Complement-dependent cytotoxicity of anti-human osteogenic sarcoma monoclonal antibodies.

Two mouse monoclonal antibodies against the human osteogenic sarcoma 791T were examined for their capacity to exert complement-dependent cytotoxicity against a panel of human tumour cell lines. Cytotoxicity was most evident against the immunizing tumour 791T although significant reactivity was directed against other osteogenic sarcomas. In admixture, the 2 antibodies displayed synergism in their cytotoxicity although this was only demonstrable with defined ranges of antibody concentration. The cytotoxicity of these antibodies was dependent upon the use of rabbit serum as complement and no tumour-cell lysis was produced using human, guinea-pig or mouse serum complement. The more potent cytotoxic antibody failed to modify the outgrowth of 791T tumour xenografts in immunodeprived mice even though localization of antibody at the tumour site has been demonstrated (Pimm et al., 1982).

, monoclonal antibodies have been produced which show preferential reactivity with malignant cells, although their potential as effective therapeutic agents has yet to be realized (reviewed by Baldwin et al., 1981). The powerful cytotoxicity of IgM and IgG2 monoclonal antibodies in the presence of complement suggests a direct role for these antibodies in limiting tumour development or controlling residual disease. Already, cytotoxic murine antibodies have been prepared against a number of human tumours, and therapeutic manipulations using the passive administration of antibody have been attempted in immunodeficient mice bearing human tumour xenografts (Herlyn et al., 1980;Herlyn & Koprowski, 1981) and with leukaemia in mice (Bernstein et al., 1980) and humans (Ritz et al., 1981;Miller et al., 1981) with varying degrees of success.
In preliminary tests, it was evident that the 2 anti-human osteogenic sarcoma monoclonal antibodies described by Embleton et al. (1981a, b) exhibited complement-dependent cytotoxicity against the immunizing osteogenic sarcoma cell line, 791T. In the present investigation, the capacity of these antibodies to mediate complement-dependent cytotoxicity against a panel of osteogenic sarcomas and other tumours has been evaluated using a short-term 51Cr-release test. An important aspect of this work was to determine whether the anti-tumour cytotoxic reactions were demonstrable using mouse and human sera as sources of complement, since these antibodies have therapeutic potential in model studies with human tumour xenografts in immunodeprived mice or in patients.
Antibody was isolated from culture supernatants by passage through a 5 ml packed bed volume of Sepharose-linked Protein A (Pharmacia, Uppsala, Sweden) at 50 ml/h. The nonbound fraction was discarded and the column was extensively washed with phosphatebuffered saline (PBS) pH 7-3, containing 0.02% (w/v) sodium azide. This column was then connected to a column of Sephadex G25 (Pharmacia) and elutionl of the bound antibody was achieved by application of 3M NaSCN. The eluted protein peak separated from the NaSCN by Sephadex G25 gel filtration was concentrated by positive-pressure membrane ultrafiltration using a PM1O Amicon membrane (Amicon, High Wycombe). Separate Sepharose-Protein A columns were used for the purification of anti-791T/36 and anti-791T/48 antibodies in order to avoid cross-contamination. The recovery of purified anti-791T/36 and anti-791T/48 antibodies was 16-8 + 8-0 and 4-3 + 2-9 jig/ml respectively of original hybridoma supernatants. Antibody concentrations were determined using the protein assay of Lowry or spectrophotometricallv assuming an E'% of 14-3 (Hudson & Hay, 1980). Both monoclonal antibodies were determined as belonging to the mouse IgG2b subclass as defined in immunodiffusion tests using mouse immunoglobulin-typing antisera (Miles Laboratories, Stoke Poges, U.K.).
Anti-HLA-A, B, C (shared determinant) Clone W6/32 HL monoclonal antibody was obtained as serum/ascites fluid (Ig content 2 mg/ml) from Sera-Lab. (Crawley Down, Sussex). Complement.-Lyophilized rabbit serum (Buxted Rabbit Co. Ltd, Sussex) was reconstituted with distilled water immediately before use. Guinea-pig (Dunkin-Hartley strain) serum, CBA/Ca mouse serum and human serum from laboratory personnel was stored at -70°C until use.
