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Increased streptavidin uptake in tumors pretargeted with biotinylated antibody using a conjugate of streptavidin-Fab fragment
ISSN 0969-8051/98/$19.00 1 0.00 PII S0969-8051(98)00018-3
Nuclear Medicine & Biology, Vol. 25, pp. 557–560, 1998 Copyright © 1998 Elsevier Science Inc.
Increased Streptavidin Uptake in Tumors Pretargeted with Biotinylated Antibody Using a Conjugate of Streptavidin-Fab Fragment Zhengsheng Yao,1 Meili Zhang,1 Harumi Sakahara,1 Tsuneo Saga,1 Hisataka Kobayashi,1 Yuji Nakamoto,1 Sakuji Toyama2 and Junji Konishi1 1
DEPARTMENT OF NUCLEAR MEDICINE, FACULTY OF MEDICINE, AND 2INSTITUTE FOR VIRUS RESEARCH, KYOTO UNIVERSITY, KYOTO, JAPAN
ABSTRACT. Radiolabeled streptavidin accumulated in tumors pretargeted with biotinylated antibody. However, the absolute delivery of radioactivity was limited. To increase the tumor uptake of radioactivity further, we conjugated streptavidin with a mouse monoclonal antibody (MAb) fragment, OST6Fab, which recognizes antigen on human osteosarcoma. Another mouse MAb, OST7, which also reacts with the same tumor but recognizes an epitope different from the OST6 epitope, was biotinylated. The radioiodinated streptavidin-OST6Fab conjugate was administered to tumor-bearing mice after the biotinylated OST7 pretargeting. The uptake of the conjugate in tumors pretargeted with the biotinylated antibody was significantly higher than that of streptavidin and that of the conjugate of streptavidin and irrelevant Fab fragment. Renal uptake of radioactivity was decreased markedly, and the blood clearance was retarded by the conjugation with Fab fragment. In conclusion, the conjugate of streptavidin with specific Fab fragment increased the accumulation of radioactivity in tumors pretargeted with biotinylated antibody. NUCL MED BIOL 25;6:557–560, 1998. © 1998 Elsevier Science Inc. KEY WORDS. Tumor targeting, Streptavidin, Biotinylated antibody, Fab fragment
INTRODUCTION To solve the problem of slow uptake in tumor and slow clearance from circulation of radiolabeled antibodies, an avidin-biotin system with a pretargeting technique has been introduced in tumor imaging (4, 6, 8, 11–14, 21, 24). In this system, antibodies and radiolabeled substances are administered separately, and radioactivity is selectively accumulated in tumors with simultaneous reduction of radioactivity in nontumor tissues. In a two-step method using biotinylated antibody and radiolabeled streptavidin, tumor uptake and/or nontumor clearance of radioactivity was accelerated and a more uniform intratumoral distribution of radioactivity was obtained in comparison with the conventional one-step method using radiolabeled antibody (8, 12, 14, 16, 21–23). Tumor accumulation of radiolabeled streptavidin in the two-step method depends, to a great extent, on the amount of biotinylated antibody on the cell surface. As antigens continuously undergo modulation or internalization after binding with antibody, the amount of biotin on the tumor surface delivered by the antibody decreases with time because of metabolism of the antigen-antibody complex. By using the two-step method, the tumor uptake of radiolabeled streptavidin was higher at 4 h than at 24 h after injection of the radiolabel (16, 18). Another study revealed that the tumor accumulation of radiolabeled streptavidin decreased with the pretargeting time after 2 days (8). Our previous study on the chase of biotinylated antibody with avidin before radiolabeled streptavidin administration showed accelerated tumor uptake and blood Address correspondence to: Zhengsheng Yao, MD, Department of Nuclear Medicine, Faculty of Medicine, Kyoto University, Shogoin, Sakyo-ku, Kyoto 606-8507, Japan; e-mail: ^[email protected]&. Received 9 January 1998. Accepted 14 March 1998.
clearance but no increase in the tumor radioactivity delivery (21). These findings suggested that the number of binding sites of streptavidin in the tumor produced by biotinylated antibody was limited. To increase the binding sites of streptavidin in tumors, we conjugated a specific Fab fragment with streptavidin. The conjugate, which had both biotin and antigen binding activities, increased the accumulation of radioactivity in tumors pretargeted with the biotinylated antibody. MATERIALS AND METHODS
Monoclonal Antibodies Monoclonal antibodies (MAbs) OST6 and OST7, both of which are murine IgG1 isotype, were raised against a human osteogenic sarcoma. The antibodies have been shown to react with human osteogenic sarcoma cells, but the epitopes are different from each other (5, 19). Radiolabeled OST6 and OST7 localized well in KT005 osteosarcoma xenografts in vivo (17). Isotype-matched control monoclonal antibody, 56C, reacts with human chorionic gonadotropin. The antibodies were purified from ascitic fluid of hybridoma-bearing mice using Protein A column chromatography (Bio-Rad, Richmond, CA). Fab fragments were generated by papain digestion of the whole IgG of OST6 or 56C. Papain was added to IgG in 0.075 M phosphate-buffered saline, pH 7.0, to give an enzyme-to-IgG weight ratio of 1:33. After incubation at 37°C for 1 h, the reaction was stopped by adding 10% volume of 0.5 M iodoacetamide. The Fab fragments were separated by Superdex 200 column chromatography (Pharmacia Biotech, Uppsala, Sweden), and were further purified from contaminating Fc fragment by Protein A affinity chromatography.
