~

Cancer Immunol Immunother (1991) 33:165 - 170 034070049100047K

ancer mmunology mmunotherapy

© Springer-Verlag 1991

Two monoclonal antibodies against small-cell lung cancer show existence of synergism in binding Shin'iehi Saito 1, T a m o t s u I n o u e 2, Iehiro K a w a s e I , Hideki H a r a I , Yoshiro Tanio 1, Isao T a e h i b a n a 1, Seiji H a y a s h i l , M a s a t o s h i W a t a n a b e 1, M a c h i k o M a t s u n a s h i 1, Tadashi Osakil, T o m i y a M a s u n o I , and S u s u m u K i s h i m o t o 1 1The Third Department of Internal Medicine, Osaka University School of Medicine, 1-1-50 Fukushima, Fukushima-ku, Osaka-553, Japan 2 Faculty of Health and Sport Science, Osaka University, Toyonaka,Osaka-560, Japan Received 6 December 1990/Accepted 24 January 1991

S u m m a r y . Murine IgG1 monoclonal antibodies (mAbs), ITK-2 and ITK-3, were generated against a small-cell lung cancer (SCLC) cell line. Enzyme-linked immunosorbent assay using a variety of established cell lines as substrates, immunoperoxidase staining of freshly frozen tissue sections, and fluorescence-activated cell sorter analysis of peripheral blood leukocytes showed that these mAbs recognize a part of the SCLC-associated cluster 1 antigen. In immunoprecipitation studies, both ITK-2 and ITK-3 bound to a 145-kDa glycoprotein of SCLC cell membrane extracts, as did MOC-1 and NKH-1, which both recognize the cluster 1 antigen. However, because the binding of 125I-labeled ITK-2 to SCLC cells was not inhibited by MOC-1 or NKH-1, the binding site of ITK-2 on SCLC cells appeared to be different from that of either MOC- 1 or NKH-1. Unexpectedly, binding of 125I-labeled ITK-2 to SCLC cells increased in the presence of ITK-3. This ITK3-induced increase in ITK-2 binding was due partly to an increase in the number of binding sites for ITK-2 on SCLC cells. Addition of ITK-3 may, therefore, improve the effectiveness of ITK-2-based tumor detection or therapy.

good [11]. Thus new treatment protocols are needed to improve the prognosis of patients with SCLC. Monoclonal antibodies (mAb) that bind specifically to tumor-associated antigens represent a promising approach to improving tumor detection and therapy. Immunotherapy using antitumor mAb as an adjuvant for conventional cancer therapy may be particularly useful in overcoming two major limitations of systemic chemotherapy: lack of specificity and drug resistance. Recently, we generated two mAb, ITK-2 and ITK-3, which both distinguish SCLC from non-SCLC, and recognize the cluster 1 antigen of SCLC [1]. In this article, we demonstrate that ITK-2 binds to a different epitope of the cluster 1 antigen from that bound by the MOC-1 or NKH-1 mAb, which both recognize this antigen, and that the binding of ITK-2 to SCLC cells is significantly increased by ITK-3, partly because of an increase in the number of binding sites for ITK-2 on SCLC cells.

K e y words: Monoclonal antibodies - Small-cell lung cancer - Synergy of mAb binding

