Int. J. Cancer: 52,631-635 (1992) 0 1992 Wiley-Liss, Inc.
Publicationof the InternationalUnion Against Cancer Publicationde I Union lnternationaleContre le Cancer
IMMUNOTOXINS DIRECTED AGAINST THE HIGH-MOLECULAR-WEIGHT MELANOMA-ASSOCIATED ANTIGEN. IDENTIFICATION OF POTENT ANTIBODY-TOXIN COMBINATIONS Aslak GODAL.', Brita KUMLE,Alexander PIHL,Siri JUELLand Oystein FODSTAD~ Department of Tumor Biology, The Norwegian Radium Hospital, Montebello, 0310 Oslo 3, Norway. To study factors influencingthe cytotoxicity of immunotoxins (ITS),we compared the in vitro cytotoxicity of conjugates in which the plant toxin abrin and the bacterialtoxin Pseudornonas exotoxin A (PE) were coupled by 2 different procedures to 2 MAbs, 9.2.27 and NR-ML-05, which bind to different epitopes on the melanoma-associated antigen p250. The individual target cell lines differed widely in sensitivity to the different ITS,as assessed by measurement of protein synthesis inhibition. The action of the ITSwas highly specific, as the toxicity of abrin and PE conjugates was respectively20-540 and 2,200-550,000 times higher in antigen-positive cell lines (FEMX, SESX, OHS) than in the antigen-negative line KPDX. The PE conjugates prepared with the 2 different MAbs differed in potency by factors of 16-1 26 in the target-cell lines, but were consistently more toxic than the abrin ITS.The results demonstrate that the cytotoxicity of ITSvaries with the nature of both of its moieties and that optimal results require that toxins and MAbs be matched. Moreover, the 2 coupling procedures affected differentially the binding and potency of some ITS. Each of the 2 toxins was conjugated to a sheep anti-mouse antibody (SAM) and the toxicity of these 2 conjugates was tested in an indirect approach using 9.2.27 and NR-ML-05 as primary MAbs. The results showed that the indirect procedure would have correctly predicted the most potent antibody-toxin pair, indicating that the approach may be valid for selecting suitable combinationsof MAbs and toxins for use as direct ITS.
o 1992 Wiley-Liss, Inc. Immunotoxins (ITS) are conjugates of MAbs and highly potent plant or bacterial toxins or their active subunits. Their construction, properties and possible applications have been discussed in a number of reviews (Olsnes and Pihl, 1982b, 1986; Vitetta et al., 1983, 1987; Pastan et al., 1986; Blakey et al., 1988; Blakey and Thorpe, 1988; Frankel, 1988; Olsnes and Sandvig, 1988; Hall and Fodstad, 1992). ITS are designed to bind to and specifically kill target cells with minimal toxicity to normal cells, the ultimate goal being to use such constructs in cell-specific treatment of human disease. However, attempts to develop antibody-mediated toxin therapy into a clinically useful modality have so far met with limited success. Many ITS with high in vitro potency have been prepared, but their effects in vii90 have often been disappointing for pharmacodynamic and other reasons (Frankel, 1988). The cytotoxicity of ITS depends on a number of factors and is difficult to predict. In the present study, we have compared conjugates in which the holotoxins abrin and Pseudomonas exotoxin A (PE) were coupled to 2 MAbs, 9.2.27 and NR-ML05, directed against different epitopes on the same antigen, the high-molecular-weight melanoma-associated antigen p250 (Bumol and Reisfeld, 1982). Abrin consists of 2 disulfide-linked polypeptide chains which have different functions (Olsnes and Pihl, 1976, 1Y82a). It is bound via its B-chain to cellular receptors and this, after internalization of the toxin by endocytosis, somehow facilitates the translocation of the active A-chain into the cytosol. PE is a single-chain protein with 3 structural domains having different functions, viz. binding, translocation and intoxication (see Wawrzynczak, 1991). Both toxins kill cells by shutting off their protein-synthesizing machinery; abrin by inactivating the large ribosomal subunit through splitting off a specific adenine residue from the 28s rRNA (Endo et al., 1988), and PE by ADP-ribosylating
