Technical Notes
Semiquantitative In Vitro Binding Assay of Indium- 111-Labeled Monoclonal Antibodies to Human Cancer and Nonnal Tissues Yuji Watanabe, Keigo Endo, Mitsuru Koizumi, Harumi Sakahara, Yasutaka
Kawamura,
Tsuneo Saga, Junji Konishi,
Gakuji Ohshio,
Tadao Manabe, and Ryukichi Tobe Department ofNuclear Medicine, First Department ofSurgery and Pathology, Kyoto University Hospital, Kyoto 606, Japan
We havedevelopeda simplein vitro methodfor the semiquantitativeassessmentof the radiolabeledantibodybindingto cancerand normaltissues.Indium-i11-labeledF(ab')@ fragments of 17-1A and I 9-9 monoclonal antibodies with well-characterized specificity for
gastrointestinalcancerdemonstratedsimilarbindingpropertiesbetweenculturedcancercells and membranefractionsof homogenatespreparedfrom tumortissues.All of the 17 colon cancerspecimensandseven(64%)of 11 gastriccancerspecimensobtainedby surgery showedpositivebindingwith 17-1A. Specificbindingof 19-9 was observedin 9 (53%)colon cancersand4 (36%)gastriccancers.However,somenormalcolontissueswerealsopositive with ‘11ln-labeled 17-lA. Relativelevelsof CA 19-9antigenexpression,determinedby the bindingwith radiolabeledantibodies,correlatedwith percentpositivecellsdeterminedby the immunohistochemical assays. Furthermore, membrane fractions could be cryopreserved
without losingantibody-bindingactivity.Theseresultsindicatethat this assaycan be usedfor testing the immunoreactivityof radiolabeled anti-tumor antibodies and in vitro binding propertiesto cancerand normaltissues. J Nuci Med 29: 1436—1442,1988
ybridoma technology has provided a large num ber of monoclonal antibodies available for cancer de
expressed on the surface membranes (6-8). Antigenic expression at tumor sites has been mainly investigated
tection and therapy. Koprowski
by immunohistochemical assays using the indirect im munofluorescence or immunoperoxidase technique (7,
et al. obtained hybri
domas secreting monoclonal antibodies that showed a specific binding to human gastrointestinal
adenocarci
nomas(1). One ofthese antibodies is 17—lA,developed against the human colorectal cancer cell line SW 1083
9,10). However, preservation
in formalin or other fix
atives may alter antigen expression. Immunohisto chemical assays have limitations for the quantitation of
and reported to be effective for the treatment of some antibody binding to cancer tissues (7), unlike binding patients with gastrointestinal cancers (2,3). CA 19—9 assays using radiolabeled antibodies in which radioac antigen, defined by another antibody 19—9, is a useful tivity bound to tissues may be readily and objectively
cancer marker for pancreatic cancer (4). Furthermore,
determined (11). We described here a simple in vitro
successful tumor imaging has been reported by i.v. administration of each radiolabeled antibody to visu
assay for the semi-quantitative assessment of antigenic expression on cancer and normal tissues by using radi
alize primary tumors, recurrences and metastases of
olabeled monoclonal antibodies and the results were
gastrointestinal
compared with those obtained by immunohistochemi
cancers (5). The clinical work with ra
diolabeled antibodies has indicated that the imaging of a given tumor may be correlated to antigen contents
cal assays. MATERIALS AND METhODS
ReceivedJuly 29, 1987;revision accepted Mar. 8, 1988. For reprints contact: Yuji Watanabe, MD, Dept. of Nuclear Medicine,Kyoto University Hospital, 54 Kawahara-cho,Shogoin, Sakyo-ku, Kyoto 606, Japan.
1436
Watanabe,Endo,Koizumiet al
Indium-ill-Labeled Monoclonal Antibodies Three murine antibodies were used in this study. Mono clonal antibodies 17—iA(1083-17-iA) and 19—9 (1 1l6-NS
The Journal of Nuclear Medicine
19-9) were thought to be specific for gastrointestinal cancer (1-3) and normal murine immunogiobuiin wasalso usedas a control antibody. All antibodies were obtained as F(ab'h fragments. Diethylenetriaminepentaacetic acid (DTPA) was covalently coupled to monoclonal antibodies through the cyclic DTPA anhydride technique (12,13). DTPA-coupled antibodies were radiolabeled by chelation with indium-i 11 (‘ ‘ ‘In) chloride yielding a specificactivity of―
[email protected]/@g. Label ing efficiency was more than 90% without further purification step, and the modified Lineweaver-Burk plot demonstrated that about 0.65 and 0.60 of ‘ ‘ ‘In-labeled F(ab')@fragments of 17—lAand 19—9 were immunoreactive, respectively (14). Human Colorectal Cancer Cell Lines Three colorectal cancer cell lines SW1 i 16, LS18O and LoVo were cultured in RPMI 1640 medium supplemented with 10% fetal calfserum. BALB/C nude mice were injected subcutaneously with 2 x i0@—i07 tumor cells. Surgical Specimens Seventeen patients with colorectal cancer and 11 patients with gastric cancer were included in this study. Tables 1 and 2 show the patients' characteristics. Immediately following the operation, surgical specimens were stored frozen at —20°C for up to 30 days. For in vitro binding studies, tissues were cut into small pieces with scissors, homogenized with a polytron homogenizer and suspended to 10 mM Tris/HC1 pH 8.0 buffer. Homogenates were filtered through two layers of cheese cloth and centrifuged at 800 g for 10 mm to remove cell debris and the nuclei fraction. The supernatant was pelleted at 8,000 g for 20 mm and the sediment was resuspended in 50 mM phosphate buffered saline (PBS) pH 7.5 buffer, with the vol ume adjusted to 0.5 g wet weight/mi. Crude membrane frac
tions (800—8,000 g) were used for the following binding stud ies. All preparations were performed at 4°C. Binding Studies Membrane fractions containing 50 mg wet weight/0. I ml
PBSbufferor 1 x i0@cancer cellswere incubated with “In labeled F(ab')@fragments of 17—iAor 19—9 (25,000 cpm/20 ng) in a microcentrifugetube for 60 mm at 4°C. After centrif ugation at 10,000 g for 5 mm, the supernatant was aspirated and the pellet was cut. The radioactivity of the pellet was measured by a well-type gamma-counter. Nonspecific binding was determined by incubating ‘ ‘ ‘In-labeled F(ab')@fragments of normal murine immunoglobulin with membrane fractions which were treated precisely like those in which total binding
wasmeasured.Normal immunoglobulinbinding,