Cell lines. The SCLC cell lines OS1, OS2 and OS3, were established in our laboratory, and their characteristics have been described previously [15]. Other SCLC lines, H69, N231 and N857, and the cell lines of giant-cell carcinoma of the lung, Lu65 and Lu99, were the kind gift of Dr. Y. Shimosato, National Cancer Center Research Institute, Tokyo, Japan. The PC9 lung adenocarcinomacell line was a generous gift of Dr. Y. Hayata, Tokyo Medical College, Tokyo, Japan. The cell lines of squamous cell carcinoma of the lung, QG56, adenocarcinoma of the lung, A549, neuroblastomas,IMR32 and NB 1, a lymphoma,CEM, and a leukemia, HL60, were obtained from the American Type Culture Collection, Rockville, Md. The human fibroblast cell line, OS1-F, was established from the lung tissue of the same patient from whom OS1 was derived. All cell lines except OS1, OS2 and OS3 were serially passaged in RPMI-1640 medium (Nissui Pharmaceutical Co., Tokyo, Japan) supplemented with 10% heat-inactivatedfetal bovine serum (FBS) (General Scientific Laboratories, Los Angeles, Calif.), 2 mM L-glutamine (Flow Laboratories, North Lyde, Australia), 100 units/ml penicillin (Meiji Seika, Tokyo, Japan) and 100 gg/ml streptumycin(Meiji Seika). This medium was designated complete medium. OS1, OS2 and OS3 were maintained in complete medium supplemented with 10 nM hydrocortisone (Sigma Chemical Co., St. Louis, Mo.), 5 btg/ml insulin (Shionogi Pharmaceutical Co., Osaka, Japan), 10 ~tg/ml transferrin

Introduction Small-cell lung cancer (SCLC) is a highly aggressive malignant neoplasm, which exhibits a rapid growth rate and which frequently invades and metastasizes [12]. The high rate of tumor recurrence and the frequent presence of subclinical metastases may preclude the possibility of surgical cure even at early stages of SCLC. SCLC may relapse rapidly and develop resistance to chemotherapeutic agents, whereas its initial response to chemoradiotherapy has been

Offprint requests to: Ichiro Kawase

Materials and m e t h o d s

166 (Sigma), 10 nM 17[3-estradiol (Sigma) and 30 nM sodium selenite (Sigma).

mixture was washed and centrifuged three times in the medium used for diluting the antibodies, and the radioactivity of the final pellet was counted with a 7 conter.

Antibodies. Anti-SCLC mAbs, MOC-1 [2] and NKH-1 [5], were purchased from Bio-Science Products, Emmenbrucke, Switzerland, and Coulter Immunology, Hialeah, Fla., respectively. 1lG1, an IgG1 mAb against a murine hepatoma [8], also was used as a control.

Enzyme-linked immunosorbent assay (EL1SA). ELISA was carried out with a mAb-screen P Kit (Zymed Laboratories, South San Francisco, Calif.) as described previously [8]. The reaction was quantified by reading the absorbance of test wells at 405 nm by spectrophotometer.

Generation ofmAb. Spleen cells of a BALB/c mouse immunized with OS 1 cells were fused with P3-X63-Ag8-U 1 murine myeloma cells using polyethyleneglycol(Mr = 3350) (Sigma), and incubated in complete medium supplemented with 0.1 mM hypoxanthine (Sigma), 0.4 btM aminopterin (Sigma), and 16 btM thymidine (Sigma) (HAT medium) [8]. Hybridomas growing in HAT medium were selected by ELISA, and subcloned twice by limiting dilution. The isotype of mAb was determined with a mAb-isotyping kit (Amersham, Buckinghamshire, England). To obtain large quantities of mAbs, hybridoma cells were inoculated intraperitoneally into pristane-primed BALB/c mice. The resulring ascitic fluid was precipitated by 33% saturated ammonium sulfate (Wako Pure Chemicals, Osaka, Japan). The precipitated immunoglobulin was purified by ion-exchange chromatography with DEAE-Sephacel (Pharmacia, Uppsala, Sweden) followed by gel filtration using Sephacryl S-300 (Pharmacia). The concentration of the purified mAb was determined by the radial immunodiffusion technique (Quantitative Immunodiffusion Kit, Serotec, Kidlington, England). lmmunoperoxidase staining. Immunoperoxidase staining was performed on frozen tissue sections by the avidin-biotin complex method (Vecstain Elite ABC Kit, Vector Laboratories, Burlingame, Calif.) as reported elsewhere [7]. Target tissues were snrgical operation or autopsy specimens. mAbs were used at a concentration of 20 btg/ml.

