Eur. J. Immunol. 1990. 20: 2505-2508

J chain deficiency in human IgM monoclonal antibodies

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Short paper Y. Gloria Meng, Anne B. Criss and Katy E. Georgiadis

Cutter Biological, Miles Inc., Berkeley

J chain deficiency in human IgM monoclonal antibodies produced by Epstein-Barr virus-transformed B lymphocytes Six human IgM monoclonal antibodies against Pseudomonas aeruginosa were purified and characterized. On agarose-acrylamide sodium dodecyl sulfate (SDS) gels run under nonreducing conditions, IgM monoclonal antibodies showed variable amounts of a slower migrating form of IgM in addition to the one co-migrating with plasma IgM. Protein blotting with anti-J chain antibody showed that the slower migrating form did not contain J chain. Analysis of one of the monoclonal antibodies by sucrose gradient centrifugation showed that the J chain-deficient form sedimented faster than the complete IgM. It is known that IgM preparations lacking J chain sediment faster by sucrose gradient centrifugation analysis and tend to form hexamers. The slower migrating form of IgM we observed on SDS gels under nonreducing conditions could be hexameric IgM. Further evaluation of this monoclonal antibody demonstrated that both forms of IgM had the same antigen-binding activity. Glycosylation of the light chain was demonstrated in two of the monoclonal antibodies.

1 Introduction

2 Materials and methods

Pseudomonas aeruginosa is an opportunistic pathogen and often causes fatal infections in immune compromised patients. Studies have shown that antibodies against LPS of Pseudomonas aeruginosa are protective in a murine burn wound sepsis model [l]. Development of IgM mAb for clinical use was therefore initiated.This report describes the biochemical evaluation of six different human IgM mAb against specific Fisher-Devlin-Gnabasik (FDG) immunotype LPS of Pseudornonas aeruginosa.

2.1 Purification and activity assessment

It was reported that when IgM was reduced and reassociated in the absence of J chain, a faster sedimenting component was observed by density gradient ultracentrifugation analysis (21. Eskeland and Christensen [3] reported that IgM preparations without J chain from patients with Waldenstrom’s macroglobulinemia sedimented faster by density gradient ultracentrifugation and tended to form hexamers when examined by EM. Davis et al. [4,51 also reported similar results of an IgM mutant, which lacked J chain, and showed that it had a higher molecular weight when analyzed by SDS-PAGE under nonreducing conditions. IgM secreted by glioma cells transfected with H chain and L chain-expressing vectors was not associated with J chain, and existed as both pentamers and hexamers in approximately equal amounts, while IgM secreted by transfected plasmacytoma cells was almost exclusively pentameric [6]. Here, we report our observations on a J chain-deficient IgM which appears to be in hexameric form.

mAb producing cell lines, derived from EBV transformed human B lymphocytes, were provided by Genetic Systems Corporation (Seattle, WA). Unless described otherwise, cell cultures were grown in 50% DMEM, 50% nutrient mixture F12 (Ham; JR Scientific Inc., Woodland, CA) supplemented with albumin, transferrin and insulin. IgM mAb were purified on an anti-IgM immunoaffinity column as described previously [7] and stored in 0.1 M Tris-HC1(pH 7.8), 0.5 N NaCl (TBS). The activity of the purified IgM from each cell line was assessed by ELISA. For antigen-binding activity, plates were coated with individual LPS preparations (5 pg/ml for antibodies from cell lines 2SC, 6Fll,9D10, 13C1,5G2 and 10 pg/ml for the antibody from cell line lCl).Twofold serial dilutions in TBS containing 1% BSA of purified IgM and non-purified IgM were titrated in parallel on an antiIgM-coated plate for IgM quantitation and on an LPScoated plate for measuring LPS-binding activity [ 7 ] . The binding of specific antibody was assessed by sequential addition of biotinylated goat anti-IgM (Vector, Burlingame, CA), pre-incubated biotinylated alkaline phosphatase (Vector), avidin (Sigma, St. Louis, MO), and finally p-nitrophenyl phosphate (Sigma) as substrate. Absorbances from the linear part of the titration curves were analyzed by a linear regression program to calculate the IgM concentration and LPS-binding activity of the purified IgM relative to the non-purified IgM from the cell culture SN [7]. The relative specific LPS-binding activity was calculated as the ratio of the relative LPS-binding activity to the relative IgM concentration.

