Radioimmunoimaging of colon cancer xenografts with anti-Tn monoclonal antibody

Pergamon

0969-8051(94)00092-l

Nud. Mrd. Bd. Vol. 22. No. 2, pp. 199-203. 1995 Copyright X“ 1995 Elstwer Science Ltd Prrnted in Great Britain. All rights reserved 0969.805 I,,95 $9.50 + 0.00

Radioimmunoimaging of Colon Cancer Xenografts with Anti-Tn Monoclonal Antibody ZHENGSHENG YAO’ , HARUMI SAKAHARA’*, HISATAKA KOBAYASHI’, HIROSHI NAKADA’, and JUNJI KONISHI’

MEILI IKUO

ZHANG’ , YAMASHINA’

‘Department of Nuclear Medicine, Faculty of Medicine, Kyoto University. Shogoin, Sakyo-ku, Kyoto 606, Japan and ‘Department of Biotechnology, Faculty of Engineering, Kyoto Sangyo University, Kita-ku, Kyoto 603, Japan (Acwppred 7 July 1994)

Tn antigen is a glycosylated tumor associated antigen and a murine monoclonal antibody, MLS128. has been identified to react with it. The potential of MLS128 for the radioimmunoimaging of colorectal cancer was studied. MLS128 was labeled with radioiodine by the chloramine-T method or indium-I 11 (“‘In) by using isothiocyanatobenzyl EDTA, and was injected into nude mice hearing human colon cancer xenografts. Radiolabeled MLS128 showed a high and specific localization in xenografted tumor. At 48 h after injection, the %ID/g of ‘251-labeledMLS128 in the tumor was 34.69,whereas that of isotype matched control antibody, FLOPC21, was 5.58 and the tumor-to-nontumor radioactivity ratios of ‘251-labeled MLS128 reached to 4.56, 17.84and 23.62 for the blood, liver and bone, respectively. “‘In-labeled MLS128 showed similar results. High accumulation of MLS128 in xenografted tumors suggested that the monoclonal antibody MLS128 is promising for radioimmunoimaging of colorectal cancer.

Introduction Tn antigen (GalNAc alphal-0-Ser/Thr) is a disease related O-linked (mucin-type) carbohydrate neoantigen, first discovered on erythrocytes of a patient with hemolytic anemia by Dausset et al. (1959) being responsible for the polyagglutination of erythrocytes owing to an anti-Tn antibody (Tn syndrome). Thereafter, the antigen was found on cells of T-cell lymphoma, leukemia and many carcinomas including cancers of pancreas, colon and breast (Springer, 1984; Springer et al., 1985, 1993; Nakada et al., 1991a; Zhuang et al., 1991; Huang et al., 1992; Osako et al., 1993; Inoue et al., 1994). Comparative immunohistochemical study with anti-Tn monoclonal antibodies demonstrated that Tn antigen was positive in 72-81% of colon cancer (Itzkowitz et al., 1989). Since Tn antigen is hardly secreted from cells, it is worthwhile to test the possibility to use it as a target antigen for tumor imaging and therapy. A monoclonal antibody (MoAb), designated MLS128, was produced by immunizing mice with LSl80 cells, a human colorectal cancer cell line (Numata et ul., 1990). MLS128 was then identified as

anti-Tn antibody, its binding to any Tn antigens being competitively inhibited by other anti-Tn antibodies such as NCC-LU-35 (Hirohashi et al., 1985) or CA3239 (Springer et al., 1988). MLS128 reacted with ovine submaxillary mucin (OSM) as well as mucin type glycopeptides prepared from LSlRO cells, Tn glycophorin A and lenkosialins. The epitopic structures have been identified as a cluster of GalNAc tl -Ser/Thr for those antigenic glycoproteins (Nakada et al., 1991a, b; 1993; Inoue et al., 1994). It reacted only a certain limited areas of normal colon and other tissues (Numata et al., 1990). Radiolabeled antitumor MoAbs have been successfully used for the diagnosis and therapy of cancers (Goldenberg and Larson, 1992; Kaminski et al., 1993). However, to our knowledge, radiolabeled anti-Tn antibodies have not been reported. In this paper, we studied localization of radiolabeled MLS128 in colon cancer xenografts for the evaluation of anti-Tn antibody in radioimmunoimaging and radioimmunotherapy.

