Medicine & Biology, Vol. 0 1997 Elsevier Science
24, pp. 61-64, Inc.
0969-805 l/97/$1 7.00 + 0.00 PII SO969(96)00162-X
Intravenous Avidin Chase Improved Localization of Radiolabeled Streptavidin in Intraperitoneal Xenograft Pretargeted with Biotinylated Antibody Meili Yuji Nakamoto, ‘DEPARTMENT
Zhung,’ Harumi I Noriko Sato,’
Sakahura,’ Zhenpheng Yao,’ Tsuneo Saga,’ Hiroshi Nakada, lkuo Yamashina2 and Junji Konishi’
OF NUCLEAR MEDICINE, FACULTY OF MEDICINE, OF BIOTECHNOLOGY, FACULTY OF ENGINEERING,
KYOTO UNIVERSITY, KYOTO, KYOTO SANGYO UNIVERSITY,
AND *DEPARTMENT JAPAN
In the present study, we examined the effect of avidin administered intravenously (i.v.) on the biodistribution of radiolabeled streptavidin in mice bearing intraperitoneal (IP) xenografts pretargeted with biotinylated antibody. Tumors were established in nude mice by IP inoculation of LS180 human colon cancer cells. Monoclonal antibody MLS128, which recognizes Tn antigen on mucin, was biotinylated and injected IP into the IP tumor-bearing mice. Radioiodinated streptavidin was administered IP or i.v. 48 h after pretargeting of biotinylated antibody. Avidin was administered i.v. 30 min prior to streptavidin injection. The localization of radioiodinated streptavidin in the tumor pretargeted with biotinylated antibody was significantly higher than that without pretargeting and that of radioiodinated MIS128 by the one-step method. Avidin administration significantly accelerated the clearance of radioiodinated streptavidin in blood and other normal tissues and increased the tumor-to-blood radioactivity ratio regardless of administration route of streptavidin. The i.v. avidin chase improved tumor localization of radiolabeled streptavidin in the IP xenografts pretargeted with biotinylated antibody. Co&right 0 1997 Elsevier Science Inc. NUCL MED BIOL 24;l: 61-64, 1997.
INTRODUCTION Targeting therapy using anti-tumor monoclonal antibodies conjugated with radionuclides, chemotherapeutic agents, or toxins may increase the cytotoxic effects on tumors with reduced normal tissue toxicity. Therapeutic trials for malignant ascites using intraperitoneal (IP) injection of antibody conjugates have been done in animals (3, 14) and patients (2, 10). Although biodistribution studies with radiolabeled antibodies showed a specific accumulation in tumors, blood radioactivity and nonspecific uptake of radiolabels in normal tissues were still high (4, 15, 18) and the therapeutic dose delivery limited. Pretargeting techniques in which antibodies and radiolabels are administered separately provide an alternative way for highly selective accumulation of radioactivity in tumors with simultaneous reduction of nontumor tissue background (5). The avidin-biotin system has been used for this purpose (7, 8, 13, 16, 20). A two-step approach using biotinylated antibody and radiolabeled streptavidin has shown improved tumor delivery and/or blood clearance of radioactivity (7, 8, 13, 16, 20). However, circulating biotinylated antibody may form complexes with radiolabeled streptavidin and interfere with its accumulation in the tumor and its clearance from circulation (16, 20). Previously, we have shown that avidin IP administration was effective to reduce the biotinylated antibody in blood and increase
Address reprint requests to: Hammi Sakahara; MD., Department of Nuclear Medicine, Faculty of Medicine, Kyoto University, Shogoin, Sakyo-ku, Kyoto 606-01 Japan; Fax: 81-75-771-9709; E-Mail: [email protected]
Received 8 August 1996. Accepted 9 September 1996.
the tumor-to-nontumor bearing subcutaneously biotinylated antibody continuous chase of biotin bound in the avidin is not practical administered avidin tions of radiolabeled
ratios of radiolabeled streptavidin in mice (s.c.) xenografted tumors pretargeted with (20). Avidin was administered IP for slow and biotinylated antibody without affecting the tumor. However, this time, IP injection of because of the IP tumor we studied. We i.v. to examine the effect on the biodistribustreptavidin in IP xenografted nude mice.
