Monoclonal Antibody A7 Tumor Localization Enhancement by Its F(abO2 Fragments to Colon Carcinoma Xenografts in Nude Mice
Departments of 'Surgery and 2Microbiology, Kyoto Prefectural University of Medicine, 465, Kawaramachihirokojikajii-cho, Kamikyo-ku, Kyoto 602 The monoclonal antibody A7 (MoAb A7), which belongs to IgGi, was digested with pepsin to yield Hab'h fragments. The maximum binding to the human colon cancer cell line, SW1116, was 27% with 125-1 labeled whole MoAb A7 and 24% with 125-1 labeled Rab'k fragments using an in v'rtro binding assay. The results showed that the binding activity of Rab'h to SW1116 was practically the same as that of whole MoAb A7. The prefered localization of the fragments to tumor tissue, compared with normal mouse tissue, was demonstrated in mice carrying SYV1116 xenografts. The tumor: blood ratio three days after injection was 2.64:18.5 for whole MoAb A7:F(ab')2, respectively. The tissue:blood ratios for the Ffab'h fragments showed a value of 18.5 in tumors, whereas its was a value < 1.0 in normal organs. The tumor accumulation of F(abO2 fragments was also dependent on the antigenic expression of each tumor among xenografts of colon carcinoma SVV1116 and WiDr, and squamous cell carcinoma KB. In kinetic experiments with whole MoAb A7 and its Rab'h fragments, whole MoAb A7 was lost, with a half-life of 4 days, in both blood and tumors, whereas FfabO? fragments were rapidly lost, with a half-life of 1.5 days. These results suggested that the F(abO2 fragments were cleared from the blood faster than was whole MoAb A7.
(Jpn. J. Clin. Oncol. 20: 139—144, 1990) Key words:
Monoclonal antibody A7— F(ab'>2 fragments—Biodistribution
Introduction Some reports on the application of monoclonal antibodies for the radioimmunodetection of human tumors have been presented, from which much information about the biodistribution of monoclonal antibodies has been accumulated.lJl) Kotanagi et al. produced MoAb A7 against human colon cancer." They demonstrated that MoAb A7 reacted with high specificity to colorectaJ carcinoma in immunostaining experiments using surgically resected human specimens.5' In addition, we have reported that the antigen detected by MoAb A7 to be distinct from other colon cancer-associated antigens which have already been characterized.61 In view of these points, it appears that the new monoclonal antibody, MoAb A7, may be useful in cancer diagnoReceived: November 11, 1989 Accepted: February 19, 1990 *For reprints and all correspondence
sis and therapy. We have produced immunoconjugates using MoAb A7 and have applied these conjugates clinically although the results showed some limitations in usage because of mouse-derived whole antibody.71 In order to enhance the in vivo specificity of MoAb A7 and eliminate the antimouse reaction derived from the presence of the Fc portion of the antibody, we attempted to produce F(ab')2 fragments. The purpose of the present investigation was to examine the biodistribution of F(ab'>2 fragments of the MoAb A7, for its application to the radioimaging of colorectal carcinomas and the targeting cancer chemotherapy. Materials and Methods Monoclonal Antibody and Its Purification Murine MoAb A7, which belongs to the class of I g d antibodies, was used in this series. The MoAb A7 was purified from murine ascitic fluid by affinity chromatography on immobilized Protein 139
Downloaded from https://academic.oup.com/jjco/article-abstract/20/2/139/818660 by University of Winnipeg user on 22 January 2019
Kazuya Kitamura1*, Toshio Takahashi1, Toshiharu Yamaguchi1, Shozo Kitai1, Takashi Amagai2 and Jiro Imanishi2
KITAMURA ET AL.