Materials

and Methods

Cells *Author for correspondence.

LS180 human colon cancer cells were grown in RPM1 1640 medium (Nissui Pharmaceutical Co.,

2110

Zhengsheng silo <‘I
Tokyo, Japan) supplemented with 5% fetal calf serum (GIBCO Laboratories, Grand Island. NY, U.S.A.) and 0.03% L-glutamine. Subconfluent cells were removed using calcium- and magnesium-free phosphate buffered saline containing 0.02% ethylenediaminetetraacetic acid (EDTA) to preserve protein antigen. Monoclonai anfihndies MLS128 is a mouse IgG3 antibody with k light chain, produced by immunizing mice with LSI 80 cells (Numata et al., 1990).The antibody (Ab) was purified from ascitic Ruid of hybridoma-daring mice using protein A affinity chromatography (Bio-Rad, Richmond, CA, U.S.A.). FLOPC 21 was used as an isotype matched control antibody (Cappel Products, West Chester, PA, U.S.A.). Radiolabeling

of‘ MoAbs

MoAbs were radioiodinated using the chloramineT method (Sakahara et al., 1987). Purified Abs (40 fig) in 0.2 mL of 0.3 M phosphate buffer, pH 7.5, and lz51 or ? (7.4 MBq) (Du Pont, Wilmington, DE, U.S.A.) were mixed with 2.5 pig of chloramin~-T (Nacalai tesque, Kyoto, Japan) dissolved in 25 [IL of 0.3 M phosphate buffer. After 5 min, radiolabeled Abs were separated from free iodine through chromatography on PD-10 gel (Pharmacia LKB Biotechnology, Uppsala, Sweden). Specific activities of radioiodinated Abs were about 74 MBqjmg. MoAbs were also labeled with “‘In using 1-(4-isothiocyanatobenzyl)ethylenediaminetetraacetic acid (SCN-Bz-EDTA) as a bifunctional chelating agent, according to the method of Brechbiel et al. (1986) and Esteban et tz!. (1987) with some modi~~t~ons, Briefly, MoAb solutions (8.4-10.07 mg/mL) in 0.05 M borate buffered saline, pH 8.5, were mixed with SCN-Bz-EDTA dissolved in dimethylformamide in molar ratio of 1:5 and incubated overnight at 30°C. The excess agent was removed by applying the mixture to PD-IO gel chromatography. Conjugated antibodies with a concentration of 100pg/SOp I. in 20 mM 2[N-morpholino] ethanesulfonic acid buffered saline, pH 6.0, were labeled with 3.7 MBq of “‘In, which was prepared by adding 20 f*L of 1.75 M HCl Table I. Biodistribution

TiSSW Blood Liver Kidney Intestine Stomach Spleen Lung MUSCk Bone

Tllm0r

~~ ~-6h

--

Il.81 f 5.25 f 5.00 f 1.98 + 10.29 + 7.95 i 8.76 i 0.88 i 2.30 +10.61 f

2.21 0.27 0.47 0.19 4.59 2.54 1.50 0.1 I 0.26 2.24

of “‘1. and “‘In-MLSl2~

24 h 1.18 0.40 0.58 I .06 i 0.24 2.5 I * 0.94 3.07 :f I .07 4.79 + 0.90 l.OOt0.16 I.80 f 0.35 20.15;4.23

99%. Cell binding assyl

?- or ‘l”In-labeled MoAbs (10 ngi 100/ALj were incubated with an increasing number of LSI80 cells (1 x 104--1 x 107) in 5.7 x 46mm microcentrifuge tubes for 1 h at 4C. After centrifugatioi~ at lO,OOOg, the supernatant was aspirated, and the tubes were cut. The radioactivity bound to cells was counted. To calculate the affinity constant of the binding using Scatchard plot analysis (Scatchard, 1949), radiolabeled Abs and increasing amounts of unlabeled Abs were incubated with 5 x IO5 LSl80 cells for 1 h at 4”C, and the radioactivity bound to cell was counted.