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TABLE 1. Experimental Protocol
Biotinylation of IgG and Fab OST7 antibody or the Fab fragment of OST6 or 56C antibody was conjugated with biotin using NHS-LC-biotin (Pierce, Rockford, IL). Freshly prepared NHS-LC-biotin solution (10 mg/mL) was added to 5 mg/mL of antibody or Fab fragment in 0.05 M phosphate-buffered saline, pH 7.5, at a molar ratio of biotin-to-IgG of 9:1 or biotin-to-Fab of 5:1. After incubation for 2 h at 4°C, unconjugated biotin was removed by chromatography on a PD10 column (Pharmacia Biotech). The number of biotin molecules coupled to antibody or fragment was determined by the method of Green (2) using HABA solution (Pierce).
Conjugation of Streptavidin with Fab Fragment Streptavidin (Pierce) was added to biotinylated Fab fragment at a molar ratio of 2.5:1 and incubated for 30 min at room temperature. The mixture was applied to Superdex 200 column chromatography, and the effluent was monitored by UV absorption at 280 nm. Three peaks were observed, and the conjugate from the second peak had a molecular weight of ;110 kDa, suggesting one-to-one coupling of streptavidin with Fab fragment, and was used in the experiment.
Radioiodination of Proteins Proteins, including OST6Fab fragment, streptavidin, and streptavidin-Fab conjugates, were radioiodinated with 125I using the chloramine T method (17). Forty micrograms of protein in 0.3 M phosphate buffer, pH 7.5, and 125I (22.2–29.6 MBq) (Du Pont, Wilmington, DE) were mixed with 2.5 mg of chloramine T (Nacalai Tesque, Kyoto, Japan) dissolved in 0.3 M phosphate buffer. After 5 min, radiolabeled proteins were separated from free iodine by chromatography through PD10 column, and the radiochemical purity was determined as being more than 97% by high-performance liquid chromatography. Specific activities of radiolabeled proteins were 370 –740 MBq/mg.
In Vitro Reactivity The biotin binding ability of the conjugates was tested using polystyrene beads coated with biotinylated antibody or immobilized D-Biotin gel (Pierce). Polystyrene beads of 6.4 mm in diameter were coated with biotinylated 145-9, a mouse MAb against CA125, as reported previously (15). The radioiodinated conjugates were incubated with the beads or the biotin gel for 30 min at room temperature and the bound percentage of radioactivity was determined. The antigen binding ability of streptavidin-OST6Fab conjugate was examined by cell binding assay. Radioiodinated OST6Fab fragment or the streptavidin-Fab conjugates (5 ng/100 mL) were incubated with KT005 cells (1 3 104 2 3 3 106/100 mL) in 5.7 3 46 mm microcentrifuge tubes for 1 h at 4°C. After centrifugation at
Time (h)
Group 1
Group 2
SA
BT-Ab Avidin SA
224 20.5 0 24
Group 3
Group 4
BT-Ab BT-Ab Avidin Avidin SA-OST6Fab SA-56CFab Biodistribution
BT-Ab: biotinylated antibody OST7; SA: radiolabeled streptavidin; Fab: antibody Fab fragment.
10,000 g, the supernatant was aspirated and the tubes were cut. Then, the radioactivity bound to cells was counted. BIODISTRIBUTION STUDY Osteosarcoma xenograft was established by subcutaneous inoculation of KT005 cells into female BALB/c-nu/nu mice, and the xenograft was maintained by serial subcutaneous transplantation. The experimental protocol is shown in Table 1. In Group 1, 125 I-labeled streptavidin (5 mg) alone was injected into the tumorbearing mice. In Groups 2, 3 and 4, 125I-labeled streptavidin (5 mg), streptavidin-OST6Fab conjugate (9 mg), or streptavidin-56CFab conjugate (9 mg) was injected, respectively, into the tumor-bearing mice 24 h after IV administration of 30 mg of biotinylated OST7 for pretargeting. Also in these three groups, avidin (80 mg) was administered intraperitoneally 30 min before injection of radiolabeled streptavidin or streptavidin-Fab conjugates for chasing biotinylated antibody in the circulation (9, 21). All of the radiolabels were given intravenously. Groups of 6 mice were killed 24 h after injection of the radiolabeled streptavidin or streptavidin-Fab conjugates and the biodistribution of radioactivity was determined. Administered dose was adjusted by the addition of unlabeled counterparts to radiolabels. Data were expressed as percentage of the injected dose per gram of tissue (%ID/g). Statistical analysis was performed using Student’s t-test. All procedures involving animal controls were carried out in accordance with the regulations for animal welfare in Japan. RESULTS The average number of biotin molecules coupled to each antibody or fragment was determined to be 2.5 and 0.9, respectively. Conjugation of streptavidin with Fab fragment resulted in some decrease of biotin binding ability (Table 2). The antigen binding ability of OST6Fab fragment was also affected by the conjugation with streptavidin, as shown in Figure 1. Table 3 shows the biodistribution of radiolabels at 24 h postinjection. The tumor uptake of radiolabeled streptavidin was higher in mice with the biotinylated antibody pretargeting than in those
TABLE 2. Biotin-Binding Capacity of Streptavidin and Streptavidin-Fab Fragment Conjugates % Bounda
Beads coated with biotinylated antibody Biotin gel a
Mean 6 SD of triplicate.
