Int. J Cancer 52,588-593 (1992) (a 1992 Wiley-Liss. Inc
%
Publication of the International Union Against Cancer Publication de I Union lnternationale Contre le Cancer
CHARACTERIZATION OF A MOUSE-HUMAN CHIMERIC ANTIBODY TO A CANCER-ASSOCIATED ANTIGEN R. ORLANDI, M. FIGINI, A. TOMASSETTI, S. CANEVARI and M.1 C O L N A G H I ~ Istituto Nazionale per lo Studio e la Cura dei Tumon, Division of Expenmental Oncology E, Via G. Venezian I , 20133 Milan,Italy. In an attempt to obtain a therapeutic antibody, the murine monoclonal antibody (MAb) MBr I (IgM,k), directed against human carcinomas, was converted in a mouse/human chimeric MAb of y l isotype. The chimeric MAb, y l CHI-MBrl, retains the ability to specifically bind tumor cells and tissues with no modification in its binding to the normal material tested. yl CHI-MBr I recognizes mucins and high-molecular-weight glycoproteins carrying the antigenic determinant and stains a neutral glycolipid extracted from MCF-7 cells. The chimeric and the murine MBrl efficiently cross-inhibit each other on the reference cell line MCF-7 and the calculated affinity constants amount to 3.8 X I O7 and I .7 x I O8 M-I, respectively. The human constant region allows ylCHI-MBrl to bind with the FcR on the human monocytic cell line U937 and to efficiently mediate antibody-dependent cellular cytotoxicity in the presence of human lymphocytes activated by IL2. In addition, y l CHI-MBr I, like the murine MBr I, mediatescomplement-dependent turnorcell lysis. Thus, by modelling a molecule with reduced size and increased functional characteristics, we have obtained a reagent which is more suitable for in vivo therapeutic approaches. ci
1992 Wiley-Liss,Inc.
The full exploitation of cancer immunotherapy with murine MAbs has been severely limited by the development of human anti-mouse antibodies (HAMA), which lead to a shortening of thc half-life of the administered MAbs and may reduce their therapeutic effect (Dillman, 1990). Since considerable difficulties are encountered in producing human MAbs by hybridoma technology, protein engineering has been used to convert mouse MAbs into mouseihuman chimeric MAbs by joining the immunoglobulin variable domains from mouse MAbs to h u man constant domains (Winter and Milstein, 1991). With the aim of developing new reagents which are more suitable for cancer immunotherapy, chimeric and reshaped MAbs directed against tumor-associated antigens have been produced and characterized (Sahagan et al., 1986; Riechmann et ul., 1988). Some have been administered to cancer patients, and a marked reduction in the HAMA response and a longer half-life have been observed, as expected (Hale at al., 1988; LoBuglio rt al., 1989). The mouse MAb MBrl (IgM, K), raised against the human mammary carcinoma line MCF-7 (Mtnard et al., 1983), recognizes a carbohydrate epitope carried by various glycoconjugates (Bremer et al., 1984; Miotti et al., 1989). The immunohistochemical reactivity of MBrl has been described (MarianiCostantini et al., 1984 a,b). MBrl has been cxtensively used in in vitro immunodiagnosis (Colnaghi et al., 1987) and active immunotoxins were obtained by coupling the MAb with the ricin A chain and restrictocin (Canevari et al., 1985; Orlandi et nl., 1988). In an attempt to exploit in iivo the thcrapeutic potcntial of the murine MBrl, the human version was generated by expressing in myeloma cells the variable domains as simple mouse/human chimeric MAb (Orlandi et al., 1989). We report here the biochemical and biological characterization of the chimeric MAb. MATERIAL AND METHODS
Transfected cells The transfected cells which produce the chimeric MBrl were maintained in Dulbecco’s modified Eagle’s minimum esscntial medium (DMEM; GIBCO, Grand Island, NY) con-
taining 10% FCS, 10 mM sodium pyruvate, 100 pgiml penicillin and streptomycin, 5 pgiml mycophenolic acid, 250 pg/ml xanthine and 20 pgiml hypoxanthine.