AND METHODS Antibody
MLS128 is a mouse IgG3 monoclonal antibody with a kappa light chain, which recognizes Tn antigen, a cluster of tri-GalNAc a-Ser/ Thr (11, 12). The antibody was purified from ascitic fluid of hy bridoma-bearing mice using protein A affinity chromatography (Bio-Rad, Richmond, CA). Radiolabeled MLS128 bound to human colon cancer cell line LS180 in vitro and accumulated well in LS180 tumors S.C. inoculated in nude mice (19).
MLS128 was conjugated with biotin using NHS-LC-biotin (Pierce, Rockford, IL) (6). Freshly prepared NHS-LC-biotin was added to 5 mg/ml of antibody in 0.05 M phosphate buffered saline, pH7.4, at a molar ratio of 5:l and incubated for 2 h at 4°C. Then unconjugated biotin was removed by chromatography on a PDlO column (Pharmacia Biotech, Uppsala, Sweden). The number of biotin molecules coupled to each antibody was determined by the method of Green et al. (6) using HABA solution (Pierce).
MLS128 and streptavidin (Pierce) were radioiodinated with “‘1 using chloramine T method (17). Forty micrograms of protein in 200 ~1 of 0.3 M phosphate buffer, pH 7.5, and “‘1 (29.6 -59.2 MBq) (DLI Pont, Wilmington, DE) were mixed with 2.5 kg 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 a PDlO column. Specific activities of radiolabeled proteins were 370-1230 MBq/mg.
In the two-step method, unlabeled biotinylated MLS128 (50 kg, 0.33 nano mole) was injected IP for pretargeting followed, 48 h later, by radioiodinated streptavidin (5 kg, 0.08 nano mole) IP or i.v. administration (Table 1). In the chase group, avidin (80 p,g, 1.21 nano mole) was i.v. injected 30 min before radiolabeled streptavidin administration. Radioiodinated MLS128 (20 p,g) or streptavidin (5 pg) was injected IP as a control. The dose of the antibody from 10 p.g to 300 p,g did not affect its biodistribution (unpublished data). Groups of 4-6 mice were sacrificed 24 h after injection of radiolabeled proteins and the distribution of radioactivity was determined. The protein doses of radiolabeled antibody and streptavidin were adjusted by adding unlabeled proteins. Data were expressed as the percentage of the injected dose per gram of tissue (%ID/g). Statistical analysis was performed using Student’s r-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 was determined to be 1.75. Tumor uptake of radiolabeled streptavidin after pretargeting of biotinylated antibody was significantly higher than those of the antibody alone (p < 0.02) or streptavidin alone (p < 0.01, Table 1). However, radioactivities in some normal
TABLE 1. Biodistribution h after IP Injection Bt.MLS128 Avidin Radiotracer Blood Liver Kidney Intestine Stomach Spleen Lung Muscle Bone Tumor Mean
tissues also increased compared to mice without pretargeting. Therefore, the biotinylated antibody was chased by i.v. injection of avidin before the radiolabeled streptavidin administration. The radioactivity distribution after the avidin chase was similar to those in mice without pretargeting (Table 1) except for a higher tumor uptake (c, = 0.0002). Although tumor radioactivity slightly decreased, the tumor-to-blood radioactivity ratio was improved significantly (Fig. 1, p < 0.01). The chase performed in mice receiving radiolabeled streptavidin i.v. gave similar results (Table 2).
LS180 cells (3 x lo6 cells in 0.2ml) were injected IP into female BALB/c-nu/nu mice. Many small nodules were found in the peritoneal cavity ten to twelve days after injection.
