Int. J . Cancer: 47, 267-273 (1991) 0 1991 Wiley-Liss, Inc.

Publication of the International Union Against Cancer Publication de I'Union lnternationale Contre le Cancer

PREPARATION AND CHARACTERIZATION OF MONOCLONAL ANTIBODIES SPECIFIC TO SYNTHETIC PEPTIDE OF CARCINOEMBRYONIC ANTIGEN Masayuki TSUJISAKI',Kohzoh IMAI~, Noriyulu HISHIKAWA', Shigeru TOKUCHI',Yuji HINODA',Hirokazu MATSUKAWA~, shim0 O I K A W A ~Hiroshi , NAKAZAT03 and Akira YACHI"~ 'Department of Internal Medicine (Section 1 ), Sapporo Medical College, S-1, W-16, Chuo-ku, Sapporo 060; 20riental Yeast Institute, Suita, Osaka; and 3Suntory Institute, Mishima-gun, Osaka, Japan. Monoclonal antibodies (MAbs) were prepared to a synthetic peptide (PI : I I94 40 amino acids) in domain I of the carcinoembryonic antigen (CEA) molecule. The majority of the amino acids in peptide P I show a hydrophilic character, and similar sequences are repeated in domains I, II and 111 of this molecule. These MAbs appear to recognize new epitopes of CEA, since representative conventional MAbs to CEA do not show reactivity to synthetic peptide PI. The resulting MAbs were divided into 2 groups; group I (MAb PI-706) reacted with peptide PI, but not with purified CEA preparation,while group 2 (MAb P 1-234 and P 1-255) reacted with either type of antigen, in the immunoblotting assay using purified CEA and in immunostaining of colonic carcinoma tissues. Furthermore, group-l MAb P 1-706 was reactive to purified CEA after treatment with periodate.

CEA has been one of the most frequently used tumor markers in clinical medicine during the last 20 years, since Gold and Freedman (1965) described it in colonic carcinoma and fetal colonic tissues. The level of circulating CEA in the serum of cancer patients changes in association with tumor progression, with recurrences after operation and with metastasis. Numerous studies (Krupey et al., 1972; Imai et al., 1984) have demonstrated that CEA is a M, 180,00CL200,000 glycoprotein and is expressed not only in colonic adenocarcinoma but also in other adenocarcinomas, such as carcinomas of the stomach, pancreas, bile duct, lung, breast and ovary. CEA is also expressed in normal epithelial tissues, as well as in malignant transforming tissues, although the amount of CEA appears to be low (Fritsche and Mach, 1978; Yachi et al., 1984). Immunological and biochemical studies have shown that many CEA-related antigens exhibit cross-reactivity to CEA, including non-specific cross-reacting antigen (NCA) (von Kleist et al., 1972), tumor-extracted CEA-related antigen (TEX) (Kessler et al., 1978), normal fecal antigen (NFA) (Matsuoka et al., 1982), meconium antigen (MA) (Primus et al., 1983) and biliary glycoprotein I (BGP I) (Svenverg, 1976). Gene cloning of CEA (Oikawa et al., 1987; Zimmermann et al., 1987), NCA (Tawaragi et al., 1988; Neumaier et al., 1988), BGP I (Hinoda et al., 1988) and pregnancy-specific P,-glycoprotein (PSPG) (Oikawa et al., 1988) has been successfully performed and the structure of these antigens has been determined. Interestingly, CEA-related antigens have revealed similar domain structures and high homology in amino-acid (AA) sequences. Studies on gene cloning also indicate that CEA and NCA belong to the immunoglobulin supergene family (Paxton et al., 1987). The results of molecular cloning have suggested that it is very difficult to prepare MAbs specific for a given antigen of the CEA family if one uses ordinary antigens, such as soluble CEA, or cell extracts from cultured cells as an immunogen, since the amino-acid sequences are very similar among CEA family antigens. Therefore, we decided to use synthetic peptides as immunogens in order to generate specific MAbs to epitopes which are unique for a given antigen. This study should further enable us to analyze conformational and structural changes in CEA molecule and the influence of the carbohydrate moiety on CEA.

