Effects of mixtures and complexes of chlorambucil and antibody on a human melanoma cell line

Europ. J. Cancer Vol. 11, pp. 725-732. Pergamon Press 1975. Printed in Great Britain

Effects of Mixtures and Complexes of Chlorambucil and Antibody on a Human Melanoma Cell Line C. VENNEGOOR, D. VAN SMEERDIJK and Ph. RI~MKE Department of Immunology, Antoni van Leeuwenhoekhuis, The Netherlands Cancer Institute, Sarphatistraat 108, Amsterdam 1004, The Netherlands Abstract--Cultured human melanoma cells were incubated with chlorambueil, antibody, a mixture of the two, or a complex of chlorambucil and antibody. Antibody increased the cytotoxic action of chlorambueil when the incubation was simultaneous. The effect was dependent on the concentration of both drug and antibody. When incubation with antibody wasfollowed by incubation with chlorambueil, or the sequence of incubations was reversed, no synergistic effect on the tumour cells was noticed. The cytotoxic effect of the complex of chlorambueil and antibody was not higher than that of the mixture of chlorambueil and antibody.

unlikely that chlorambucil is sufficiently firmly attached to immunoglobulins under these conditions [13, 14]. Moreover, it has been shown that a mixture of drug and antibody or a separate administration of these agents can already be effective in vivo [11, 15-17] as well in vitro [14]. In this paper, we report our results of the binding ofchlorambucil to gamma-globulin, and also the effect of chlorambucil and antibody on h u m a n melanoma cells in vitro.

INTRODUCTION

CYTOTOXIC drugs are frequently used in the treatment of cancer. However, there are often limitations in their application as a result of an accompanying systemic toxicity. For this reason attempts have been made to bind cytotoxic agents to plasma proteins [1-4] which seem to be taken up rather selectively by cancer cells without prior extracellular hydrolysis [5-7]. A particularly attractive concept is the binding of a cytotoxic drug to specific, tumour-directed antibodies. Several reports have now been made of favourable effects of chlorambucil bound to antibodies in animal tumour systems in vivo [8-10], and it has also been possible to study the effect in vitro [11]. In addition, in a patient with disseminated melanoma, regression of all the metastatic nodules has been described after treatment with chlorambuciMabelled antibody directed to the tumour [9]. For linkage of drug to antibody the authors applied a method described by Linford et al. [12] for physical binding of chlorambucil to serum proteins without loss of its alkylating activity. However, recent studies indicated that it is

MATERIAL AND METHODS Phosphate buffered saline

Phosphate buffered saline (PBS), p H 7"2, consisted of NaCI, 8.0gfl; KC1, 0.2g/l; KH2PO4, 0"2 g/1 and Na2HPO4"2H20, 1-15 g/l, Melanoma cells

H u m a n melanoma cells, NKI-2 and NKI-4, have both been developed to cell lines in our laboratory [18]. The NKI-2 cells were used for immunisation. The cytotoxicity tests were performed with NKI-4 cells which were subcultured

Accepted 6 June 1975. 725

726

C. Vennegoor, D. van Smeerdijk and Ph. Riimke

in a CO2-incubator at 37°C. The medium consisted of MEM-Eagle containing 10% (v/v) foetal bovine serum (Gibco Biocult, Glasgow, Great Britain), and a mixture of antibiotics, namely Na-penicillin G (100U/ml), streptomycin (0.1 mg/ml), pimaricin (2"5 mg/ml) and kanamycin (0.1 mg/ml). All antibiotics were obtained from Mycofarm (Delft, The Netherlands).

Antiserum preparation The cultured melanoma cells (NKI-2) to be used for immunisation were detached from the tissue culture flasks by incubation with 0.54 m M EDTA in PBS for 3 min at 37°C. Immediately after the incubation an equal volume of PBS containing 0.65mM CaC12 and 0 . 5 4 m M MgSO4 was added. The cells were washed five times in PBS. A suspension of 108 living cells in 1 ml PBS (without adjuvant) was injected three times, with weekly interfalls, into the ear vein of a rabbit. One week after the last injection blood was collected by cardiac puncture.