A.8) The eluted antibody was dialyzed against phosphate buffered saline (PBS), pH 7.2, and stored in a deep freezer at -70°C. Normal mouse I g d control was purchased from Boehringer Mannheim Biochemical (Mannheim F.R.G,). Cells
Preparation of F(ab')2 Fragments and Fab The F(ab')2 fragments were obtained from purified MoAb A7 by pepsin digestion. Pepsin was obtained from the Sigma Chemical Co. (St. Louis, MO, USA). The optimal conditions employed were an enzyme-antibody ratio of 1:50 and incubation at 37°C for 8h in 100 mM acetate buffer at pH 3.6." The fragment preparation was dialyzed against PBS and purified by gel filtration using a Sephacryl S-200 column. The Fab fragments were obtained by papain digestion of MoAb A7. The optimal conditions employed were those described by Stanworth.10' The purity of the F(ab')2 and Fab fragments was analyzed by Sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDSPAGE, 9%).
Tumor Xenografts Athymic nude mice (Balb/c, nu nu), 4-5 weeks old, were purchased from Shimizu Inc., Kyoto. They were given a subcutaneous injection in the flank of 5x10* cells of SW1116, WiDr and KB in 0.5 ml RPMI 1640 medium containing 10% FCS. Tumors began to appear within 1-2 weeks of the injection. Experiments were performed using nude mice bearing tumors of between 0.5 and 1.0 g. Biodistribution Radiolabeled whole MoAb A7 or its F(ab')2 fragments, and normal Igd were intraperitoneally injected into nude mice. Between 1 and 7 days after injection, the mice were killed by cervical dislocation and dissected. Tumors, blood and visceral organs were weighed, and their radioactivities were counted using a gamma counter. In the organ and tumor distribution studies, the results were expressed as ratios of the specific activity of the antibody in tissue to that in blood [(cpm/g in tissue)/(cpm/g in blood)]. Specific activities in tumor tissue were also compared among whole MoAb A7, F(ab')2 fragments and normal IgGi. To examine the kinetics of radiolabeled whole MoAb A7 and its F(ab')2 fragments, the radioactivities of the tumors and blood were measured over time. The results, expressed as cpm/g in tissue, for whole MoAb A7 and its F(ab') 2 fragments were compared.
Radiolabeling Purified MoAb A7 together with its F(ab')2 fragments and normal I g d were labeled with 125-1 using the chloramine T method.1" The unbound iodine was removed by filtration on an anion exchange column. The resulting specific activities were 5-10 nCi/fig. Cell Binding Tests The immunological reactivity of the labeled preparation was assayed by a quantitation binding test with target tumor cells.4' 125-1 labeled whole MoAb A7 and its F(ab')2 fragments were diluted in RPMI-1640 medium with 10% FCS in a twofold serial dilution. Trypsin harvested antigen positive cells (SW1116) and antigen negative cells (KB) were diluted in RPMI-1640 medium, and 100 xlO3 cells in 0.5 ml were placed into an Eppendorf tube. An equal volume of each dilution of antibody was added in triplicate to the cells. After the tubes containing the cells had been incubated at 4°C with agitation for 2h, the cells were pelleted by centrifugation. The cells were then washed three times with 140
Results F(ab')2 and Fab Fragment Production and Purity The product of whole MoAb A7, digested by pepsin and followed by gel filtration, yielded a single band on polyacrylamide gel electrophoresis under non-reducing conditions. The apparent molecular weights of the whole antibody, its F(ab')2 fragments and the Fab fragment were approximately 150xl0\ 120X103 and 60x10* daltons respectively (Fig. 1). Under the conditions adopted, the digested whole MoAb A7 solution contained a single fraction of F(ab')2 fragments with a large amount of small sized proteins which could not be recognized as apparent bands by SDSPAGE. The digested products under these conditions yielded F(ab')2 fragments at 20% of the total protein amount (data not shown).
Jpn. J. Clin. Oncol. 20(2) 1990
Downloaded from https://academic.oup.com/jjco/article-abstract/20/2/139/818660 by University of Winnipeg user on 22 January 2019
Colon cancer cell lines, SW1116 and WiDr, were used as target cells for the in vitro binding assay and in vivo biodistribution studies. The squamous carcinoma cell line, KB, was used to provide antigen negative cells in vitro and in vivo. All these cell lines were maintained in Roswell Park Memorial Institute's Medium 1640 (RPMI 1640) supplemented with 10% heat inactivated fetal calf serum (FCS).
