HYBRIDOMA Volume 10, Number S, 1991 Mary Ann Liebert, Inc., Publishers

Immunological and Biochemical Characterization of the Nonspecific Cross-Reacting Antigen Epitopes Using Twenty-three Monoclonal Antibodies MASAHIDE KUROKI, FUMIKO ARAKAWA, HIROSHI HIGUCHI, MASATORA HARUNO, MAMIE WAKISAKA, and YUJI MATSUOKA First

Department of Biochemistry, School of Medicine,

Fukuoka

University, 7-45-1 Nanakuma,

Jonan-ku, Fukuoka 814-01, Japan

ABSTRACT

Twenty-three monoclonal antibodies (MAbs) reactive with nonspecific crossreacting antigen (NCA) were prepared and used for constructing a serological map of the NCA molecule. The MAbs were generated using purified NCA or carcinoembryonic antigen (CEA) as immunogen. The MAbs could be divided into two groups: Group X, 10 clones reactive with NCA and CEA; and Group Y, 13 clones specific for NCA. Cross-competition enzyme immunoassays between MAbs of the individual groups revealed that at least 8 different subgroups can be defined i. e., 5 and 3 subgroups in Groups X and Y, respectively. The chemical nature of the epitopes recognized by those MAbs was tested using chemically or enzymatically treated antigens; all MAbs reacted with periodate-treated NCA and deglycosylated NCA, indicating that all the epitopes identified appeared to be protein in nature. Reduction and alkylation, pepsin digestion or pronase

differential results with respect to MAb and biochemical studies reported here thus provide information as to the range and nature of the epitopes on the NCA molecule and help form the basis for selecting the anti-NCA MAbs for use in biological and immunological study of NCA. treatment of

NCA, however, gave

some

binding. The sérologie mapping

INTRODUCTION

Nonspecific cross-reacting antigen (NCA) (1) is a glycoprotein antigen that partially shares antigenic determinants with carcinoembryonic antigen (CEA) (2). The CEA

is

one

of the most useful human tumor markers for the post

surgical monitoring of patients with various malignancies (3), and is a highly glycosylated protein with a molecular weight 180,000 (4). NCA was originally found in normal lung and spleen (1). About 15 NCA-like antigens with various molecular sizes from 26,000 to 160,000 have since been reported to exist in normal or malignant colon, lung, granulocytes, and plasma (5-12). Following successful cDNA cloning of CEA (13), the primary structures of several NCAs have also been determined by cloning of cDNAs libraries of tumor cell lines (14, 15) and of normal or leukemic leukocytes (16, 17). 557

have clarified membrane anchoring device some NCAs as well as CEA. remain to be elucidated and their is of the interrelations between little known biological distributions and functions. Although numerous monoclonal antibodies (MAbs) to CEA have been prepared to characterize the CEA molecule and their cross-reactivity with NCA have been reported (6-8, 10, 24, 25), only a few reports described the MAbs raised against NCA itself (26, 27) and there seem to be no publications describing the epitope mapping of the NCA molecule. We have previously generated a series of anti-CEA MAbs which were classified into five groups in terms of the reactivity with CEA and CEA-related antigens and found that at least 25 epitopes were present on the CEA molecule (28-31). Four of the 25 epitopes were identified on the sites shared between CEA and NCA from normal lungs (29). In the present study, we describe (a) preparation of 17 new antl-NCA MAbs using purified NCA-50 as immunogen;(W the use of the 17 antiNCA MAbs and 6 previously described anti-CEA MAbs cross-reacting with NCA to help define a sérologie mapping of the NCA molecule; and (c) biochemical characterization of the epitopes recognized by these MAbs.