Sera and gamma-globulinfractions Sera. Sera were inactivated by incubation at 56°C for 30 min, and stored in small aliquots at - 20°C. The antiserum was used unabsorbed, since absorption with normal tissue reduced the titer to such an extent that antibodies against melanoma cells could hardly be detected. Moreover, for the present in vitro studies it was unimportant whether the antibodies were directed to normal or to tumour associated antigens. Detection of antibodies was performed with the membrane immunottuorescence test. With NKI-4 cells the titer of the antiserum, expressed as the serum dilution that stained 50% of the cells, was 800. The control serum diluted 1:10 was negative. Preparation of gamma-globulin. All steps were carried out at 0 - 4 ° C . The sera were diluted two-fold with PBS. Fractions containing largely IgG were prepared by adding (NH4) zSO4 to a final concentration of 1.6 M (NH4) zSO4 (pH 7.2). The precipitate was washed two times with a 1.6 M (NHg)zSO 4 solution, dissolved, and dialysed against PBS. In some cases the gamma-globulin fraction was further purified by passage through a column of DEAE-Sephadex A-50 equilibrated in 0.41% NaC1, 0.08% Na2HPO4.2H20 and 0.02% NaH2PO4"HzO, pH 7.4119]. The protein containing fractions, which appeared to

be pure IgG after examination with immunoelectrophoresis, were concentrated by ultrafiltration under high pressure (membrane PM 10, Amicon Co., Lexington, Mass., U.S.A.) to concentrations of 10 mg protein per ml. The gammaglobulin fractions were stored in small aliquots at - 70°C.

Physical binding of chlorambucil to gamma-globulin Chlorambucil was linked to the gammaglobulin fraction of antiserum by a procedure described by Blakeslee and Kennedy [20]. Chlorarnbucil (Sigma, St. Louis, Mo., U.S.A.) was dissolved in 0.06 M N a O H and immediately added to an equal volume of either the gammaglobulin or the more purified IgG fraction of antiserum in PBS, previously diluted 1:2 in distilled water. The concentration of protein in the reaction mixture was 5 mg/ml and the protein to .chlorambucil ratio (w/w) was 1 : 1"52. In case of pure IgG, this ratio corresponds to a molar ratio of protein to chlorambucil of 1 : 750. The pH of the mixture was adjusted to 9.0 or 10.5 with 0.1 N NaOH. After incubation for 10 min at 37°C the sample was subjected to gelfiltration on a column of Sephadex G-25 (coarse), equilibrated in 0.15 M NaC1 and 0.01 M TrisHC1 (pH 8.5) at 4°C. The protein containing fractions were dialysed against PBS at 4°C for 24 hr and stored in small quantities at -70°C.

Determination of the active and hydrolysed form of chlorambucil A method described by Linford was applied [21]. Samples containing free or physically bound chlorambucil were extracted with ethanol. After centrifugation the supernantant fractions were dried by evaporation under reduced pressure. The residues were dissolved in a 0-1 M potassium phosphate buffer (pH 3.6) and extracted with either benzene, for isolation of the active form of the drug, or with ethyl acetate, in which both the active and hydrolysed (inactive) forms of chlorambucil are isolated. The extracts were evaporated till dry under reduced pressure and the residues were dissolved in absolute ethanol. The estimation of the dissolved residues was measured in the spectrophotometer in the region 230-280 nm with 2 nm interfalls. The amount of chlorambucil was determined from the absorbance at 258 nm after correction for protein [21], scattering [22] and recovery of chlorambucil after extraction [21 ]. As a reference a solution of 30/~g chlorambucil/ml ethanol was used with an absorbance of 1.55 corrected for scattering.

E,fects of Chlorambucil and Antibody on Human Melanoma In a number of experiments the active form of chlorambucil was measured by its alkylating activities in a modified Epstein technique [23]. Both methods for determination of the active form of chlorambucil gave the same results.

