Abstract:
Antibodies to Tumor Necrosis Factor receptors (TNF-Rs) which inhibit the cytocidal effect of TNF but not its binding to the TNF-Rs, and ligands interacting with other receptors of the TNF/NGF family, are provided together with methods of producing them. The antibodies preferably bind to the fourth cysteine rich domain of the p 75  TNF receptor or to the region between said fourth cysteine rich domain and the cell membrane.

Description:
CROSS REFERENCE TO RELATED APPLICATIONS 
     The present application is a division of U.S. application Ser. No. 08/476,862, filed Jun. 7, 1995, now U.S. Pat. No. 6,262,239, which is a continuation-in-part of U.S. application Ser. No. 08/321,685, filed Oct. 12, 1994, now abandoned, the entire contents of which is hereby incorporated herein by reference. 
    
    
     FIELD OF THE INVENTION 
     The present invention relates to ligands to Tumor Necrosis Factor receptors (TNF-Rs) which inhibit the effect of TNF but not its binding to the TNF-Rs, as well as to ligands interacting with other receptors of the TNF/NGF family. 
     BACKGROUND OF THE INVENTION 
     Tumor necrosis factor (TNF) is a pleiotropic cytokine, produced by a number of cell types, mainly by activated macrophages. It is one of the principal mediators of the immune and inflammatory response. Interest in its function has greatly increased, recently, in view of evidence of the involvement of TNF in the pathogenesis of a wide range of disease states, including endotoxin shock, cerebral malaria and graft-versus-host reaction. Since many of the effects of TNF are deleterious to the organism, it is of great interest to find ways of blocking its action on host cells. An evident target for such intervention are the molecules to which TNF has to bind in order to exert its effects, namely the TNF-Rs. These molecules exist not only in cell-bound, but also in soluble forms, consisting of the cleaved extra-cellular domains of the intact receptors (see Nophar et al., EMBO Journal, 9(10):3269-78, 1990). The soluble receptors maintain the ability to bind TNF, and thus have the ability to block its function by competition with surface receptors. 
     Another method of TNF inhibition based on the principle of competing with cell-bound molecules, is the use of antibodies recognizing TNF receptors and blocking the ligand binding. 
     The cell surface TNF-Rs are expressed in almost all cells of the body. The various effects of TNF, the cytotoxic, growth-promoting and others, are all signaled by the TNF receptors upon the binding of TNF to them. Two forms of these receptors, which differ in molecular size: 55 and 75 kilodaltons, have been described, and will be called herein p 55  and p 75  TNF-R, respectively. It should be noted, however, that there exist publications which refer to these receptors also as p 60  and p 80 . 
     The TNF-Rs belong to a family of receptors which are involved in other critical biological processes. Examples of these receptors are the low affinity NGF receptor, which plays an important role in the regulation of growth and differentiation of nerve cells. Several other receptors are involved in the regulation of lymphocyte growth, such as CDw40 and some others. Another member of the family is the FAS receptor also called APO, a receptor which is involved in signaling for apoptosis and which, based on a study with mice deficient in its function, seems to play an important role in the etiology of a lupus-like disease. Herein, this family of receptors is called “TNF/NGF receptor family”. 
     One of the most striking features of TNF compared to other cytokines, thought to contribute to the pathogenesis of several diseases, is its ability to elicit cell death. The cell-killing activity of TNF is thought to be induced by the p 55  receptor. However, this p 55  receptor activity can be assisted by the p 75  receptor, through a yet unknown mechanism. 
     Parent application No. 07/524,263 and European Patent publications 398,327 and 412,486 disclose antibodies to the soluble TNF-Rs. These antibodies were found to recognize the soluble TNF-Rs and to inhibit the binding of TNF to the TNF-Rs on the cell surface. Monovalent F(ab) fragments blocked the effect of TNF, while intact antibodies were observed to mimic the cytotoxic effect of TNF. European patent publication 585,939 describes ligands interacting with a certain region in TNF-Rs. 
