jurnal ab monoklonal

7/28/2019 Jurnal Ab Monoklonal

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Vol. 189, No. 2, 1992 BIOCHE MICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONSDecem ber 15, 1992 Pages 625-632

GENERATION OF SPECIFICITY-VARIANT ANTIBODIES BYALTERATION OF CARBOHYDRATE IN LIGHT CHAIN OF HUMAN

MONOCLONAL ANTIBODIES

Hirofumi Tachibana*, Sanetaka Shirahata and Hiroki MurakamiGraduate School of Genetic Resources Technology, Kyushu Univers ity,Hakozaki, Higashi-ku, Fukuoka, 812, Japan

Received October 13, 1992

SUMMARY: A hybridoma line, CSTN, produced human monoclonal antibody ofwhich light chain had N-linked carbohydrate chain within the variable region. Somemolecular-weight variants of light chain of the antibody were produced by CSTNvariants resistant to cytotoxic effect of concanavalin A. The variant antibodiessignificantly altered the original cross-reactivity with antigens or lost the ability ofantigen binding. The variants various ly trimmed their carbohydrate chains byglycosidases,showed the changed reactivity or acquired the ability to bind for antigens.The carbohydrate-deficient antibodies from tunicamycin-treated CSTN and the variantclones behaved in a similar manner on antigen-binding reactivity. Furthermore,comparison of antibodies of which light chains have carbohydrate chains sensitive andresistant to some glycosidases showed that carbohydrate chain in variable region of lightchain can influence their reactivity with antigen. 0 1992 cademic Press, nc.

Immunoglobulin (Ig) molecules are glycoproteins of which carbohydrates are usuallylocated in their constant regions of heavy chain (1). Such carbohydrates have beenindicated to be of signi ficant importance in complement fixation, secretion of Igmolecules (2,3). Studies on a large number of human myeloma protein have revealedthat the light chain of some Ig molecules (15% of human myeloma proteins examined)contains additional asparagine-linked carbohydrates attached to the variab le region of thechain (4,5). Such carbohydrates have been found in variab le regions in both )c and Kchains. However, the role of the carbohydrate for antigen recognition has not beenestablished yet (6). It has been wel l known that variations in amino acid sequence ofvariable region contribute to many different binding specificities, and immunochemicalstudies of the antibodies have given insights into the diversification in structure of Igvariable regions in Ig.

* To whom correspondence should be addressed.Abbreviations used: Con A, concanavalin A; Ig, immunoglobulin; bovinecarboxypeptidase A, Cpase; Candida cytocrome C, Cyt C; double-stranded DNA, dsDNA; FCS, fetal calf serum; End H, end-P-N-acetylglucosaminidase H.

0006-291X/92 $4.00625 Copyright 0 1992 by Academic Press, inc.All rights of reproduction in any form reserved.


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immobilized with 0.001% protamine sulfate having been trapped to immunoplate. Theassay of concentration of antibodies were measured by use of anti human B chainantibody.Western Blotting. The antibodies from original and Con A-resistant clones wereapplied to SDS-polyacrylamide gel electrophoresis (lO%T) followed by Western blottingfor assay of +e molecular weight (13). The blotted nitrocellulose filter was blocked withBlock Ace (blocking reagent, Dainippon Pharmaceuticals, Japan) overnight at 4C. Afterwashing with TPBS, the blotted filter was incubated with peroxidase-conjugated antihuman @and h chain antibodies (TACO, CA, USA). The color reaction was developedusing 4-chloro-1-naphthol as substrate.

RESULTS AND DISCUSSIONWe have analyzed the role of carbohydrate chains bound for variable region in light

chains of antibodies with regard to their reactivity to antigens. To our knowledge, it isthe firs t case to show that antibody-antigen reactivity is affected by the carbohydrate chainon variable region in the light chain. The CSTN i s a subclone of HB4C5 producingantibody specific to human lung cancer, and produces monoclonal antibody cross-reactive to Cpase, Cyt C and dsDNA. N-linked carbohydrate chain has been found atposition 25 in CDR 1 of the light chain of HB4CS antibody (unpublished data). Toobtain antibodies altered in their carbohydrate chains, carbohydrate-variants were isolatedby screening of Con A-resistance of CSTN. Somatic mutants with defects in the abilityto glycosylate proteins have been isolated from the resistants to cytotoxic plant lectinssuch as Con A (8,9). The CSTN cells were cultured with serum-free ERDF mediumcontaining Con A in 96 well plates, and survived cells were further cultured in themedium. Of 60 wells selected at first screening, two clones, C5TN-C4 (C4) and CSTN-9T (9T) were obtained. The molecular sizes of both heavy and light chains of Ig derivedfrom C4 (C4Ig) and that derived from 9T (9TIg) were examined by SDS-PAGE followedby Western blotting. Differences in migration pattern of their heavy and light chains wereseen on the SDS-PAGE. Both heavy and light chains of C4Ig migrated slightly lowerthan those of C5TNIg. On the other hand, the heavy chain of 9TIg migrated identicallywith that of C41g, and the molecular size of 9T light chain was smaller than that of CSTNbut bigger than the light chain derived from CSTN having been treated with tunicamycin(Figure 1). However, heavy and light chains of C4Ig produced by treating C4 withtunicamycin migrated at the same rate as those of CSTNIg. These results indicate that thedifference in molecular sizes result from alterations of carbohydrate structure on the Igmolecules. Although tunicamycin-treated 9TIg heavy chain migrated faster than that ofnormal one, light chain of 9TIg produced by treating with or without even 20 pg/mltunicamycin was indistinguishable. The light chain of 9TIg was detected withperoxidase-conjugated Con A, a mannose binding lectin, indicating that the chain wasglycosylated. To obtain these variants, we did not use any mutagenesis in thisexperiment, and the variants were not isolated for the alterations of the reactivi ty toantigen but only for resistance to cyto toxic effect of Con A. Therefore, this samplesuggests that some variants with regard to alteration of glycosylation could have existedat a lower frequencies and that the different molecular sizes of the antibodies including 9T