Target cells The human tumor-cell lines listed in Table I were obtained from the ATCC (Rockville, MD), with the exception of I G R O V l and OVCA432, which were kindly provided respectively by Dr. Benard (Institute G. Roussy, Villejuif) and Dr. Knapp (Dana Farber Institute, Boston, MA). The cells were maintained in RPMI-1640 medium (Microbiological Associates, Walkersville, MD) supplemented with 10% FCS, penicillin and streptomycin (100 Fgiml). The cells were checked for Mycoplasma contamination by electron microscopy and/or by bisbenzimide (Sigma, St Louis, MO) staining and were always found to be negative. Human peripheral blood mononuclear cells (PBMC) were obtained from healthy donors by fractionation on FicollHypaque (Pharmacia, Uppsala, Sweden) density-gradient centrifugation. The PBMC used in the irnniunofluorescence tests were activated by 3 different protocols: (1) 30 Uiml IL2 (Cetus, Emeryville, CA) for 6 days; (2) 200 uiml yIFN (Genenzyme. Cambridge, MA) for 6 days; (3) 1 pg/ml PHA (Wellcome, Beckenham, UK) for 3 days. Monocytes were obtained by adhesion to plastic: the PBMC were incubated for 1 hr at 37°C and thc adherent cells were harvested by vigorous pipetting. U937 cells wcre stimulated with 500 u/ml yIFN for 6 days. Antibody characteristics and purification The A3B10 MAb, raised against the variable domain of the murine MBrl, is an Ab2 p anti-idiotypic antibody since it rcacts with the binding site of MBrl and blocks the binding of MBrl to its relevant target antigen (Vialc et al., 1987). It was kindly provided by Dr. G. Viale (Dipartimento di Biologia e Genetica per le Scienze Mediche, Universita degli Studi, Milan) as ascitic fluid and was purified by Protein A chromatography. The ylCHI-MBR1 was purified by immunoaffinity on an anti-idiotypic column. The immunoabsorbent was prepared by coupling the purified A3B10 MAb to CNBr-activated Sepharose 4B (Pharmacia) according to the manufacturer’s instructions. The ascitic fluid or the supernatant from cultured cells containing the chimeric MAb were applied to a 2-ml A3B10Sepharose 4B column. The ascitic fluid was diluted 1:2 in 100 mM Tris HCI, p H 8, whereas the supernatant was concentrated by precipitation with 60% ammonium sulphate solution. After 30 ml washing with 100 mM Tris-HCI, p H 8, the ylCHI-MBR1 was directly eluted with the phosphatelcitratel borate universal buffer (6.7 mM citric acid, 6.7 mM orthophosphate acid and 11.4 mM orthoboric acid) at p H 3. Fractions (0.5 ml) were collected in tubes containing an appropriate volume of 0.5 M Tris HCI, p H 9.5, to neutralize the pH of the solution, and assayed by solid-phase RIA (see below) on MCF-7 cells. The fractions containing antigenically reactive immunoglobulin were pooled, concentrated and analyzed by ‘To whom correspondence and reprint requests should be addressed. Received: March 11,1992 and in revised form June S. 1992
589
CHIMERIC MBrl CHARACTERIZATION
SDS-PAGE. The yICHI-MBR1 showed in reducing conditions 2 bands with the expected electrophoretic mobility and appeared to be > 95% pure as judged by SDS-PAGE densitometric tracings (Model 620 Video Densitometer, BioRad, Richmond, CA). The murine MBrl was purified as previously described (Canevari et a/., 1985). Either murine or chimeric MAbs were labelled by BoltonHunter reagent ( h e r s h a m , Aylesbury, UK), according to the manufacturer's instructions.
Antibody binding assays Indirect solid-phase radioimmunoassay (RIA) was carried out essentially as described (MCnard et al., 1983) using as target material glutaraldehyde-hed cells. The amount of murinc or chimeric MBrl bound to the surface of cells was determined. respectively, by 12sIgoat anti-mouse 7s Ig and lZsI Protein A (Amersham). Direct competitive RIA was performed by incubating different dilutions of murine or chimeric unlabelled MBrl in the presence of a fixed amount of labelled Iz5I-MBrlMAbs for 3 hr at 37°C. The ICsr,,i.e. molar concentration of unlabelled MAbs rcquired to inhibit SO% of the radiolabelled antibody binding, was calculated. To determine the MAbs' affinity constant (Kaff), data from direct competitive RIA were analyzed by the computer program BIND described by Antoni and Mariani (1985). Indirect immunofluorescence (IF) was carried out on suspended or cytospun cells. Briefly, the target material was incubated for 30 min at 3TC, repeatedly washed and further incubated with a fluorescein-labelled goat anti-mouse or antihuman immunoglobulin (Meloy, Springfield, VA). Samples were analyzed by immunofluorescence microscopy or by flow cytofluorometry using an EPICS C instrument (Coulter, Hialeah, FL). As regards the histologic sections of surgical specimens, immunofluorescence was carried out essentially as described above, whereas immunoperoxidase (IPX) was performed with an anti-human IgG kit kindly provided by Dr. J. Endl (Boehringer, Mannheim, Germany). Targel antigeti detrctiorz frnmunob/otting ana~ysis.Samples from frozen surgical specimens were obtained from 5-10 tissue slices, separated by SDS-PAGE electrophoresis and subjected to immunoblotting as described (Leoni ct a/., 1986). The ylCHI-MBRI binding was detected by 12sIProtein A (Amersham), whereas the lzsI MBrl was used directly. High-petfbnnutice thin-layer chromatography (HPTLC ) analysis. Immunostaining of total glycolipid extracts from MCF7 cells fractionated by HPTLC was performed essentially as described (Leoni et a/., 198h), using the detection system described above.