Streptavidin
Streptavidin-OST6Fab
Streptavidin-56CFab
73.8 6 5.2 56.9 6 5.5
53.1 6 3.7 52.3 6 4.3
48.5 6 4.8 48.9 6 3.2
Tumor Targeting with Streptavidin-Fab Conjugate
559
FIG. 1. Binding of 125I-labeled OST6Fab fragment or streptavidin-Fab conjugates to KT005 cells. The radiolabels were incubated with increasing concentration of cells for 1 h at 4&C. After centrifugation, radioactivity bound to cells was counted. Error bars show SD of triplicate.
without the pretargeting (Group 2 vs. Group 1, p , 0.002), indicating that the biotinylated antibody accumulated in tumors and contributed to the specific tumor uptake of streptavidin. The tumor uptake of streptavidin-OST6Fab conjugate was higher than that of streptavidin (Group 3 vs. Group 2, p , 0.05) and that of streptavidin-56CFab conjugate (Group 3 vs. Group 4, p 5 0.002), suggesting that the specific Fab portion of the conjugate directly bound the surface antigen on tumor cells. Blood clearance of the conjugates was slower than that of streptavidin, and the renal uptake of streptavidin was decreased markedly by the conjugation with Fab fragments. DISCUSSION For an effective tumor radioimmunotherapy, both absolute delivery and target-to-nontarget ratios of radioactivity must be high. The disadvantages of radiolabeled antibodies are the slow tumor uptake and low target-to-nontarget ratios. Application of the avidin-biotin
system with a pretargeting technique could partly overcome these shortcomings. However, because of the increased number of steps, the number of binding sites in tumor for the following step would be decreased as a result of metabolism of antigen-antibody complex. Our previous studies revealed that radiolabeled streptavidin accumulated rapidly in tumors pretargeted with biotinylated antibody, but the peak uptake of radioactivity in the tumor was lower than that obtained by the radiolabeled antibody (20, 21). In the present study, streptavidin was conjugated to a MAb Fab fragment. The streptavidin-Fab conjugate was radiolabeled and used as the second step in the two-step method with biotinylated antibody pretargeting. Owing to the bifunctionality of the conjugate, i.e., its ability to bind both biotinylated antibody and tumor-associated antigen in the tumor cell surface, tumor uptake of radioactivity was increased. The improvement of tumor uptake of the conjugate is not only useful to the two-step tumor-targeting technique, as in the method in this study, but also possibly useful to other multistep targeting when biotin is used as radiotracer (1, 4, 6, 24) because the amount
TABLE 3. Biodistribution of Radiolabeled Streptavidin or Streptavidin-Fab Conjugates in KT005 Tumor-Bearing Mice (24 h) Organ Blood Liver Kidney Intestine Stomach Spleen Lung Muscle Bone Tumor
Group 1
Group 2
Group 3
Group 4
0.99 6 0.13 3.44 6 0.26 79.11 6 14.4 0.74 6 0.12 0.93 6 0.13 2.33 6 0.22 1.60 6 0.31 0.58 6 0.08 0.84 6 0.09 3.33 6 0.61
1.70 6 0.26 4.18 6 0.70 85.51 6 5.13 0.65 6 0.06 0.83 6 0.22 3.29 6 0.47 1.67 6 0.17 0.49 6 0.05 0.89 6 0.07 9.14 6 2.35
3.87 6 0.34 4.79 6 0.41 19.27 6 2.41 0.78 6 0.06 1.31 6 0.38 4.21 6 0.46 2.59 6 0.36 0.53 6 0.06 1.01 6 0.09 12.48 6 2.68
4.27 6 0.48 5.11 6 0.79 16.06 6 2.25 1.23 6 0.13 3.55 6 0.91 3.52 6 0.48 2.79 6 0.34 0.52 6 0.07 1.03 6 0.13 5.71 6 0.81
Mean 6 SD of %ID/g (n 5 6). See Table 1 Protocol for more details.