Immunoprecipitation. Immunoprecipitations with mAbs or control antibodies were performed by the method of Kubo et al. [9]. Briefly, cells were radiolabeled with 12»I(New England Nuclear, Boston, Mass.) by the iodogen method, and subsequently lysed in 10 mM TRIS/HC1 (pH 7.4) containing 150 mM NaC1, 0.5% Triton X-100, 0.2 mM phenylmethylsulfonyl fluoride (Wako Pure Chemicals) and 50 units/ml aprotinin (Bayer, Leverkusen, FRG) at 4°C for 15 min. Nuclei and cell debris were removed by centrifugation at 10000 g for 10 min. Cell lysates were immunoprecipitated with rabbit anti-(mouse-Ig)-coated protein-A-Sepharose (Sigma) conjugated with each mAb. After extensive washing, immunoprecipitated materials were dissolved in 62.5 mM TRIS/HC1 (pH 6.8) containing 10% glycerol, 2.6% sodium dodecyl sulfate, 10% 2-mercaptoethanol and 0.025% bromophenol blue, and boiled for 5 min. The samples were electrophoresed on a 10% polyacrylamide gel according to the method of Laemmli [10], and autoradiographed.

Radioiodination of mAbs. mAbs were radiolabeled by iodination with 125I using the iodogen method [4]. To separate 125I-labeledmAbs from free 125I, reaction mixtures were applied to a Sephadex G-15 column (Pharmacia), and eluted with 20 mM borate buffer (pH 8.0) supplemented with 137 mM NaC1, 1 mM EDTA and 5 mg/ml ovalbumin (Sigma).

Competitive binding inhibition assay. Competing antibodies were serially diluted with RPMI-1640 medium supplemented with 25 mM HEPES buffer (Gibco), 0.1% NaN3, 0.1% heat-inactivated normal human AB serum, 0.5% bovine serum albumin (BSA) (Sigma) and 50 units/ml aprotinin (Bayer). Target cells (2 x 10») were suspended in 50 btl serially diluted antibody solution/well of a Linbro microtiter plate (no. 76-321-05, Flow Laboratories) and incubated, in triplicate, at 4°C for 1 h with occasional shaking. After incubation, 20 btl t25I-labeledmAb solution (approximately 5 x 104 cpm) was added to each well, and the plate was incubated further at 4 ° C for 1 h with occasional shaking. The

Quantification of binding site number. A living-cell radioimmunoassay was performed using I25I-labeledmAbs [3]. Briefly, 2 x 105 tumor cells suspended in 50 gl serially diluted 125I-labeledantibody solution were added to a well of a Linbro microtiter plate (no. 76-321-05) which had been blocked ovemight with BSA (10 mg/tal) in phosphate-buffered saline (PBS; pH 7.4), incubated at 4°C for 1 h, and washed three times with PBS (pH 7.4) supplemented with 0.5% BSA. The amount of bound and unbound antibody was determined by counting the radioaetivity of the precipitated cells and the supernatant, respectively. In some experiments, tumor cells were first incubated in the presence of an unlabeled mAh at 4 ° C for 30 min before the addition of an 12»I-labeledmAb, and then subjected to the radioimmunoassay described above.