MATERIAL AND METHODS

Synthesis of CEA peptides According to the amino-acid sequences of CEA determined by gene cloning (Oikawa et al., 1987; Zimmermann et al., 1987), 22 amino acids (peptide P1) from 119 to 140 in domain I of CEA were synthesized by an amino-acid synthesizer (LKB, Bromma, Sweden, BIOLYNX). Synthetic peptides P2 (AA 181-194), P3 (AA 202-221), P4 (AA 367-386), P5 (AA 558-577) and P6 (AA 1-20) were also prepared and used as controls. The purity of each peptide was confirmed by use of reverse-phase liquid chromatography (ODs 120 T column, TOSHO model CCPM, Tokyo, Japan). The amino-acid composition of peptide P1 was examined by an amino-acid analyzer (Waters, Milford, MA, PICO-TAG). Preparation of purijied CEA and NCA Purified CEA and NCA were prepared from colonic cancer tissues and from normal lung tissues, respectively, by an immunoperchloric extraction method followed by successive gel filtration (Imai et al., 1984). Purified CEA and NCA were used as antigens in the binding and blotting assays. Recombinant domains of CEA Recombinant domains N, I, I1 and I11 were prepared as follows: peptides virtually identical, except for termini, to the domains of CEA were synthesized in E . coli transfected with the vector carrying the appropriate fragment of the CEA cDNA (data not shown). N-domain (AA 1-106) was the product of direct expression and was soluble upon disruption of E . coli by sonication. Domains I (AA 107-262), I1 (AA 301-478) and I11 (AA 479-6681, including membrane portion, were expressed as chimeric peptides fused to N-terminal 210 AA of Pgalactosidase, insoluble upon sonication, and were solubilized with SDS, diluted and used for analysis. Animals Eight- to 12-week-old female BALB/c mice were purchased from Charles River, Wilmington, MA. Development of MAbs to synthetic CEA peptide PI The MAbs are secreted hybridomas constructed with myeloma cells and splenocytes from BALB/c mice which were immunized once a week with 3 intraperitoneal injections of 100 kg of synthetic peptide PI suspended in Freund's complete adjuvant. Hybridization with murine myeloma cells P3X63-Ag8.653 and subcloning were performed according to standard procedures (Imai et al., 1984); MAbs from the resulting hybridomas were purified either by caprylic acid preparation (Russo et al., 1983), by affinity chromatography on pro-

4 T whom ~ correspondence and reprint requests should be sent, at the Department of Internal Medicine (Section I), Sapporo Medical College, S-1, W-16, Chuo-ku, Sapporo 060, Japan. Received: June 26, 1990 and in revised form September 24, 1990.

'

268

TSUJISAKI ET AL.

