Infern. Rev. Immunol. 5 . 1989. pp. 131-137 Reprints availahle directly from the publisher Photocopying permitted by license only .c 1989 Harwood Academic Publishers GmbH Printed in the United States of America

Auto-Anti-Idiotypy, Autoimmunity and Some Thoughts on the Structure of Internal Images BERNARD F. ERLANGER Department of Microbiology/Comprehensive Cancer Center, Columbia University, New York, NY 10032

Application of principles proposed by N . K . Jerne has led to the development of an auto-anti-idiotypic protocol for the preparation of monoclonal antibodies specific for receptors for acetylcholine, adenosine (A,), TSH. glucocor- ticoids and aldosterone. The properties otthese antibodies are described with reference to their behavior as internal images of auto-antibodies found in patients with autoimmune diseases or of ligands of the various receptors. The molecular basis of "internal imagery" is discussed in the context of specific antibodies and. in particular, with respect to the hemoglobin-myoglobin family of proteins. We venture the conclusion that immunoglobulins that mimic other biologically active polypeptides need not share primary sequence homologies.

KEYWORDS: anti-idiorypic, internal images. outoimmuniry, molecular mimicry. idiotypir nemwk. receptors, structurd homoloKies

The approaches used in our research on receptors and autoimmunity depend on the existence of the idiotypic network described by Jerne, in particular three of his postulates [ I , 21:

I . Antibodies produced by B cells can recognize any foreign or self epitope. 2. Antibody molecules have immunogenic idiotopes 3. The idiotypes ( i . ~ . , arrangement of idiotopes on a single antibody) can mimic ( i . e . , be

internal images of) any foreign or self epitope. Acceptance of these postulates leads to the conclusion that properties of all molecules, in

the outside world or in an organism, can be mimicked by immunoglobulins. This means immunoglobulin molecules can substitute for nucleic acids. sugars, carbohydrates, drugs, and or course, for other biologically active polypeptides in defined biological systems. This has not been an easy concept to accept, especially by immunologists. It still has not gained general acceptance even though our laboratory I31 and others [4] have used the concept successfully. We have tried to deal with this problem in a recent review [ 5 ) .

It follows from the postulates of Jerne that anti-idiotypic antibodies can also mimic the activities of antibodies to self antigens, which circulate in individuals with autoimmune diseases such as myasthenia gravis (MG), Graves disease, etc. Indeed, there is evidence that antiidiotypic antibodies may play a role in the etiology of autoimmune diseases [6 ,7] . In the case of MG. the antibodies need only bind to the receptor to producc signs of the disease. Binding prevents the action of acetylcholine on its receptor and. as well, promotes destruc- tion of the receptor by phagocytosis [S] . In Graves disease, mimicry can be exhibited by

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132 B. E ERLANGER

antibodies that stiniulate the TSH receptor or inhibit the stimulation of the receptor by TSH 19, 101. Anti-idiotypic antibodies can also stimulate the insulin receptor [ II, 121.

In our early work on antibodies to the acetylcholine receptor (AChR) [13], we were not aware of our indebtedness to Jerne. We had been studying the activity of a highly potent agonist of the receptor, rruns-3,3’-bis [(a-trimethylammonio)-methyl] azobenzene (BisQ). On making antibodies to Bis Q (in rabbits) we noted that the antibodies resembled AChR in its activated state in that they recognized and bound strongly to agonists but not so well to antagonists. This was consistent with the fact that agonists stabilize the activated receptor (ion channels open) 1141.

We were stimulated to make anti-idiotypic antibodies to anti-BisQ because we reasoned that if anti-BisQ mimicked AChR. anti-idiotypic antibodies would mimic antibodies to AChR. We immunized rabbits with anti-BisQ in complete Freund’s adjuvant and observed the production of antibodies that bound receptor. Moreover, we observed transient signs of myasthenia gravis in the rabbits. It could be shown that the antibodies inhibited the binding of 13H]-BisQ to anti-BisQ and hcncc had properties of antiidiotypic antibodies.