elongation factor 2 (Ogata et al., 1990). High IT potency requires that the surface-bound conjugates should be efficiently internalized and routed to a compartment permitting translocation of the toxic moiety to the cytosol. Our conjugates, when tested against 3 different antigenpositive cell lines, differed greatly in their cytotoxic potency, depending on the MAb and toxin used, as well as on the conjugation procedure. These data reflect the complexity of developing effective ITS, and the advantage of being able to select suitably matched antibody-toxin combinations by means of a reasonably simple procedure. We found that the indirect approach described by Weltman et al. (1987) and Till et al. (1988), and adapted in our laboratory to include the use of holotoxins (Fodstad et al., 19886), would indeed have selected the most active antibody-toxin conjugate among the possible combinations of toxins and antibodies studied here. MATERIAL AND METHODS
Antibodies and toxins MAbs 9.2.27 (Morgan et al., 1981) and NR-ML-05 (Woodhouse et al., 1989), directed against different epitopes on a human melanoma-associated proteoglycan, were kindly provided by Dr. A.C. Morgan (NeoRx, Seattle, WA). Affinitypurified sheep anti-mouse immunoglobulin (SAM) was a gift from Dr. T. Michaelsen (Institute for Public Health, Oslo) and Dr. K. Nustad (The Norwegian Radium Hospital, Oslo). The immunoreactive fractions of the 2 MAbs were compared to those of the corresponding conjugates according to the method of Lindmo et al. (1984). Briefly, a constant amount of 1251-labeledligand was incubated with increasing concentrations of melanoma cells (FEMX). After washing, the bound radioactivity was measured, and the immunoreactive fraction determined from a plot of inverse cell concentration vs. added ligandibound ligand by extrapolation to infinite antigen excess. Abrin was extracted and purified according to Olsnes (1978), whereas Pseudornonas exotoxin A was obtained from the Swiss Serum and Vaccine Institute, Bern, Switzerland. Preparation of immunotoxins Two different methods were used for the preparation of immunotoxins. In the SPDP method, abrin or PE (1 mg in 1 ml of PBS) was mixed with a 1 5 molar ~ excess of SPDP (in 50 pl 'Present address: Nycomed Imaging. Hafslund-Nycorned AIS, P.O. Box 4220 Torshov, 0401 Oslo 4, Norway.
2Towhom correspondence and reprint requests should be sent. Ahhrciwrionc: SPDP, N-Succinimidyl-3-(2-pyridvIdithio)-proplonate: PDP. 2-pyridyldithio propionate; PBS, phosphate-buffered snlinc; DMSO. dimrthylsulfoxide: DTT. dithiothrritol; SMCC. succinimidyl-3-(N-rnalt.imid~~methyl)cyclohcxaneI -cnrboxylate; MCC, malcimidomethvl cyclohcxanr- 1-carboxylate: FITC. fluorescein isot hiocyanate: MAb. monoclonal antibody.
Received: April 13. 1YY2 and in r e v i d form June 19. l Y Y 2 .
632
GODAL E T A L .
of DMSO) and incubated for 1 hr at room temperature. The antibody (1 mg in 1 ml of PBS) was mixed with a lox molar excess of SPDP in 50 p1 of DMSO and incubated as above. Toxin and antibody were dialyzed against PBS overnight at 4"C, and the PDP-toxin was treated with 50 mM D T T for 20 min at room temperature, followed by gel filtration to remove the reducing agent. The eluted toxin was immediately mixed with the PDP-substituted antibody and the mixture was concentrated by ultrafiltration to approximately 1 ml and incubated overnight at 4°C. In the SMCC method, the toxin (1 mg in 1 ml of PBS) was ~ excess of the SMCC reagent (Pierce, mixed with a 1 5 molar Rockford, USA) in 50 pl of DMSO and incubated for 1 hr at room temperature before gel filtration to remove excess coupling agent. The antibody moiety [ l mg in 1 ml of 100 mM Na-phosphate (pH 6.0) containing 5 mM EDTA] was treated with 15 mM DTT for 90 min at 37°C followed by gel filtration. The substituted toxin was mixed with the reduced antibody, concentrated as described above and incubated overnight at 4°C. All the resulting conjugates were purified by gel filtration and the fractions were analyzed by SDS-PAGE. The fractions used contained predominantly material of MW 200-300 kDa. The amount of free antibody was always less than lo%, as judged by visual examination of the gels. Before use, the abrin conjugates were passed through a column of acid-treated Sepharose 4B to remove molecular species with exposed B-chain binding sites (Godal et al., 19866).