     SUMMARY OF THE INVENTION 
     The present invention provides a ligand to a member of the TNF/NGF receptor family, which binds either to the region of the fourth cysteine rich domain of such a receptor, or to the receptor between it and the cell membrane. 
     The region of the fourth cysteine rich domain will be called herein, for simplicity&#39;s sake, the “67 epitope” and the antibodies recognizing it the “group 67” antibodies. This region may extend between about amino acids pro-141 and thr-179 in the p 75  TNF-R (residues 163-201 of SEQ ID NO:2) or a corresponding region in another member of the TNF/NGF family. More particularly, the region may extend between about amino acids pro-141 and cys-163 of the p 75  TNF-R (residues 163-185 of SEQ ID NO:2) or a corresponding region in another member of the TNF/NGF family. The ligand downstream of the fourth cysteine rich domain includes the amino acid sequence between about thr-179 and about the end of the extracellular domain of the receptor (residues 201-257 of SEQ ID NO:2) or a corresponding region in another member of the TNF/NGF family. 
     Preferably, the receptor is the TNF-R, in particular the p 75  TNF-R. 
     One such ligand includes the amino acid sequence for the CDR region of the heavy chain of monoclonal antibody no. 67, and/or of the light chain thereof. 
     Another such ligand includes the amino acid sequence for the CDR region of the heavy chain of monoclonal antibody no. 81, and/or the light chain thereof. 
     Yet another such ligand includes the amino acid sequence or antibody against the “stalk” region, i.e., from about amino acid thr-181 to about amino acid 235-asp. 
     The ligands may comprise, for example, proteins, peptides, immunoadhesins, antibodies or other organic compounds. 
     The proteins may comprise, for example, a fusion protein of the ligand with another protein, optionally linked by a peptide linker. Such a fusion protein can increase the retention time of the ligand in the body, and thus may even allow the ligand-protein complex to be employed as a latent agent or as a vaccine. 
     The term “proteins” includes muteins and fused proteins, their salts, functional derivatives and active fractions 
     “Functional derivatives” as used herein cover derivatives of the ligands and their fused proteins and muteins, which may be prepared from the functional groups which occur as side chains on the residues or the N- or C-terminal groups, by means known in the art, and are included in the invention as long as they remain pharmaceutically acceptable, i.e., they cannot destroy the activity of the ligand and do not confer toxic properties on compositions containing it. These derivatives may, for example, include polyethylene glycol side-chains which may mask antigenic sites and extend the residence of the ligands in body fluids. Other derivatives include aliphatic esters of the carboxyl groups, amides of the carboxyl groups by reaction with ammonia or with primary or secondary amines, N-acyl derivatives of free amino groups of the amino acid residues formed with acyl moieties (e.g., alkanoyl or carbocyclic aroyl groups) or O-acyl derivatives of free hydroxyl groups (for example, that of seryl or threonyl residues) formed with acyl moieties. 
     As “active fractions” of the ligands, its fused proteins and its muteins, the present invention covers any fragment or precursors of the polypeptide chain of the ligand alone or together with associated molecules or residues linked thereto, e.g., sugar or phosphate residues, or aggregates of the protein molecule or the sugar residues by themselves, provided said fraction has the same biological and/or pharmaceutical activity. 
     As used herein the term “muteins” refers to analogs of the proteins, peptides and the like in which one or more or the amino acid residues of the protein found to bind are replaced by different amino acid residues or are deleted, or one or more amino acid residues are added to the original sequence, without changing considerably the activity of the resulting product. These muteins are prepared by known synthesis and/or by size-directed mutagenesis techniques, or any other known technique suitable therefor. 
     The term “fused protein” refers to a polypeptide comprising the ligands or a mutein thereof fused with another protein which has an extended residence time in body fluids. The ligands may thus be fused to another protein, polypeptide or the like, e.g., an immunoglobulin or a fragment thereof. 