tein A Sepharose or by ion exchange chromatography on DEAE. Antibodies were radiolabelled with lZ5I, utilizing the chloramine T method. Conventional MAbs to CEA Representative anti-CEA MAbs MA208 (IgG,) (Kitagawa et a/., 1986), CEA281 (IgG,) (Hammarstrom et al., 1989), 12140-7 (IgG,,) and 12-140-1 (IgG,) which were selected for this study, were established by 14 research groups organized by the International Workshop on the epitope reactivity of MAbs against CEA (chaired by Drs. J.E. Shively and S. Hammarstrom). As described elsewhere (Hammarstrom et al., 1989), it is suggested that the epitopes on CEA recognized with these MAbs are distinct according to competition assay. Binding assay Polyvinyl chloride 96-well microtiter plates (Dynatech, Alexandria, VA) were coated with synthetic peptide P1, P2, P3, P4, P5, P6, purified CEA or recombinant domains of CEA by incubating 100 p.1 of each solution (100 pg/ml) in 0. I M bicarbonate buffer, PH 9.5 for 16 hr at 4°C. Following 3 washings with phosphate buffered saline (PBS), PH 7.4, containing 0.05% Tween 20 (PBS-Tween 20), 100 p.1 of hybridoma supernatants were added. Plates were then incubated for 3 hr at 4°C. Following 5 washings with PBS-Tween 20, 1251-labelled anti-murine IgG Fc xeno-antibodies (Jackson ImmunoResearch, Avondale, PA, 1 X lo5 cpm/well) were added. After incubation for 4 hr, plates were washed 5 times with PBS-Tween 20 and the bound radioactivity was counted in a gamma-counter (Tsujisaki et al., 1986). In order to check the influence of the carbohydrate moiety of CEA to the binding activity of the antibody, binding assays to periodate-treated CEA were performed. After being coated to the plate, CEA was treated with 1% periodate for 5, 10, 20 and 30 min. Following 3 washings, 12sI-labelled MAbs were added and incubated for 4 hr. After washing, bound radioactivity was counted. Cross-blocking assay Microtiter plates were coated with synthetic peptide PI by incubation for 16 hr at 4°C. After washing, excess cold MAbs were added and incubated for 3 hr at 4°C. Following 3 washings, 1251-labelledMAbs (1 x lo5 cpm/well) were added to test for their ability to bind. After incubation for 4 hr, plates were washed 5 times and bound radioactivity was counted. Results are calculated as percentage blocking, as compared with negative control. Tissue processing and fmation Normal tissues as well as benign and malignant tissues were obtained from patients who underwent surgery. A portion of each specimen was fixed with 10% buffered formaldehyde. Three consecutive 5-pm-thick paraffin sections were obtained from each sample. One of the paraffin sections was stained with hematoxylin and eosin and studied for histologic features, while the other sections were used as a substrate for immunoperoxidase analysis. For the cryostat sections, the technique described elsewhere (Jephthah-Ochola et a l . , 1988) was em-

ployed. Briefly, small pieces of tissue were embedded in Tissue-Tec (Miles, Naperville, IL), snap-frozen in liquid nitrogen, and stored at - 70°C. Cryosections (5 pm) were fixed in acetone. Immunoperoxidase technique A conventional procedure for the immunoperoxidase technique was employed for the staining reactions with MAbs (Yachi et al., 1984). The sections were first incubated with 0.3% H,O, in methanol for 20 min to block endogenous peroxidase and then overlaid with MAbs for 30 rnin at room temperature in a moist chamber. After washing 3 times for 5 rnin in cold PBS, peroxidase-conjugated rabbit anti-mouse Ig antiserum (Dako, Copenhagen, Denmark) was applied for another 30 min. After a final wash with cold PBS, the sections were reacted for 60 sec in a solution containing 40 mg 3.3'diaminobenzidine and 0.01% hydrogen peroxide in 100 ml 0.05 M Tris-HCl buffer (PH 7.6). They were stained with 0.2% methyl green for 3 to 5 min and then observed with a light microscope. Periodic acid treatment One hundred microliters of 1 % periodic acid solution in PBS (PH 7.2) were placed on each section for 30 to 60 rnin at room temperature. After 3 washes with PBS, immunoperoxidase staining was performed as described above.

SDS-PAGE and Western blotting Purified antigens were separated by SDS-PAGE on a 7.5% polyacrylamide slab gel (Imai et al., 1982). Transfer to nitrocellulose membrane was achieved, essentially as described by Towbin et al. (1979), in a transblotting chamber (Bio Rad, Richmond, CA) at 170 mA overnight, using 25 mmol/liter Tris and 192 mmol/liter glycine in 20% methanol, PH 8.3. The nitrocellulose membrane was then sliced vertically into strips. These were incubated in 3% bovine serum albumin (Sigma, St. Louis, MO) in PBS at room temperature for 6 hr. Each strip was then incubated with hybridoma supernatant at room temperature for 2 hr. Strips were washed 3 times for 10 min each, in PBS containing 11400 dilution of peroxidase-conjugated rabbit anti-mouse immunoglobulins (Dako). After being washed as above, the strips were incubated in a freshly prepared mixture of 60 mg of chloronaphtol in 20 ml of methanol and 60:l of H,0,/100 ml of 20 m~ Tris, PH 7.5. After a 15-min incubation at room temperature, the strips were transferred to water. Antigen-antibody binding was identified by the presence of black stain. To check the binding activity of MAbs to periodate-treated CEA, immunoblotting was performed. Purified CEA separated by SDS-PAGE was transferred to nitrocellulose and then treated with 1% periodate for 10 rnin at room temperature. The procedure that followed was the same as described above. RESULTS