Xenografted tumor was established by the S.C. inoculation of 5 x IO6LSI 80 cells in female BALB$nu/nu mice and the tumor was maintained by seria1 subcutaneous transptantation. Potassium iodide solution was administered to mice one day before the injection of radioiodinated Abs throughout the experiment. The radiolabeled Abs (37 kBq) were injected into the tail vein of nude mice bearing LS180 xenografts. The Ab doseswere adjusted to IO ;lg per mouse by addition of corresponding unlabeled Ab. At 6, 24, 48, and 96 h after injection, groups of four mice were killed by ether inhalation, organs were removed and weighed, and radioactivity was counted. Data were expressedas the percentage of the injected dose per gram of tissue (%IDjg). For the imaging of tumor-bearing nude mice, 3.7 MBq of “iI-MLS128 was administered iv. with 3Opg of total Ab dose. At 6, 24, 48. and 96 h after injection, mice were anesthetized by i.p. injection 01 sodium pentobarbital, and scintigrams were obtained using a gamma camera equipped with a pin-hole collimator. All procedures involving animal controls were carried out in accordance with the regulations for animal welfare in Japan.

in LS180 xenografted

‘waLS128

-.

and 80 i.rL of 1 M sodium acetate to 100jrL 01 “‘InCl, (Nihon Mediphysics. Takarazuka. Japan) The mixture was allowed to stand for 1 h at 1oon1 temperature. The labeling ellicicncy was more than

-

~48 h

7.72 f I .99 k 2.71 & 0.81 + 2.56 i 4.34 + 4.261 0.90 * I .48 + 34.69 z

1.70 0.49 0.62 0.09 0.66 I .29 1.19 0.14 0.26 7.44

96 h 4.72 + 1.05 f 1.51 + 0.50 f 2.02 i 3.43 * 2.80 rt 0.44 * 0.83 + 23.59 +

nude mice (means t SD for four mice)

1.23 0.22 0.40 0.1 I 0.59 I .76 0.63 0.08 0.24 4.27

---

...~~

“11n-MLS12X ~~~~ ~~~~~

-.

24 h

48 h

96h

IO.68 + 0.66 I I .37 * 0.20 5.X8 * 0.34 1.90f0.31 0.48 + 0.06 8.25 & 1.91 6.28 k 0.49 0.77 5 0.08 1.65 I 0.34 24.16 k 6.24

7.93 * 0.71 IO.71 f 0.39 4.93 ir 0.39 1.35 + 0.15 0.24 _+0.03 8.06 2 1.70 4.25 & 0.29 0.63 & 0.09 1.48+_0.12 31.05 + 3.02

5.01 2. 0.60 7.64 rt 0.60 3.34 F 0.28 0.96 _+0.02 0.21 i 0.05 6.15 I7.12 3.25 + 0.31 0.37 i 0.02 1.02FO.l2 23.78 -t 3.08

Immunoscintigraphy

with anti-Tn

MoAb

20 1

60 m .-0 s LT

50

; E 3 E 0 c c6 L g

40

Blood Liver

30

m

Kidney

q

Intestine

0

Spleen

n Lung q Muscle

20

El

Bone

13 I 10

0 25I-MLS128

125I-FLOPC21

“’ In-MLS128

“‘hFLOPC21

Fig. I. Comparison of radiolabeled MLSl28 with FLOPC21 with respect to tumor-to-nontumor radioactivity ratios at 48 h after injection in LS180-bearing nude mice. The number of mice is four in each group.