     The term “salts” herein refers to both salts or carboxyl groups and to- acid addition salts of amino groups of the ligands, muteins and fused proteins thereof. Salts of a carboxyl group may be formed by means known in the art and include inorganic salts, for example, sodium, calcium, ammonium, ferric or zinc salts, and the like, and salts with organic bases as those formed, for example, with amines, such as triethanolamine, arginine or lysine, piperidine, procaine and the like. Acid addition salts include, for example, salts with mineral acids such as, for example, hydrochloric acid or sulfuric acid, and salts with organic acids such as, for example, acetic acid or oxalic acid. 
     The peptides include peptide bond replacements and/or peptide mimetics, i.e., pseudopeptides, as known in the art (see, e.g., Proceedings of the 20th European Peptide Symposium, ed. G. Jung, E. Bayer, pp. 289-336, and references therein), as well as salts and pharmaceutical preparations and/or formulations which render the bioactive peptide(s) particularly suitable for oral, topical, nasal spray, ocular, pulmonary, I.V. or subcutaneous delivery, depending on the particular treatment indicated. Such salts, formulations, amino acid replacements and pseudopeptide structures may be necessary and desirable to enhance the stability, formulation, deliverability (e.g., slow release, prodrugs), or to improve the economy of production, as long as they do not adversely affect the biological activity of the peptide. 
     Besides substitutions, three particular forms of peptide mimetic and/or analogue structures of particular relevance when designating bioactive peptides, which have to bind to a receptor while risking the degradation by proteinases and peptidases in the blood, tissues and elsewhere, may be mentioned specifically, illustrated by the following examples: Firstly, the inversion of backbone chiral centres leading to D-amino acid residue structures may, particularly at the N-terminus, lead to enhanced stability for proteolytical degradation without adversely affecting activity. An example is given in the paper “Tritriated D-ala 1 -Peptide T Binding”, Smith C. S. et al., Drug Development Res. 15, pp. 371-379 (1988). Secondly, cyclic structure for stability, such as N to C interchain imides and lactams (Ede et al. in Smith and Rivier (Eds.) “Peptides: Chemistry and Biology., Escom, Leiden (1991), pp. 268-270), and sometimes also receptor binding may be enhanced by forming cyclic analogues. An example of this is given in “Confirmationally restricted thymopentin-like compounds”, U.S. Pat. No. 4,457,489 (1985), Goldstein, G. et al. Thirdly, the introduction of ketomethylene, methylsufide or retroinverse bonds to replace peptide bonds, i.e., the interchange of the CO and NH moieties are likely to enhance both stability and potency. An example of this type is given in the paper “Biologically active retroinverso analogues of thymopentin”, Sisto A. et al in Rivier, J. E. and Marshall, G. R. (eds) Peptides, Chemistry, Structure and Biology”, Escom, Leiden (1990), pp. 722-773). 
     The peptides of the invention can be synthesized by various methods which are known in principle, namely by chemical coupling methods (cf. Wunsch, E: “Methoden der organischen Chemiet”, Volume 15, Band 1+2, Synthese von Peptiden, thime Verlag, Stutt (1974), and Barrany, G.; Marrifield, R. B.: “The Peptides”, eds. E. Gross, J. Meienhofer, Volume 2, Chapter 1, pp. 1-284, Academic Press (1980)), or by enzymatic coupling methods (cf. Widmer, F. Johansen, J. T., Carlsberg Res. Commun., Vol.44, pp. 37-46 (1979), and Kullmann, W.: “Enzymatic Peptide Synthesis”, CRC Press Inc. Boca Raton, Fla. (1987), and Widmer, F., Johansen, J. T. in “Synthetic Peptides in Biology and Medicines”, eds. Alitalo, K., Partanen, P., Vatieri, A., pp.79-86, Elsevier, Amsterdam (1985)), or by a combination of chemical and enzymatic methods if this is advantageous for the process design and economy. 
     A cysteine residue may be added at both the amino and carboxy terminals of the peptide, which will allow the cyclization of the peptide by the formation of a disulphide bond. 
     Any modifications to the peptides of the present invention which do not result in a decrease in biological activity are within the scope of the present invention. 