Antihoc!v-depewdent cellular cytoto.uicity (ADCC) assay A standard 4-hr "Cr-release assay was carried out. PBMC maintained in 30 Uiml IL2 for 2 days were used as effector cells. Different dilutions of the MAbs were mixed with 5 x lo3 "Cr-labelled target cells and effector cells at an effector:targetcell ratio of 5O:I. Maximum release was measured by incubating the target cells in 2% Triton X-100 (BioRad), and spontaneous release was determined by incubating the target cells in the medium alone. The percentage of specific lysis was calculated as IOOX (experimental release-spontaneous relcasei maximum release-spon taneous release). Complrment-dependei7t cytotoxicity (CDC) assa.v "Cr-labelled target cells (5,00O/well) were plated in 96-well microtiter plates. MAbs were added at different concentrations and incubated for 1 hr at 37°C. After washine. ~ oundiluted -~~ , .-
AB human serum (Whittaker, Walkersville, MD) was added as a complement source. The plates were incubated for 1 hr at 37°C and then centrifuged at 1,OOOg for 5 min. A 100-p1 aliquot of each supernatant was analyzed for radioactivity. Maximum release, spontaneous release and the percentage of specific lysis were determined as described above.
RESULTS
The cDNA corresponding to the murine MBrl VH and VK domains were cloned and expressed in NSO cells as a simple chimeric antibody of human IgGI isotype (Orlandi et al., 1989). The transfected cells were cloned by limiting dilution and a clone producing the human/mouse chimeric MBrl was established and maintained in selective medium. This antibody was termed chimeric MBrl and is designated ylCHI-MBrl. Reactivity of ylCH1-MBrl and MBrl on cells and tissues The binding reactivity and specificity of ylCHI-MBrl was first examined by different techniques on tumor and normal cells and tissues. As shown in Table I, identical patterns of reactivity were observed when the murine and chimeric MAbs were tested by IF or indirect RIA on a panel of tumor-cell lines. Both antibodies bound to the relevant target cell MCF-7, whereas they did not react with the other tumor-cell lines. The 2 antibodies showed the same pattern of reactivity on normal and tumor tissues by IF or IPX analysis. They showed a strong reactivity on breast carcinoma and on normal mammary ducts and they both recognized ovarian carcinoma and SCLC, but did not recognize testicular and renal carcinomas, in agreement with the reactivity pattern previously reported for MBrl (Mariani-Costantini et ul., 19846). Moreover, the y1CHIMBrl MAb, like the original murine MAb, did not recognize other normal adult tissues or cells, including a ovarian specimen, monocytes and resting or activated PBMC. TABLE 1 REACTIVITY OF CHIMERIC AND MURINE MBrl ON HUMAN TUMORS AND NORMAL CELLS AND TISSUES ~
Target cell
Tumor-cell lines MCF-7, mammary carcinoma DU4475, mammary carcinoma HSO578T. mammary carcinoma HT-20, colon carcinoma IGROVl, ovarian carcinoma OvCa432, ovarian carcinoma U937, lymphoma A431, epidermoid carcinoma MeWo, melanoma JAR, choriocareinoma BeWo, choriocarcinoma JEG-3, choriocarcinoma Tumor tissues' Mammary carcinoma (3) SCLC (3) Ovarian carcinoma (2) Testicular carcinoma (1 ) Renal carcinoma (1) Normal cells and tissues? Breast (2)
%2#I)
Activated PBMC' (4) Monocytes (1)
Reactivity' ylCHlMBrl
MBrl
+ + +-
+ + +-
-
-
+-
+-
-
-
-
-
-
-
'Analyzed by IF, indirect RIA and/or IPX.-?The number of analyzed samples is indicated in parentheses.-3PBMC were aetivated by PHA. IL2 or yIFN, as described in the text.