Results In vitro reactivity

of’ MLSI28

The data obtained from determining the binding of radiolabeled MLS 128 to tumor cells according to the method of Lindmo et al. (1984) indicated that the immunoreactive fractions of ‘251-MLS128 and “‘InMLS128 were 52.6 and 53.1%, respectively. The binding of radiolabeled MLS128 to LS180 cells was inhibited by the addition of unlabeled MLS128 and the affinity constant calculated according to the Scatchard plot analysis was 9.0 x 10’ Mm’. In vice biodistribution

study

After the injection of “‘I-labeled MLS128 into tumor bearing nude mice, the antibody localized well in LS180 xenografts as shown in Table 1. The accumulation of the ‘251-labeled MLS128 in tumor was rapid and high, being 10.61 in %ID/g at 6 h and Table 2. Biodistribution of radiolabeled FLOPCZI in LSIXO xenografted nude mice at 4X h after injection (%dose/g, means k SD for four mice) Tissue Blood

LlVW Kidney Intestine Stomach Spleen Lung MUSCk Bone TUll0r

‘*‘I-FLOPC2I 9.2X i- I.81 2.00 & 0.52 3.04 f 0.84 0.8X + 0.10 2.08 * 0.13 I .74 * 0.55 5.12 & 1.70 I .06 i 0.49 1.27*0.14 5.5x + 0.38

“‘IwFLOPC21 9.52 7.18 5.23 I .53 I .04 3.73 6.66 I .02 4.62 6.43

k 2.74 kO.54 + 1.26 * 0.20 i 0. I7 IO.14 I4.24 + 0.54 + 0.X1 + 0.32

reaching a peak, 34.69, at 48 h after injection, The ratios of tumor-to-nontumor radioactivity reached 4.56, 17.84 and 23.62 for blood, liver and bone, respectively, at 48 h (Fig. 1) and became larger thereafter. “‘In-labeled MLS128 also showed a high tumor uptake. The biodistribution of lZ51-and “‘In-labeled control antibody, FLOPC21 in normal organ, was similar to that of MLS128 labeled with corresponding radionuclide, but the tumor uptake of FLOPC21 was very low (Table 2). Scintigraphic imagings (Fig. 2) using “‘I-MLS128 were consistent with the results of biodistribution data. A high tumor uptake was obtained as rapidly as 6 h after injection and only xenografted tumor was discernible after 48 h in the scintigrams of tumorbearing mice.

Discussion Radioimmunoimaging of colorectal carcinoma has been done with several antibodies recognizing different tumor associated antigens, such as CEA and TAG72 (Goldenberg and Larson, 1992; Gallinger et al., 1993). Radioimmunotherapy has also been well documented with these antibodies in animal models (Blumenthal et al., 1992; Schlom et al., 1992). Comparing the in vivo animal biodistribution data of the present study with those of MoAb NPseries and B72.3 and its second generation MoAbs (Esteban et al., 1987; Sharkey et al., 1990; Colcher et a/., 1988), MLS128 was almost the same with respect to tumor localization, as indicated by the values 30-40% of

207

Zhcngsheng Yao CI r/l.

2. Scintigrams of a nude mouse bearing LSl80 xenograft. Images of the mouse wcrc obtdtt!ed
Fig.

injected

dose

per

gram

of

tissue.

But

the

low

nontumor localization for MLS128 has resulted in higher tumor-to-nontumor radioactivity ratios. The in rive tumor accumulation of radiolabeled MoAb is not directly related with in vitro binding activity (Sakahara et al., 1988). Furthermore, the results obtained from human study using MoAbs against TAG72 has shown that MoAb with higher K, does not result in higher tumor localization than MoAb with lower K, (Gallinger et al., 1993). The low K, and high tumor localization of MLS128 seems to suggest that even low K, MoAb could be used for radioimmunoimaging and radioimmunotherapy. For the clinical

convenience,

the MoAb

was first

labeled with “‘In through DTPA conjugation. Since the hepatic uptake of “‘In-labeled MLS128 prepared by the DTPA method was very high (data not shown), the chelating reagent was replaced by SCN-Bz-EDTA (Brechbiel e/ al., 1986; Esteban of “‘In et ul.. 1987). Nonspecific accumulation reduced dramatically while the immunoreactivity in vitro and tumor uptake in riw were the same as radioiodinated antibody.