We lurther observed, however, that certain of the sera could enhance the binding of [ ”1- BisQ to anti-RisQ and that, indeed. they bound [-‘H]-BisQ on their own. It was at this time that we became convinced that Jerne’s postulated “network” was operating, i . e . , that these antibodies were anti-idiotypic antibodies stimulated by anti-anti-BisQ.

In all of our subsequent studies we exploited Jerne’s proposed network to prepare monoclonal antibodies to various receptors. The strategy called for immunization of mice with a ligand of the receptor and screening for antiidiotypic antibodies using rabbit antibodies (or Fab fragments) specilic for the ligand. We chose t o screen for binding to an heterologous ( i . e . , rabbit) anti-ligand antibody in order to select for antibodies directed at public idiotopcs. Public idiotopes would more likely be involved in specificity and hence, be shared by epitopes o n the receptor. Subsequent screening was with receptor preparations.

This strategy was used successfully to prepare monoclonal antibodies to receptors for AChR [IS]. adenosine (A, ) [I61 glucocorticoids [ 171. thyroid stimulating hormone (TSH) [91 and, most recently for aldosterone 1281. In the case of AChR, one of the monoclonal antibodies inhibited ion flux in a reconstituted vesicle system, produced signs of MG in mice and produced the same fluorescence pattern in Torpedo tissue as did a rabbit antibody raised by immunization with receptor 13. 71. In these respects it mimicked antibodies circulating in individuals with MG.

The “internal image” properties o f the antibodies to the A, adenosine receptor included inhibition of adenylate cyclase in ;I brain membrane system, mimicking the activity of 1,-PIA, an agonist of the receptor. The antibodies to the TSH and glucocorticoid receptors did not act as agonists of their respective receptors. The former inhibited adenylate cyclase activation by TSH in FRTL-S cells 191. The latter bound some distance from the steroid binding site of the rcccptor at an epitope that apparently is shared by the B-chain of insulin 13, 291.

The auto-anti-idiotypic strategy has also been used successfully by others for the prepara- tion of monoclonal anti-receptor antibodies [18, 191.

Raising anti-reccptor receptor antibodies by an anti-idiotypic procedure has the advan- tage that purified receptor preparations need not be isolated for use as immunogens. This was not particularly important for anti-AChR; AChR is available in quantity in the pure state [ 201. The adenosine receptor, however, is present at very low levels in various tissues. On the other hand. adenosine is available i n quantity. In principle, an antibody to the adenosine

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AUTO-ANTI-IDIOTYPY AND AIJTOIMMUNITY 133

receptor obtained by immunization with ligand could be used to obtain workable quantities of receptor viu cDNA cloning procedures. allowing characterization of the receptor and its use as an immunogen. We are, presently, in the process of attempting to obtain the appropriate cDNAs.

The auro-antiidiotypic (one step) route has an important advantage over the two step procedure in which idiotypic antibodies are first isolated and used to raise anti-idiotypic antibodies. In the one step proccdurc there is no need to select and immunize with the “correct” idiotypic antibody, i . e . , the one whose binding site shares properties (epitopes) with the binding site of the receptor.’ The mouse does this; all the researcher must do is screen for antibody that binds receptor. This was brought home forcefully to us recently. A highly specific monoclonal anti-aldosterone antibody was used to raise monoclonal anti- idiotypic antihodies. The latter behaved as internal images of aldosterone in the idiotype- antiidiotype system but not in the receptor system. In fact, they did not bind receptor.

With respect to the aldosterone system. we were recently successful in producing an idiotype and receptor specific monoclonal internal image antibody by the auto-anti- idiotypic procedure [ 2 8 ] . Immunization was with aldosterone conjugated to bovine serum albumin via a 3-0-carboxymethyl oxime of aldosterone 1211.