Characterization of synthetic CEA peptide PI Synthetic CEA peptide PI was selected as a immunogen firstly because of its hydrophilic character and secondly be-

TABLE I - AMINO-ACID SEQUENCE OF SYNTHETIC PEPTIDE (PI-PEPTIDE, 119-140) USED FOR IMMUNIZATION. SIMILAR SEQUENCES ARE PRESENT IN DOMAIN I1 AND 111 OF THE CARCINOEMBRYONIC ANTIGEN Amino acid sequence

Domain

I I1

I11

$I9N S 2Y7N S 47sN S

K P fl P K P

V E D K D A V A F T C E P V E D E D A V A L T C E P V E D K D A V A F T C E P

E T Q D AIM E I Q T3lX E & Q & T496

The underlined amino-acids are different from those in 119-140 in domain I. Figures denote amino-acid numbers from the N-terminus of the CEA molecule.

269

ANTI-CEA PEPTIDE ANTIBODIES TABLE 11 - COMPOSITION ANALYSIS OF AMINO-ACID IN PI PEFTIDE Asn

Theoretical Exoerimental

Asp

1 -

3 -

Asx

Gln

Glu

1 3 4 - -

Glx

4

Ser

I 1

Gly

-

His

-

Arg

-

Thr

Ala

2 2

3 3

Ro

2 2

Tyr

Val

2 2

-

Met

Ile

Leu

- - _

- - -

cause of its common presence in sequences of domain I, I1 and 111 of the CEA molecule (Table I). The resulting synthetic CEA peptide P1 was tested for amino-acid composition by an aminoacid analyser in order to confirm its sequence. The experimental composition completely agreed with the theoretical one (Table 11).

Phe

1 1

Trp

-

Lys

2 2

Comparison of anti-CEA peptide MAbs with conventional anti-CEA MAbs An International Workshop on the epitope reactivity of MAbs against CEA has concluded that conventional anti-CEA MAbs, which were prepared by immunizing mice with CEAproducing cells or purified CEA, are divided into 5 groups according to serological studies, mainly by competition assay Reactivity of anti-CEA peptide MAbs to CEA peptides and (Hammarstrom et al., 1989). In this study, representative conpur@ed CEA ventional anti-CEA MAbs of each group were tested for reacThe cell fusion experiment resulted in 13 out of 922 (1.4%) tivity with purified CEA and synthetic peptides of CEA. None clones whose supernatants showed strong reactivity with pep- of them reacted with synthetic peptides or recombinant CEA tide P1. From these, 3 MAbs, designated P1-706, P1-234 and domains, while all reacted with purified CEA, suggesting that P1-255, were established and used in this study. The reactiv- the epitopes recognized with these conventional MAbs do not ities of these MAbs were tested for binding to 6 kinds of exist either in P1, P2, P3, P4, P5 or P6 (Table VI). In other synthetic peptide of CEA. As shown in Table 111, anti-CEA words, anti-CEA peptide MAbs established in our laboratory peptide MAbs P1-706, P1-234 and P1-255 were reactive with appeared different from the conventional ones and recognized PI peptide specifically but not with P2, P3, P4, P5 or P6. MAb epitopes with which conventional MAbs do not react. Pl-234 and Pl-255 strongly reacted with purified CEA preparation, whereas MAb P1-706 did not (by binding assay). Reactivity to purijied CEA in immunoblotting assay In order to confirm the serological results of anti-CEA pepReactivity of MAbs to recombinant CEA domain produced in tide MAbs, an immunoblotting assay was performed, utilizing E. coli purified CEA as an antigen. Anti-CEA peptide MAbs P1-234 Recombinant domains N, I, I1 and 111 of CEA were con- and P1-255 showed reactivity to purified CEA, demonstrating structed with amino-acid sequences as described in “Material a broad band of M, 200,000 in immunoblotting, while MAb and Methods”. MAb PI-706 reacted with domain I only, while P1-706 did not (Fig. 1). MAb P1-234 reacted with domains I and 111 and MAb P1-255 reacted with domains I, I1 and 111 (Table IV). Characterization of the epitope recognized by MAb P1-706 Difference of epitopes In order to determine the sieric hindrance of carbohydrate Cross-blocking assay (Table V) showed that MAbs P1-706 moiety in CEA to the epitope recognized with MAb P1-706, an and P1-255 inhibited the binding of the other antibodies to immunoblotting assay was carried out using periodate-treated peptide PI, and MAbs P1-234 and P1-255 showed similar CEA as an antigen. As shown in Figure 2, MAb P1-706 was results. MAbs PI-706 and P1-234, however, completely failed reactive with CEA treated with 1% periodate for 30 min, but not with non-treated CEA, while conventional anti-CEA MAb to affect the reactivity of the other antibody. TABLE 111- REACTIVITY OF ANTI-CEA PEFTIDE MONOCLONAL ANTIBODIES WITH VARIOUS PREPARATIONS OF SYNTHETIC CEA PEPTIDES Amino-acid sequence

