioactivity was similarly divided between cytoplasmic and nuclear fractions.

DISCUSSION Daunomycin is known to react with polynucleotide compounds. The intercalation and adlineation of daunomycin and similar derivatives to DNA was established by various physico-chemical methods. Thus, the cellular activity of the drug is assumed to be mainly nuclear in nature, causing the unavailability of template DNA both to DNA and R N A polymerases [11, 12]. Whether this describes the full scope of the drug activity is not entirely clear. Various data and experimental observations seem to indicate that a wider spectrum of activities could be attributed to daunomycin, including undesirable effects such as cardiac toxicity and carcinogenesis [19], in addition to the observed beneficial effects, i.e. cytotoxicity to tumors. Antimitotic activity and cell destruction was observed with doses ineffective as inhibitors of macromolecular synthesis. In uptake studies most

of the drug was accumulated in the nuclei and only a small amount in the mitochondria and microsomal fractions. However, chemical modifications, such as those shown by Israel et al. [20], showed that very active derivatives were mostly located in the cytoplasm [21] and not in the nucleus. In the experiments carried out in this study tritium-labeled daunomycin and its conjugates were used. The stability of the label in all the compounds was checked in order to ensure that the extent of tritium exchange during the 4 h r required for uptake experiments, will not interfere with the experiments or affect their interpretation. The conjugates were found to be stable in the presence of cells during the experiment (Table

2). The binding of daunomycin to various macromolecules affects its capacity to be taken up by the tumor cells and their nuclei (Fig. 2). Direct daunomycin antibody conjugates were taken up more readily than those prepared via a dextran bridge. Both of the latter were taken up better than their controls, daunomycin normal Ig and daunomycin-dextran (daunomycin-dextran was completely excluded from the cells in some preparations). In preliminary experiments with double labeled anti-Yacdaunomycin, where the anti-Yac was labeled by 12si and the drug by 3H-, both labels were tound to be located on the nucleus. The extent of label tbund on the nuclei amounted to 50q50~Io of the total intracellular drug content. The per cent of drugconjugates taken up by the cells did not change appreciably with increasing concentrations. The uptake was more dependent on concentration with free tritiated daunomycin, similarly to observations of uptake with Hela cells [22]. Free drug uptake is maximal alter 1 hr incubation and declines gradually alter 3hr. This is in a g r e e m e n t with results observed with L1210 and 3T12 cell lines [11, 13]. Daunomycin bound to anti-tumor antibodies was taken up more effectively by respective tumor cells than by normal rat splenocytes. With the normal cells no difference was observed between daunomycinanti-tumor antibodies or daunomycin-immunoglobulin. These findings emphasize the contribution of the surface contact to the uptake by the cells. No close correlation is observed between the uptake capacity and the inhibitory activity (Fig. 3). All conjugate types retained

Daunomycin-Immunoglobulin Conjugates, Uptake and Activity In Vitro a significant amount of the original drug activity. The daunomycin-dextran conjugate was a better inhibitor of nucleotide incorporation than daunomycin-dextran-antibody conjugates (Table 1). All conjugates prepared were at least as active as the free drug in the suppression of tumour development in vivo, and daunomycin~textran and daunomycin-dextran-antibody were in many instances even better [24]. Thus, the cytotoxic activity of the conjugates is not directly related to the penetration of the conjugates into the cells. The mechanism by which the covalently bound daunomycin conjugates exert their intracellular activity is probably similar to that believed to operate for the non-covalent d a u n o m y c i n - D N A complexes. The first step in this process is pinocytosis, for which affinity of the complex to the cell surface plays an important role and facilitates the entrapment by the cells. Hence, daunomycindextran or daunomycin-dextran-lg which have no surt~tce affinity to either tumor or normal cells are taken up at lower levels than daunomycin linked to specific antibodies. Daunomycin directly linked to antibody could be envisaged to enter the nucleus in the conjugate tbrm, or, alternatively, it might be released from the carrier prior to its penetration into the nucleus. The

1219

different labeling methods used in our study suggest that the latter process takes place. When most of the radioactive label was in the dextran bridges between daunomycin and antibody, incorporation studies showed that about half of the incorporated radioactivity was tbund in the cytoplasm and only half to two thirds attached to the nucleus. On the other hand, when tritiated daunomycin was bound to the conjugate so that all the radioactivity was located on the drug, most of the label was found in the nucleus after 3hr of incubation. When the two labeling methods were combined, uptake was again divided between cytoplasmic contents and nucleus (Fig. 4). Furthermore, linking daunomycin to antibodies by stable peptide bonds, such as direct coupling with carbodiimide between antibody and drug [13] or between an N-succinyl derivative of daunomycin coupled to the antibody via N-hydroxysuccinimide ester, no daunomycin activity could be demonstrated. The succinyl-daunomycin itself was as active as the original drug (unpublished results). Theretbre, it seems that daunomycin, in order to convey its inhibitory activity, is released fi'om its conjugates by acid or enzymatic hydrolysis. Further studies to elucidate the mechanism of action of daunomycin conjugates are in progress.

REFERENCES

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