Aniong the anti-idiotypic antibodies was one H10E4C9E that: 1 ) Bound to Fab fragments of affinity purified rabbit anti-aldosterone antibody. Binding

2 ) Inhibited the binding of [3H1 aldosterone to rabbit polyclonal antibodies and to several

3) Could be purified on a column of immobilized monoclonal anti-aldosterone antibody. 4) Inhibited binding of [3H]-aldosterone to rabbit kidney cytosolic aldosterone receptors

but had no effect on glucocorticoid receptors. 5) Displaced [.3H]-aldosterone from receptor with kinetics that were similar to displace-

ment by “cold” aldosterone (Fig. I ) . 6) Displaced [3H] aldosterone from both the 9s (Fig. 2) and the 4s receptor (not shown),

as demonstrated by glycerol gradient sedimentation. Thus, here we have a case in which an immunoglobulin mimics a steroid. The structural

basis of this mimicry is not easily determined. I n fact, an understanding of the way in which an immunoglubulin can act as an internal image of a non-polypeptide can only be deter- mined by x-ray crystallography.

There are cases, however, in which immunoglobulins act as internal images of other polypeptides. For example. there is considerable activity in the field of vaccines. Anti- idiotypic antibodies are being sought which mimic regions of a viral coat that make contact with the viral receptor [cf. 221. In such cases. an investigation of commonalities in the primary structure of the antibody and the viral coat is feasible.

Perhaps the most successful experiments have been reported by Greene and his col- leagues [23, 241. They have identified an area o f sequence similarity shared by the reovirus type 3 cell attachment protein and an anti-idiotypic antibody that can bind to the same cell- surface receptor. Sequence identities were seen in short stretches (3 and 4 amino acid sequences) in the CDRll region of the anti-idiotypic antibody. A tetradecapeptide identical to the V, , sequence and a haptadecapeptide identical to the V, sequence. both containing the homologous stretches of short peptide sequences. were synthesized and shown to inhibit binding of antiidiotypic antibody and reovirus type 3 to the type 3 receptor. I t is proposed, therefore. that the basis of the molecular mimicry of the antibody is local sequence

was inhibited by aldosterone.

murinc monoclonal antibodies raised during the same fusion.

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I34 R . F. ERLANGER

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TIME (Hours) FIGURE I Diswciation kinetic studies of [ '111-aldosterone receptor complexes with HIOE4C9F and aldosterone. Aldosterone receptors were preincubated with 5 nM [ZH]-aldosterone for 24 hours at 4°C. A 1.OOO fold excess of unlabeled aldostcrone (A). diluted ascites H10E4C9F ( + ) or an IgGl control (LH9G4) (U) were added and the rcniaining [ 'HI-aldostcrone binding was nieaured after various periods of time at 4°C. Results are expressed as the percentage of aldusterone binding (7% cpni).

homology to the reovirus. Computer modelling procedures support the proposal. It would appear from the above experimental evidence, therefore, that an explanation of internal imagery might be sequence homology, at least in the reovirus system.

There is considerable evidence, however, that entirely different sequences can assume almost identical conformations. Moreover, these identical conformations can function in an identical manner.

A recent exaniple is the finding that different Vl- and V,, genes can code for similar combining sites with the same specificity for (1-6) dextrans ( 2 5 ) . Sequence data showed major differences in amino acid sequences in all three complementarity-determining re- gions.

Perhaps the most striking example of protein conformations with the same biological activity but made up of entirely different primary sequences is in the hemoglobin, myo- globin family. Both of these protein\. in order to be successful carriers of oxygen, must have a structure that (a) prevents the oxidation of Fe' + to Fe' ' ' , (b) allows Fe + ' to bind molecular oxygen and (c) undergoes a conformational change that releases the molecular

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AUTO-ANTI-IDIOTYPY AND AUTOIMMUNITY 135

c B a s

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0 0 1 0 20 3c

FIGURE 2 Glycerol density gradient analysis of aldosterone receptors. Rabbit kidney cytosol was incubated with 5 nM [211]-aldosterone in the presence ofthe auto-anti-idiotypic antibody HIOE4CYF (diluted ascites 11150) ( + ) o r an IgGl control LHYG4 (il) for 24 hours at 4°C. The mixture (0.25 ml) was layered onto the top of a 5 ml 15-20% glycerol gradient in 20 mM Tris-HCI. ImM EDTA. 2OmM stdium tungstate. pH 7 . 4 buffer. External standards were myoglobin (2s). bovine serum albumin (4.6s) and aldolase (7 .8s ) .

oxygen under appropriate conditions. Thus the requirements are rather stringent and dependent upon conformational properties of the protein.