Fluorescent

727

ceils (~) O/^.,j~,,~..~O 0~.=,.',-~////~ "~ 100

100

80

- 80

60

/ / /X

Cytotoxicity tests These tests were carried out under sterile conditions. Melanoma cells (NKI-4) were detached from the tissue culture flasks with the aid of a phosphate buffered saline solution containing 0.54 m M EDTA as described under the immunisation procedure. The cells were centrifuged, resuspended in tissue culture medium and seeded in microtest plates No. 3034 (Falcon, Oxnard, Ca., U.S.A.) so that each well would have 100-150 cells attached to the bottom. After incubation for 18 hr at 37°C the culture medium was removed and freshly prepared solutions of chlorambucil and]or rabbit globulin fractions in culture medium which had been sterilised by passage through Millipore filters (pore size 0-22 #m, MiUipore, Bruxelles, Belgium) were added. The volumes of the incubation mixtures were 20 #1. The number of wells in a test or a control was usually 8, and occasionally 4 or 12. After incubation for 24 or 48 hr at 37°C the plates were carefully washed with PBS containing 0.65 m M CaC12 and 0 . 5 4 m M MgSO4. The cells which had neither died nor become detached were fixed in methanol, stained with crystal violet and counted under a microscope. When chlorambucil was tested, the drug was dissolved to a concentration of 10 mg/ml in 0 . 5 M N a H C O 3 . To this solution 4vol of 0.07 M NaCI were added and 5 vol of medium for tissue culture containing 20% foetal bovine serum. Control experiments with a 0.5 M solution of N a H C O 3, diluted like those containing chlorambucil, showed no significant reduction in cell number.

Membrane immunofluorescence Membrane immunofluorescence was carried out essentially as described by Lewis and Phillips [24], except that incubations were at 37°C for 30 min. As conjugate FITC-labelled horse anti rabbit immunoglobulin was used (PK-17, Red Cross Blood Transfusion Service, Amsterdam, The Netherlands). The percentage of stained cells was scored and as an end-point of titration the concentration of the test serum which gave 50% positive cells was used. As the percentage of positive cells was proportional to the logarithm of the serum concentration in 80-20% of positive cells (see Fig. 1), the titer could be easily D

40 0 20

t Xl

-

60

- 40

iI

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-

20

/ya/#//o 10.4

"-

~ 10-3~

10.2

10-1

C o n c e n t r a t i o n of gamma globelins (relatiYe 1o serum)

Fig. l. Membrane immunofluorescence o f a partially purified gamma-globulin fraction direc~d against melanoma cells o n the cell line N K I - 4 . © , © untreated gamma-globulin x - - - × chlorambucil linked to gamma-globulin

evaluated. The percentage of loss of antibody activity due to the chlorambucil binding procedure was calculated using the formula (1-B[A) xlO0, in which A represents the concentration of the globulin fraction, incubated with chlorambucil, which gives 50% fluorescent cells and B the concentration of the untreated globulin fraction giving 50% staining.

Statistical evaluation To test the significance of the difference in cell number between the test and the control in each microplate, we applied Student's t-test. A value o f P < 0.05 was considered significant.

RESULTS

Binding active chlorambudl to gamma-globulin and to serum proteins Attempts to link chlorambucil to the gammaglobulin fraction of serum, without loss of the alkylatlng activity of the drug, were made according to three methods described in the literature. These procedures have in common that linkage is acquired by incubation of protein with drug, whereafter the uncombined drug is removed by dialysis or gel filtration. The incubations were performed at a protein-chlorambucil ratio of 10:1 in the presence of 0.1 M NaHCO3 without adjusting the pH (pH 9.7) [11, 12, 25], the same after adjusting the pH to 3.5 [11], or at a protein--chlorambucil ratio of 1 : 1.52 at pH 9.0-10.5 [20]. The results are summarised in Table 1. Physically bound chlorambucil was

728

C. Vennegoor, D. van Smeerdijk and Ph. Riimke Table 1. Binding of chlorambucil to gamma-globulin and serum Incubation

Chlorambucil incubated with Partially purified gamma globulin fraction IgG Serum

Ratio protein to chlorambueil (pg/ml) 10: 1

1• 1"52 1: 1.52 10:1

Chlorambucil

Time

Temperature (°C)

pH

Physically bound Active Hydrolysed (%) (%)