[I 80291 Correspondence: Y. Gloria Meng, Cutter Biological, Miles Inc., F? 0. Box 1986, Berkeley, CA 94701, USA

2.3 Analysis of IgM mAb

NaCl

L chain types of the purified mAb were determined by the Ouchterlony double diffusion method using anti-h and

0 VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1990

0014-2980/90/1111-2505$3.50+ .25/0

Abbreviation: TBS: 0.1 M Tris-HC1 (pH 7.8), 0.5

N

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Eur. J. Immunol. 1990. 20: 2505-2508

Y. G. Meng, A . B. Criss and K. E. Georgiadis

from pooled human plasma by the same method used for purification of the mAb. IgM mAb and plasma IgM were analyzed on agarose-acrylamide SDS gels under nonreducing conditions. On Coomassie blue-stained gels, while plasma IgM exhibited only one band, one additional slower migrating band was seen clearly in antibodies from cell lines 5G2 and 9D10 and faintly in antibodies from cell lines 1C1, 13C1 and 6 F l l (Fig. 1A).

anti-x antisera supplied by ICN (Lisle, IL). To check for aggregation, purified antibodies at a concentration of 80 to 100 pg/ml were chromatographed on a Superose 6 gel filtration column by FPLC (Pharmacia, Pitscataway, NJ) in TBS. For gel electrophoresis analyses, antibodies were run either on 0.5 % agarose , 2.5 YO acrylamide-N ,N '-bis-met hylene acrylamide gels under nonreducing conditions or on 15% acrylamide SDS gels under reducing conditions as described previously [7]. For protein blotting, proteins were transferred electrophoretically onto nitrocellulose paper and probed with anti-human J, IgG(H+L) and p chain antibodies as described previously [7].

A duplicate gel was transferred to nitrocellulose paper and probed sequentially with anti-J chain and anti-IgG(H+L) antibodies. Probing with the anti-J chain antibody detected only the bands co-migrating with the plasma IgM (Fig. 1 B). The slower migrating bands showed up clearly when the blot was further probed with anti-IgG(H+L) antibody (Fig. 1C).When a separate blot containing these antibodies was probed with anti-p chain antibody, the slower migrating band was confirmed to be IgM. Therefore, our blotting results suggested that the slower migrating protein was IgM lacking J chain. Although the slower migrating form of IgM was not seen in 2SC IgM in Fig. 1 C, three other preparations of 2SC IgM (two from cultures grown in RPMI 1640 medium containing 10% FBS) showed small amounts of this slower migrating band as well as an even more slowly migrating band which was likely aggregated IgM molecules.

For sucrose gradient ultracentrifugation analyses, monoclonal IgM from 6 F l l cells (0.4 A280nrnunit in 2 ml) was concentrated fivefold by ultrafiltration using a Centricon 30 unit (Amicon Corporation, Danvers, MA), loaded onto two 10%-40% (wh) sucrose gradients in TBS and centrifuged in a SW40 rotor (Beckman Instruments, Irvine, CA) at 27000 rpm for 20 h at 5°C. Fractions (0.5 ml) were collected from the bottom of the tube and the absorbance at 280 nm was measured after addition of 0.5 ml of water.

3 Results 3.1 Purification of IgM mAb Six IgM mAb produced by cell lines 2SC, 6F11,1C119D10, 13C1 and 5G2 were purified by chromatography on an anti-IgM column.They are against LPS of FDG type 1 , 2 , 3 (and 7), 4, 5 and 6, respectively. Analysis by ELISA demonstrated that all six mAb retained full LPS binding activity after purification. mAb produced by 2SC and 1C1 cell lines had h type L chains and the rest had x type L chains. All six antibodies showed retention volumes similar to that of plasma IgM when analyzed by FPLC.

3.2 Electrophoretic analysis of IgM mAb under nonreducing conditions Since the mAb were intended for clinical use, they were compared with plasma IgM. Polyclonal IgM was purified

M P

1 2 3 4 5 6

t 2 3 4 5 6

P

To characterize further the two bands observed on gels under nonreducing conditions, 6 F l l IgM (from a culture grown in medium containing 1% FBS) was run on a preparative agarose acrylamide gel. Strips of the gel were stained to locate the two IgM bands. Proteins were eluted from the two bands and run on an agarose-acrylamide gel. Both isolated proteins retained the original electrophoretic mobility; therefore, the two IgM species were not exchangeable nor in equilibrium with each other. We then fractionated by density gradient ultracentrifugation a preparation of 6 F l l IgM which contained approximately equal amounts of the two forms of IgM. Fractions were collected from the bottom of the tube. Fractions 17, 18 (the peak) and 19 of the IgM containing fractions were run on an agarose-acrylamide gel under nonreducing conditions (Fig. 2). Fraction 17 contained predominantly the slower migrating form and fraction 19 contained predominantly the form co-migrating with plasma IgM. Comparison of