Furthermore, several

recent

findings

(12, 18-20) and cell adhesion activity (21-23) of The molecular identities of those NCAs, however,

MATERIALS AND METHODS

Generation and

source

of monoclonal antibodies (MAbs)

Six anti-CEA MAbs cross-reacting with NCAs; F33-108, F34-1, -187, F36-9, 54, and -81. were described elsewhere (28. 29). MAb F34-1 is IgG2a(ic) and the remainder are IgGl(ic). Seventeen anti-NCA MAbs were prepared using NCA-50 as immunogen. Briefly, 5-week-old BALB/c mice were immunized by i.p. inoculation of 20 to 50 |ig of NCA-50 in complete Freund's adjuvant and boosted with the same dose of antigen in saline 5 weeks later. Spleen cells were harvested 3 days later for cell fusion. Hybridomas were prepared as previously described (28). One thousand cultures were assayed for reactivity to NCA-50 using anti-mouse Ig reagents; 60 were found to produce antibodies binding to NCA-50. Further cloning and assaying yielded 17 clones that produced antibodies which bound to purified NCA-50. These cultures were then recloned and reassayed to yield 17 anti-NCA MAbs, F63-19, -49, F105-5, 34, -35, F106-19. -23, -30, -37, -39, -54, -62, -68, -78, -81, -88. and -92. Immunoglobulin isotypes were determined as previously described (28). All 17 anti-NCA MAbs are IgGl(ic). Purified myeloma proteins MOPC 21 (IgGl) and UPC 10 (IgG2a) were used as negative controls (Organon Teknika, Durham, -

-

NC).

Purification of MAbs

purified from ascitic fluids by ammonium sulfate by gel filtration on a TSKgel G3000SW column (21.5 x 600 mm) (Tosoh, Tokyo, Japan) connected to a fast protein liquid chromatography system (Pharmacia, Uppsala, Sweden). Column fractions were analyzed by SDS-PAGE, and the protein concentrations of the purified IgG were determined by the method of Lowry et al. (32). MAb

IgGs

were

precipitation followed

558

Antigens Two representative forms of NCA; NCA-50 and NCA-90, were highly purified from crude perchloric acid extracts of pooled normal lungs and spleens as CEA was also highly purified from liver described previously (33, 34). métastases of a colon carcinoma (35). The purity of the antigen preparations was confirmed by immunoelectrophoresis and SDS-PAGE, as well as NH2terminal amino acid sequence analysis (36, 37). The Western blotting patterns of each antigen used are shown in Fig. 1, which were visualized by using a MAb reacting with all the purified antigens. The apparent mol. wts. of CEA, NCA-50, and NCA-90 were 180,000, 50,000, and 90,000, respectively. The NCA-50 used here is probably corresponding to NCA-55 of Buchegger et al. (6) or to NCA-50 of Grunert et al. (7) and the NCA-90 is probably corresponding to NCA-95 of Buchegger et al. (6) or to NCA-97 of Grunert et al..{7).

blotting analysis of CEA, NCA-50, NCA-90, and hundred-fifty nanograms of each antigen were deglycosylated antigens. added to each lane (4-20% polyacrylamide gel). The amounts of protein were determined by the method of Lowry et al (32). MAb used for the detection was F34-187 (Group X). Antigens used were as follows: lanel, CEA; lane 2, DG-

FIGURE 1.

Western

Two

CEA; lane 3, NCA-50; lane 4, DG-NCA-50; lane 5, NCA-90; lane 6, DG-NCA-90. Vertical scales, molecular weight markers (x 10-3). Chemical and enzymatic treatment of

Purified NCA-50

was

purified antigens

reduced and

559

alkylated

with dithiothreitol and

iodoacetamide as described previously (24). Heat treatment was done by boiling purified NCA-50 in 0.01 M borate-buffered saline, pH 8.0 (BBS) for 10 min. Pepsin (Sigma) or pronase (from Streptomyces griseus, Sigma) digestion of NCA-50 was performed as described previously (24). Control NCA-50 samples were incubated similarly but without enzymes. Each of the treated NCA-50 preparations (50 ng/50 (il/well) was dried onto each well of 96-well plates on the basis of the concentration before treatment, and nonspecific The remaining protein binding was blocked with 5% BSA in BBS.