1 hr 2 hr 2 hr 30 min 10 min 10 min 10 min 1 hr 2 hr 10 rain 2 hr

4 20 37 20 37 37 37 4 4 37 37

9.7 9.7 9.7 3.5 10.5 10.5 9.0 9.7 9.7 9.7 9.7

0 0.08 0 5"2I" 8.9 13.5 9.2 23 14 17 14

N.D.* N.D. 0 0 0 0.3 0 N.D. 0 0 0

Binding by alkylation (%) N.D. N.D. 49 27 2.2 N.D. N.D. N.D. 35 35 61

*Not determined. tThis figure is too high since chlorambucil precipitated at low pH.

recovered after extraction with benzene, indicating that it was present in its active form. This was confirmed by determination of chlorambucil with the Epstein technique. In the buffer used for dialysis only the total content ofchlorambucil was measured, without discrimination between the active or hydrolysed (inactive) form. The amount of the added chlorambucil that could not be removed by dialysis, nor be extracted with ethyl acetate from the dialysed incubation mixtures, was considered covalently bound to protein by alkylation. Occasionally, samples were investigated before and after they had been stored at - 2 0 ° C for 12 days. Virtually the same contents of active chlorambucil were found. This indicates that chlorambucil when physically bound to protein is preserved from both hydrolysis and alkylation at temperatures below zero. Comparison of the results of binding the active form of chlorambucil to gamma-globulin with that to whole serum, using a protein-chlorambucil ratio of 10: 1 at pH 9.7 (Table 1), indicated that chlorambucil could not be bound to the gamma-globulin fraction of serum by the procedure used for whole serum [12]. We also attempted to bind chlorambucil to gammaglobulin at low pH [11]. The major difficulty encountered consisted in the poor solubility of chlorambucil at low pH. When the gamma-globulin fraction of serum was incubated at a considerably lower proteinchlorambucil ratio 1 : 1.52 at high pH, binding of active chlorambucil to the protein occurred. The data presented were obtained after gel filtration of the incubation mixture on Sephadex G-25. After subsequent dialysis against PBS some 80%

of the chlorambucil initially bound to the gamma-globulin was still present in the active form. However, a considerable proportion of the protein precipitated in the majority of the samples, apparently in the form of a chlorambucil-protein complex since on centrifugation both the chlorambucil and protein contents of the samples were lowered, while the chlorambucil to protein ratio of the supernatant remained constant.

Determination of antibody activity The titer of antibody in the unabsorbed gamma-globulin fraction of the antiserum before and after incubation with chlorambucil was measured in the indirect membrane immunofluorescence test. In case chlorambucil was bound to gamma-globulin loss of antibody activity was about 50% (Fig. 1). The loss of antibody activity appeared not to be due to the presence of chlorambucil in the protein but to the exposure to high pH.

Effects of chlorambucil and antibody on melanoma cells in vitro Effect of chlorambucil alone. We first examined the cytotoxic effect of chlorambucil on human melanoma cells. Figure 2 shows the doseresponse. After incubation for 1 hr with chlorambucil no reduction in cell number was noticed. After prolongation of incubation to 24 hr or more, significant effects of chlorambucil were detectable. When the results were plotted on logprobability papei [27], the concentration of

Effects of Chlorambucil and Antibody on Human Melanoma chlorambucil resulting in 20% reduction in cell number was found to be 100 #g ml in case the incubation time after addition of the drug was 24 hr, and 50 #g/ml when the incubation time with the drug was 48 hr. Reduction (%)

100

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/'/ I 'X

8O

40

,

|

20

,

,

,

40 60 80 100

Concentration

,

200.

i

|

400 600

of chlorambucil (/tg/ml)

Fig. 2. Dose response on human melanoma cells ( NKI-4) of ehlorambucil. The vertical lines indicate twice the s.c. of the mean in both directions [26] 0 0 effect after 24 hr of incubation in the presence of chlorambucil x - - - x effect after 48 hr of incubation in the presence of chlorambucil.

Effect of chlorambucil bound to antibody. Chlorambucil linked to the unabsorbed antimelanoma gamma-globulin fraction was tested on melanoma cells in a 48 hr incubation period. As controls chlorambucil, gamma-gobulin, and the combination of free chlorambucil and gammaglobulin were used at the same concentrations as in the complex of chlorambucil and gammaglobulin (Table 2). Often no cytotoxicity of the complex was found and the effect was always equal to, or less than, that of the mixture of

729

chlorambucil and antibody. In some experiments the reduction in cell number obtained with the mixture of drug and gamma-globulin was significantly higher than that of each of the controls of chlorambucil and gamma-globulin. For this reason we further studied the effect of different combinations of chlorambucil and gamma-globulin on melanoma cells.