MLS128 6.88 iz 1.65 1.88 * 0.36 2.43 f 0.51 1.02 f 0.25 4.21 f 0.54 2.35 + 0.52 3.33 * 0.66 0.52 k 0.15 0.82 c 0.21 9.34 + 3.43
k SD of %ID/g
The purpose of the present study was to investigate whether i.v. injected avidin would improve the tumor targeting with the twostep method in the animal model of carcinomatous peritonitis we developed. By using the two-step method in the IP tumor xenograft, both tumor delivery and blood clearance of radioactivity were improved in comparison with the radiolabeled antibody alone (13), which is similar to the results using S.C. or metastatic tumor model (8, 16, 20). Because the binding affinity between biotin and streptavidin is much higher than that of antigen-antibody interaction and streptavidin could penetrate easier in the tumor as the result of its smaller molecule size, it is likely that streptavidin accumulated in the tumor more rapidly than antibody. These results suggested that IP injection in both steps is one option of many pretargeting strategies for IP tumor. However, the biotinylated antibody remaining in the circulation prolonged the blood clearance of radiolabeled streptavidin by forming complexes and increased the radioactivity in normal organs (16, 20). In the previous study, we showed that i.v. avidin injection was effective for the chase of radiolabeled biotinylated antibody (9). We also reported that IP avidin administration could accelerate tumor uptake and blood clearance of radiolabeled streptavidin with the two-step method (20) in mice bearing S.C. tumor xenografts. The present study revealed that i.v. administration of avidin was also effective for the two-step targeting of IP tumors. Although IP injection of avidin did not affect the radiolabeled streptavidin delivery to the tumor, i.v. injection of avidin decreased tumor uptake of streptavidin. Avidin injected IP may be absorbed gradually into the circulation and accumulate in the liver rapidly. However, the very high concentration of avidin in the blood im-
for 4-6 mice.
MLS 128 or Streptavidin
50 I% IP Streptavidin
5ol% IP 80 pg, i.v. Streptavidin
1.73 k 0.08 3.84 f 0.10 106.57 + 3.59 1.22 & 0.19 2.16 + 1.34 2.62 + 0.38 2.19 k 0.14 0.49 + 0.03 0.76 k 0.10 4.71 f 0.88
4.77 + 0.96 5.73 iz 0.92 81.77 f 8.55 1.30 * 0.10 1.99 * 0.15 5.06 f 2.15 3.12 zt 0.37 0.65 + 0.03 1.04 * 0.10 15.50 k 0.96
1.98 f 0.25 4.43 f 0.28 92.89 f 4.33 1.13 f 0.28 1.89 f 0.99 3.12 f 0.38 2.25 + 0.18 0.53 + 0.04 0.66 f 0.09 11.24 zt 1.93
of IP Tumor
FIG. 1. Tumor-to-organ radioactivity ratios in mice bearing LS180 intraperitoneal xenografts 24 h after II’ ‘z51-streptavidin (hatched column), ‘Z51-streptavidin with biotinylated injection of 1251-MLS128 (filled column), ML!3128 pretargeting (dotted column) and ‘Z51-streptavidin with biotinylated MLS128 pretargeting and avidin chase (light hatched column). The bars represent mean and SD for 4-6 mice.
mediately after i.v. administration may result in the binding of avidin to biotin in the tumor before being localized to the liver. The difference in tumor uptake of radiolabeled streptavidin between i.v. and IP avidin administration routes suggests that avidin should be administered slowly, which could be realized by slow iv. dropping in humans. Then, avidin chase would accelerate blood clearance of radioactivity without decreasing tumor localization of streptavidin whether in IP or in systemic tumors. Another important consideration is the proper dose of avidin which is related to its administration route, number of biotin mol-
TABLE 2. Effect of Avidin Chase on the Biodistribution of Radioiodinated Streptavidin 24 h after i.v. Injection in Mice Bearing LS180 II? Xenografts Pretargeted II? with Biotinylated Antibody (50 ug, 48 h) Avidin
Blood Liver Kidney Intestine Stomach Spleen Lung Muscle Bone Tumor
Mean + SD of%ID/g.