Results

Binding specificity of mAbs In p r e l i m i n a r y experiments, h y b r i d o m a selection was carried out b y E L I S A using OS1, O S 1 - F and P C 9 as target cells. T w o h y b r i d o m a s were f o u n d to p r o d u c e antibodies that b o u n d to OS 1 but not to either OS 1-F or PC9. A f t e r cloning b y limiting dilution, the m A b s p r o d u c e d by these h y b r i d o m a s were n a m e d I T K - 2 and ITK-3. The i s o t y p e s o f the m A b s were both IgG1. The b i n d i n g specificity o f these m A b s was e x a m i n e d further b y E L I S A using a variety o f established cell lines (Table 1). Both I T K - 2 and I T K - 3 b o u n d to all S C L C cell lines u s e d in this study, but did not react with cells o f either n o n - S C L C or h e m a t o p o i e t i c t u m o r cell lines. O n the other hand, these two m A b s exhibited strong reactivity with n e u r o b l a s t o m a cell lines. The b i n d i n g specificity o f the m A b s was quite similar to those shown b y M O C - 1 and N K H - 1 . F l u o r e s c e n c e - a c t i v a t e d cell sorter ( F A C S ) analysis using f l u o r e s c e i n - c o n j u g a t e d rabbit anti-(mouse Ig) antibodies ( T A G O , Blurlingame, Calif.) r e v e a l e d that I T K - 2 can b i n d to a significant n u m b e r o f i n t e r l e u k i n - 2 - s t i m u l a t e d h u m a n p e r i p h e r a l b l o o d l y m p h o c y t e s , and the positive shift o f the F A C S pattern b y I T K - 2 is identical to that shown b y N K H - 1 (data not shown). I T K - 2 was purified f r o m ascites o f B A L B / c mice inj e c t e d with the relevant h y b r i d o m a and used for imm u n o p e r o x i d a s e staining o f cryostat sections. I T K - 2 b o u n d only to S C L C a m o n g h u m a n lung c a r c i n o m a s (Table 2). It also e x h i b i t e d strong b i n d i n g to tissue specimens o b t a i n e d f r o m brain, spinal cord and adrenal gland, but not to any other organs tested.

Biochemical analysis of antigens recognized by the mAbs W h e n tested b y E L I S A , the b i n d i n g o f I T K - 2 to OS 1 cells was shown to be m o d e r a t e l y sensitive to 0.25% trypsin, 0.05% pronase E and 0.7 m g / t a l m i x e d glycosidase, and strongly sensitive to 20 m M periodate. Furthermore, 5-day culture o f OS1 cells with tunicamycin, an inhibitor of sugar-chain synthesis, at a non-toxic concentration ( 0 . 2 5 - 0 . 5 btg/ml) also resulted in an apparent inhibition (more than 50% inhibition) o f I T K - 2 binding to OS 1 cells

167

-- 200 KD

--

200 KD

Q ~

--116 KD

A

B

C

"

97

KD

--

66

KD

- - 1 1 6 KD

A

D

Fig. 1. Immunoprecipitation of extracts of SCLC cell lines by ITK-2. t25I-labeledproteins from Triton X-100 lysates of OS 1 (A), OS3 (B), H69 (C) and PC9 (D) were immunoprecipitated by ITK-2, and analyzed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions

B

C

D

KD

--

97

--

66KD

E

Fig. 2. Immunoprecipitation of an extract of OS3 cells by anti-SCLC mAbs. 125I-labeledprotein from Triton X-100 lysates of OS3 cells was immunoprecipitated by ITK-2 (A), 1TK-3 (B), MOC-1 (C), NKH-1 (D) or 11G 1 (E), and analyzed by SDS-PAGE under reducing conditions

Table 1. Reactivity of ITK-2 and ITK-3 against cultured cell lines, measured by enzyme-linked immunosorbent assaya Absorbance at 405 nm

Target cell lines

MOC- 1

NKH- 1

ITK-2

ITK-3

Small-cell carcinoma of the lung

OS1 OS2 OS3 H69 N231 N857

1.381 NTb 1.326 0.550 0.785 0.514

1.458 NT 1.468 0.719 1.401 1.170

1.114 0.485 0.961 0.514 0.631 0.716

1.052 NT 0.774 0.481 0.548 0.625

Adenocarcinoma of the lung

A549 PC9

NT 0.003

NT 0.009

0.026 0.036

NT 0.0l 1

Squamous-cell carcinoma of the lung

QG56

NT

NT

0.045

NT

Giant-cell carcinoma of the lung

Lu65 Lu99

0.039 0.030

0.021 0.046

0.009 0.023

0.004 0.026

Lymphoma

CEM

0.026

0.066

0.044

0.053

Leukemia

HL60

0.093

0.082

0.052

0.058

Neuroblastoma

IMR32 NB 1

0.715 0.665

1.297 0.934

0.746 0.783

0.515 NT

Fibroblast

OS 1-F

NT

NT

0.016

NT

a Dilution of antibodies: MOC- 1, 1 : 40 dilution of the supernatant; NKH- 1, 1.25 btg/ml; ITK-2 and ITK-3, undiluted culture supernatants of the relevant hybridomas b NT, not tested