Synthetic peptide P1 P2 P3 P4 P5 P6 Purified CEA Control (bovine serum albumin)

119-140 18 1 - 194 202-221 367-386 558-577 1-20 1-668 + CH02

Monoclonal antibody PI-706

PI-234

PI-255

21445 cpm’ 1324 1587 1439 1665 1258 2649 1591

26628 cpm 1041 1164 1004 1022 864 25549 I775

23143 cpm 967 1080 1216 934 832 23788 852

‘Mean cpm of triplicate values.-*CHO carbohydrate moiety. TABLE IV - REACTIVITY OF ANTI-CEA PEPTIDE MONOCLONAL ANTIBODIES WITH RECOMBINANT CEA DOMAINS Amino-acid sequence

CEA domain N I I1 111 Control (bovine serum albumin) ’Mean cpm of triplicate values

1-106 107-262 301-478 479-668

’251-labelledanti-CEA peptide MAb PI-706

PI-234

1910 cpm’

1226 cpm 11710 1413

4573 1941 1876 1264

I1980 1 loo

P1-255

1487 cpm

12505 13689 1844

270

TSUJISAKI ET AL TABLE V - EPITOPE ON P1 PEF'TIDE RECOGNIZED WITH MAbs IN BLOCKING ASSAY' '251-labelled anti-CEA peptide MAbs

Cold inhibitor

MAb P1-706 MAb P1-234 MAb Pl-255

PI-706

PI-234

PI-255

81% 12 79

6% 85 87

53% 41 90

'Microtiter plates coated with P 1 peptide were incubated with an excess of cold MAbs and then tested for their ability to bind 'Z51-labelled MAbs. Results are expressed as percentage blocking.

CEM 010 reacted with both. Binding assay using periodatetreated CEA had shown the same results as those of the immunoblotting assay. MAb P1-706 displayed a reactivity with CEA in a time-dependent manner after treatment with periodate, but not with non-treated CEA, while MAb Pl-234 and P1-255 reacted with both (Fig. 3).

Staining pattern of anti-CEA peptide MAbs in tissues using the immunoperoxidase method Immunostaining in colonic and gastric carcinoma tissues, which were fixed with 10% buffered formaldehyde, was carried out utilizing anti-CEA peptide MAbs and conventional anti-CEA MAb CEM 010. Representative data are shown in Table VII and Figure 4. MAbs P1-234 and P1-255 exhibited a similar distribution in carcinoma tissues to that of anti-CEA MAb CEM 010, showing apical labelling of the adenocarcinoma glands. They did not show positive staining for the noncancerous colonic and gastric tissues except for the surface epithelial cells of the colon, with which they faintly reacted. Cryostat sections of colonic cancer tissue gave similar results (data not shown). On the other hand, MAb P1-706 failed to stain any of 16 colonic and 9 gastric carcinoma tissues. Periodic acid treatment of the tissue sections was done to investigate whether the epitope recognized by MAb PI-706 is exposed. MAb PI-706

TABLE VI - REACTIVITY PATTERN OF CONVENTIONAL ANTI-CEA MONOCLONAL ANTlBODlES WITH VARIOUS PREPARATIONS OF SYNTHETIC CEA PEWIDES