adhere. Appropriate amounts of toxin or IT were added and the cells were incubated for 20 hr at 37°C. The protein synthesis was thcn measured using the 3H-leucine incorporation assay as described by Sandvig and Olsnes (1982). In the indirect approach, 2 x 106cells were incubated with 10 pg of primary antibody in 0.5 ml of RPMI/10% FCS for 30 min at 4°C. The cells were washed twice in cold PBS and resuspended in RPMI/10% FCS to a final concentration of 5 x lo4per ml. One ml of the cell suspension was then added to each well of a 24-well tray already supplied with the appropriate amounts of either SAM-PE or SAM-abrin and incubated for 20 hr at 37°C. Protein synthesis was then measured as described for the direct assay. The results presented arc averages of 2 to 3 experiments. RESULTS
Cell binding of the antibodies Binding of the 2 melanoma-associated MAbs to the 4 cell lines was measured by incubating the cells with radiolabeled antibodies. The results (Table I) show that the antigen-positive cell lines differ by factors of 2 4 in their ability to bind the 2 antibodies. The relative binding to the different cell lines was roughly the same for the 2 antibodies, the OHS sarcoma line binding most and the FEMX cells least. The KPDX cell line used as a negative control did not bind measurable amounts of the antibodies. To see whether binding measured by an indirect method would give the same binding profile as that obtained by the direct method, we performed flow cytometric analysis with the Cell lines The melanoma cell lines FEMX (Fodstad et a/., 1980,1988~) NR-ML-05 antibody. The relative binding to the different cell and SESX (Hwanget al., 1985) and the osteosarcoma cell lines lines tested (Table I) was similar to that seen with radiolabeled antibodies. OHS (Fodstad et al., 1986; Godal et a/., 1986a) and KPDX (Kjanniksen et al., 1991) were all established in our laboratory Role of conjugation method and grown as monolayer cultures in RPMI medium supplePrevious studies have shown that the type of antibody-toxin mented with 10% fetal calf serum (FCS). Before use in the linkage may affect not only the in vivo stability of ITS (Morgan experiments, the cells were brought into suspension by treat- et al., 1990), but also their potency and specificity in vitro. Our ment with 10 mM EDTA in phosphate-buffered saline (PBS) purpose was to explore whether 2 conjugation methods that supplemented with 0.05% KCI. and washed twice in PBS. involve different treatments of the antibody moiety may differentially affect the binding of the conjugates to the target Assay of antigen expression cells. Abrin was coupled to each of the MAbs NR-ML-05 and The level of surface antigen expression on the cell lines 9.2.27 using either the SMCC reagent, which involves a partial studied was determined by measuring their ability to bind the reduction of disulfides in the hinge region of the antibody, or '2SI-labcled MAbs 9.2.27 and NR-ML-05, as previously de- the SPDP reagent, which involves reaction with amino groups. scribed (Godal et al., 19866). In the case of NR-ML-05, the The immunoreactive fraction and the cytotoxicity of the various antigen expression was also measured by flow cytometry. The conjugates were then studied in OHS sarcoma cells which excells were then incubated with primary antibody (lo6 cells in 1 pressed the highest number of antigenic binding sites (Table I). ml PBSi0.175 HSA containing 1 pg of NR-ML-05) washed, The results presented in Table I1 show that the cytotoxic and further incubated with FITC-conjugated SAM before effect of the NR-ML-05 conjLgate prepared by the SPDP being analysed in a Coulter Epics V flow cytometer equipped method was less than 10% of that of the corresponding SMCC with an argon laser. conjugate. The corresponding immunoreactive fractions were 20% and 56%, suggesting that the lower activity of the Assay of cytotoxicity The cytotoxic effect of the toxins and ITS was assessed by SPDP-conjugate was due to a reduced ability to bind to the measuring their ability to inhibit cellular protein synthesis cells. With the 9.2.27 conjugates, only minor differences were (Godal et al., 1986b). In the direct assay, 5 X lo4cells in 1 ml of observed. In the case of the secondary SAM-abrin conjugate, the RPMI/10% FCS were added to each well of a 24-well (Falcon) plate and incubated for 3 hr at 37°C to allow the cells to SPDP method produced a highly active conjugate, as tested on TABLE I - EXPRESSION OF EPITOPES BINDING THE MONOCLONAL ANTIBODIES 9.2.27 AND NR-ML-05' Assaved bv