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of its binding site, streptavidin, in tumor is increased. Conversely, by using antibodies OST7 and OST6, which recognize different epitopes on the same tumor, for pretargeting and targeting, respectively, binding inhibition would be avoided. The avidin-biotin system resulted in signal amplification in vitro with the increase in steps because each avidin molecule can bind four biotin molecules and protein can be polybiotinylated. This amplification was expected to occur in vivo with increase of steps or cycles. Unfortunately, the in vivo results are not satisfactory (7). Also, the binding sites could be increased by the use of a larger dose or higher biotinylation rate of biotinylated antibody. However, in that case, the amount of biotinylated antibody in circulation would also be large, resulting in a low tumor-to-nontumor ratio as large amounts of streptavidin-antibody complex formed and cleared slowly (24). In the approach described in our study, tumor delivery of radioactivity should be effectively increased by increasing the number of binding sites of the tracer only in tumor, as in the use of interferon, which increased tumor antigen expression and enhanced tumor localization of radiolabeled antibody (3, 10). High accumulation of streptavidin in kidney is a problem in its clinical application. Interestingly, the uptake of the conjugates in the kidney decreased markedly, suggesting that kidney uptake of streptavidin could be reduced through modification. Both the biotin binding ability of streptavidin and the antigen binding ability of Fab fragment were reduced after conjugation. The antigen binding ability was probably decreased by the steric hindrance from streptavidin because of its larger molecular size. The steric hindrance from Fab fragments may also contribute to the decreased biotin binding ability of streptavidin, but another possibility is the reduction of biotin binding sites of streptavidin as it was coupled to the fragments through biotin. In contrast, the conjugate showed a slower blood clearance than streptavidin, possibly as a result of enlarged molecule and significantly reduced kidney uptake. Therefore, a conjugate with a smaller molecular weight and full reactivity against both biotin and antigen is worthy of being developed.
7.
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10. 11.
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13. 14.
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18.
The authors are grateful to Kato Asao International Scholarship Foundation for its kind help. This work was supported in part by Grants-inAid for Scientific Research (07670995, 08266230) from the Ministry of Education, Science and Culture, and a Grant-in-Aid from the Sankyo Foundation of Life Science, Japan.
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20.
References 1. Alvarez-Diez T. M., Polihronis J. and Reilly R. M. (1996) Pretargeted tumor imaging with streptavidin immunoconjugates of monoclonal antibody CC49 and 111In-DTPA-biocytin. Nucl. Med. Biol. 23, 459 – 466. 2. Green N. M. (1975) Avidin. Adv. Protein Chem. 29, 85–133. 3. Greiner J. W., Ullmann C. D., Nieroda C., Qi C.-F., Eggensperger D., Shimada S. et al. (1993) Improved radioimmunotherapeutic efficiency of an anticarcinoma monoclonal antibody (131I-CC49) when given in combination with r-interferon. Cancer Res. 53, 600 – 608. 4. Hnatowich D. J., Virzi F. and Rusckowski M. (1987) Investigations of avidin and biotin for imaging applications. J. Nucl. Med. 28, 1294 – 1302. 5. Hosoi S., Nakamura T., Higashi S., Yamamuro T., Toyama S., Shinomiya K. et al. (1982) Detection of human osteosarcoma-associated antigen (s) by monoclonal antibodies. Cancer Res. 42, 654 – 659. 6. Kalofonos H. P., Rusckowski M., Siebecker D. A., Sivolapenko G. B., Snook D., Lavender J. P. et al. (1990) Imaging of tumor in patients with
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indium-111-labeled biotin and streptavidin-conjugated antibodies: Preliminary communication. J. Nucl. Med. 31, 1791–1796. Kassis A. I., Jones P. L., Matalka K. Z. and Adelstein S. J. (1996) Antibody-dependent signal amplification in tumor xenografts after pretreatment with biotinylated monoclonal antibody and avidin or streptavidin. J. Nucl. Med. 37, 343–352. Khawli L. A., Alauddin M. M., Miller G. K. and Epstein A. L. (1993) Improved immunotargeting of tumors with biotinylated monoclonal antibodies and radiolabeled streptavidin. Antibody Immunoconjug. Radiopharmaceut. 