Effect of co-incubation with chlorambudl and antibody. Table 3 summarises the results when the tumour cells were incubated in the presence of different concentrations of chlorambucil and/ or gamma-globulin. Though normal gammaglobulin fraction never showed any immunofluorescence staining or complement dependent cytotoxicity, a cytotoxic effect was recorded in the combination with chlorambucil. The effect of the combination ofchlorambucil and gammaglobulin from antiserum instead of normal serum was only slightly higher. To compare reductions in cell number in the different combinations, we calculated the difference between the reduction in cell number after incubation with antibody and the reduction with control globulin. To obtain information as to whether the effects of chlorambucil and antibody were synergistic or only additive, we compared the effects of interaction between chlorambucil and gammaglobulins. The effect of interaction was calculated by substracting the sum of the data of the controls (i.e., reduction in cell number when the tumour cells were respectively incubated with only ehlorambucil and with only gamma-globulin) from the data obtained after incubation with the mixture of drug and gamma-globulin. Table 4 shows that, at a dilution of the gammaglobulin fraction of antiserum comparable with a serum dilution of 1 : 80, only a concentration of chlorambucil of 12.5 #g/rnl showed a significant effect on the turnout cells as compared with the controls, i.e. gave a significant value both for

Table 2. Effectof the complex of chlorambucil ana antiboay on human melanoma cells (NKI-4) ; time of inoubation 48 hrs

Agent Medium control Chlorambucil Antibody Chlorambucil + antibodyt Chlorambucil/antibody+ +

Chlorambucil concentration (pg/ml)

Antibody concentration Cell number (pg protein/ml) + s.d.

21 21 21

*Compared with the medium control. 1"Mixture of chlorambucil and antibody. ++Chlorambucil linked to antibody.

75 75 75

66.1 + 17.6 54.1 + 10.2 85.7 + 20-2 24"6 + 3.7 65-0 + 9.3

P-value*

> > < >

0.10 0.10 0-001 0"50

730

C. Vennegoor, D. van Smeerdijk and Ph. Riimke Table 3. Summary of the observed reduction in cell number of human melanoma cells after incubation with chlorambacil, gamma-globulin or a mixture of chlorambucil and gamma-globulin; time of incubation 48 hr Variation in reduction in cell number compared with the untreated control % ±s.d.

Incubation with

16-6±3.6"-- 20.3±5-1" Chlorambucil (12.5 #g/ml to 50/~g/ml) -10.7±6.0 --3.7±6.8 Normal gamma-globulin (diluted 1: 80 to 1: 20) - 8.1 ±6.2 - - 19.9+4-7" Gamma-globulin from antiserum (diluted 1: 80 to 1: 20) 21.3±4"6"-- 39.6+2.6* Mixture of chlorambucil and normal gamma-globulin Mixture of chlorambucil and gamma- 31.1 ± 3"3*-- 53.3_+2.5* globulin from antiserum *Significant; P < 0.05

Table 4. Effect of.interaction of chlorambucil with antibody during incubation with human melanoma cells; time of incubation 48 hr

1:80 Chlorambucil concentration (/tg/ml) 0 12.5 25 50

Dilution ofthe gamma-globulin ~action of antiserum 1:40

1:20

Interaction (% ± s.d.)

Difference in reduction* (% ± s.d.)

Interaction (% ± s.d.)

Difference in reduction (% ± s.d.)

Interaction (% ± s.d.)

Difference in reduction (% ± s.d.)