Cell line
Origin
9.2.21
FEMX SESX OHS
KPDX
Melanoma Melanoma Sarcoma Sarcoma
Flow cytometly (rel. fluorescence)'
'2s1-laheledantibody (ng bound/lOScells)
2.3 5.0 5.7
Publicationof the InternationalUnion Against Cancer Publicationde I Union lnternationaleContre le Cancer
IMMUNOTOXINS DIRECTED AGAINST THE HIGH-MOLECULAR-WEIGHT MELANOMA-ASSOCIATED ANTIGEN. IDENTIFICATION OF POTENT ANTIBODY-TOXIN COMBINATIONS Aslak GODAL.', Brita KUMLE,Alexander PIHL,Siri JUELLand Oystein FODSTAD~ Department of Tumor Biology, The Norwegian Radium Hospital, Montebello, 0310 Oslo 3, Norway. To study factors influencingthe cytotoxicity of immunotoxins (ITS),we compared the in vitro cytotoxicity of conjugates in which the plant toxin abrin and the bacterialtoxin Pseudornonas exotoxin A (PE) were coupled by 2 different procedures to 2 MAbs, 9.2.27 and NR-ML-05, which bind to different epitopes on the melanoma-associated antigen p250. The individual target cell lines differed widely in sensitivity to the different ITS,as assessed by measurement of protein synthesis inhibition. The action of the ITSwas highly specific, as the toxicity of abrin and PE conjugates was respectively20-540 and 2,200-550,000 times higher in antigen-positive cell lines (FEMX, SESX, OHS) than in the antigen-negative line KPDX. The PE conjugates prepared with the 2 different MAbs differed in potency by factors of 16-1 26 in the target-cell lines, but were consistently more toxic than the abrin ITS.The results demonstrate that the cytotoxicity of ITSvaries with the nature of both of its moieties and that optimal results require that toxins and MAbs be matched. Moreover, the 2 coupling procedures affected differentially the binding and potency of some ITS. Each of the 2 toxins was conjugated to a sheep anti-mouse antibody (SAM) and the toxicity of these 2 conjugates was tested in an indirect approach using 9.2.27 and NR-ML-05 as primary MAbs. The results showed that the indirect procedure would have correctly predicted the most potent antibody-toxin pair, indicating that the approach may be valid for selecting suitable combinationsof MAbs and toxins for use as direct ITS.
o 1992 Wiley-Liss, Inc. Immunotoxins (ITS) are conjugates of MAbs and highly potent plant or bacterial toxins or their active subunits. Their construction, properties and possible applications have been discussed in a number of reviews (Olsnes and Pihl, 1982b, 1986; Vitetta et al., 1983, 1987; Pastan et al., 1986; Blakey et al., 1988; Blakey and Thorpe, 1988; Frankel, 1988; Olsnes and Sandvig, 1988; Hall and Fodstad, 1992). ITS are designed to bind to and specifically kill target cells with minimal toxicity to normal cells, the ultimate goal being to use such constructs in cell-specific treatment of human disease. However, attempts to develop antibody-mediated toxin therapy into a clinically useful modality have so far met with limited success. Many ITS with high in vitro potency have been prepared, but their effects in vii90 have often been disappointing for pharmacodynamic and other reasons (Frankel, 1988). The cytotoxicity of ITS depends on a number of factors and is difficult to predict. In the present study, we have compared conjugates in which the holotoxins abrin and Pseudomonas exotoxin A (PE) were coupled to 2 MAbs, 9.2.27 and NR-ML05, directed against different epitopes on the same antigen, the high-molecular-weight melanoma-associated antigen p250 (Bumol and Reisfeld, 1982). Abrin consists of 2 disulfide-linked polypeptide chains which have different functions (Olsnes and Pihl, 1976, 1Y82a). It is bound via its B-chain to cellular receptors and this, after internalization of the toxin by endocytosis, somehow facilitates the translocation of the active A-chain into the cytosol. PE is a single-chain protein with 3 structural domains having different functions, viz. binding, translocation and intoxication (see Wawrzynczak, 1991). Both toxins kill cells by shutting off their protein-synthesizing machinery; abrin by inactivating the large ribosomal subunit through splitting off a specific adenine residue from the 28s rRNA (Endo et al., 1988), and PE by ADP-ribosylating