immunoreactivity of the NCA-50

was estimated using MAbs and biotin-labeled second Ab as described in solid-phase enzyme immunoassays (SPEIAs). Periodate treatment and neuraminidase treatment were performed against NCA-50 on 96-well plates. Purified NCA-50 (50 ng/50 |ñ/well) was dried onto each well. After blocking the remaining protein-binding sites with 5% BSA in BBS, the plates were washed 3 times with buffers used for each of the following treatments. Periodate treatment was done at concentrations ranging from 0.1 to 100 mM in 50 mM sodium acetate buffer, pH 4.5, for a period of 30 min at room temperature in the dark. Treatment with neuraminidase from Clostridium perfringens (Sigma) was carried out as described previously (24). After washing the wells 4 times with 1% BSA in BBS, the remaining immunoreactivity of the NCA-50 was determined by SPEIAs. Deglycosylation of NCA-50, NCA-90 or CEA was done as previously described (24, 38). Briefly, each purified antigen (2 mg) was thoroughly dialyzed against deionized and distilled water, lyophilized, and chemically deglycosylated with trifluoromethane-sulfonic acid (TFMS):anisole, 2:1 (v/v), under a nitrogen atmosphere and incubated at 4°C for 4 hr. The deglycosylated NCA-50, NCA-90 or CEA was designated as DG-NCA-50, DG-NCA-90 or DG-CEA, respectively. The Western blotting patterns of deglycosylated antigens are shown in Fig. 1 in comparison with the respective intact antigens, which were also visualized by using a MAb (F34-187). The apparent molecular weights of DG-NCA-50, DGNCA-90 or DG-CEA were about 35,000, 35,000, and 77,000, respectively. These values were in close agreement with those of the respective molecules obtained by others (18, 39-41). The protein concentration of the determined was deglycosylated antigens by the method of Lowry et al (32). Each of the deglycosylated antigens (50 ng/50 (il/well) was dried onto each well of 96-well plates. The remaining immunoreactivity was compared in SPEIAs with NCA-50, NCA-90 or CEA in which the concentration was similarly determined by the Lowry method. The remaining immunoreactivity was also determined by Western blotting procedure.

Solid-phase

enzyme

immunoassays (SPEIAs)

The reactivities of MAbs with antigens were estimated by SPEIAs by using the antigens immobilized on 96-well polyvinyl chloride microtiter plates (Dynatech, Alexandria, VA) (42). Fifty (il of each antigen solution were dried in each well at 37°C. Nonspecific protein absorption was blocked with 5% BSA in BBS. After washing the plates with 0.05% Nonidet P-40 in BBS, varying amounts of purified antibodies (in 100 jxl of 1% BSA in BBS) were added and incubated for 1 h at 37°C. The unbound IgG was removed and biotinylated horse anti-mouse IgG(H + L) antibody (Vector, Burlingame, CA) was then added (50 ng in 100 id of 1% BSA in BBS). After an additional 1 h incubation at 37°C, the biotinylated antibody was removed and horseradish peroxidase-avidin D (Vector) was added (25 ng in 100 \il of 1% BSA in BBS), and then the plates were incubated for 30 min at room temperature. The plates were washed and 150 fj.1 of 0.05 M citrate/0.1 M phosphate buffer, pH 5.0, containing 4% o560

and 0.006% H2Q2 was added. After incubation for 25 min at room temperature, the reaction was terminated by adding 20 id of 8 N H2SO4 and the absorbance at 492 nm of each well was measured by a microtiter plate reader. The absorbance obtained with MOPC 21 (IgGl) or UPC 10 (IgG2a) instead of MAbs was subtracted from the absorbance obtained with MAbs.

phenylenediamine

Western

blotting analyses

Electrophoresis was carried out in SDS-polyacrylamide gel. The electrophoresed proteins were transferred to hydrophobic Durapore filter (Millipore, Bedford, MA) as previously described (24, 42). The blots were then

treated with 10% skim milk, washed with BBS containing 0.05% Nonidet P-40, and incubated with each MAb at a concentration of 1 |ig/ml for 2 h at room temperature with gentle agitation. After washing, the blots were successively incubated at room temperature with biotinylated horse anti-mouse IgG (H+L) or goat anti-rabbit IgG (H+L) (0.5 ng/ml) for 1 h, with horseradish peroxidaseavidin D (0.25 ng/ml) for 1 h, and then with 10 mM Tris-HCl buffer, pH 7.4, containing 0.03% 3, 3'-diaminobenzidine and 0.01% H2O2 for 20 min.