1 2 3 4 5 6

P

Figure 1. Electrophoretic analysis of IgM antibodies under nonreducing conditions. Purified IgM mAb were run on duplicate 2.5% acrylamide, 0.5% agarose SDS gels. One gel was stained with Coomassie blue (A), and the other was transferred to nitrocellulose paper and probed sequentially with anti-J chain (B) and antiIgG(H+L) (C) antibodies. Samples are: IgM antibodies purified from cell lines 1C1. 5G2, 13C1, 6Fl1, 2SC and 9D10 (lanes 1 to 6) and affinitypurified plasma IgM (lane P). The molecular mass of the markers on the stained ael (lane M) is shown in kDa.

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Eur. J. Immunol. 1990. 20: 2505-2508

J chain deficiency in human IgM monoclonal antibodies

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3.3 Electrophoretic analysis of IgM mAb under reducing conditions

Figure 2. Electrophoretic analysis of fractionated 6 F l l IgM under nonreducing conditions. Fractions 17, 18 and 19 (lanes 2, 3 and 4) and unfractionated 6 F l l IgM (lanes 1and 5) were run on an agarose-acrylamide SDS gel and stained with Coomassie blue.

1 2 3 4 5

fractions 17,19 and unfractionated 6 F l l IgM by ELISA on an FDG immunotype 2 LPS-coated plate and on an anti-IgM-coated plate showed that they all had the same specific LPS-binding activity. Titration curves of fractions 17 and 19 are shown in Fig. 3.These results indicate that the two forms of 6 F l l IgM have the same antigen-binding activity. The 6 F l l IgM in Fig. 2 contained more of the J chain deficient form of IgM than that in Fig. 1, which was purified from a different culture. This suggests that culture conditions can affect the relative ratio of complete and J chain-deficient forms of IgM.

All IgM mAb were analyzed on 15% acrylamide SDS gels under reducing conditions. On silver stained gels, the H chains of all six IgM mAb showed the same mobility as that of plasma IgM. The mobility of L chains of some mAb differed from that of plasma IgM (Fig. 3 A). A low mobility L chain band and an additional minor L chain band of normal mobility were seen in 1ClIgM.The L chain of 13C1 IgM had a slightly higher mobility. A smear was seen under the L chain band in 2SC IgM. The amount of the smear varied between 2SC IgM antibodies purified from different cultures which could be partially degraded L chains. 9D10 IgM showed two L chain bands of similar intensity, both migrating slower than that of plasma IgM. A duplicate gel was transferred to nitrocellulose paper and was probed sequentially with anti-J chain (Fig. 4B) and anti-IgG(H+L) antibodies (Fig. 4C).The J chain of all the IgM mAb had the same mobility as that of the plasma IgM and the J chain band in 5G2 IgM was very faint. In order to visualize L chains without the interference of the previously probed J chain bands, a separate blot was probed with anti-IgG(H+L) antibody only. Both putative L chain bands of 9D10 IgM and 1C1 IgM seen on the stained gel were observed by blotting thus confirming their identity. Glycosylation of the L chain has been reported for some human myeloma IgG antibodies [ 8 ] .When 9D10 IgM was treated with N-glycanase (0.45 unit of enzyme/0.0042 A280,,,,, unit of IgM) the lower mobility L chain band was converted to the higher mobility species [7]. Treatment of 1C1 IgM with N-glycanase (0.45 unit of enzyme/0.015 Am,,,,, unit of IgM) also converted most of the lower mobility L chain band to the L chain species exhibiting normal mobility. Therefore, the high molecular weight L chains seen in 9D10 and 1C1 IgM antibodies are glycosylated L chains. N-glycanase treatment of 2SC and 13C1IgM mAb did not change the L chain mobilities.