elongation factor 2 (Ogata et al., 1990). High IT potency requires that the surface-bound conjugates should be efficiently internalized and routed to a compartment permitting translocation of the toxic moiety to the cytosol. Our conjugates, when tested against 3 different antigenpositive cell lines, differed greatly in their cytotoxic potency, depending on the MAb and toxin used, as well as on the conjugation procedure. These data reflect the complexity of developing effective ITS, and the advantage of being able to select suitably matched antibody-toxin combinations by means of a reasonably simple procedure. We found that the indirect approach described by Weltman et al. (1987) and Till et al. (1988), and adapted in our laboratory to include the use of holotoxins (Fodstad et al., 19886), would indeed have selected the most active antibody-toxin conjugate among the possible combinations of toxins and antibodies studied here. MATERIAL AND METHODS
Antibodies and toxins MAbs 9.2.27 (Morgan et al., 1981) and NR-ML-05 (Woodhouse et al., 1989), directed against different epitopes on a human melanoma-associated proteoglycan, were kindly provided by Dr. A.C. Morgan (NeoRx, Seattle, WA). Affinitypurified sheep anti-mouse immunoglobulin (SAM) was a gift from Dr. T. Michaelsen (Institute for Public Health, Oslo) and Dr. K. Nustad (The Norwegian Radium Hospital, Oslo). The immunoreactive fractions of the 2 MAbs were compared to those of the corresponding conjugates according to the method of Lindmo et al. (1984). Briefly, a constant amount of 1251-labeledligand was incubated with increasing concentrations of melanoma cells (FEMX). After washing, the bound radioactivity was measured, and the immunoreactive fraction determined from a plot of inverse cell concentration vs. added ligandibound ligand by extrapolation to infinite antigen excess. Abrin was extracted and purified according to Olsnes (1978), whereas Pseudornonas exotoxin A was obtained from the Swiss Serum and Vaccine Institute, Bern, Switzerland. Preparation of immunotoxins Two different methods were used for the preparation of immunotoxins. In the SPDP method, abrin or PE (1 mg in 1 ml of PBS) was mixed with a 1 5 molar ~ excess of SPDP (in 50 pl 'Present address: Nycomed Imaging. Hafslund-Nycorned AIS, P.O. Box 4220 Torshov, 0401 Oslo 4, Norway.
2Towhom correspondence and reprint requests should be sent. Ahhrciwrionc: SPDP, N-Succinimidyl-3-(2-pyridvIdithio)-proplonate: PDP. 2-pyridyldithio propionate; PBS, phosphate-buffered snlinc; DMSO. dimrthylsulfoxide: DTT. dithiothrritol; SMCC. succinimidyl-3-(N-rnalt.imid~~methyl)cyclohcxaneI -cnrboxylate; MCC, malcimidomethvl cyclohcxanr- 1-carboxylate: FITC. fluorescein isot hiocyanate: MAb. monoclonal antibody.
Received: April 13. 1YY2 and in r e v i d form June 19. l Y Y 2 .
632
GODAL E T A L .
of DMSO) and incubated for 1 hr at room temperature. The antibody (1 mg in 1 ml of PBS) was mixed with a lox molar excess of SPDP in 50 p1 of DMSO and incubated as above. Toxin and antibody were dialyzed against PBS overnight at 4"C, and the PDP-toxin was treated with 50 mM D T T for 20 min at room temperature, followed by gel filtration to remove the reducing agent. The eluted toxin was immediately mixed with the PDP-substituted antibody and the mixture was concentrated by ultrafiltration to approximately 1 ml and incubated overnight at 4°C. In the SMCC method, the toxin (1 mg in 1 ml of PBS) was ~ excess of the SMCC reagent (Pierce, mixed with a 1 5 molar Rockford, USA) in 50 pl of DMSO and incubated for 1 hr at room temperature before gel filtration to remove excess coupling agent. The antibody moiety [ l mg in 1 ml of 100 mM Na-phosphate (pH 6.0) containing 5 mM EDTA] was treated with 15 mM DTT for 90 min at 37°C followed by gel filtration. The substituted toxin was mixed with the reduced antibody, concentrated as described above and incubated overnight at 4°C. All the resulting conjugates were purified by gel filtration and the fractions were analyzed by SDS-PAGE. The fractions used contained predominantly material of MW 200-300 kDa. The amount of free antibody was always less than lo%, as judged by visual examination of the gels. Before use, the abrin conjugates were passed through a column of acid-treated Sepharose 4B to remove molecular species with exposed B-chain binding sites (Godal et al., 19866).