Competition

RIAs

Cross-competition assays among MAbs for NCA-50 binding were performed by a solid-phase RIA as described previously (28). To each of polystyrene beads previously coated with a given MAb, 1 to 5 ng of i25T-iabeled NCA-50 in 100 ill of 1% BSA in BBS and increasing amounts of competitor MAbs (up to 100 Mg/ml) in 100 |il of the same buffer were added. After a 2 h incubation at 37°C, each bead was washed with saline and binding of i25i-iabeled NCA-50 was counted in a gamma counter. The amounts of competitor Abs required to inhibit binding of i2 5i-iabeled NCA-50 by 50% were determined. The concentrations of MAbs used for coating the beads were those that were capable of binding 30-40% of labeled NCA-50 in the absence of competitor antibody. Determination of

affinity

constant (Ka)

The affinity constants of MAbs to NCA-50 Farr assay as described previously (28).

or

CEA

were

determined

by

the

RESULTS

Reactivity of MAbs with NCA-50. NCA-90. and CEA Six anti-CEA MAbs cross-reactive with NCA-50 and 17 anti-NCA MAbs

generated using NCA-50 as immunogen were used in this study. The reactivity of all 23 MAbs with purified NCA-50, NCA-90 and CEA was determined in SPEIAs (Table I).

Titration

curves were

carried out for all 23 MAbs

versus

the 3

purified antigen preparations; representative curves are shown in Fig. 2. All 23 MAbs were reactive with NCA-50 and NCA-90. Ten MAbs including five anti-

CEA MAbs were found to be reactive with CEA. Thus, the MAbs could be divided into two groups: Group X, 10 clones reactive with NCA-50, NCA-90, and CEA; and Group Y, 13 clones specific for NCA-50 and NCA-90 (Table 1). Of the two forms of NCA, NCA-50 was primarily used for the following study, because it 561

B F36-81

A F36-81

0.3

1.6

8.0

40

200 1,000

C F106-88

.3

1.6

8.0

0.3

1.6

8.0

40

200 1,000

40

200 1,000

D F106-88

40

200

ANTIBODY ADDED

1,000

0.3

1.6

8.0

ANTIBODY ADDED

(ng)

(ng)

, CEA; , DG-CEA; X, Reduced and alkylated NCA-50 • DG-NCA-50; , Pepsin-treated NCA-50; , Pronase-treated NCA-50 A, NCA-90; A, Heat-treated NCA-50; +, Neuraminidase-treated NCA-50

O, NCA-50; ,

Reactivity of MAb F36-81 (Group X) and F106-88 (Group Y) with CEA, NCA-50, NCA-90, and chemically modified antigens in SPEIA. Fifty nanograms of each antigen per 50|il were dried onto wells of 96-well plates and FIGURE 2.

assayed for each MAb as described in "Materials and Methods". was

used

as

immunogen for generation of the anti-NCA MAbs and because

deglycosylation of NCA-50 and NCA-90 showed almost the same mol. wt. of 35,000 (Fig. 1, lanes 4 and 6), suggesting that the two forms of NCA used in this study are different only in their glycosylation patterns. The affinity constants of all 23 MAbs uersus NCA-50 and those of 10 MAbs

determined (Table 1). The values of the 23 MAbs to NCA-50 0.4 x 10» M-i to 5.0 x 108 m-i. When comparing the Ka for NCA-50 with that for CEA of Group X MAbs, six anti-CEA MAbs (F33-108, F34-1, -187, F36-9, -54, and -81) showed 2 or more times higher values for CEA than those for NCA-50. On the other hand, the remaining four MAbs (F63-49, F106-19, versus

CEA

were

ranged from

562

TABLE 1

Reactivity of 23 MAbs with NCA-50, NCA-90 and CEA, and affinity constants (Kas) vs. NCA-50 and CEA

Group Clone

Class

X X

F106-19 F106-39 F106-68

Gl (k) G2a (k) Gl K) Gl K) Gl K) Gl K) Gl K) Gl K) Gl k) Gl K)

Y Y Y Y Y Y Y Y Y Y Y Y Y

F63-19 F105-5 F105-34 F105-35 F106-23 F106-30 F106-37 F106-54 F106-62 F106-78 F106-81 F106-88 F106-92

Gl Gl Gl Gl Gl Gl Gl Gl Gl Gl Gl Gl Gl

X X X X X

X X X

Ka

Antigen

MAb

F33-108 F34-1 F34-187 F36-9

F36-54 F36-81 F63-49

vs.