'251-labeIIed conventional anti-CEA MAb

Amino acid sequence

Synthetic peptide P1 P2 P3 P4 P5 P6 CEA domain N I I1 111

Purified CEA Control (bovine serum albumin)

119-140 181-194 202-221 367-386 558-577 1-20 1-106 107-262 301-478 479-668 1-668 + CH02

__

MA208

CEA281

12-140-7

12- 140-1

459 cpm' 423 419 419 484 47 1 412 423 801 413 20416 297

516 cpm 48 1 513 515 420 460 522 766 867 688 6065 316

378 cpm 304 355 327 368 294 358 354 541 396 19157 266

390 cpm 347 387 330 35 1 354 1488 432 398 1030 16943 275

'Mean cprn of triplicate values.-'CHO: carbohydrate moiety

FIGURE 1 - Western blot analysis of the binding of anti-CEA MAb 010 (lane 1) and anti-CEA peptide MAb P1-706 (lane 2), P1-234 (lane 3) and P1-255 (lane 4) to purified CEA under non-reduced conditions.

FIGURE 2 - Western blot analysis of non-treated CEA (lanes 1, 3 and 5) and periodate-treated CEA (lanes 2, 4 and 6) reacted with anti-CEA MAb CEM 010 (lanes 1 and 2) and anti-CEA peptide MAb P1-706 (lanes 3 and 4) and P1-255 (lanes 5 and 6).

27 1

ANTI-CEA PEPTIDE ANTIBODIES

the synthetic peptides, but none of them showed reactivity to purified CEA preparation or to cancerous tissues. It is of interest that MAb P1-706 reacted with domain I of CEA, but not with domain N, 11, I11 or purified CEA, suggesting that the carbohydrate moiety may affect the reactivity of J MAb P1-706 with a particular epitope. To investigate this possibility, periodic acid treatment was performed. The data ob&.."' -Atained from binding assay, Western blotting and the immuno40000 , peroxidase method may suggest that the epitope is hindered by ---p.PI-255 the carbohydrate moiety, although further study will be needed , PI-234 for a firm conclusion to be made regarding the effect of car-..-&-PI-706 bohydrate moiety, because periodate can also destroy certain 20000 amino-acids. In any event, these results further suggest that it Negative would be rather difficult to develop MAbs which react with the epitope recognized by MAb PI-706. Therefore, MAb P1-706 is a unique antibody and might be used as a serological assay to detect CEA in the serum after treatment of a sample with periodic acid. On the other hand, anti-CEA peptide MAbs Pl-234 and P1-255 recognize distinct epitopes with which conventional MAbs do not react, since none of conventional ones are reactive with synthetic P1 peptide of CEA. Therefore, these MAbs to the peptide should be used to establish an assay for measuring serum CEA in order to evaluate these antibodies from the clinical viewpoint. demonstrated positive staining of colonic adenocarcinoma cells The different reactivities of each MAb with purified CEA after the sections were treated with 1% periodic acid for 30 min and recombinant CEA domains N, I, I1 and I11 enable us to at room temperature, whereas MAb CEM 010 showed a similar analyse possible amino-acid sequences in synthetic peptide P 1 staining pattern in intensity and extent before and after treat- recognized by each MAb. The amino-acid differences between ment with periodic acid. recombinant CEA domains I and I1 are 121, 126, 131, 137, 139 and 140, and those between domains I and 111are 137, 139 and 140 (Table I). It shows that the same continuous sequences which were included in domain I, I1 and 111 should be 5 amino DISCUSSION acids at most. Cross-blocking assay to examine the spatial Using synthetic CEA peptide as an immunogen, we have relationship among epitopes recognized with MAbs P1-706, established 3 MAbs consisting of 2 groups based on reactivity. P1-234 and PI-255 indicates that each epitope is distinct on the The MAb belonging to group 1 (MAb P1-706, IgG,) reacted P1 peptide: the epitope of MAb P1-255 was close to both that with immunizing CEA peptide P1, but failed to react with of MAb P1-706 and that of P1-234, while that of P1-706 and purified CEA preparation, which was revealed by binding as- that of P1-234 were spatially distant. Based on those results say, immunoblotting assay and the immunoperoxidase method. (Tables I11 to V), computer analysis was done to define the The MAbs belonging to group 2 (MAbs P1-234 and P1-255, MAbs. We suggest that 5 amino-acids within CEPETQDA both IgG,) reacted not only with immunizing CEA peptide P1 (133-140) are for MAb P1-706, 5 amino-acids within NSKbut also with purified CEA preparation: both showed strong PVEDKDAVAFTCEP (119-135) for MAb P1-234 and positive staining for colonic adenocarcinomas with a pattern TCEPE (132-136) for MAb P1-255, when each MAb recogsimilar to that of conventional anti-CEA MAb. As far as the nizes 5 amino-acids. The 4 possible amino-acid sequences as MAbs to synthetic CEA peptide are concerned, our report dem- epitope of MAb P1-706 are 4 amino-acids selected within onstrates the establishment of MAbs reacting with purified EPETQDA (134-140), while those for MAb P1-234 are within CEA preparation and with tissue sections of freshly resected NSKPVE (119-124) or VEDKDAV (123-129) when that of carcinoma. Schwaibold et al. (1988) prepared several MAbs to MAb P1-255 is DAVA (127-130). Further possible sequences