590
OKLANDl E T A L .
Reactivity of ylCHI-MBrI and MBrl 011 the different nioleculur fomis of the target antigen It was previously reported that the murine MBrl MAb recognizes a saccharide epitope shared by a glycolipid, glycoproteins and mucins (Miotti et al., 1989). To investigate further and to biochemically evaluate whether the ylCHI-MBrl can recognize all the antigenic glycoconjugates carrying the epitope, 2 different approaches were applied, according to the chemical nature of the antigen. Soluble extracts from different carcinomas and human milk werc analyzed by immunoblotting in order to evaluate the reactivity of the 2 MAbs on the glycoprotein molecules (Fig. 1). The y ICHI-MBrl recognized high-molecular-weight glycoproteins, which did not enter the separation gel. on mammary carcinoma (lane 1) or SCLC (lane 2) samples. Bands which were separated in the gel were also stained on samples from human milk (lane 3) and ovarian carcinoma (lane 4). Murine MBrl showed a comparable pattern of reactivity (Fig. 1. lanes 5,6,7). The second approach involved the immunostaining of total glycolipid extracts from MCF-7 cells fractionated by HPTLC. In Figure 1, lane 8, the band stained by the ylCHI-MBrl MAb showed the same mobility as that exhibited by the GL6 glycolipid (Bremer et al., 1984) recognized by the '2sI-MBrl (lane 9). Both MAbs wcre found to be negative on HPTLC of the glycolipid extract from A431 cells. Competition studies To comparc the antigen-binding activity and the relative affinities of ylCHI-MBrl and MBrl for the tumor-associated antigen present on MCF-7 cells, a competitive direct solidphase RIA was carried out. As shown in Figure 2, both ylCHI-MBrl and MBrl were able to compete with the binding of both labelled MAbs. No competition was observed with a MAh of IgG isotype directed against an irrelevant antigen. The IC50sof the competitor MAbs, as reported in Table 11, were, in both the combinations, higher for the chimeric MAb. The conipetitive RIA data werc used to calculate the Kaff of the YlCHI-MBrl and MBrl MAbs and the obtained values are reported in Table 11. According to the ICso, the chimeric MAb exhibited a lower binding affinity than the murine one. The Kaff values are comparable to those obtained by the Scatchard analysis of binding of radiolabclled MAbs (data not shown). y l CHI-MBrl arid MBrl binding to FcR The ability of the human y l and murine p constant regions of rlCHIMBrl and MBrl to bind to the FcRs of the monocytic
line U937 was evaluated. The ylCHI-MBrl strongly labelled over 90% of U937 cells (Fig. 3a) and the binding level was similar to that observed with a murine control MAb of IgG2a isotype and with a pool of human IgG. No binding was detected when the cells were treated with the murine MBrl (Fig. 36).
BioIogical uctivity of y l CHI-MBrl and MBrl The ability of chimeric and murine MBrl antibodies to mediate by ADCC and CDC the tumor-cell lysis of 51Crlabelled MCF-7 cells is shown in Figure 4. As regards the ADCC, in the presence of the ylCHI-MBrl MAb, a significant level of target-cell lysis (60%) was observed. As a control, MCF-7 cells were incubated with PBMC alone, MAbs alone or 100
1
80
I I
D
C
0
Z 60 .-
z c .-C
.c
0 40
ae
20
0 '
' -9
10
I
10
-8
'
-7
10
M
100
b 80 c
0
'L .- 60
Pc .-K c
0
FIGURE1 -Binding reactivity of chimeric and murine MBrl as analyzed by immunoblotting (lines 1-7) and by HPTLC (lanes 8 and 9). Samples were obtained from: mammary carcinoma (lanes 1 and 5). SCLC (lanes 2 and 6), human milk fat globules (lanes 3 and 7), ovarian carcinoma (lane 4) and total glycolipid extract from MCF-7 cell line (lanes 8 and 9). SDS-PAGE was carried out on 10% acrylamide slab gel under reducing conditions. Molecular weight markers: phosphorylase B, 92,000; bovine serum albumin, 69.000: ovalbumin, 46,000; carbonic anhydrase, 30,000, The arrow indicates the beginning of the separation gel. Immunoreaction on nitrocellulose papers or silica-gel plates was carried out with ylCHI-MBrl detected by 12SI-ProteinA (lanes 1-4 and 8) or with l2