25-9±7"5t 21.0±7"6t 27-4±7-4t

2.6±7.7 10.5±5.0 t 9.8±5.2 2.5±4.4

24-6±7"2t 17"1±7"3t 31.2±6"9~

3.2±7-2 9-7±4"6t 10"2±5"0t 15.9±4.2~

2"1±6-5 3.4±6.4 13"1±6"1t

26.8±6.7 t 13.4±4.8 t 7.8±4.4 20"8±4"2t

*Difference in reduction in cell number compared with the control containing normal gamma-globulin. ~'Significant; P < 0.05. Interested readers may get the statistical treatment by writing the author C. Vennegoor directly.

difference in reduction and for interaction. With gamma-globulin from antiserum diluted to 1 : 40 significant effects were obtained at all three concentrations of chlorambucil tested. When the gamma-globulin fraction was diluted to 1:20 only significant results were obtained with a concentration of chlorambucil of 50 #g/ml. T h e effect of chlorambucil and antibody appears therefore to be dependent on the concentration of both chlorambucil and antibody.

Effect of separate incubation with chlorumbucil and antibody. T o obtain more information as to the necessity of simultaneous presence of drug and antibody, we examined the effects of incubation with chlorambucil, followed by incubation with gamma-globulin, and also the reversed sequence. In one type of experiment the melanoma cells were incubated for 24 hr with chlorambucil. Hereafter the medium was removed

and replaced by the gamma-globulin fraction from either normal serum or antiserum. After a second incubation for 2 4 h r the cells were washed, fixed and counted. In other types of experiments the incubations were first with the gamma-globulin fraction from either normal or antiserum for 24 hr and then with chlorambucil for 24 or 48 hr respectively. In all these experiments the concentrations of chlorambucil and gamma-globulin were as used in the experiment represented in Table 4. T h e reductions in cell number were in both types of experiments comparable with those obtained after co-incubation with drug and antibody (Table 3). However, there was no indication of any interaction between chlorambucil and gamma-globulin from antiserum when the incubations were separately. This indicates that the reduction in cell number. in the latter experiments could only be the result of additive effects of the drug and antibody.

Effects of Chlorambucil and Antibody on Human Melanoma DISCUSSION

Serum proteins readily bind or envelope chlorambucil and other alkylating agents without loss of their alkylating ability [4, 12]. The results of Linford and coworkers [12] suggested that the forces which bind chlorambucil to the serum proteins have their origin in the mutual attraction of benzene rings in the drug and the protein. The nature of a distinct protein will therefore determine the quantity ofchlorambucil which can non-covalently be bound in an active form. In our experiments active chlorambucil could only be linked to gamma-globulin or IgG when a considerably higher drug to protein ratio was used than is necessary for binding total serum proteins. The molar ratio of active chlorambucil linked to IgG varied from 55 to 100, which is approximately five to ten times higher than reported [20]. In the in vitro test system, however, the complex of chlorambucil and antibody never showed an increase in cytotoxicity compared with the control containing a mixture of drug and antibody. Several causes could underlie the failure of the complex to augment cytotoxicity in our test. It is most likely that during incubation with tumour cells the drug has become inactive as a result of alkylation with the carrier protein. Our results (Table 1) indicated that alkylation of chlorambucil to gamma-globulins occurred easily and we were not able to prevent this by masking the sulphydryl groups in the gamma-globulin fraction with etyleneimine [28].

Further possible events which might have occurred are dissociation of drug and antibody or hydrolysis of the active groups of chlorambucil during incubation with the tumour cells. When melanoma cells were incubated with a mixture of chlorambucil and antibody, a reduction in cell number could be observed, which appeared to be significant compared with the controls. This indicates that the cytotoxicity of chlorambucil was augmented specifically by antibody. The results are comparable with the in vitro studies of Rubens and Dulbecco [14], who used polyoma virus-transformed baby hamster kidney cells, and of Davies and O'Neill [11] with theta antigens of the mouse. It is an interesting feature that a combination of free chlorambucil and free antibody can also increase the life span of mice which are injected with lethal doses of turnout cells [11, 17]. In conclusion we can say that mixtures of chlorambucil and antibody were more cytotoxic for melanoma cells than complexes of chlorambucil and antibody. When, however, inactivation of the drug by alkylation with the carrier could be prevented, better results than reported in this paper might be obtained. Acknowledgements--The authors are obliged to Ir.

G. Hart for statistical evaluations, to Mrs. S. Swen for providing facilities for tissue culture, to Dr. C.

Benekhuijsen for determinations of chlorambucil with the Epstein technique and for fruitful discussions and to J. de Vries for gift of the sera and the melanoma cell line NKI-4.

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