Conclusion High

accumulation

of

MLS128

in xenografted

tumors suggested that the monoclonal antibody MLS128 is promising for radioimmunoimaging of colorectal cancer. Acknowledgements-We are grateful to the Sasagawa Memorial Health Foundation and Japan-China Medical Association for their kind help. This work is also supported

in part by the Grant-in-Aid for Cancer Reacarch (i-31 i from the Ministry of Health and Welfare, Japan

References Blumenthal R. D., Sharkey R. M., Haywjood L.. Natale A. M., Wong G. Y., Siegel J. A., Kennel S. J. and Goldenberg D. M. (1992) Targeted therapy of athymic mice bearing GW-39 human colonic cancer micrometastases with ‘3’I-labeled monoclonal antibodies. Cuncer Kcs. 52, 6036.~6044. Brechbiel M. W., Gansow 0. A.. Atcher R. W., Schlom .I., Esteban J., Simpson D. E. and Colcher D. (1986) Synthesis of I-(p-isothiocyanatobenzyl) derivatives of DTPA and EDTA. Antibody labeling and tumor-imaging studies. Inorg. Chem. 25, 2172-278I. Colcher D., Minelli M. F.. Roselli M., Muraro R.. Simpson Milenic D. and Schlom J. (1988) Radioimmunolocalization of human carcinoma xenografts with B72.3 second generation monoclonal antibodies. C~zccr Rr.5. 48, 4597 --4603. Dausset J., Moullec J. and Benard J. (1959) Acquired hemolytic anemia with polyagglutinability of red blood cells due to a new factor present in normal human serum (Anti-Tn). Blood 14, 107991093. Esteban J. M., Schiom J., Gansow 0. A.. Atcher R. W., Brechbiel M. W.. Simpson D. E. and Colcher D. (1987) New method for the chelation of indium-I I I to monoclonal antibodies: biodistribution and imaging of athymic mice bearing human colon carcinoma xenografts. J. Nucl. Med. 28, 861&870. Gallinger S., Reilly R. M.. Kirsh J. C.. Odze R. D.. Schmocker B. J., Hay K., Polihronis J., Damani M. T.. Shpitz B. and Stern H. S. (1993) Comparative dual label study of first and second generation antitumor-associated glycoprotein-72 monoclonal antibodies in colorectal cancer patients. Cancer Res. 53, 271~-278. Goldenberg D. M. and Larson S. M. (1992) Radioimmunodetection in cancer identification. J. Nucl. Med. 33, 803-814. Hirohashi S., Clausen H., Yamada T.. Shimosato Y. and Hakomori S. 1. (1985) Blood group A cross-reacting

Immunoscintigraphy epitope defined by monoclonal antibodies NCC-LU-35 and -81 expressed in cancer of blood group 0 or B individuals: its identification as Tn antigen. Proc. Nutl. Acud. Sci. U.S.A. 82, 7039-7043. Huang J.. Byrd J. C., Siddiki B., Yuan M., Lau E. and Kim Y. S. (1992) Monoclonal antibodies against partially deglycosylated colon cancer mucin that recognize Tn antigen. Dis. Markers 10, 81-94. lnoue M., Nakada H., Tanaka N. and Yamashina I. (1994) Tn antigen is expressed on leukosialin from T-lymphoid cells. Cancer Res. 54, 85.-88. ltzkowitz S. H.. Yuan M., Montgomery C. K.. Kjeldsen T., Takahashi H. K.. Bigbee W. L. and Kim Y. S. (1989) Expression of Tn, sialosyl-Tn, and T antigens in human colon cancer. Cancer Res. 49, 197-204. Kaminski M. S., &sadny K. R.. Francis I. R., Milik A. W.. Ross C. W., Moon S. D., Crawford S. M., Burgess J. M.. Petrv N. A.. Butchko G. M.. Glenn S. D. and Wahl R. L. (i993) Radioimmunotherapy of B-cell lymphoma with [“‘I] anti-B1 (anti-CD20) antibody. N. Engl. J. Med. 329, 459 465. Lindmo T., Boven E., Cuttitta F.. Fedorko J. and Bunn P. A., Jr. (1984) Determination of the immunoreactive fraction of radiolabeled monoclonal antibodies by linear extrapolation to binding at infinite antigen excess. J. Immunol. Meth. 72, 77-89. Nakada H., Inoue M., Tanaka N.. Numata Y., Kitagawa H., Fukui S. and Yamashina I. (1991a) Expression of the Tn antigen on T-lymphoid cell line Jurkat. Biochem. BiophJs. Rrs. Commun. 179, 762 767. Nakada H.. Numata Y.. Inoue M., Tanaka N., Kitdgawa H.. Funakoshi 1.. Fukui S. and Yamashina I. (1991b) Elucidation of an essential structure recognized by an anti-GalNAc r-Ser(Thr) monoclonal antibody (MLSl28). J. Biol. Chwn. 266, 12402-12405. Nakada H., Inoue M., Numata Y., Tanaka N., Funakoshi 1.. Fukui S., Mellors A. and Yamashina I. (1993) Epitopic structure of Tn glycophorin A for an anti-Tn antibody (MLS128). Proc. Natl. Acud. Sci. U.S.A. 90, 2495-2499. Numata Y.. Nakada H.. Fukui S., Kitagawa H., Ozaki K.. Inoue M., Kawasaki T., Funakoshi I. and Yamashina I. (1990) A monoclonal antibody directed to Tn antigen. Biochem. Bioplzys. Res. Commun. 170, 981-985. Osako M., Yonezawa S., Siddiki B.. Huang J., Ho J. J., Kim Y. S. and Sato E. (1993) lmmunohistochemical study of mucin carbohydrates and core proteins in human pancreatic tumors. Crrncrr 71, 2191--2199.