Complement-dependent cytotoxicity assay.-The complement-dependent cytotoxicity of anti-791T monoclonal antibodies was measured using a 2 h 51Cr-release assay (Price, 1978). Briefly, tumour target cells were labelled with 125,tCi Na251CrO4 (Radiochemical Centre, Amersham) for 60 min at 37°C in a volume of 0 5-1 0 ml Eagle's HEPES containing 5% FCS. After washing cells 3 x by centrifugation, cells were aliquoted (50 ,ul containing 104 cells) in quadruplicates into round-bottomed Sterilin M24A microtest plates which contained media, diluted heatinactivated (56°C for 1 h) sera or diluted monoclonal antibody preparations at 100 ,ul/well. As source of complement, rabbit, mouse, human or guinea-pig serum (100 ,ul aliquots) was added to each well at concentrations which had been predetermined for each cell line to be non-toxic for the cells alone. All dilutions were performed using Eagle's HEPES medium containing 5% FCS. After incubation at room temperature for 2 h, plates were centrifuged at 280 g for 10 min, and lOO1I aliquots of the supernatants were collected and counted for radioactivity using an LKB-Wallac gamma counter. The percentage release of 51Cr was calculated for each test sample and medium control samples. The maximum percentage release of 51Cr ( > 90% with all cell lines) was determined by the addition of 1% sodium dodecyl sulphate to labelled target cells, and the percentage release in samples exposed to medium alone did not exceed 15%. The percentage cytotoxicity values were calculated as [(% 51Cr release in test samples -% 51Cr release in medium control samples)/(maximum % 51Cr release -% 51Cr release in medium control samples)] x 100%.
Mice and xenografts.-CBA/Ca mice (Bantin and Kingman, Hull) were thymectomized at 3-4 weeks of age and 3-6 weeks later received 9 Gy whole-body y-irradiation from a 60Co source. The lethal effect of irradiation was prevented by i.p. injection of 200 mg/kg cytosine arabinoside (Cytosar, Upjohn, Crawley) 2 days before irradiation (Steel et al., 1978). Xenograft growths were initiated by s.e. injection of 106-107 tumour cells harvested withl trypsin from in vitro cultures and washed in serum-free medium. Mice were maintained on sterile bedding with sterilized food and water and held in isolator cabinets (Vickers Pathoflex Isolator, Basingstoke). They were exsanguinated by heart puncture, and the serum was collected and stored at -20°C. Mice were found to be free of macroscopically visible thymic remnants at the time of blood collection.

RESULTS
(omplement-dependent cytotoxicity of antihuman osteogenic sarcoma 791T monoclonal antibodies for various target tumour cell lines The anti-human osteogenic sarcoma monoclonal antibodies anti-791T/36 and anti-791T/48 belong to the IgG2b subclass with mouse K light chain. Murine IgG2b antibodies are complement-fixing (Stanworth & Turner, 1978) and the data in Fig. I confirm that both antibodies mediate complement-dependent cytotoxicity against 791T target cells employing rabbit serum as the source of complement.
At the highest antibody concentration tested (125 .tg/ml), anti-791T/36 monoclonal antibody reacted strongly with the 4 osteogenic sarcoma cell lines 791T, 788T, 278T and 208, and the cross-reactive prostate carcinoma cell line EB33. This antibody displayed no reactivity for the cell lines A549 (lung carcinoma) and PAl (ovarian carcinoma). At the lower antibody concentrations, down to 1 ,ug/ml, it was evident that the cell lines 791T and 788T were the most susceptible to antibody-induced, complement-dependent cell lysis.
The other anti-human osteogenic sarcoma 791T monoclonal antibody, anti-791T/48, exhibited much lower reactivity in these tests although significant levels of cytotoxicity were recorded against the line 791T. This finding is in accord with those of preliminary experiments in which anti-791T/48 hybridoma supernatants failed to induce complement-dependent cytotoxicity. In such supernatants, antibody concentrations are approximately 5 ,ug/ml, which clearly is insufficient for demonstrable cytotoxicity (Fig. 1). All cytotoxic reactions against the various cell lines in Fig. 1 were dependent upon the presence of active rabbit serum complement, and all cytotoxic values determined in the presence of heatinactivated complement (56TC for 1 h) were in the range -0 5 to 1 9% cytotoxicity. The pattern of reaction of the 2 monoclonal antibodies with these cell lines parallels those demonstrated using the 125I-Protein A cell-binding assay (Embleton et al., 1981a, b).