in E L I S A . T h e s e results strongly suggest that I T K - 2 recognizes a g l y c o p r o t e i n , p r o b a b l y the oligosaccharide, on the cell surface o f the S C L C line (data not shown). The sizes o f the antigens r e c o g n i z e d b y I T K - 2 and I T K - 3 were a n a l y z e d b y i m m u n o p r e c i p i t a t i o n o f 125I-lab e l e d cell lysates f o l l o w e d b y s o d i u m d o d e c y l sulf a t e / p o l y a c r y l a m i d e gel electrophoresis. I T K - 2 b o u n d to a 145-kDa p r o t e i n f r o m OS1, OS3 and H69 cells (Fig. 1). This b a n d d i d not a p p e a r in i m m u n o p r e c i p i t a t e s o f PC9. W h e n OS3 cells were used as a target, both I T K - 2 and I T K - 3 precipitated the positive b a n d at 145 kDa, as did M O C - 1 and N K H - 1 (Fig. 2). N o positive b a n d was seen w h e n 1 l G 1 , a m A b against an unrelated tumor, was used as the first a n t i b o d y in this assay.

Competitive inhibition of the binding of mAb to SCLC The b i n d i n g o f 125I-labeled I T K - 2 to H69 cells was inhibited b y I T K - 2 but not b y either M O C - 1 or N K H - 1 , indicating that I T K - 2 binds to a different epitope from those r e c o g n i z e d by M O C - 1 and N K H - 1 (Fig. 3). U n e x p e c t e d l y , I T K - 3 strongly a u g m e n t e d the binding o f 125I-labeled I T K - 2 to H69 cells in a c o n c e n t r a t i o n - d e p e n d e n t manner. S i m i l a r results were obtained when I T K - 2 and N K H - 1 were used as competitors for the b i n d i n g o f 125I-labeled I T K - 3 to OS3 cells (Fig. 4). The binding o f ~25I-labeled I T K - 3 was inhibited b y I T K - 3 and was not affected b y N K H - 1 , but it was i n c r e a s e d b y ITK-2. H o w e v e r , the increase in b i n d i n g o f 125I-labeled I T K - 3 in the presence o f

168

200 300

150 2OO 2

g~

,õ 100 I00

50 10-5

10-d

10-3

10-2

10-1

1

Diluti0n of MoAb Fig. 3. Competitive inhibition of binding of ITK-2 to H69 cells. H69 cells (2 × 105) were suspended in serial dilutions of ITK-2 ( • ) ; ITK-3 (©), MOC-1 ([3) and NKH-1 (A), and incubated at 4°C for 1 h. Samples of 20 gl 125I-labeledITK-2 were then added to the mixtures, and the plate was incubated further at 4°C for 1 h. After three washings, radioactivity of the pellets was counted. Cells incubated without competitor antibodies served as a control. Bars represent SE. Serial dilution of competitor antibodies began with 5 gg/tal for ITK-2 and NKH-1, and with undiluted culture supematants for ITK-3 and MOC-1

10-6

10-5

10-4

10-3

10-2

10"1

1

Dilution of MoAb Fig. 4. Competitive inhibition of binding of ITK-3 to OS3 cells. Samples of 2 x 105 OS3 cells were suspended in serial dilutions of ITK-2 ( • ) , ITK-3 (O) and MOC-1 ( 5 ) . x2»I-labeled ITK-3 was then added as described in the legend to Fig. 3

20 ITK-2 was small, and it was insensitive to the concentration of ITK-2. A similar small increment in 125I-labeled ITK-3 binding by ITK-2 also occurred when H69 cells were used as a target (data not shown).