%
Publication of the International Union Against Cancer Publication de I Union lnternationale Contre le Cancer
CHARACTERIZATION OF A MOUSE-HUMAN CHIMERIC ANTIBODY TO A CANCER-ASSOCIATED ANTIGEN R. ORLANDI, M. FIGINI, A. TOMASSETTI, S. CANEVARI and M.1 C O L N A G H I ~ Istituto Nazionale per lo Studio e la Cura dei Tumon, Division of Expenmental Oncology E, Via G. Venezian I , 20133 Milan,Italy. In an attempt to obtain a therapeutic antibody, the murine monoclonal antibody (MAb) MBr I (IgM,k), directed against human carcinomas, was converted in a mouse/human chimeric MAb of y l isotype. The chimeric MAb, y l CHI-MBrl, retains the ability to specifically bind tumor cells and tissues with no modification in its binding to the normal material tested. yl CHI-MBr I recognizes mucins and high-molecular-weight glycoproteins carrying the antigenic determinant and stains a neutral glycolipid extracted from MCF-7 cells. The chimeric and the murine MBrl efficiently cross-inhibit each other on the reference cell line MCF-7 and the calculated affinity constants amount to 3.8 X I O7 and I .7 x I O8 M-I, respectively. The human constant region allows ylCHI-MBrl to bind with the FcR on the human monocytic cell line U937 and to efficiently mediate antibody-dependent cellular cytotoxicity in the presence of human lymphocytes activated by IL2. In addition, y l CHI-MBr I, like the murine MBr I, mediatescomplement-dependent turnorcell lysis. Thus, by modelling a molecule with reduced size and increased functional characteristics, we have obtained a reagent which is more suitable for in vivo therapeutic approaches. ci
1992 Wiley-Liss,Inc.
The full exploitation of cancer immunotherapy with murine MAbs has been severely limited by the development of human anti-mouse antibodies (HAMA), which lead to a shortening of thc half-life of the administered MAbs and may reduce their therapeutic effect (Dillman, 1990). Since considerable difficulties are encountered in producing human MAbs by hybridoma technology, protein engineering has been used to convert mouse MAbs into mouseihuman chimeric MAbs by joining the immunoglobulin variable domains from mouse MAbs to h u man constant domains (Winter and Milstein, 1991). With the aim of developing new reagents which are more suitable for cancer immunotherapy, chimeric and reshaped MAbs directed against tumor-associated antigens have been produced and characterized (Sahagan et al., 1986; Riechmann et ul., 1988). Some have been administered to cancer patients, and a marked reduction in the HAMA response and a longer half-life have been observed, as expected (Hale at al., 1988; LoBuglio rt al., 1989). The mouse MAb MBrl (IgM, K), raised against the human mammary carcinoma line MCF-7 (Mtnard et al., 1983), recognizes a carbohydrate epitope carried by various glycoconjugates (Bremer et al., 1984; Miotti et al., 1989). The immunohistochemical reactivity of MBrl has been described (MarianiCostantini et al., 1984 a,b). MBrl has been cxtensively used in in vitro immunodiagnosis (Colnaghi et al., 1987) and active immunotoxins were obtained by coupling the MAb with the ricin A chain and restrictocin (Canevari et al., 1985; Orlandi et nl., 1988). In an attempt to exploit in iivo the thcrapeutic potcntial of the murine MBrl, the human version was generated by expressing in myeloma cells the variable domains as simple mouse/human chimeric MAb (Orlandi et al., 1989). We report here the biochemical and biological characterization of the chimeric MAb. MATERIAL AND METHODS
Transfected cells The transfected cells which produce the chimeric MBrl were maintained in Dulbecco’s modified Eagle’s minimum esscntial medium (DMEM; GIBCO, Grand Island, NY) con-
taining 10% FCS, 10 mM sodium pyruvate, 100 pgiml penicillin and streptomycin, 5 pgiml mycophenolic acid, 250 pg/ml xanthine and 20 pgiml hypoxanthine.