6, 13–27. Kobayashi H., Sakahara H., Hosono M., Yao Z., Toyama S., Endo K. et al. (1994) Improved clearance of radiolabeled biotinylated monoclonal antibody following the infusion of avidin as a “chase” without decreased accumulation in the target tumor. J. Nucl. Med. 35, 1677–1684. Nakamura K., Kubo A., Hosokawa S., Nagaike K. and Hashimoto S. (1993) Effect of alpha-interferon on anti-alpha-fetoprotein-monoclonal-antibody targeting of hepatoma. Oncology 50, 35– 40. Ngai W. M., Reilly R. M., Polihronis J. and Shpitz B. (1995) In vitro and in vivo evaluation of streptavidin immunoconjugates of the second generation TAG-72 monoclonal antibody CC49. Nucl. Med. Biol. 22, 77– 86. Paganelli G., Belloni C., Magnani P., Zito F., Pasini A., Sassi I. et al. (1992) Two-step tumour targetting in ovarian cancer patients using biotinylated monoclonal antibodies and radioactive streptavidin. Eur. J. Nucl. Med. 19, 322–329. Paganelli G., Magnani P., Zito F., Villa E., Sudati F., Lopalco L. et al. (1991) Three-step monoclonal antibody tumor targeting in carcinoembryonic antigen-positive patients. Cancer Res. 51, 5960 –5966. Paganelli G., Pervez S., Siccardi A. G., Rowlinson G., Deleide G., Chiolerio F. et al. (1990) Intraperitoneal radiolocalization of tumors pre-targeted by biotinylated monoclonal antibodies. Int. J. Cancer 45, 1184 –1189. Saga T., Endo K., Nakashima T., Awaji T., Koizumi M., Kawamura Y. et al. (1990) Construction of an immunoradiometric assay for ovarian cancer associated antigen CA125 recognizing different antigenic determinant. Acta Obstet. Gynecol. Scand. 69, 175–181. Saga T., Weinstein J. N., Jeong J. M., Heya T., Lee J. T., Le N. et al. (1994) Two-step targeting of experimental lung metastases with biotinylated antibody and radiolabeled streptavidin. Cancer Res. 54, 2160 – 2165. Sakahara H., Endo K., Koizumi M., Nakashima T., Kunimatsu M., Watanabe Y. et al. (1988) Relationship between in vitro binding activity and in vivo tumor accumulation of radiolabeled monoclonal antibodies. J. Nucl. Med. 29, 235–240. Sung C., van Osdol W. W., Saga T., Neumann R. D., Dedrik R. L. and Weinstein J. N. (1994) Streptavidin distribution in metastatic tumors pretargeted with a biotinylated monoclonal antibody; theoretical and experimental pharmacokinetics. Cancer Res. 54, 2166 –2175. Tanaka C., Yamamuro T., Masuda T., Tanaka H., Matsumoto M., Kotoura Y. et al. (1986) Recognition of serum alkaline phosphatase by murine monoclonal antibodies against human osteo-sarcoma cells. Cancer Res. 46, 4853– 4867. Yao Z., Sakahara H., Zhang M., Kobayashi H., Nakada H., Yamashina I. et al. (1995) Radioimmunoimaging of colon cancer xenografts with anti-Tn monoclonal antibody. Nucl. Med. Biol. 20, 199 –203. Yao Z., Zhang M., Kobayashi H., Sakahara H., Nakada H., Yamashina I. et al. (1995) Improved targeting of radiolabeled streptavidin in tumors pretargeted with biotinylated monoclonal antibodies through an avidin chase. J. Nucl. Med. 36, 837– 841. Zhang M., Sakahara H., Yao Z., Saga T., Nakamoto Y., Sato N. et al. (1997) Intravenous avidin chase improved localization of radiolabeled streptavidin in intraperitoneal xenograft pretargeted with biotinylated antibody. Nucl. Med. Biol. 24, 61– 64. Zhang M., Yao Z., Saga T., Sakahara H., Nakamoto Y., Sato N. et al. (1998) Improved intratumoral penetration of radiolabeled streptavidin in intraperitoneal tumor pretargeted with biotinylated antibody. J. Nucl. Med. 39, 33–36. Zhang M., Yao Z., Sakahara H., Saga T., Nakamoto Y., Sato N. et al. (1998) Effect of administration route and dose of streptavidin or biotin on the tumor uptake of radioactivity in intraperitoneal tumor with multistep targeting. Nucl. Med. Biol. 25, 101–105.
Nuclear Medicine & Biology, Vol. 25, pp. 557–560, 1998 Copyright © 1998 Elsevier Science Inc.
Increased Streptavidin Uptake in Tumors Pretargeted with Biotinylated Antibody Using a Conjugate of Streptavidin-Fab Fragment Zhengsheng Yao,1 Meili Zhang,1 Harumi Sakahara,1 Tsuneo Saga,1 Hisataka Kobayashi,1 Yuji Nakamoto,1 Sakuji Toyama2 and Junji Konishi1 1
DEPARTMENT OF NUCLEAR MEDICINE, FACULTY OF MEDICINE, AND 2INSTITUTE FOR VIRUS RESEARCH, KYOTO UNIVERSITY, KYOTO, JAPAN
ABSTRACT. Radiolabeled streptavidin accumulated in tumors pretargeted with biotinylated antibody. However, the absolute delivery of radioactivity was limited. To increase the tumor uptake of radioactivity further, we conjugated streptavidin with a mouse monoclonal antibody (MAb) fragment, OST6Fab, which recognizes antigen on human osteosarcoma. Another mouse MAb, OST7, which also reacts with the same tumor but recognizes an epitope different from the OST6 epitope, was biotinylated. The radioiodinated streptavidin-OST6Fab conjugate was administered to tumor-bearing mice after the biotinylated OST7 pretargeting. The uptake of the conjugate in tumors pretargeted with the biotinylated antibody was significantly higher than that of streptavidin and that of the conjugate of streptavidin and irrelevant Fab fragment. Renal uptake of radioactivity was decreased markedly, and the blood clearance was retarded by the conjugation with Fab fragment. In conclusion, the conjugate of streptavidin with specific Fab fragment increased the accumulation of radioactivity in tumors pretargeted with biotinylated antibody. NUCL MED BIOL 25;6:557–560, 1998. © 1998 Elsevier Science Inc. KEY WORDS. Tumor targeting, Streptavidin, Biotinylated antibody, Fab fragment