adhere. Appropriate amounts of toxin or IT were added and the cells were incubated for 20 hr at 37°C. The protein synthesis was thcn measured using the 3H-leucine incorporation assay as described by Sandvig and Olsnes (1982). In the indirect approach, 2 x 106cells were incubated with 10 pg of primary antibody in 0.5 ml of RPMI/10% FCS for 30 min at 4°C. The cells were washed twice in cold PBS and resuspended in RPMI/10% FCS to a final concentration of 5 x lo4per ml. One ml of the cell suspension was then added to each well of a 24-well tray already supplied with the appropriate amounts of either SAM-PE or SAM-abrin and incubated for 20 hr at 37°C. Protein synthesis was then measured as described for the direct assay. The results presented arc averages of 2 to 3 experiments. RESULTS
Cell binding of the antibodies Binding of the 2 melanoma-associated MAbs to the 4 cell lines was measured by incubating the cells with radiolabeled antibodies. The results (Table I) show that the antigen-positive cell lines differ by factors of 2 4 in their ability to bind the 2 antibodies. The relative binding to the different cell lines was roughly the same for the 2 antibodies, the OHS sarcoma line binding most and the FEMX cells least. The KPDX cell line used as a negative control did not bind measurable amounts of the antibodies. To see whether binding measured by an indirect method would give the same binding profile as that obtained by the direct method, we performed flow cytometric analysis with the Cell lines The melanoma cell lines FEMX (Fodstad et a/., 1980,1988~) NR-ML-05 antibody. The relative binding to the different cell and SESX (Hwanget al., 1985) and the osteosarcoma cell lines lines tested (Table I) was similar to that seen with radiolabeled antibodies. OHS (Fodstad et al., 1986; Godal et a/., 1986a) and KPDX (Kjanniksen et al., 1991) were all established in our laboratory Role of conjugation method and grown as monolayer cultures in RPMI medium supplePrevious studies have shown that the type of antibody-toxin mented with 10% fetal calf serum (FCS). Before use in the linkage may affect not only the in vivo stability of ITS (Morgan experiments, the cells were brought into suspension by treat- et al., 1990), but also their potency and specificity in vitro. Our ment with 10 mM EDTA in phosphate-buffered saline (PBS) purpose was to explore whether 2 conjugation methods that supplemented with 0.05% KCI. and washed twice in PBS. involve different treatments of the antibody moiety may differentially affect the binding of the conjugates to the target Assay of antigen expression cells. Abrin was coupled to each of the MAbs NR-ML-05 and The level of surface antigen expression on the cell lines 9.2.27 using either the SMCC reagent, which involves a partial studied was determined by measuring their ability to bind the reduction of disulfides in the hinge region of the antibody, or '2SI-labcled MAbs 9.2.27 and NR-ML-05, as previously de- the SPDP reagent, which involves reaction with amino groups. scribed (Godal et al., 19866). In the case of NR-ML-05, the The immunoreactive fraction and the cytotoxicity of the various antigen expression was also measured by flow cytometry. The conjugates were then studied in OHS sarcoma cells which excells were then incubated with primary antibody (lo6 cells in 1 pressed the highest number of antigenic binding sites (Table I). ml PBSi0.175 HSA containing 1 pg of NR-ML-05) washed, The results presented in Table I1 show that the cytotoxic and further incubated with FITC-conjugated SAM before effect of the NR-ML-05 conjLgate prepared by the SPDP being analysed in a Coulter Epics V flow cytometer equipped method was less than 10% of that of the corresponding SMCC with an argon laser. conjugate. The corresponding immunoreactive fractions were 20% and 56%, suggesting that the lower activity of the Assay of cytotoxicity The cytotoxic effect of the toxins and ITS was assessed by SPDP-conjugate was due to a reduced ability to bind to the measuring their ability to inhibit cellular protein synthesis cells. With the 9.2.27 conjugates, only minor differences were (Godal et al., 1986b). In the direct assay, 5 X lo4cells in 1 ml of observed. In the case of the secondary SAM-abrin conjugate, the RPMI/10% FCS were added to each well of a 24-well (Falcon) plate and incubated for 3 hr at 37°C to allow the cells to SPDP method produced a highly active conjugate, as tested on TABLE I - EXPRESSION OF EPITOPES BINDING THE MONOCLONAL ANTIBODIES 9.2.27 AND NR-ML-05' Assaved bv
Cell line
Origin
9.2.21
FEMX SESX OHS
KPDX
Melanoma Melanoma Sarcoma Sarcoma
Flow cytometly (rel. fluorescence)'
'2s1-laheledantibody (ng bound/lOScells)
2.3 5.0 5.7