Target cells The human tumor-cell lines listed in Table I were obtained from the ATCC (Rockville, MD), with the exception of I G R O V l and OVCA432, which were kindly provided respectively by Dr. Benard (Institute G. Roussy, Villejuif) and Dr. Knapp (Dana Farber Institute, Boston, MA). The cells were maintained in RPMI-1640 medium (Microbiological Associates, Walkersville, MD) supplemented with 10% FCS, penicillin and streptomycin (100 Fgiml). The cells were checked for Mycoplasma contamination by electron microscopy and/or by bisbenzimide (Sigma, St Louis, MO) staining and were always found to be negative. Human peripheral blood mononuclear cells (PBMC) were obtained from healthy donors by fractionation on FicollHypaque (Pharmacia, Uppsala, Sweden) density-gradient centrifugation. The PBMC used in the irnniunofluorescence tests were activated by 3 different protocols: (1) 30 Uiml IL2 (Cetus, Emeryville, CA) for 6 days; (2) 200 uiml yIFN (Genenzyme. Cambridge, MA) for 6 days; (3) 1 pg/ml PHA (Wellcome, Beckenham, UK) for 3 days. Monocytes were obtained by adhesion to plastic: the PBMC were incubated for 1 hr at 37°C and thc adherent cells were harvested by vigorous pipetting. U937 cells wcre stimulated with 500 u/ml yIFN for 6 days. Antibody characteristics and purification The A3B10 MAb, raised against the variable domain of the murine MBrl, is an Ab2 p anti-idiotypic antibody since it rcacts with the binding site of MBrl and blocks the binding of MBrl to its relevant target antigen (Vialc et al., 1987). It was kindly provided by Dr. G. Viale (Dipartimento di Biologia e Genetica per le Scienze Mediche, Universita degli Studi, Milan) as ascitic fluid and was purified by Protein A chromatography. The ylCHI-MBR1 was purified by immunoaffinity on an anti-idiotypic column. The immunoabsorbent was prepared by coupling the purified A3B10 MAb to CNBr-activated Sepharose 4B (Pharmacia) according to the manufacturer’s instructions. The ascitic fluid or the supernatant from cultured cells containing the chimeric MAb were applied to a 2-ml A3B10Sepharose 4B column. The ascitic fluid was diluted 1:2 in 100 mM Tris HCI, p H 8, whereas the supernatant was concentrated by precipitation with 60% ammonium sulphate solution. After 30 ml washing with 100 mM Tris-HCI, p H 8, the ylCHI-MBR1 was directly eluted with the phosphatelcitratel borate universal buffer (6.7 mM citric acid, 6.7 mM orthophosphate acid and 11.4 mM orthoboric acid) at p H 3. Fractions (0.5 ml) were collected in tubes containing an appropriate volume of 0.5 M Tris HCI, p H 9.5, to neutralize the pH of the solution, and assayed by solid-phase RIA (see below) on MCF-7 cells. The fractions containing antigenically reactive immunoglobulin were pooled, concentrated and analyzed by ‘To whom correspondence and reprint requests should be addressed. Received: March 11,1992 and in revised form June S. 1992
589
CHIMERIC MBrl CHARACTERIZATION
SDS-PAGE. The yICHI-MBR1 showed in reducing conditions 2 bands with the expected electrophoretic mobility and appeared to be > 95% pure as judged by SDS-PAGE densitometric tracings (Model 620 Video Densitometer, BioRad, Richmond, CA). The murine MBrl was purified as previously described (Canevari et a/., 1985). Either murine or chimeric MAbs were labelled by BoltonHunter reagent ( h e r s h a m , Aylesbury, UK), according to the manufacturer's instructions.
Antibody binding assays Indirect solid-phase radioimmunoassay (RIA) was carried out essentially as described (MCnard et al., 1983) using as target material glutaraldehyde-hed cells. The amount of murinc or chimeric MBrl bound to the surface of cells was determined. respectively, by 12sIgoat anti-mouse 7s Ig and lZsI Protein A (Amersham). Direct competitive RIA was performed by incubating different dilutions of murine or chimeric unlabelled MBrl in the presence of a fixed amount of labelled Iz5I-MBrlMAbs for 3 hr at 37°C. The ICsr,,i.e. molar concentration of unlabelled MAbs rcquired to inhibit SO% of the radiolabelled antibody binding, was calculated. To determine the MAbs' affinity constant (Kaff), data from direct competitive RIA were analyzed by the computer program BIND described by Antoni and Mariani (1985). Indirect immunofluorescence (IF) was carried out on suspended or cytospun cells. Briefly, the target material was incubated for 30 min at 3TC, repeatedly washed and further incubated with a fluorescein-labelled goat anti-mouse or antihuman immunoglobulin (Meloy, Springfield, VA). Samples were analyzed by immunofluorescence microscopy or by flow cytofluorometry using an EPICS C instrument (Coulter, Hialeah, FL). As regards the histologic sections of surgical specimens, immunofluorescence was carried out essentially as described above, whereas immunoperoxidase (IPX) was performed with an anti-human IgG kit kindly provided by Dr. J. Endl (Boehringer, Mannheim, Germany). Targel antigeti detrctiorz frnmunob/otting ana~ysis.Samples from frozen surgical specimens were obtained from 5-10 tissue slices, separated by SDS-PAGE electrophoresis and subjected to immunoblotting as described (Leoni ct a/., 1986). The ylCHI-MBRI binding was detected by 12sIProtein A (Amersham), whereas the lzsI MBrl was used directly. High-petfbnnutice thin-layer chromatography (HPTLC ) analysis. Immunostaining of total glycolipid extracts from MCF7 cells fractionated by HPTLC was performed essentially as described (Leoni et a/., 198h), using the detection system described above.