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1 2 3 4 5 6 7

Figure 1. SDS- PAG E analysis of lg molecules by Con A-resistant lines.Culture superna~~nts from the cells treated with or without tunicamvcinwere analyzed on reducing gel and by Western blotting .Ig heavy and ligh t chains were detected with peroxidase-conjugatedanti human m and I chain antibodies. Lane I: CSTN Ig, Lane 2: C4 Ig,Lanes 3 and 7: 9T Ig, Lanes 4,s and 6: CSTN, C4 and 9T lgs aftertunicamycin treatment, respectively.

light chain result from alteration of carbohydrate chain, bu t not from the alteration ofprimary structure.

The antibodies produced by the variants were assessed for the reactivities to Cpase,Cyt C and dsDNA. C4 and 9TIgs showed dramatical alteration in cross-reactivities toantigens (Figure 2). C4 Ig reacted to Cyt C with very lower strength and completely lostthe abil ity to bind Cpase and dsDNA. The 9TIg showed an elevated reactivity for Cyt C.The reactiv ity for Cpase was lower than that of CSTNIg, and could not bind to dsDNA.If the alterations of reactivity result from differences in the amino acid sequence of mutantantibodies, the alteration of the reactivities cou ld have been observed when carbohydrate-deficient antibodies were compared. In order to elucidate whether there are alterations inprimary structure or carbohydrate chain, deglycosylated Igs were obtaine d by treatingCSTN and C4 cells with tunicamycin. The deglycosylated C5TNIg reacted to Cpase andCyt C with much lower strength, and did not bind to dsDNA (Figure 3). On the otherhand, the C4Ig without carbohydrate gained the abil ity to bind to Cpase and Cyt C, andthe patterns of reactivities were simila r with that of CSTN deglycosylated-Ig. Theseresults indicate that the antigen binding alterations are due to the alterations on thecarbohydrate structure. This was recently substantiated by assessing that nucleotidesequences in the variable regions in these Igs were completely identical (data not shown).

According ly, correlation between the antigen binding activity and the structuralchanges of carbohydrate chain in the antibody molecule was examined in detail. Apreliminary experiment using glycosidase-digestion followed by Western blot analysisrevealed that carbohydrate chain on light chain of CSTNIg was sensitive to neuraminidaseand End H. Based on the finding, we selected several glycosidases to modify thecarbohydrate moieties. The CSTN, C4 and 9TIgs were treated with different

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..l 1Ig ConcernZion (n$l) 11

1.5

5$ 1.0-285-f 0.5

40.00

ds DNA

1 10 100 11Ig Concentration (@ml)

Ig Concentration (@ml)Figure 2. ELISA analysis comparing the reactivitiesof Igs from C4 and 9T with CST N lg. The Igs wereassayed for the react iv ity to Cpase, Cyt C and ds DNAAssays were done as descr ibed in MATERIALS ANDMETH ODS . CSTNlg (0); C4lg (A); OTlg (m)

1.52 1.5 - Cpase A 5 ds DNA

3 3 LO-I l.O- z

$e 0.5 -

00-A10 100 1000 I 10 100Ig Concentration (rig/ml) Ig Concentration (q/ml)

Ig Concentration (@ml)Fi ure 3 ELBA analysis of N-linked carbohydrate.-+-y+c am e lclent Igs produced by tunicamycin-treatedcel ls. Assays were done as descr ived in MATERIALSAND METHODS.C5TN Ig from tunucamycin treated cel ls(O);C4Ig from tunicamycin treated cells(A).

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antigens with simila r reac tivity. This finding supports the idea that altered glycosylationon light chain could induce altered antigen binding affinity and specificity .