INTRODUCTION To solve the problem of slow uptake in tumor and slow clearance from circulation of radiolabeled antibodies, an avidin-biotin system with a pretargeting technique has been introduced in tumor imaging (4, 6, 8, 11–14, 21, 24). In this system, antibodies and radiolabeled substances are administered separately, and radioactivity is selectively accumulated in tumors with simultaneous reduction of radioactivity in nontumor tissues. In a two-step method using biotinylated antibody and radiolabeled streptavidin, tumor uptake and/or nontumor clearance of radioactivity was accelerated and a more uniform intratumoral distribution of radioactivity was obtained in comparison with the conventional one-step method using radiolabeled antibody (8, 12, 14, 16, 21–23). Tumor accumulation of radiolabeled streptavidin in the two-step method depends, to a great extent, on the amount of biotinylated antibody on the cell surface. As antigens continuously undergo modulation or internalization after binding with antibody, the amount of biotin on the tumor surface delivered by the antibody decreases with time because of metabolism of the antigen-antibody complex. By using the two-step method, the tumor uptake of radiolabeled streptavidin was higher at 4 h than at 24 h after injection of the radiolabel (16, 18). Another study revealed that the tumor accumulation of radiolabeled streptavidin decreased with the pretargeting time after 2 days (8). Our previous study on the chase of biotinylated antibody with avidin before radiolabeled streptavidin administration showed accelerated tumor uptake and blood Address correspondence to: Zhengsheng Yao, MD, Department of Nuclear Medicine, Faculty of Medicine, Kyoto University, Shogoin, Sakyo-ku, Kyoto 606-8507, Japan; e-mail: ^[email protected]&. Received 9 January 1998. Accepted 14 March 1998.
clearance but no increase in the tumor radioactivity delivery (21). These findings suggested that the number of binding sites of streptavidin in the tumor produced by biotinylated antibody was limited. To increase the binding sites of streptavidin in tumors, we conjugated a specific Fab fragment with streptavidin. The conjugate, which had both biotin and antigen binding activities, increased the accumulation of radioactivity in tumors pretargeted with the biotinylated antibody. MATERIALS AND METHODS
Monoclonal Antibodies Monoclonal antibodies (MAbs) OST6 and OST7, both of which are murine IgG1 isotype, were raised against a human osteogenic sarcoma. The antibodies have been shown to react with human osteogenic sarcoma cells, but the epitopes are different from each other (5, 19). Radiolabeled OST6 and OST7 localized well in KT005 osteosarcoma xenografts in vivo (17). Isotype-matched control monoclonal antibody, 56C, reacts with human chorionic gonadotropin. The antibodies were purified from ascitic fluid of hybridoma-bearing mice using Protein A column chromatography (Bio-Rad, Richmond, CA). Fab fragments were generated by papain digestion of the whole IgG of OST6 or 56C. Papain was added to IgG in 0.075 M phosphate-buffered saline, pH 7.0, to give an enzyme-to-IgG weight ratio of 1:33. After incubation at 37°C for 1 h, the reaction was stopped by adding 10% volume of 0.5 M iodoacetamide. The Fab fragments were separated by Superdex 200 column chromatography (Pharmacia Biotech, Uppsala, Sweden), and were further purified from contaminating Fc fragment by Protein A affinity chromatography.
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TABLE 1. Experimental Protocol
Biotinylation of IgG and Fab OST7 antibody or the Fab fragment of OST6 or 56C antibody was conjugated with biotin using NHS-LC-biotin (Pierce, Rockford, IL). Freshly prepared NHS-LC-biotin solution (10 mg/mL) was added to 5 mg/mL of antibody or Fab fragment in 0.05 M phosphate-buffered saline, pH 7.5, at a molar ratio of biotin-to-IgG of 9:1 or biotin-to-Fab of 5:1. After incubation for 2 h at 4°C, unconjugated biotin was removed by chromatography on a PD10 column (Pharmacia Biotech). The number of biotin molecules coupled to antibody or fragment was determined by the method of Green (2) using HABA solution (Pierce).
Conjugation of Streptavidin with Fab Fragment Streptavidin (Pierce) was added to biotinylated Fab fragment at a molar ratio of 2.5:1 and incubated for 30 min at room temperature. The mixture was applied to Superdex 200 column chromatography, and the effluent was monitored by UV absorption at 280 nm. Three peaks were observed, and the conjugate from the second peak had a molecular weight of ;110 kDa, suggesting one-to-one coupling of streptavidin with Fab fragment, and was used in the experiment.