Antihoc!v-depewdent cellular cytoto.uicity (ADCC) assay A standard 4-hr "Cr-release assay was carried out. PBMC maintained in 30 Uiml IL2 for 2 days were used as effector cells. Different dilutions of the MAbs were mixed with 5 x lo3 "Cr-labelled target cells and effector cells at an effector:targetcell ratio of 5O:I. Maximum release was measured by incubating the target cells in 2% Triton X-100 (BioRad), and spontaneous release was determined by incubating the target cells in the medium alone. The percentage of specific lysis was calculated as IOOX (experimental release-spontaneous relcasei maximum release-spon taneous release). Complrment-dependei7t cytotoxicity (CDC) assa.v "Cr-labelled target cells (5,00O/well) were plated in 96-well microtiter plates. MAbs were added at different concentrations and incubated for 1 hr at 37°C. After washine. ~ oundiluted -~~ , .-
AB human serum (Whittaker, Walkersville, MD) was added as a complement source. The plates were incubated for 1 hr at 37°C and then centrifuged at 1,OOOg for 5 min. A 100-p1 aliquot of each supernatant was analyzed for radioactivity. Maximum release, spontaneous release and the percentage of specific lysis were determined as described above.
RESULTS
The cDNA corresponding to the murine MBrl VH and VK domains were cloned and expressed in NSO cells as a simple chimeric antibody of human IgGI isotype (Orlandi et al., 1989). The transfected cells were cloned by limiting dilution and a clone producing the human/mouse chimeric MBrl was established and maintained in selective medium. This antibody was termed chimeric MBrl and is designated ylCHI-MBrl. Reactivity of ylCH1-MBrl and MBrl on cells and tissues The binding reactivity and specificity of ylCHI-MBrl was first examined by different techniques on tumor and normal cells and tissues. As shown in Table I, identical patterns of reactivity were observed when the murine and chimeric MAbs were tested by IF or indirect RIA on a panel of tumor-cell lines. Both antibodies bound to the relevant target cell MCF-7, whereas they did not react with the other tumor-cell lines. The 2 antibodies showed the same pattern of reactivity on normal and tumor tissues by IF or IPX analysis. They showed a strong reactivity on breast carcinoma and on normal mammary ducts and they both recognized ovarian carcinoma and SCLC, but did not recognize testicular and renal carcinomas, in agreement with the reactivity pattern previously reported for MBrl (Mariani-Costantini et ul., 19846). Moreover, the y1CHIMBrl MAb, like the original murine MAb, did not recognize other normal adult tissues or cells, including a ovarian specimen, monocytes and resting or activated PBMC. TABLE 1 REACTIVITY OF CHIMERIC AND MURINE MBrl ON HUMAN TUMORS AND NORMAL CELLS AND TISSUES ~
Target cell
Tumor-cell lines MCF-7, mammary carcinoma DU4475, mammary carcinoma HSO578T. mammary carcinoma HT-20, colon carcinoma IGROVl, ovarian carcinoma OvCa432, ovarian carcinoma U937, lymphoma A431, epidermoid carcinoma MeWo, melanoma JAR, choriocareinoma BeWo, choriocarcinoma JEG-3, choriocarcinoma Tumor tissues' Mammary carcinoma (3) SCLC (3) Ovarian carcinoma (2) Testicular carcinoma (1 ) Renal carcinoma (1) Normal cells and tissues? Breast (2)
%2#I)
Activated PBMC' (4) Monocytes (1)
Reactivity' ylCHlMBrl
MBrl
+ + +-
+ + +-
-
-
+-
+-
-
-
-
-
-
-
'Analyzed by IF, indirect RIA and/or IPX.-?The number of analyzed samples is indicated in parentheses.-3PBMC were aetivated by PHA. IL2 or yIFN, as described in the text.