A

E

Augmentation of lTK-2 binding by ITK-3 To investigate the effect of ITK-3 on the binding of ITK-2 to SCLC cells, a living-cell radioimmunoassay was performed using OS3 cells as a target. Figure 5 shows the binding of serially diluted 125I-labeled ITK-2 in the absence and presence of ITK-3. The ITK-3-induced increase in ITK-2 binding was apparent only at higher concentrations of 125I-labeled ITK-2. Scatchard plot analysis of the radioimmunoassay data showed that the addition of ITK-3 increased the number of binding sites for ITK-2 on OS3 cells slightly from 8.16 x 104 to 1.05 x 105 per cell, while the equilibrium or affinity constant (K) also changed from 5.2 × 107 M -1 to 4.05 × 107 M- ~ (Fig. 6). These results suggest that the ITK3-induced increase in ITK-2 binding to SCLC cells is due partly to an increase in the number of binding sites that possess a slightly decreased binding affinity.

i0

i

62.5

I

125

250

i

500

ng of Ab added 1 well Fig. 5. Effect of added ITK-3 on the binding of 1TK-2 to OS3 cells. OS3 cells (2 × 105) were suspended in medium containing either no antibody (O) or ITK-3 at 10 gg/tal ( • ) , and incubated at 4°C for 30 min. Serial dilutions of 125I-labeled ITK-2 were added to the mixtures, which were then incubated at 4 ° C for 1 h. After three washings, the radioactivity of the pellets was counted. Bars represent SE

169

Discussion

~4 ,°Pc,

~3 tl-

~2

1

--

1

2

3

4

5

~i

Bound Ab ( ng I 2x105 cells) Fig. 6. Scatchard plot analysis of the binding of ITK-2. Data from the binding of 125I-labeled ITK-2 to OS3 cells in the absence ( © ) or presence ( 0 ) of ITK-3 were replotted in the Scatchard form. Linear regression yielded the following values for the slopes, the affinity constant (K) of the binding and the number of binding sites on OS3 cells. In the absence of 1TK-3: slope = -1.04 × 10 • M -~ (r = -0.995), K = 5.2 x 107 M -1, n u m ber of binding sites = 8.16 x 104/cell; and in the presence of ITK-3: slope = -8.09 × 107 M -1 ( r = -0.997), K = 4.05 × 107 M 1, number of binding sites = 1.05 x 105/cell

Table 2. Reactivity of ITK-2 against frozen tissue specimens as measured by immunoperoxidase staininga Tissue

No. staining positive/ n o tested b

Human lung cancer Adenocarcinoma Small-cell carcinoma Squamous-cell carcinoma Large-cell carcinoma

0/5 a 3/3 0/5 0/1

Normal human tissues Adrenal gland Brain Spinal cord Esophagus Stomach Duodenum Small intestine Colon Gall bladder Pancreas Liver Kidney Spleen Aorta Cardiac muscle Pericardium Placenta Umbilical cord

2/2 1/1 3/3 0/1 0/1 0/1 0/1 0/1 0/1 0/1 0/4 0/4 0/2 0/1 0/1 0/1 0/1 0/1

a ITK-2 purified from ascitic fluids was used at a concentration of 20 gg/ml b A case with a proportion of positively stained cells in the tissue specimen of more than 50% was defined as a positive staining