NCA-50

NCA-90

CEA

NCA-50

CEA

+++a

+++

+++

+++

+++

++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

++

+++

+++

++

+++

+++

++

++

1.0 1.2 1.9 1.4 1.4 1.2 1.4 4.6 4.6 2.7

2.2 2.4 8.0 7.8 2.8 2.6 0.2 0.3 0.4 0.2

+++

++

++

+++

++

++

K) K) K) K)

+++

+

+++

+++

+++

+++

+++

+++

K)

+++

++

K) K) K)

+++

++

+++

++

+++

++

K) K)

+++

++

++ +

++

K) K) K)

(xl08 M-i)

+++

++

0.4 4.6 4.1 3.9 4.0 3.5 4.3 4.0 4.1 4.2 4.2

+++

++

5.0

+++

+++

2.7

reactivity with NCA-50, NCA-90, and CEA was determined by SPEIA (see "Materials and Methods"). Fifty nanograms of each antigen were dried

«The

onto wells of 96-well plates. The degree of the absorbance at 492 nm obtained at the highest concentration of MAb used (1 |ig/100 |xl/well) was classified into 5 groups. The code is as follows: +++, A 492 >2.0; ++, 1.0< X!

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is rational that they showed higher values for immunizing antigen than those for their cross-reacting antigens, respectively (Table 1). To further define relationships among epitopes recognized by the MAbs in each group, cross-competition RIAs were carried out by using a purified radiolabeled antigen. The results revealed that at least eight different epitopes can be identified on the NCA molecule: five (X-a to X-e) identified by Group X MAbs on the part common to NCA and CEA (Table 2), and three (Y-a to Y-c) recognized by Group Y MAbs on the part specific for NCA (Table 3), respectively. When the biochemical features of epitopes were compared, however, some differences in their sensitivity to the protease or neuraminidase digestions (Tables 4 and 5) were seen among the cross-competing MAbs in the three subgroups (X-e, Y-a, and Y-b), suggesting that some fine structural The differences may exist among the epitopes in those subgroups. nonreciprocal cross-competition observed could result from (a) steric hindrance of an epitope by a second antibody bound to a different site and/or (b) conformational change in the antigen molecule by binding of one antibody, which may affect binding of the second antibody (24, 44). Although some of Group X MAbs and one of Group Y MAbs showed partially reduced reactivities after deglycosylation of antigens and pepsin or pronase digestion could not completely destroyed the reactivities of antigens with either Group X or Y MAbs, the results listed in Tables 4 and 5, taken together, may indicate that all the epitopes recognized by 23 MAbs tested are of protein nature. A molecular weight of 35,000 of DG-NCA-50 (Fig. 1, lane 4) is in agreement with those of the molecules obtained with TFMS by others (39, 41), and with that of NCA calculated from the peptide sequence (14) since the Asnlinked N-acetylglucosamine is not removed by TFMS. Thus, the chemical deglycosylation with TFMS seems to be almost complete, and may damage some peptide epitopes in some way, resulting in reduced reactivities of DG-NCA-50 (see below). All 10 Group X MAbs, which are reactive with CEA, also reacted with DG-CEA. Since the DG-NCA-50 and DG-NCA-90 showed almost the same mol. wt. of 35,000 (Fig. 1, lanes 4 and 6), it seems likely that the two forms of NCA used in this study are different only in their glycosylation patterns as suggested by others (15, 41). This possibility was also supported by the fact that all 23 MAbs used reacted with DG-NCA-90 (data not shown). The reactivities of 6 of 10 Group X MAbs more or less decreased after reduction and alkylation of NCA-50, indicating that the epitopes recognized by them are dependent on a conformation stabilized by disulfide bonds. It is worthy of notice that the reactivities of these 6 Group X MAbs reduced after deglycosylation of NCA-50 or CEA (Table 4). It may be that these antibodies, similar to antibodies to human chorionic gonadotropin-ß C-terminal peptide (45), recognized the tertiary structures of peptide chains, which are significantly influenced by carbohydrate moieties attached to the peptide chain. However, our results do not rule out the possibility that carbohydrates may play some role in the immunological recognition. The decreased reactivities of all 13 Group Y MAbs with reduced and alkylated NCA-50 also indicate that the epitopes recognized by them are conformation-dependent. All but one Group Y MAb retained almost the same reactivities after deglycosylation of NCA-50, suggesting that the epitopes recognized by them are independent of carbohydrate moieties. Immunohistologic studies have shown that NCA is present in two types of cells: gastrointestinal epithelial cells with the same apical localization as CEA (46); and hematocytes such as granulocytes, macrophages, monocytes and lymphocytes (47-50). A large number of granulocytes and macrophages in lung and spleen explains the relative abundance of NCA in the extracts of these