PBS and the radioactivities of the pellets determined using a gamma counter.
F(ab')2 FRAGMENTS OF MOAB A7
2
22 23 24 25 26 27
Dilution of Labeled Preparations ((Powers of | ) x - ^ - )
Fig. 1: SDS-PAGE (9%) of whole MoAb A7, its F(ab')2 and Fab fragments. Samples were run under non-reducing conditions, lane A, Fab fragment; lane B, F(ab')2 fragments; lane C, whole MoAb A7
Binding Activities of MoAb A7 and Its F(ab')2 Fragments The maximum binding to cell line SW1116 was 27% with 125-1 labeled whole MoAb A7, and that of 125-1 F(ab')2 showed 24% binding (Fig. 2). Neither preparation showed any significant binding to antigen negative control cells of squamous cell carcinoma, KB. Kinetics of Radiolabeled MoAb A7 and Its F(ab')2 Fragments in Mice Fig. 3 shows the radioactivities of the blood and tumors of mice after intraperitoneal injection of MoAb A7 and its F(ab'>2 fragments. Whole MoAb A7 was lost, with a half-life of 4 days, in the blood and tumors, whereas F(ab')2 was rapidly lost, with a half-life of 1.5 days. According to the tissue distribution data, whole MoAb A7 showed high accumulations not only in tumors but in organs such as the lung, liver and spleen which have reticuloendothelial systems, whereas F(ab')2 fragments showed less accumulation than whole MoAb A7 in such organs (data not shown). These results suggest the greater localization of F(ab')2 fragments in tumors to have been due to the rapid clearance
Fig. 2: Binding activities of radiolabeled MoAb A7 and its F(ab') 2 fragments to antigen positive cells (SW1116) and antigen negative cells (KB). Cells were incubated with radiolabeled whole MoAb A7 or F(ab') 2 fragments for 1 h at various concentrations of the labeled antibody. After washing, the pellets were subjected to gamma counting. — o — Whole MoAb A7 vs SW1116; _ A — F(ab')2 vs SW1116; — o — Whole MoAb A7 vs KB; — A — F(ab') 2 vs KB
of fragments from the blood and their lower accumulation in the reticuloendothelial system. Tumor and Organ Distribution Studies The prefered localization of the MoAb A7 fragments to SW1116 tumor tissue compared with normal mouse tissue was demonstrated in xenografted mice (Fig. 4). The tumonblood value for the F(ab')2 fragments reached 18.5 three days after injection, whereas tissuerblood values were < 1.0 in all other organs. The tumonblood value was also much higher in the F(ab')2 fragments compared to those of whole MoAb A7 and normal IgG three days after injection, which were 18.6, 2.6 and 0.3, respectively, as indicated in Fig. 5. Radiolabeled F(ab')2 localizations were highly specific for SW1116 and WiDr xenografts, although less so in WiDr xenografts. Control studies in mice with KB squamous carcinoma cell xenografts showed no tumor localization (Fig. 6). The results of the experiment using three cancer cell lines showed the
Downloaded from https://academic.oup.com/jjco/article-abstract/20/2/139/818660 by University of Winnipeg user on 22 January 2019
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KITAMURA ET AL.
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Fig. 3: Kinetics of radiolabeled whole MoAb A7 and its F(ab')2 fragments in both tumor and blood. Groups of five mice were intraperitoneally injected with 125-1 labeled MoAb A7 and 125-1 labeled F(ab') 2 fragments, and the radioactivities were counted. A 125-1 MoAb A7 in blood; A 125-1 MoAb A7 in SW1116; o 125-1 F(ab') 2 in blood; • 125-1 F(ab') 2 in SW1116
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Fig. 4: Tissue:blood ratio value of F(ab') 2 fragments. Five mice were intraperitoneally injected with 125-1 labeled F(ab'>2 fragments, and the radioactivities in organs counted three days after injection (n = 5). Values are means with their standard deviations represented by vertical bars.