Based on the binding specificity of a large panel of antiSCLC mAb, a cluster classification of the cell-surface antigens of SCLC was proposed at the First International Workshop on SCLC Antigens [1], and six clusters were defined. Cluster 1, a neuroendocrine-system-associated antigen, is the most important antigen for distinguishing SCLC from non-SCLC. This antigen is expressed specifically in small-cell carcinoma among lung cancers, but has also been detected on carcinoids, neuroblastomas, specimens of brain, nerve, adrenal gland, thyroid gland, and on a subset of peripheral blood leukocytes, including large granular lymphocytes. Antibodies detecting the cluster 1 antigen include MOC-1 [2], NKH-1 [5], NE25 [14], TSF-4 [16], 123C1 [13], and NCC-Lu-243, -244, and -246 [6]. In this study, two routine IgG1 mAbs, ITK-2 and ITK-3, were raised against OS1, a cultured SCLC line established in our laboratory. The binding specificity shown by ELISA, immunoperoxidase staining and FACS analysis clearly suggests that both of these mAbs, and particularly ITK-2, should be included among the cluster1-recognizing antibodies. Biochemical data suggest that the cluster 1 antigen is a glycoprotein composed of at least two chains, one of 25-29 kDa [13, 14] and the other of 124-145 kDa [6, 17]. The results of the present study showed that ITK-2 and ITK-3 bind to a 145-kDa glycoprotein, which is consistent with a cluster 1 target, though they seem to bind a different epitope from MOC-1. Unexpectedly, the competitive-binding-inhibitionassay revealed that the presence of ITK-3 significantly increased the binding of ITK-2 to SCLC cells. Conversely, ITK-2 augmented the binding of ITK-3 to SCLC cells, but to a lesser extent. The 1TK-3 effect was analyzed further by a living-cell radioimmunoassay using 1251-1abeled ITK-2. ITK-3-induced increase in ITK-2 binding occurred olny when 125I-labeled ITK-2 was present at a high concentration, suggesting that ITK-3 increases a low-affinity binding of ITK-2 to SCLC cells. Scatchard plot analysis of the radioimmunoassay data indicated that the ITK-3-induced increase in the number of binding sites for ITK-2 on SCLC cells paralleled a decrease in the affinity constant. Therefore, the ITK-3-induced increase in ITK-2 binding to SCLC cells may be due partly to an increase in the numbers of a binding site with slightly decreased binding affinity. It is also of interest whether ITK-3 could enhance the binding of other anti-SCLC mAbs besides ITK-2, such as MOC-1 and NKH- 1, while MOC-1 cansed no influence on the binding either ITK-2 or ITK-3. This possibility could be clarified by testing effects of ITK-3 on the binding of 125I-labeled MOC-1 and NKH-1. The mechanism of the ITK-3-induced increase in ITK-2 binding remains unclear. In preliminary experiments, no further increases in ITK-2 binding were produced by prolonging the incubation of SCLC cells with ITK-3 for up to 2 h at 4 ° C prior to the addition of 125I-labeled ITK-2 (data not shown). One possible mechanism is that the binding of ITK-3 to SCLC cells may canse structural or biochemical modifications at the cell surface, which occur within 30 min at 4 °C, and result in an increased exposure of

170 b i n d i n g sites for ITK-2. In this study, the preincubation of SCLC cells with ITK-3 was performed at 4 ° C to prevent shedding or pinocytosis of the target antigen of ITK-3. However, the effects of preincubation of cells with ITK-3 at 37°C on I T K - 2 b i n d i n g to SCLC cells will also be interesting. These results hint that ITK-3 m a y a u g m e n t the therapeutic efficacy of I T K - 2 when conjugated to cytotoxic agents. W e have investigated this possibility using in vitro cytotoxicity assays. The results of these studies will be reported in detail in a future paper.

Acknowledgements. This work was supported by a grant-in-aid for Cancer Research from the Ministry of Health and Welfare.