+ (n 1.75 4.92 89.55 1.12 1.86 3.57 2.08 0.54 0.80 7.49
= 6) + k ? * * k k k k r
0.16 0.19 11.1 0.14 0.38 0.39 0.26 0.08 0.16 1.12
- (n 6.44 5.86 76.69 1.08 1.36 4.64 3.76 0.73 1.28 9.72
= 4) f f + f + f f + f f
1.84 0.43 10.7 0.12 0.19 0.69 0.45 0.06 0.23 1.37
ecules per antibody molecule, the dose and pretargeting time of biotinylated antibody as described in several studies (9, 13, 20). In the two-step method, the avidin chase should be optimized to produce the greatest effects of removing circulating biotinylated antibody without decreasing the tumor accumulation of streptavidin. Based on our previous experiments and considered different administration route of chase, the dose of avidin in this study was determined as 80 pg, which showed effective chasing but a little decrease in tumor accumulation of radiolabeled streptavidin. Streptavidin showed a high uptake in the kidney, liver and spleen which is probably due to RYD sequence contained in streptavidin (1) and the complex formation between streptavidin and biotinylated antibody. The high renal uptake may cause toxic effect when this approach is applied for therapy but will be less significant for imaging because SPECT is helpful to identify the lesion which overlaps with kidney on planar images. Modification of streptavidin has reduced the localization of radioactivity in kidney (unpublished data). In conclusion, iv. administered avidin reduced the radiolabeled streptavidin in blood and other normal tissues and improved the tumor-to-blood radioactivity ratio in the IP tumor pretargeted with biotinylated antibody.
The authors are grateful to Kate Asao International Scholarship Foundation for the kind help. This work was also supported in part by Gruntsin-Aid for Scientific Research (07670995, 08266230) from the Ministry of Education, Science and Culture, and a Grant-in Aid from the Fuguku Trust for Medical Research, Japan.
M. Zhang et al.
References 1 Alon R., Bayer E. A. and Wilchek M. (1990) Streptavidin contains an RYD sequence which mimics the RGD receptor domain of fibronectin. Biochem. Biophys. Res. Comm. 170, 1236-1241. 2 Crippa F., Bolis G., Seregni E., Gavoni N., Scarfone G., Ferraris C., Buraggi G. L. and Bombardieri E. (1995) Single-dose intraperitoneal radioimmunotherapy with the murine monoclonal antibody I-131 MOv18; clinical results in patients with minimal residual disease of ovarian cancer. Eur. .I. Cancer 31A, 686-690. 3. Esteban J. M., Hyams D. M., Beatty B. G., Merchant B. and Beatty J. D. (1990) Radioimmunotherapy of human colon carcinomatosis xenograft with 90Y-ZCE025 monoclonal antibody; toxicity and tumor phenotype studies. Cancer Res. (Suppl.) 50, 989s-992s. 4. Chatal J. F., Saccavini J. C., Gestin J. F., Thedrez I’., Curtet C., Kremer M., Guerreau D., Nolibe D., Fumoleau P. and Guillard C. (1989) Biodistribution of iridium-1 1 l-labeled OC 125 monoclonal antibody intraperitoneally injected into patients operated on for ovarian carcinomas. Cancer Res. 49, 3087-3094. 5. Goodwin D. A. (1995) Tumor pretargeting: almost the bottom line. J. Nucl. Med. 36, 876-879. 6. Green N. M. (1975) Avidin. Adu. Protein Chem. 29, 85-133. 