590
OKLANDl E T A L .
Reactivity of ylCHI-MBrI and MBrl 011 the different nioleculur fomis of the target antigen It was previously reported that the murine MBrl MAb recognizes a saccharide epitope shared by a glycolipid, glycoproteins and mucins (Miotti et al., 1989). To investigate further and to biochemically evaluate whether the ylCHI-MBrl can recognize all the antigenic glycoconjugates carrying the epitope, 2 different approaches were applied, according to the chemical nature of the antigen. Soluble extracts from different carcinomas and human milk werc analyzed by immunoblotting in order to evaluate the reactivity of the 2 MAbs on the glycoprotein molecules (Fig. 1). The y ICHI-MBrl recognized high-molecular-weight glycoproteins, which did not enter the separation gel. on mammary carcinoma (lane 1) or SCLC (lane 2) samples. Bands which were separated in the gel were also stained on samples from human milk (lane 3) and ovarian carcinoma (lane 4). Murine MBrl showed a comparable pattern of reactivity (Fig. 1. lanes 5,6,7). The second approach involved the immunostaining of total glycolipid extracts from MCF-7 cells fractionated by HPTLC. In Figure 1, lane 8, the band stained by the ylCHI-MBrl MAb showed the same mobility as that exhibited by the GL6 glycolipid (Bremer et al., 1984) recognized by the '2sI-MBrl (lane 9). Both MAbs wcre found to be negative on HPTLC of the glycolipid extract from A431 cells. Competition studies To comparc the antigen-binding activity and the relative affinities of ylCHI-MBrl and MBrl for the tumor-associated antigen present on MCF-7 cells, a competitive direct solidphase RIA was carried out. As shown in Figure 2, both ylCHI-MBrl and MBrl were able to compete with the binding of both labelled MAbs. No competition was observed with a MAh of IgG isotype directed against an irrelevant antigen. The IC50sof the competitor MAbs, as reported in Table 11, were, in both the combinations, higher for the chimeric MAb. The conipetitive RIA data werc used to calculate the Kaff of the YlCHI-MBrl and MBrl MAbs and the obtained values are reported in Table 11. According to the ICso, the chimeric MAb exhibited a lower binding affinity than the murine one. The Kaff values are comparable to those obtained by the Scatchard analysis of binding of radiolabclled MAbs (data not shown). y l CHI-MBrl arid MBrl binding to FcR The ability of the human y l and murine p constant regions of rlCHIMBrl and MBrl to bind to the FcRs of the monocytic
line U937 was evaluated. The ylCHI-MBrl strongly labelled over 90% of U937 cells (Fig. 3a) and the binding level was similar to that observed with a murine control MAb of IgG2a isotype and with a pool of human IgG. No binding was detected when the cells were treated with the murine MBrl (Fig. 36).
BioIogical uctivity of y l CHI-MBrl and MBrl The ability of chimeric and murine MBrl antibodies to mediate by ADCC and CDC the tumor-cell lysis of 51Crlabelled MCF-7 cells is shown in Figure 4. As regards the ADCC, in the presence of the ylCHI-MBrl MAb, a significant level of target-cell lysis (60%) was observed. As a control, MCF-7 cells were incubated with PBMC alone, MAbs alone or 100
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FIGURE1 -Binding reactivity of chimeric and murine MBrl as analyzed by immunoblotting (lines 1-7) and by HPTLC (lanes 8 and 9). Samples were obtained from: mammary carcinoma (lanes 1 and 5). SCLC (lanes 2 and 6), human milk fat globules (lanes 3 and 7), ovarian carcinoma (lane 4) and total glycolipid extract from MCF-7 cell line (lanes 8 and 9). SDS-PAGE was carried out on 10% acrylamide slab gel under reducing conditions. Molecular weight markers: phosphorylase B, 92,000; bovine serum albumin, 69.000: ovalbumin, 46,000; carbonic anhydrase, 30,000, The arrow indicates the beginning of the separation gel. Immunoreaction on nitrocellulose papers or silica-gel plates was carried out with ylCHI-MBrl detected by 12SI-ProteinA (lanes 1-4 and 8) or with l2