References 1. Beverley PCL, Souhami RL, Bobrow L (1988) Results of the central data analysis. Lung Cancer 4 [Suppt]: 1 2. De Leij L, Poppema S, Nulend JK, TerHaar A, Schwander E, Ebbens F, Postmus PE, The TH (1985) Neuroendocrine differentiation antigen on human lung carcinoma and Kulchitski cells. Cancer Res 45:2192 3. Epstein AL, Marder RJ, Winter JN, Stathopoulos E, Chen F-M, Parker JW, Tailor CR (1987) Two new monoclonal antibodies, Lym-1 and Lym-2, reactive with human B-lymphocytes and derived tumors, with immunodiagnostic and immunotherapeutic potential. Cancer Res 47:830 4. Fraker PJ, Speck GC Jr (1978) Protein and cell membrane iodinations with a sparlingly soluble chloramide, 1, 3, 4, 6-tetrachloro-3-6diphenylglycoluril.Biochem Biophys Res Commun 80:849 5. Hercend T, Griffin JD, Bensussan A, Schmidt RE, Edoson MA, Brennan A, Murray C, Daley JF, Schlossman SF, Ritz J (1985) Generation of monoclonal antibodies to a human natural killer clone. J Clin Invest 75:932 6. Hirano T, Hirohashi S, Kunii T, Noguchi M, Shimosato Y, Hayata Y (1989) Quantitative distribution of cluster 1 small cell lung cancer antigen in cancerous and non-cancerous tissues, cultured cells and sera. Jpn J Cancer Res 80:348

7. Hsu S, Raine L, Fanger H (1981) Use of avidin-biotin-complex (ABC) in immunoperoxidase techniques. J Histochem Cytochem 29:577 8. Kawase I, Komuta K, Ogura T, Fujiwara H, Hamaoka T, Kishimoto S (1985) Murine tumor cell lysis by antibody-dependent macrophage-mediatedcytotoxicityusing syngeneicmonoclonal antibodies. Cancer Res 45:1663 9. Kubo R, Pellanne ML (1983) Tunicamycin inhibits the expression of membrane IgM in the human lymphoblastoid cell line Daudi. Mol Immuno120:67 10. Laemmli UK (1970) Cleavage of structurai proteins during the assembly of the head of bacteriophage T4. Nature 227:680 11. Morstyn G, Ihde DC, Lichter AS, Bunn PA, Carney DN, Glatstein E, Minna JD (1984) Small cell lung cancer 1973-1983: early progress and recent obstacles. Int J Radiat Oncol Biol Phys 10:515 12. Mountain CF (1978) Clinical biology of small cell lung carcinoma: Relationship to surgical therapy. Semin Oncol 5:272 13. Schol DJ, Mool WJ, Van der Gugten AA, Wagenaar SS, Hilger J (1988) Monoclonal antibody 123C3, identifying small cell lung carcinoma phenotype in lung tumors, recognizes mainly, but not exclusively, endocrine and neuron-supporting normal tissues. Int J Cancer [Suppl] 2:34 14. Takahashi T, Ueda R, Song X, Nishida K, Shinzato M, Namikawa R, Ariyoshi Y, Ohta K, Kato K, Nagatsu T, Imaizumi M, Abe T, Abe T, Takahashi T (1986) Two novel cell surface antigens on small cell lung carcinoma defined by mouse monoclonal antibodies NE25 and PE35. Cancer Res 46:4770 15. Tanio Y, Watanabe M, Inoue T, Kawase I, Shirasaka T, Ikeda T, Hara H, Masuno T, Saito S, Kawano K, Kitamura H, Kubota K, Kodama N, Kawahara M, Sakatani M, Furuse K, Yamamoto S, Kishimoto S (1990) Chemo-radioresistance of small cell lung cancer cell lines derived from untreated primary tumors obtained by diagnostic bronchoscopy. Jpn J Cancer Res 81: 289 16. Watanabe J, Okabe T, Fujisawa M, Takaku F, Hirohashi S, Shimosato Y (1987) Monoclonal antibody that distinguishes small-celIlung cancer from non-small-cell lung caneer. Cancer Res 47:826 17. Watanabe J, Okabe T, Fujisawa M, Takaku F, Fukayama M (1987) Isolation of small cell lung cancer-associated antigen from human brain. Cancer Res 47:960