Radioiodination of Proteins Proteins, including OST6Fab fragment, streptavidin, and streptavidin-Fab conjugates, were radioiodinated with 125I using the chloramine T method (17). Forty micrograms of protein in 0.3 M phosphate buffer, pH 7.5, and 125I (22.2–29.6 MBq) (Du Pont, Wilmington, DE) were mixed with 2.5 mg of chloramine T (Nacalai Tesque, Kyoto, Japan) dissolved in 0.3 M phosphate buffer. After 5 min, radiolabeled proteins were separated from free iodine by chromatography through PD10 column, and the radiochemical purity was determined as being more than 97% by high-performance liquid chromatography. Specific activities of radiolabeled proteins were 370 –740 MBq/mg.
In Vitro Reactivity The biotin binding ability of the conjugates was tested using polystyrene beads coated with biotinylated antibody or immobilized D-Biotin gel (Pierce). Polystyrene beads of 6.4 mm in diameter were coated with biotinylated 145-9, a mouse MAb against CA125, as reported previously (15). The radioiodinated conjugates were incubated with the beads or the biotin gel for 30 min at room temperature and the bound percentage of radioactivity was determined. The antigen binding ability of streptavidin-OST6Fab conjugate was examined by cell binding assay. Radioiodinated OST6Fab fragment or the streptavidin-Fab conjugates (5 ng/100 mL) were incubated with KT005 cells (1 3 104 2 3 3 106/100 mL) in 5.7 3 46 mm microcentrifuge tubes for 1 h at 4°C. After centrifugation at
Time (h)
Group 1
Group 2
SA
BT-Ab Avidin SA
224 20.5 0 24
Group 3
Group 4
BT-Ab BT-Ab Avidin Avidin SA-OST6Fab SA-56CFab Biodistribution
BT-Ab: biotinylated antibody OST7; SA: radiolabeled streptavidin; Fab: antibody Fab fragment.
10,000 g, the supernatant was aspirated and the tubes were cut. Then, the radioactivity bound to cells was counted. BIODISTRIBUTION STUDY Osteosarcoma xenograft was established by subcutaneous inoculation of KT005 cells into female BALB/c-nu/nu mice, and the xenograft was maintained by serial subcutaneous transplantation. The experimental protocol is shown in Table 1. In Group 1, 125 I-labeled streptavidin (5 mg) alone was injected into the tumorbearing mice. In Groups 2, 3 and 4, 125I-labeled streptavidin (5 mg), streptavidin-OST6Fab conjugate (9 mg), or streptavidin-56CFab conjugate (9 mg) was injected, respectively, into the tumor-bearing mice 24 h after IV administration of 30 mg of biotinylated OST7 for pretargeting. Also in these three groups, avidin (80 mg) was administered intraperitoneally 30 min before injection of radiolabeled streptavidin or streptavidin-Fab conjugates for chasing biotinylated antibody in the circulation (9, 21). All of the radiolabels were given intravenously. Groups of 6 mice were killed 24 h after injection of the radiolabeled streptavidin or streptavidin-Fab conjugates and the biodistribution of radioactivity was determined. Administered dose was adjusted by the addition of unlabeled counterparts to radiolabels. Data were expressed as percentage of the injected dose per gram of tissue (%ID/g). Statistical analysis was performed using Student’s t-test. All procedures involving animal controls were carried out in accordance with the regulations for animal welfare in Japan. RESULTS The average number of biotin molecules coupled to each antibody or fragment was determined to be 2.5 and 0.9, respectively. Conjugation of streptavidin with Fab fragment resulted in some decrease of biotin binding ability (Table 2). The antigen binding ability of OST6Fab fragment was also affected by the conjugation with streptavidin, as shown in Figure 1. Table 3 shows the biodistribution of radiolabels at 24 h postinjection. The tumor uptake of radiolabeled streptavidin was higher in mice with the biotinylated antibody pretargeting than in those
TABLE 2. Biotin-Binding Capacity of Streptavidin and Streptavidin-Fab Fragment Conjugates % Bounda
Beads coated with biotinylated antibody Biotin gel a
Mean 6 SD of triplicate.
Streptavidin
Streptavidin-OST6Fab
Streptavidin-56CFab
73.8 6 5.2 56.9 6 5.5
53.1 6 3.7 52.3 6 4.3
48.5 6 4.8 48.9 6 3.2
Tumor Targeting with Streptavidin-Fab Conjugate
559
FIG. 1. Binding of 125I-labeled OST6Fab fragment or streptavidin-Fab conjugates to KT005 cells. The radiolabels were incubated with increasing concentration of cells for 1 h at 4&C. After centrifugation, radioactivity bound to cells was counted. Error bars show SD of triplicate.