The role of carbohydrate chains on constant region of Ig heavy chains have beenestablished for Ig assembly, transport, secretion and maintenance of Ig cornformation.Some Ig heavy chains contain N-linked carbohydrate chain attached to the variableregions (4,5). Wa llick et al. demonstrated that the presence of carbohydrate chain inCDR2 of variable region of heavy chain is critical for the high-affinity binding of antidextran antibody (16). On the other hand, the role of the carbohydrate chains found inthe variable regions of some light chain (15% of human myeloma proteins) has not beenestablished yet. In this study, carbohydrate chain on variable region of light chain ofCSTN Ig is shown to play important roles in its antigen binding affinity and specificity .With regard to specif icity of antibody, Ohno et al. have proposed that structure of heavychain is critical for the specific ity of the antibody, and light chain is destined to play asubsidiary role for to heavy chain (17). Our findings demonstrate that not only aminoacid sequence of variable region of heavy chain but also carbohydrate moiety on variableregion of light chain can contribute to determine the specific ity of the antibody.

Recently, several animal ce ll lines have been used for the production ofglycoproteins, and their carbohydrate structure have been reported (18,19,2(l). Thesereports indicate that glycosylation is highly host cell-dependent. Therefore, it isimportant to select host cell which can properly glycosylate each glycoprotein. In theview point, isolation of lectin-resistant cells may be useful in selection of suitable hostcells with regard to glycosylation. In addition, it would be an useful technique forgeneration of specifici ty-variant antibodies that alteration of carbohydrate moieties bycombination the production using lectin-resistant variants of the host cells and digestionof the products using several glycosidases.

REFERENCES1.2.3.4.5.6.7.8.9.

Spiro, R. G. (1973) Advances in Protein Chemistry 27, ~349-467.Academic Press, New York.Koide, N., Nose, M. and Muramatsu, T. (1977) Biochem. Biophys.Res. Commun. 75,838-844.Sibley, C. H. and Wagner, R. A. (1981) J. Immunol. 126, 1868-1873.Spiegelberg, H. L., Abel, C. A., Fishkin, B. G. and Grey, H. M.(1970) Biochemistry 9,4217-4223.Sox, H. C. and Hood, L. (1970) Proc. Natl. Acad. Sci. USA 66,975-982.Taniguchi, T., Mizuochi, T., Beale, M., Dwek, R. A., Rademacher,T. W. and Kobata, A. (1985) Biochemistry 24,555 l-5557.Tachibana, H., Shirahata, S. and Murakami, H. (1992) In AnimalCell Technology: Bas ic & Applied Aspects (H. Murakami et. al. Ed)Vol. 4, ~~547-55 1, Kluwer Academic Publishers, Netherlands.Cifone, M. A., Hynes, R. 0. and Baker, R. M. (1979) J. Cell. Physiol.loo, 39-54.Stanley, P. and Siminovitch, L. (1977) Somat. Cell Genet. 3, 391-405.

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Murakami, H., Hashizume, S., Ohashi, H., Shinohara, K., Yasunoto,K., Nomoto, K. and Omura, H. (1985) In Vitro Cell. Develop. Bio l.21,593-596.Hashizume, S., Kamei, M., Mochizuki, K., Sato, S., kuroda, K.,Kato, M., Yasumoto, K., Nakahashi, H., Hirose, H., Tai, H., Okano,H., Nomoto, K. and Murakami, H. (1991) Hum. Antibod.Hybridomas 2, 142- 147.Murakami, H., Masui, H., Sato G. H., Sueoka, N., Chow T. P. andkano-Sueoka T. (1982) Proc. Nat]. Acad. Sci. USA 79,575-583.Kate, M., Mochizuki, K., Kuroda, K., Sato, S., Murakami, H.,Yasumoto, K., Nomoto, K. and Hashizume, S. (1991) Hum.Antibod. Hybridomas 2,94-101.Tachibana, H., Akiyama, K., Shirahata, S. and Murakami, H.(1991) Cytotechnology 6,219-226.Tachibana, H., Shirahata, S., Kawahara, H. and Murakami, H.(1991) Cytotechnology 7, l-6.Wallick, S. C., Kabat, E. A. and Morrison S. L. (1988) J. Exp. Med.168, 1099-1109.Ohno, S., Mori, M. and Matsunaga, T. (1985) Proc. Natl. Acad. Sci.USA 82,2945-2949.Takeuchi, M., Takasaki, S., Miyazak i, H., Kato, T., Hoshi, S.,Kochibe, N. and Kobata, A. (1988) J. Biol. Chem. 263,3657-3663.Tsuda, E., Goto, M., Murakami, A., Akai, K., Ueda, M., Kawanishi,G., Takahashi, N., Sasaki, R., Chiba, H., Ishihara, H., Mori, M.,Tejima, S., Endo, S. and Arata, Y . (1988) Biochemistry 27,5646-5654.Kagawa, Y., Takasaki, S., Utsumi, J., Hosoi, K., Shimizu, J.,Kochibe, N. and Kobata, A. (1988) J. Biol. Chem. 263, 17508-17515.

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