When one examines the hemoglobins and myoglobins of various species, marked differ- ences in their amino acid sequence are seen [26] . Thus, out of 141 positions, the beta chain of shark hemoglobin can differ from the human by 96 amino acids as analyzed by pairwise comparisons. Leghemoglobin differs from human by 137 amino acids when analyzed in the same way. Yet they all have very similar three dimensional structures and they all bind heme and function as oxygen carriers. Shown in Figure 3, for comparison are the polypeptide backbones of three species of hemoglobin as determined by X-ray crystallography [26] . They are startlingly similar in conformation despite large differences in primary sequence. It is of intercst to note that lamprey hemoglobin undergoes the Bohr effect even though the oxygenated form is a single polypeptide. On losing oxygen i t dimerizes, allowing the heme- heme interaction necessary for the Bohr effect [ 271.

Thus, identical function does not necessarily dictate an identical primary sequence. Nature does not select for primary sequences but for vital functions. In so doing, it gives the organisni considerable leeway in constructing functional molecules. We can cxpect therefore

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B E ERI.ANGER

Lamprey hem - , -

\ \

\\/

FIGURE 3 Comparisons of structures (polypeptide backbones) of OL chain of horse iiiethemoglobin. lamprey hemoglobin and plycrra hemoglobin. Number\ represent percent of and number o f pairwise sequence differences relative to the alpha chain of human hemoglobin [adapted from 261.

that immunoglobulins that mimic biologically active polypeptides need not share primary sequences in regions that are critical to function, just as immunoglobulins that mimic non- polypeptides (steroids, alkaloids, etc.) cunnot share them.

Acknowledgments

Work descrihed in thi5 rc'vicw was supported hy thc National Institutes of Health, the N Y. Heari A\sociation and the Muscular Dptrnphy Aswciatlon. Figure\ I and 2 are froni a paper submitted for publicatlon hy M. Lombeh. whu received \upport froin INSERM ol' France and B Stephen I . Morse Fellowship

Note

I . Thi\ IS particularl) a problem wlth an anligcn like TSH. a protein of molecular weight IX.OO(1. which ha5 many ininiunodrterniinant groups. 1iitx.t of which do not interact with the hinding bite o ! the TSH receptor

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AUTO-ANTI-IDIOTYPY AND AUTOIMMUNITY 137

References

I . Jerne. N . K . (1974) Ann. fmnrunol. (Inst. Pasteur) 12SC:373-389. 2. Jerne. N . K. (19x4) Immuml. Rev. 79:s-24. 3. Erlanger. 8. E, Cleveland, W. L.. Wassermann. N. H.. K u . H. H.. Hill. B. L.. Sarangarajan. R.. Rajagopalan.

R.. Cayanis. E., Edelman. 1. S. and Penn. A . S. (1986) Immwiol. Rev. 94:23-37. 4 , Gaulton. G . N. and Greene. M. I . (1986) Ann. Rn: /tritnutio/. 4:253-280. S. Erlanger. B. E (1985). Immunolog,v 7iodn.v 6:IO-I I. 6. Erlanger. B. E. Cleveland. W L.. Wassermann. N. H.. Hill. B. L.. Penn. A. S . . Ku. H. H . and Sarangarajan.

7. Erlanger. R. E , Cleveland. W. L. . Wassermann. N . H. , Ku. H. H.. Hill. B. L.. and Wan K . K . (IY86)Ann.

8 . Lindstrom. J. ( 1979) In: Pla.s~riupheresis and fhe fnrmu~~obiolog~ of Mwsfhetiiu Crewis. Boston: Houghton

9. Hill, B. I.. and Erlanger, B. E (1988) Ent/oc,ritiolog,v 1222840-2850.

R. (19x5) In: Moleculcrr Rusis of'Nrrrv Acfii,ify, Berlin. New York: W. de Gruyter, pp. 523-536.

N.Y. Aceid. Sci. 219-226.