tumor localization of the fragments to increase with the degree of antigen expression. Discussion
Departments of 'Surgery and 2Microbiology, Kyoto Prefectural University of Medicine, 465, Kawaramachihirokojikajii-cho, Kamikyo-ku, Kyoto 602 The monoclonal antibody A7 (MoAb A7), which belongs to IgGi, was digested with pepsin to yield Hab'h fragments. The maximum binding to the human colon cancer cell line, SW1116, was 27% with 125-1 labeled whole MoAb A7 and 24% with 125-1 labeled Rab'k fragments using an in v'rtro binding assay. The results showed that the binding activity of Rab'h to SW1116 was practically the same as that of whole MoAb A7. The prefered localization of the fragments to tumor tissue, compared with normal mouse tissue, was demonstrated in mice carrying SYV1116 xenografts. The tumor: blood ratio three days after injection was 2.64:18.5 for whole MoAb A7:F(ab')2, respectively. The tissue:blood ratios for the Ffab'h fragments showed a value of 18.5 in tumors, whereas its was a value < 1.0 in normal organs. The tumor accumulation of F(abO2 fragments was also dependent on the antigenic expression of each tumor among xenografts of colon carcinoma SVV1116 and WiDr, and squamous cell carcinoma KB. In kinetic experiments with whole MoAb A7 and its Rab'h fragments, whole MoAb A7 was lost, with a half-life of 4 days, in both blood and tumors, whereas FfabO? fragments were rapidly lost, with a half-life of 1.5 days. These results suggested that the F(abO2 fragments were cleared from the blood faster than was whole MoAb A7.
(Jpn. J. Clin. Oncol. 20: 139—144, 1990) Key words:
Monoclonal antibody A7— F(ab'>2 fragments—Biodistribution
Introduction Some reports on the application of monoclonal antibodies for the radioimmunodetection of human tumors have been presented, from which much information about the biodistribution of monoclonal antibodies has been accumulated.lJl) Kotanagi et al. produced MoAb A7 against human colon cancer." They demonstrated that MoAb A7 reacted with high specificity to colorectaJ carcinoma in immunostaining experiments using surgically resected human specimens.5' In addition, we have reported that the antigen detected by MoAb A7 to be distinct from other colon cancer-associated antigens which have already been characterized.61 In view of these points, it appears that the new monoclonal antibody, MoAb A7, may be useful in cancer diagnoReceived: November 11, 1989 Accepted: February 19, 1990 *For reprints and all correspondence
sis and therapy. We have produced immunoconjugates using MoAb A7 and have applied these conjugates clinically although the results showed some limitations in usage because of mouse-derived whole antibody.71 In order to enhance the in vivo specificity of MoAb A7 and eliminate the antimouse reaction derived from the presence of the Fc portion of the antibody, we attempted to produce F(ab')2 fragments. The purpose of the present investigation was to examine the biodistribution of F(ab'>2 fragments of the MoAb A7, for its application to the radioimaging of colorectal carcinomas and the targeting cancer chemotherapy. Materials and Methods Monoclonal Antibody and Its Purification Murine MoAb A7, which belongs to the class of I g d antibodies, was used in this series. The MoAb A7 was purified from murine ascitic fluid by affinity chromatography on immobilized Protein 139
Downloaded from https://academic.oup.com/jjco/article-abstract/20/2/139/818660 by University of Winnipeg user on 22 January 2019
Kazuya Kitamura1*, Toshio Takahashi1, Toshiharu Yamaguchi1, Shozo Kitai1, Takashi Amagai2 and Jiro Imanishi2
KITAMURA ET AL.
A.8) The eluted antibody was dialyzed against phosphate buffered saline (PBS), pH 7.2, and stored in a deep freezer at -70°C. Normal mouse I g d control was purchased from Boehringer Mannheim Biochemical (Mannheim F.R.G,). Cells
Preparation of F(ab')2 Fragments and Fab The F(ab')2 fragments were obtained from purified MoAb A7 by pepsin digestion. Pepsin was obtained from the Sigma Chemical Co. (St. Louis, MO, USA). The optimal conditions employed were an enzyme-antibody ratio of 1:50 and incubation at 37°C for 8h in 100 mM acetate buffer at pH 3.6." The fragment preparation was dialyzed against PBS and purified by gel filtration using a Sephacryl S-200 column. The Fab fragments were obtained by papain digestion of MoAb A7. The optimal conditions employed were those described by Stanworth.10' The purity of the F(ab')2 and Fab fragments was analyzed by Sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDSPAGE, 9%).