without the pretargeting (Group 2 vs. Group 1, p , 0.002), indicating that the biotinylated antibody accumulated in tumors and contributed to the specific tumor uptake of streptavidin. The tumor uptake of streptavidin-OST6Fab conjugate was higher than that of streptavidin (Group 3 vs. Group 2, p , 0.05) and that of streptavidin-56CFab conjugate (Group 3 vs. Group 4, p 5 0.002), suggesting that the specific Fab portion of the conjugate directly bound the surface antigen on tumor cells. Blood clearance of the conjugates was slower than that of streptavidin, and the renal uptake of streptavidin was decreased markedly by the conjugation with Fab fragments. DISCUSSION For an effective tumor radioimmunotherapy, both absolute delivery and target-to-nontarget ratios of radioactivity must be high. The disadvantages of radiolabeled antibodies are the slow tumor uptake and low target-to-nontarget ratios. Application of the avidin-biotin
system with a pretargeting technique could partly overcome these shortcomings. However, because of the increased number of steps, the number of binding sites in tumor for the following step would be decreased as a result of metabolism of antigen-antibody complex. Our previous studies revealed that radiolabeled streptavidin accumulated rapidly in tumors pretargeted with biotinylated antibody, but the peak uptake of radioactivity in the tumor was lower than that obtained by the radiolabeled antibody (20, 21). In the present study, streptavidin was conjugated to a MAb Fab fragment. The streptavidin-Fab conjugate was radiolabeled and used as the second step in the two-step method with biotinylated antibody pretargeting. Owing to the bifunctionality of the conjugate, i.e., its ability to bind both biotinylated antibody and tumor-associated antigen in the tumor cell surface, tumor uptake of radioactivity was increased. The improvement of tumor uptake of the conjugate is not only useful to the two-step tumor-targeting technique, as in the method in this study, but also possibly useful to other multistep targeting when biotin is used as radiotracer (1, 4, 6, 24) because the amount
TABLE 3. Biodistribution of Radiolabeled Streptavidin or Streptavidin-Fab Conjugates in KT005 Tumor-Bearing Mice (24 h) Organ Blood Liver Kidney Intestine Stomach Spleen Lung Muscle Bone Tumor
Group 1
Group 2
Group 3
Group 4
0.99 6 0.13 3.44 6 0.26 79.11 6 14.4 0.74 6 0.12 0.93 6 0.13 2.33 6 0.22 1.60 6 0.31 0.58 6 0.08 0.84 6 0.09 3.33 6 0.61
1.70 6 0.26 4.18 6 0.70 85.51 6 5.13 0.65 6 0.06 0.83 6 0.22 3.29 6 0.47 1.67 6 0.17 0.49 6 0.05 0.89 6 0.07 9.14 6 2.35
3.87 6 0.34 4.79 6 0.41 19.27 6 2.41 0.78 6 0.06 1.31 6 0.38 4.21 6 0.46 2.59 6 0.36 0.53 6 0.06 1.01 6 0.09 12.48 6 2.68
4.27 6 0.48 5.11 6 0.79 16.06 6 2.25 1.23 6 0.13 3.55 6 0.91 3.52 6 0.48 2.79 6 0.34 0.52 6 0.07 1.03 6 0.13 5.71 6 0.81
Mean 6 SD of %ID/g (n 5 6). See Table 1 Protocol for more details.
Z. Yao et al.
560
of its binding site, streptavidin, in tumor is increased. Conversely, by using antibodies OST7 and OST6, which recognize different epitopes on the same tumor, for pretargeting and targeting, respectively, binding inhibition would be avoided. The avidin-biotin system resulted in signal amplification in vitro with the increase in steps because each avidin molecule can bind four biotin molecules and protein can be polybiotinylated. This amplification was expected to occur in vivo with increase of steps or cycles. Unfortunately, the in vivo results are not satisfactory (7). Also, the binding sites could be increased by the use of a larger dose or higher biotinylation rate of biotinylated antibody. However, in that case, the amount of biotinylated antibody in circulation would also be large, resulting in a low tumor-to-nontumor ratio as large amounts of streptavidin-antibody complex formed and cleared slowly (24). In the approach described in our study, tumor delivery of radioactivity should be effectively increased by increasing the number of binding sites of the tracer only in tumor, as in the use of interferon, which increased tumor antigen expression and enhanced tumor localization of radiolabeled antibody (3, 10). High accumulation of streptavidin in kidney is a problem in its clinical application. Interestingly, the uptake of the conjugates in the kidney decreased markedly, suggesting that kidney uptake of streptavidin could be reduced through modification. Both the biotin binding ability of streptavidin and the antigen binding ability of Fab fragment were reduced after conjugation. The antigen binding ability was probably decreased by the steric hindrance from streptavidin because of its larger molecular size. The steric hindrance from Fab fragments may also contribute to the decreased biotin binding ability of streptavidin, but another possibility is the reduction of biotin binding sites of streptavidin as it was coupled to the fragments through biotin. In contrast, the conjugate showed a slower blood clearance than streptavidin, possibly as a result of enlarged molecule and significantly reduced kidney uptake. Therefore, a conjugate with a smaller molecular weight and full reactivity against both biotin and antigen is worthy of being developed.
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The authors are grateful to Kato Asao International Scholarship Foundation for its kind help. This work was supported in part by Grants-inAid for Scientific Research (07670995, 08266230) from the Ministry of Education, Science and Culture, and a Grant-in-Aid from the Sankyo Foundation of Life Science, Japan.
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