7. Hnatowich D. J., Virzi F. and Rusckowski M. (1987) Investigations of avidin and biotin for imaging applications. J. Nucl. Med. 28, 12941302. 8. 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, Immunoconj. Radiophanna. 6, 13-27. 9. Kobayashi H., Sakahara H., Hosono M., Yao Z., Toyama S., Endo K. and Konishi J. (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. 10. Muto M. G., Finkler N. J., Kassis A. J., Howes A. E., Anderson L. L., Lau C. C., Zurawski V. R. Jr., Weadock K., Tumeh S. S., Lavin I’. and Knapp R. C. (1992) Intraperitoneal radioimmunotherapy of refractory ovarian carcinoma utilizing iodine-131-labeled monoclonal antibody OC125. Gynecol. Oncol. 45, 265-272. 11. Nakada H., Inoue M., Numata Y., Tanaka N., Funakoshi I., Fukui S., Mellors A. and Yamashina I. (1993) Epitopic structure of Tn glyco-
phorin A for an anti-Tn antibody (MLS128). Proc. NatI. Acad. Sci. USA 90, 2495-2499. Nakada H., Numata Y., Inoue M., Tanaka N., Kitagawa H., Funakoshi I., Fukui S. and Yamashina I. (1991) Elucidation of an essential structure recognized by an anti-GalNAc ol-Ser(Thr) monoclonal antibody (MLS128). 1. Biol. Chem. 266, 12402-12405. Paganelli G., Pervez S., Siccardi A. G., Rowlinson G., Deleide G., Chiolerio F., Malcovati M., Scassellati G. A. and Epenetos A. A. (1990) Intraperitoneal radio-localization of tumors pre-targeted by biotinylated monoclonal antibodies. ht. J. Cancer 45, 1184-1189. Pearson J. W., Sivam G., Manger R., Wiltrout R. H., Morgan A. C. Jr. and Longo D. L. (1989) Enhanced therapeutic efficacy of an immunotoxin in combination with chemotherapy against an intraperitoneal human tumor xenograft in athymic mice. Cancer Res. 49, 49904995. Rowlinson G., Snook D., Busza A. and Epenetos A. A. (1987) Antibody-guided localization of intraperitoneal tumors following intraperitoneal or intravenous antibodv administration. Cancer Res. 47. 65286531. Saga T., Weinstein J. N., Jeong J. M., Heya T., Lee J. T., Le N., Paik C. H., Suns C. and Neumann R. D. (1994) Two-steo tareeting of exoerimental metastases with bioiinylaied antibody ind ridioladeled streptavidin. Cancer Res. 54, 2160-2165. Sakahara H., Endo K., Koizumi M., Nakashima T., Kunimatsu M., Watanabe Y., Kawamura Y., Nakamura T., Tanaka H., Kotoura Y., Yamamuro T., Hosoi S., Toyama S. and Torizuka K. (1988) Relationship between in vitro binding activity and in viva tumor accumulation of radiolabeled monoclonal antibodies. J. Nucl. Med. 29, 235-240. Thedrez P., Saccavini J. C., Nolibe D., Simoen J. P., Guerreau D., Gestin J. F., Kremer M. and Chatal J. F. (1989) Biodistribution of iridium-1 1 l-labeled OC 125 monoclonal antibody after intraperitoneal injection in nude mice intraperitoneally grafted with ovarian carcinomas. Cancer Res. 49, 3081-3086. Yao Z., Sakahara H., Zhang M., Kobayashi H., Nakada H., Yamashina I. and Konishi J. (1995) R a d’lolmmunoimaging of colon cancer xenografts with anti-Tn monoclonal antibody. Nucl. Med. Biol. 22, 199203. Yao Z., Zhang M., Kobayashi H., Sakahara H., Nakada H., Yamashina I. and Konishi J. (1995) Improved targeting of radiolabeled streptavidin in tumors pretargeted with biotinylated monoclonal antibodies through an avidin chase. J. Nucl. Med. 36, 837-841.