Tumor Xenografts Athymic nude mice (Balb/c, nu nu), 4-5 weeks old, were purchased from Shimizu Inc., Kyoto. They were given a subcutaneous injection in the flank of 5x10* cells of SW1116, WiDr and KB in 0.5 ml RPMI 1640 medium containing 10% FCS. Tumors began to appear within 1-2 weeks of the injection. Experiments were performed using nude mice bearing tumors of between 0.5 and 1.0 g. Biodistribution Radiolabeled whole MoAb A7 or its F(ab')2 fragments, and normal Igd were intraperitoneally injected into nude mice. Between 1 and 7 days after injection, the mice were killed by cervical dislocation and dissected. Tumors, blood and visceral organs were weighed, and their radioactivities were counted using a gamma counter. In the organ and tumor distribution studies, the results were expressed as ratios of the specific activity of the antibody in tissue to that in blood [(cpm/g in tissue)/(cpm/g in blood)]. Specific activities in tumor tissue were also compared among whole MoAb A7, F(ab')2 fragments and normal IgGi. To examine the kinetics of radiolabeled whole MoAb A7 and its F(ab')2 fragments, the radioactivities of the tumors and blood were measured over time. The results, expressed as cpm/g in tissue, for whole MoAb A7 and its F(ab') 2 fragments were compared.
Radiolabeling Purified MoAb A7 together with its F(ab')2 fragments and normal I g d were labeled with 125-1 using the chloramine T method.1" The unbound iodine was removed by filtration on an anion exchange column. The resulting specific activities were 5-10 nCi/fig. Cell Binding Tests The immunological reactivity of the labeled preparation was assayed by a quantitation binding test with target tumor cells.4' 125-1 labeled whole MoAb A7 and its F(ab')2 fragments were diluted in RPMI-1640 medium with 10% FCS in a twofold serial dilution. Trypsin harvested antigen positive cells (SW1116) and antigen negative cells (KB) were diluted in RPMI-1640 medium, and 100 xlO3 cells in 0.5 ml were placed into an Eppendorf tube. An equal volume of each dilution of antibody was added in triplicate to the cells. After the tubes containing the cells had been incubated at 4°C with agitation for 2h, the cells were pelleted by centrifugation. The cells were then washed three times with 140
Results F(ab')2 and Fab Fragment Production and Purity The product of whole MoAb A7, digested by pepsin and followed by gel filtration, yielded a single band on polyacrylamide gel electrophoresis under non-reducing conditions. The apparent molecular weights of the whole antibody, its F(ab')2 fragments and the Fab fragment were approximately 150xl0\ 120X103 and 60x10* daltons respectively (Fig. 1). Under the conditions adopted, the digested whole MoAb A7 solution contained a single fraction of F(ab')2 fragments with a large amount of small sized proteins which could not be recognized as apparent bands by SDSPAGE. The digested products under these conditions yielded F(ab')2 fragments at 20% of the total protein amount (data not shown).
Jpn. J. Clin. Oncol. 20(2) 1990
Downloaded from https://academic.oup.com/jjco/article-abstract/20/2/139/818660 by University of Winnipeg user on 22 January 2019
Colon cancer cell lines, SW1116 and WiDr, were used as target cells for the in vitro binding assay and in vivo biodistribution studies. The squamous carcinoma cell line, KB, was used to provide antigen negative cells in vitro and in vivo. All these cell lines were maintained in Roswell Park Memorial Institute's Medium 1640 (RPMI 1640) supplemented with 10% heat inactivated fetal calf serum (FCS).
PBS and the radioactivities of the pellets determined using a gamma counter.