MAbs, it

570

organs, which are often used for the purification of NCA. Using anti-CEA MAbs which cross-react with NCA, about 15 NCA-like antigens with various mol. wts. from 26,000 to 160,000 have since been reported in the epithelial cells and hematocytes (5-12). The interrelations among those NCAs, however, remain to be elucidated. And only a few reports described MAbs raised against NCA, which can be used for mapping of the NCA molecule. The sérologie mapping and biochemical studies reported here thus provide information as to the range and nature of the epitopes on the NCA molecule and help form the basis for selecting the anti-NCA MAbs for use in biological and immunological study of NCA. ACKNOWLEDGMENTS We thank Misses K. Fukushima and N. Okamoto for their technical and secretarial assistance. This work was supported in part by a Grant-in-Aid for Cancer Research from the Ministry of Education, Science and Culture, Japan and by Kyushu Cancer Society, Fukuoka, Japan. REFERENCES 1.

2. 3.

4.

5.

6.

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8.

9.

10.

11.

Kleist S., Chavanel G., Burtin P. (1972) Identification of an antigen from normal human tissue that crossreacts with the carcinoembryonic antígen. Proc. Nati. Acad. Sei. USA 69: 2492-2494. Gold P., Freedmann S.O. (1965) Demonstration of tumor-specific antigens in human colonie carcinomata by immunological tolerance and absorption techniques. J. Exp. Med. 121: 439-462. Goldenberg D.M. (1981) Carcinoembryonic antigen: Its role as a marker in the management of cancer. A National Institutes of Health consensus development conference. Ann. Int. Med. 94: 407-409. Yamashita K., Totani K., Kuroki Ma., Matsuoka Y., Kobata A. (1987) Structural studies of the carbohydrate moieties of carcinoembryonic antigens. Cancer Res. 47: 3451-3459. Laferte S., Krantz M.J. (1983) Purification and characterization of a 50,000 mol.wt glycoprotein antigen, and localization of determinants involved in cross-reactivity with carcinoembryonic antigen. Molec. Immunol. 20: 421-431. Buchegger F., Schreyer M., Carrel S., Mach J.-P. (1984) Monoclonal antibodies identify a CEA crossreacting antigen of 95 kD (NCA-95) distinct in antigenicity and tissue distribution from the previously described NCA of 55 kD. Int. J. Cancer 33: 643-649. Grunert F., Abuharfeil N, Schwarz K., von Kleist S. (1985) Two CEA and three NCA species, although distinguishable by monoclonal antibodies, have nearly identical peptide patterns. Int. J. Cancer 36: 357-362. Neumaier M., Fenger U., Wagener C. (1985) Delineation of four carcinoembryonic antigen (CEA) related antigens in normal plasma by transblot studies using monoclonal anti-CEA antibodies with different epitope specificities. Molec. Immunol. 22: 1273-1277. Burtin P., Chavanel G., Hendrick J.C., Frenoy N. (1986) Antigenic variants of the nonspecific cross-reacting antigen (NCA). J. Immunol. 137: 839-845. Audette M., Buchegger F., Schreyer M., Mach J.-P. (1987) Monoclonal antibody against carcinoembryonic antigen (CEA) identifies two new forms of crossreacting antigens of molecular weight 90,000 and 160,000 in normal granulocytes. Molec. Immunol. 24: 1177-1186. Kuroki Mo., Kuroki Ma., Moore J.E., Ichiki S., Matsuoka Y. (1988) The von

571

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Department of Biochemistry School of Medicine, Fukuoka University First

7-45-1 Nanakuma, Jonan-ku Fukuoka 814-01

Japan

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