A

4 Discussion

n

E

3

1.0-

The J chain has been shown to support the correct assembly of the IgM polymer [9]. We observed a slower migrating form of IgM lacking the J chain, on SDS gels run under 6 nonreducing conditions. Analysis of 6 F l l IgM by sucrose +? gradient ultracentrifugation showed that the J chain defi0.5a n cient form sedimented faster than the complete IgM. It was reported that IgM molecules lacking J chain sedimented faster and tended to form hexamers [2-61. The slower migrating form of IgM we observed on SDS gCls are likely 0.0' hexameric IgM. The J chain deficient and the complete 0 2 4 8 8 10 12 forms of 6 F l l IgM were found to have the same antigenLog 2 (reciprocal di l u t i o n l binding activity. IgM preparations lacking J chain were Figure 3. ELISA comparison of the specific antigen-binding activ- shown to be active. Davis et al. [4, 51 reported that the ity of J chain-deficient and complete 6 F l l IgM. Duplicate aliquots hexameric mutant IgM, which lacked J chain, mediated of twofold serial dilutions of Fractions 17 and 19 from the sucrose complement-dependent cytolysis and that the hexamer and gradient ultracentrifugation (Fig. 2) were added to either an anti-IgM-coated plate (-) for IgM quantitation o r to an LPS- pentamer containing fractions of a wild-type IgM produced coated plate (. . .) for LPS binding. The relative specific LPS- by a transformant showed the same avidity for antigen. binding activity of fraction 17 to fraction 19was calculated to be 1.0 Cattaneo et al. [6] also showed that a J chain deficient using absorbances from dilutions 1/32t o 11256 in duplicate for both lacking antibody secreted by glioma transfectants bound samples for both IgM ELISA and LPS ELISA. the hapten and were recognized by anti-Id antibodies. -r

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Eur. J. Immunol. 1990.20: 2505-2508

Y. G. Meng, A. B. Criss and K. E. Georgiadis

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(A 1

(C)

(B)

Figure 4. Electrophoretic analysis of IgM antibodies under

reducing conditions. Purified mAb were run on duplicate 15% acrylamide SDS gels. One gel was stained with silver (A) and the other was transferredto nitrocellulose paper and probed sequentially with anti-J chain (B) and anti-IgG(H+L) (C) antibodies. Samples are IgM mAb purified from cell lines 1C1, 5G2, 13C1, 6Fl1, 2SC and 9D10 (lanes 1to 6) and

P

1 2 3 4 5 6

1 2 3 4 5 6

P

There are several possible reasons for the presence of J chain-deficient IgM. It has been reported, for example, that the J chain of IgM is highly susceptible to protease digestion [9, 101. Extracellular proteases present under tissue culture conditions may therefore selectively degrade J chains, resulting in a fraction of IgM molecules lacking this component. Subtilisin was shown to digest the Cterminal portion of the J chain peptide without altering the structure of the IgM polymer appreciably [ 101. The possibility that the slower migrating band contained partially digested J chain which was not recognized by the anti-J chain antibody used for probing cannot be excluded. Alternatively, the EBV-transformed cell lines expressing the mAb evaluated in the present studies may have a deficiency in J chain synthesis. This possibility could perhaps be determined by comparing the amount of J chain mRNA by Northern blotting or the amount of cellulaf- J chain by RIA in cells which produce different amounts of the slower migrating form of IgM. IgM derived from a heterohybridoma established by fusion of 9D10 cells and the mouse myeloma X63Ag8.653 was found to contain mouse J chain [7] and did not show the slower migrating form of IgM. These results could indicate that J chain synthesis in the heterohybridoma cells is not limiting the expression of complete IgM molecules. Since our data suggest that the two forms of 6 F l l IgM have the same antigen-binding activity, it is likely that the slower

1 2 3 4 5 6

P

affinity-purified plasma IgM (lane P).

migrating form of IgM observed in other mAb preparations also has full antigen-binding activity and the application of these mAb for therapeutic uses will not be adversely affected. We thank Drs. K . J. Lembach and R. E. Louie for reviewing and editing this manuscript, and Dr. U.Opitz forproviding tissue culture supernatants for these studies.

Received October, 24, 1989; in revised form July 24, 1990.

5 References 1 Cryz, S. J. Jr., Fiirer, E. and Germanier, R., Infect. lmmun. 1983. 3Y: 1072. 2 Eskeland, T., Scand. J. Immunol. 1974.3: 757. 3 Eskeland,T. and Christensen,T. B., Scand. J. lmmunol. 1975. 4: 217. 4 Davis, A. C., Roux, K. H. and Shulman, M. J., Eur. J. Immunol. 1988. 18: 1001. 5 Davis, A. C., Roux, K. H., Pursey, J. and Shulman, M. J., EMBO J. 1989. 8: 2519. 6 Cattaneo, A. and Neuberger, M. S., EMBO J. 1987. 6: 2753. 7 Meng,Y. G. and Trawinski, J., J. Immunol. 1988. 141: 2684. 8 Spiegelberg, H. L., Abel, C. A., Fishkin, B. G. and Grey, H. M., Biochemistry 1970. 9: 4217. 9 Koshland, M. E., Annu. Rev. lmmunol. 1985. 3: 425. 10 Koshland, M. E., Chapuis, R. M., Recht, B. andBrown, J. C., J. lmmunol. 1977. 118: 775.