F(ab')2 FRAGMENTS OF MOAB A7
2
22 23 24 25 26 27
Dilution of Labeled Preparations ((Powers of | ) x - ^ - )
Fig. 1: SDS-PAGE (9%) of whole MoAb A7, its F(ab')2 and Fab fragments. Samples were run under non-reducing conditions, lane A, Fab fragment; lane B, F(ab')2 fragments; lane C, whole MoAb A7
Binding Activities of MoAb A7 and Its F(ab')2 Fragments The maximum binding to cell line SW1116 was 27% with 125-1 labeled whole MoAb A7, and that of 125-1 F(ab')2 showed 24% binding (Fig. 2). Neither preparation showed any significant binding to antigen negative control cells of squamous cell carcinoma, KB. Kinetics of Radiolabeled MoAb A7 and Its F(ab')2 Fragments in Mice Fig. 3 shows the radioactivities of the blood and tumors of mice after intraperitoneal injection of MoAb A7 and its F(ab'>2 fragments. Whole MoAb A7 was lost, with a half-life of 4 days, in the blood and tumors, whereas F(ab')2 was rapidly lost, with a half-life of 1.5 days. According to the tissue distribution data, whole MoAb A7 showed high accumulations not only in tumors but in organs such as the lung, liver and spleen which have reticuloendothelial systems, whereas F(ab')2 fragments showed less accumulation than whole MoAb A7 in such organs (data not shown). These results suggest the greater localization of F(ab')2 fragments in tumors to have been due to the rapid clearance
Fig. 2: Binding activities of radiolabeled MoAb A7 and its F(ab') 2 fragments to antigen positive cells (SW1116) and antigen negative cells (KB). Cells were incubated with radiolabeled whole MoAb A7 or F(ab') 2 fragments for 1 h at various concentrations of the labeled antibody. After washing, the pellets were subjected to gamma counting. — o — Whole MoAb A7 vs SW1116; _ A — F(ab')2 vs SW1116; — o — Whole MoAb A7 vs KB; — A — F(ab') 2 vs KB
of fragments from the blood and their lower accumulation in the reticuloendothelial system. Tumor and Organ Distribution Studies The prefered localization of the MoAb A7 fragments to SW1116 tumor tissue compared with normal mouse tissue was demonstrated in xenografted mice (Fig. 4). The tumonblood value for the F(ab')2 fragments reached 18.5 three days after injection, whereas tissuerblood values were < 1.0 in all other organs. The tumonblood value was also much higher in the F(ab')2 fragments compared to those of whole MoAb A7 and normal IgG three days after injection, which were 18.6, 2.6 and 0.3, respectively, as indicated in Fig. 5. Radiolabeled F(ab')2 localizations were highly specific for SW1116 and WiDr xenografts, although less so in WiDr xenografts. Control studies in mice with KB squamous carcinoma cell xenografts showed no tumor localization (Fig. 6). The results of the experiment using three cancer cell lines showed the
Downloaded from https://academic.oup.com/jjco/article-abstract/20/2/139/818660 by University of Winnipeg user on 22 January 2019
— — -!__!_ _L _L_L
KITAMURA ET AL.
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rh 1 2
3 4 5 6 Days After Injection
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8
Fig. 3: Kinetics of radiolabeled whole MoAb A7 and its F(ab')2 fragments in both tumor and blood. Groups of five mice were intraperitoneally injected with 125-1 labeled MoAb A7 and 125-1 labeled F(ab') 2 fragments, and the radioactivities were counted. A 125-1 MoAb A7 in blood; A 125-1 MoAb A7 in SW1116; o 125-1 F(ab') 2 in blood; • 125-1 F(ab') 2 in SW1116
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Fig. 4: Tissue:blood ratio value of F(ab') 2 fragments. Five mice were intraperitoneally injected with 125-1 labeled F(ab'>2 fragments, and the radioactivities in organs counted three days after injection (n = 5). Values are means with their standard deviations represented by vertical bars.
tumor localization of the fragments to increase with the degree of antigen expression. Discussion