Mifflin. pp. 3-19.

10. Islam, M. N. , Pepper. B. M , Rriones-Crbina. R. and Farid. N . R. (1983). Eur. J. Immunol 13:57-63. 1 I . Sege. K. . and Peterson. I? A. (IY7X) !'rot.. N d . Ac.trd. Sci. U.S.A. 75:2443-2447. 12. Schechter. Y.. Elias D.. Maron. R . and Cohen. I . K. (IYX4) J. Biol. Chern 259. 6416-6419. 13. Wassermann. N. H.. Penn. A. S.. Freimuth. P I . . Treptow. N.. Wentzrl. S., Cleveland. W. E and Erlanger. R .

E (1982) f r o c . Nafl. Acad. Sci. U.S.A. 79:4810-4814. 14. Heidmann. T. and Changeux. J. P (1978) Anti. Re\,. Riochem. 47:317-357. IS. Cleveland. W. L.. Wassermann. N. H.. Sarangarajan, R., Penn. A. S.. and Erlanger. B. E (1983) Ntrfure

16. Ku. H H.. Cleveland. W. L. and Erlanger. B. E (1987) J. /mnruno/. 139:2376-2384. 17. Cayanis. E.. Rajagopalan. R.. Cleveland, W. L.. Edelman. I . S. and Erlanger. B. E (1986) J. Riol. ('hrm.

26 I :50')4-S 103. 18. Ludwig. D. S.. Finkelstein. R. A, . Karu, A. E . 1)allas. W. S.. Ashby. E. R.. Schoolnik, G . K . (1YX7) P r o c

Nar. Accrd. U.S.A. 84.3673-3677. 19. I . R. ('ohen in discussion of ref. 7. 20. Reviewed in Changeux. J-P (IY81) Harvey Lecture. Series 75:85-254. 71 . Del-auzon. S.. LeTrang. N.. Moreau. M. E. Gentin, M . , Christeff. N . . Desfosses. B. and Cittanova. N. ( 1987)

J. Steroid Biorhrtn. 28:459-463. 22. Kennedy. R. C., Eichberg. J. W.. Langford. R. E. and Iheesnian. G . R . (1986) Science 232:220-223. 23. Bruck. C.. Co.. M. S.. Slaoui. M.. Gaulton. G . N., Smith. T., Fields. B. N. , Mullins. J. I . and Greene, M. 1.

( 1986) /'roc.. Nufl. Actrd. S c i . U.S.A. 83:6578-6582. 24. Willianis. W. V.. Guy. H. R.. Rubin. D. H.. Robey. E , Myers. J. N.. Kleiber-Enimons. T.. Weiner. D. B. and

Greene. M . I . (1988) Pro(.. M i l l . Acud. Sci. U.S.A. 85:6488-6492. 25. Akolkar. I? N.. Sikder. S. K , Bhattacharya. S. B.. 1-iao. J.. Gruezo. E. Morrison. S. L. and Kabat. E. A .

( 1987) J. / w n i r r i o / . 138:4472-4479. 26. Dickerson. R. E. and &is. I . ( IY83) tfc~nio~l~~brn:Sfrtcc~frtre. Fioic~rio~i . Ewluf ion. undfeuhologv. Menlo Park:

BenjaminiCunlmings. 27. Briehl. R. W. (1963) J. R i d . Chrm. 238:2361-2366. 28 Imihes . M.. Edelman. I . 5.. and Erlanger. B. E (1989) J. B i d Chem. 264:2528-2536. 29. Cayanih. E.. Sarangarajan. R.. Loniheh. M.. Nahon. E . . Edelman. 1. S. . and Erlanger. B. E ( 1989) Proc. Nor/.

305:56-S7.

A c r d Sci . U.S.A X6:213X-2142.

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