In a second experiment, using the crossreactive human osteogenic sarcoma cell line 788T, synergistic lysis of target cells was less evident, although it was increased 3-fold (from 16-2 to 43.4% cytotoxicity) with anti-791T/36 and anti-791T/48 at 1 and 125 ,g/ml respectively (Table II). The findings with 791T and 788T cells indicate that the 2 antibodies can act synergistically in complement-dependent cytotoxicity tests, although only within a fairly narrow range of concentrations.
Effect of source of complement on the cytotoxicity of anti-791T monoclonal antibodies While both anti-791T monoclonal antibodies mediated complement-dependent cytotoxicity against 791T cells with rabbit serum complement ( Fig. 1 and Tables I  and II Symbols *, t, t, as footnotes to Table I.   TABLE III.-Complement-dependent cytotoxicity of anti-791T monoclonal antibodies: screen of complement sources % cytotoxicity against
To evaluate whether the human, mouse and guinea-pig sera selected exhibited complement activity, tests were performed using the anti-HLA-A, B, C (shared determinant) monoclonal antibody W6/32. As target cells, 791T tumour cells and PHAinduced human peripheral blood lymphocyte blasts were employed (Table III). This antibody was cytototoxic for 791T target cells in the presence of rabbit serum complement, but not with mouse, human or guinea-pig serum. However, lysis of PHA-induced blasts (40.7-76.3% cytotoxicity) was produced by W6/32 using rabbit, human and guinea-pig serum complement (but again, not with mouse serum complement). In all cases, the cytotoxic reactions were shown to be complement-dependent, and all cytotoxicity values determined in the presence of heat-inactivated (56°C for 1 h) complement were between -0-8 and 1.2% (Table III). At the dilution of complementsource serum used (final dilution 1/5 in all tests), there was little or no evidence of natural cytotoxic activity (due to alloor heterophile antibodies) against 791T cells or PHA-induced blasts, except that rabbit serum produced a low, complement-dependent cytotoxicity of 8-4 + 0.8% against PHA-induced blasts (Table III). Lytic activity of serafrom mice bear'ing 791T tumour xenografts 791T tumour cells when injected s.c.
In vivo reactivity of anti-791T/36 monoclonal antibody 791T-xenografted, CBA/Ca mice were treated with the more potent monoclonal antibody anti-791T/36, using total inocula of 120 and 240 ,ug/mouse (Figs 3(a) and (b), respectively) administered in 3 equal intraperitoneal doses on Days 0, 7 and 14. However, the outgrowth of tumours in treated mice was essentially equivalent to that in untreated control animals, and there was no statistically significant difference in the incidence of tumours in test mice (8/11) and controls (10/11) (Fig. 3). DISCUSSION The pattern of complement-dependent cytotoxic reactivity of anti-791T/36 and anti-791T/48 monoclonal antibodies for various tumour target cell lines, and using rabbit serum complement, paralleled that defined using an 125I-Protein A cellbinding assay (Embleton et  reactivity against the immunizi 791T (Fig. 1). In addition, in the 2 antibodies mediated killing of 791T and 788T oste comas, although this was on within a restricted range of con (Tables I and II). The synerg observed were not as pronounc demonstrated by Hellstr6m el using 2 anti-melanoma mono( bodies which together gave up cytotoxicity at antibody dilut with either antibody alone hK no effect. The explanation for in the melanoma studies the 2 reacted with 2 distinct antig minants upon the same cell-su cule, p 97 (Hellstrom et al., 11 anti-osteogenic sarcoma antil however, considered to be rei antigenic determinants on se surface molecules. The patter] reactions of the 2 antibodies with various TEST 314 tumour target cells are distinct and clearly distinguishable, suggesting that CONROLS 5/5 they are reactive with separate surface antigens (Embleton et al., 1981a, b). It is not clear why synergistic killing occurred only with mixtures of low concentrations of anti-791T/36 and high concentrations of anti-791T/48 antibodies. It is possible that the antigen defined by anti-791T/48 displays restricted lateral mobility within -----------the plane of the surface membrane so that it represents a poor target for cytotoxic reactions which require close proximity TEST 517 of 2 cell-bound IgG antibodies to initiate ACONROLS 5/6 the complement cascade. The addition of small quantities of the more cytotoxic antibody anti-791T/36, defining a more mobile surface antigen, would then be expected to enhance cytotoxicity in a synergistic manner. 30

40