-

. I

L

- .':

---------

..-.*~-.""-.""~

--.--

TABLE VU - STAINING PA'ITERN OF ANTI-CEA PEEIDE MONOCLONAL ANTIBODY P1-255 IN COLONIC AND GASTRIC CANCEROUS TISSUES ASSESSED BY IMMUNOPEROXIDASE METHOD' Number tested

Colon Adenocarcinoma: Well differentiated Moderately differentiated Mucinous carcinoma Stomach Adenocarcinoma: Well differentiated Moderately differentiated Poorly differentiated Signet-ring-cell carcinoma Mucinous carcinoma Total

+ + : strong positive; + : positive; - : negative

Staining intensity

++

+

11

9

2

4

2 0

1

0 0 0 0 0 11

2 1 1 2 0 10

1

2 1 2 3

1 25

1

-

0 1 0

0 0 1

1 1 4

212

TSUJISAKI ET AL.

FIGURE4 - Staining pattern of colonic cancerous tissues with antiCEA peptide MAbs by the immunoperoxidase method. Conventional anti-CEA MAb CEM 010 (a), anti-CEA peptide MAb P1-234 (b), P1-255 (c) and PI-706 ( d ) were used. A periodate-treated consecutive section ( e ) was also stained by MAb PI-706. Bar = 50 p,m in consecutive sections (a-e).

of MAb P1-234 are 4 amino-acids selected within NSKPVE (1 19-124), VEDKDAV (123-129) or AVAFTCE (128-124) when that of P1-255 is TCEP or CEPE. It remains absolutely necessary to investigate the reactivity of each MAb with the above-mentioned synthetic fragments of peptide. These anti-peptide MAbs would also be useful in comparing the structural features and functional properties of the sequences in the molecule, since they could be prepared by the aimed approach to have special and functional epitopes. In this respect, CEA and NCA have been found to act as adherent molecules between cells (Benchimol et al., 1989). Finally, these anti-peptide MAbs (Abl) would be useful tools to de-

velop anti-idiotypic MAb (Ab2) and to study the internal image of antigen mimicked by Ab2, when the amino-acid sequences of epitope recognized with Abl have already determined (Tsujisaki et al., 1988). ACKNOWLEDGEMENTS

We acknowledge helpful discussions with Dr. J.E. Shively (Research Institute, City of Hope, Duarte, CA) and Dr. T. Sugiyama (Sapporo Medical College). We also appreciated the editorial assistance of Mr. J. Nolin (Sapporo Medical College) and the secretarial assistance of Ms. A. Kitamura.

ANTI-CEA PEPTIDE ANTIBODIES

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REFERENCES

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Preparation and characterization of monoclonal antibodies specific to synthetic peptide of carcinoembryonic antigen.

Monoclonal antibodies (MAbs) were prepared to a synthetic peptide (PI: 119-140 amino acids) in domain 1 of the carcino-embryonic antigen (CEA) molecul...
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