with anti-Tn

MoAb

203

Sakahara H., Endo K.. Nakashima T.. Koizumi M.. Kunimatsu M., Kawamura Y., Ohta H., Nakamura T.. Tanaka H., Kotoura Y., Yamamuro T., Hosoi S.. Toyama S. and Torizuka K. (1987) Localization of human osteogenic sarcoma xenografts in nude mice by a monoclonal antibody labeled with radioiodine and indium- 111. J. Nucl. Med. 28, 342-348. 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 oirro binding activity and in rice tumor accumulation of radiolabeled monoclonal antibodies. J. NW/. Med. 29, 235 240. Scatchard G. (1949) The attraction of proteins for small molecules and ions. Ann. N. Y. Acad. Sci. 51, 660 672. Schlom J., Eggensperger D., Colcher D., Molinolo A.. Houchens D.. Miller L. S.. Hinkle G. and Siler K. (1992) Therapeutic advantage of high-affinity anticarcinoma radioimmunoconjugates. Cuncrr Rex. 52, 1067 1077. Sharkey R. M., Gold D. V., Aninipot R.. Vagg R.. Ballance C., Newman E. S., Ostella F., Hansen H. J. and Goldenberg D. M. (1990) Comparison of tumor targeting in nude mice by murine monoclonal antibodies directed against different human colorectal cancer antigens. Cunwr Rcs 50, 828~s834s. Springer G. F. (1984) T and Tn. general carcinoma autoantigens. Scitwcr 224, 1198%1206. Springer G. F., Taylor C. R., Howard D. R., Tegtmeyer H.. Desai P. R., Murthy S. M., Felder B. and Scanlon E. F. (1985) Tn. a carcinoma-associated antigen. reacts with anti-Tn of normal human sera. Cancer 55, 561 569. Springer G. F., Chandrasekaran E. V., Desai P. R. and Tegtmeyer H. (1988) Blood group Tn-active macromolecules from human carcinomas and erythrocytes: characterization and specific reactivity with mono- and polyclonal anti-Tn antibodies induced by various immunogens. Carbohydr. RES. 178, 27 1~292. Springer G. F., Desdi P. R., Tegtmeyer H.. Spencer B. D. and Scanlon E. F. (1993) Pancarcinoma T;Tn antigen detects human carcinoma long before biopsy does and its vaccine prevents breast carcinoma recurrence. Ann. N. 1~. Acad. Sci. 690, 355-357. Zhuang D., Yousefi S. and Dennis J. W. (1991) Tn antigen and UDP-GakGalNAc alpha-R beta I3Galactosyltransferase expression in human breast carcinoma. Canwr Biochem. Biophy.~. 12, 185 198.