The 2 anti-osteogenic sarcoma monoclonal antibodies were not cytotoxic for s.C. 791T 791T cells using mouse, human or guineali-791T/36 pig serum as complement (Table III). was ms Mouse complement is known to be both doses as Lted mice weak and labile so that the failure to demonstrate cytotoxicity using this source of complement is not unexpected. Other ing cell line, studies have demonstrated that normal admixture or nude mouse serum complement is insynergistic effective in mediating lysis compared with ,ogenic sar-rabbit, human or guinea-pig serum (Drake tly evident et al., 1973;Herlyn & Koprowski, 1981), centrations although murine IgG2b monoclonal anti-:istic effects bodies have been shown to fix mouse -ed as those complement in haemolysis assays (Neut al. (1981) berger & Rajewsky, 1981. In contrast to clonal anti-the anti-human colon carcinoma monoto 70-90% clonal antibodies described by Herlyn & tions which Koprowski (1981) which utilize human ad little or complement to effect lysis, the anti-791T this is that antibodies were non-cytotoxic in the I antibodies presence of human serum (Table III), renic deter-although one obvious difference is that irface mole-the anti-colon carcinoma antibodies belong 981). The 2 to the IgM immunoglobulin class whereas bodies are, the anti-791T antibodies are IgG2b. active with Clearly, the human sera tested as com-,parate cell plement sources were not deficient in this ns of cross-activity, since PHA-induced blasts were lysed by the monoclonal antibody W6/32 and these human sera complement sources. However, even this antibody failed to lyse 791T target cells in the presence of human serum complement and again rabbit serum complement was required (Table III).
Before attempting therapy of 791Txenografted mice using these monoclonal antibodies, the sera from immunodeprived tumour-bearing mice were examined for anti-tumour activity and it was determined that about 30-40% of animals had circulating antibodies which were highly cytotoxic for 791T target cells (Fig. 2). This cytotoxic response was not a "natural" humoral reaction against tumour cells, since non-tumour-bearing immunodeprived mice did not display this activity even though T-deprivation by thymectomy and irradiation has been reported to increase natural anti-tumour activity in mice (M6nard et at., 1977;Colnaghi et al., 1977). The reactivity of the circulating antibodies was directed against surface antigens other than, or in addition to those defined by the anti-791T monoclonal antibodies since these antibodies were cytotoxic for the anti-791T/36 and anti-791T/48-negative cell line, A549 (Table V). They were not cytotoxic using mouse complement (Table  IV), so that they would not be expected to limit tumour growth in vivo by their cytotoxic reactivity. The presence of these antibodies in a proportion of tumourbearing mice also prevented the type of therapeutic manipulation attempted by Herlyn & Koprowski (1981), who administered anti-colon carcinoma monoclonal antibody and rabbit serum as complement to nude mice bearing tumour xenografts.
The monoclonal antibody anti-791T/36 was selected for the in vivo tests for 3 reasons: at a practical level, it was available in much greater quantities than anti-791T/48, it is the more cytotoxic of the 2 antibodies ( Fig. 1) and, finally, tests performed using radiolabelled anti-791T/36 have shown that this antibody localizes in vivo in 791T xenograftsinimmunodeprived mice (Pimm et A., 1982). The latter is an essential property if any direct antitumour activity is to be demonstrated although, as illustrated in Fig. 3, the administration of anti-791T/36 antibody to 791T-bearing mice modified neither tumour development nor incidence. This finding would also infer that antibodydependent cell-mediated cytotoxicity is inoperative with this antibody, tumour and treatment protocol. Such a mechanism has been invoked to account for the inhibition of growth of human colon carcinomas in nude mice by administration of an anticolon carcinoma IgG2a monoclonal antibody (Bernstein et al., 1980).
The results of this study demonstrate that anti-human osteogenic sarcoma 791T monoclonal antibodies are cytotoxic for tumour cells in vitro, although the full expression of their cytotoxicity depends very much upon the use of rabbit serum as the source of complement. With regard to continuing clinical studies which have demonstrated tumour localization ofradiolabelled anti-791T/36 antibody preparations by y-scintigraphy, the present findings predict that with tumours showing antibody localization no therapeutic benefit will be produced by complementdependent cytotoxicity reactions. However, antibodies localizing to tumours may well represent suitable vehicles for the delivery of cytotoxic drugs or bacterial or plant toxins to the site(s) of the tumour.