     There are numerous examples which illustrate the ability of anti-idiotypic antibodies (anti-Id Abs) to an antigen to function like that antigen in its interaction with animal cells and components of cells. Thus, anti-Id Abs to a peptide hormone antigen can have hormone-like activity and interact specifically with a mediator in the same way as the receptor does. (For a review of these properties see: Gaulton, G. N. and Greane, M. I. 1986. Idiotypic mimicry of biological receptors, Ann. Rev. Immunol. Vol. 4, pp. 253-280; Sege K. and Peterson, P. A., 1978, Use of anti-idiotypic antibodies as cell surface receptor probes, Proc. Natl. Acad. Sci. U.S.A., Vol. 75, pp. 2443-2447). 
     It is expected from this functional similarity of anti-Id Ab and antigen, that anti-Id Abs bearing the internal image of an antigen can induce immunity to such an antigen. (See review in Hiernaux, J. R., 1988, Idiotypic vaccines and infectious diseases, Infect. Immun., Vol. 56, pp. 1407-1413). 
     It is, therefore, possible to produce anti-idiotypic antibodies to the peptides of the present invention which will have similar biological activity. 
     Accordingly, the present invention also provides anti-idiotypic antibodies to the peptides of the present invention, the anti-idiotypic antibody being capable of inhibiting TNF toxicity, but not its binding to the receptor. 
     The individual specificity of antibodies resides in the structures of the peptide loops making up the Complementary Determining Regions (CDRs) of the variable domains of the antibodies. Since in general the amino acid sequence or the CDR peptides of an anti-Id Ab are not identical to or even similar to the amino acid sequence of the peptide antigen from which it was originally derived, it follows that peptides whose amino acid sequence in quite dissimilar, in certain contexts, can take up a very similar three-dimensional structure. The concept of this type of peptide, termed a “functionally equivalent sequence” or mimotope by Geyson is known. (Geyson, H. M. et al, 1987, Strategies for epitope analysis using peptide synthesis., J. Immun. Methods, Vol. 102, pp. 259-274). 
     Moreover, the three-dimensional structure and function of the biologically active peptides can be simulated by other compounds, some not even peptidic in nature, but which nevertheless mimic the activity of such peptides. This field is summarized in a review by Goodman, M. (1990), (Synthesis, Spectroscopy and computer simulations in peptide research, Proc. 11th American Peptide Symposium published in Peptides-Chemistry Structure and Biology, pp. 3-29; Eds. Rivier, J. E. and Marshall, G. R. Publisher Escom). 
     It is also possible to produce peptide and non-peptide compounds having the same three-dimensional structure as the peptides of the present invention. These “functionally equivalent structures” or “peptide mimics” will react with antibodies raised against the peptide of the present invention and may also be capable of inhibiting TNF toxicity. 
     Accordingly, a further embodiment of the present invention provides a compound the three-dimensional structure of which is similar as a pharmacophore to the three-dimensional structure of the peptides of the present invention, the compound being characterized in that it reacts with antibodies raised against the peptides of the present invention and that the compound is capable of inhibiting TNF toxicity. 
     More detail regarding pharmacophores can be found in Bolin et al., p. 150, Polinsky et al., p. 287, and Smith et al., p. 485, in Smith and Rivier (eds.) “Peptides: Chemistry and Biology”, Escom, Leiden (1991). 
     All of the molecules (proteins, peptides, etc.) may be produced either by conventional chemical methods, as described herein, or by recombinant DNA methods. 
     All of the molecules (proteins, peptides, etc.) may be produced either by conventional chemical methods, as described herein, or by recombinant DNA methods. 
     The invention also provides DNA molecules encoding the ligands according to the invention, vectors containing them and host cells comprising the vectors and capable of expressing the ligands according to the invention. 
     The host cell may be either prokaryotic or eukaryotic. 
     The invention further provides DNA molecules hybridizing to the above DNA molecules and encoding ligands having the same activity. 
     The invention also provides pharmaceutical compositions comprising the above ligands which are useful for treating diseases induced or caused by the effects of TNF, either endogenously produced or exogenously administered. 
     The invention also provides for using the ligands according to the invention for increasing the inhibitory effect of a soluble receptor of the TNF/NGF receptor family. As stated above, the soluble receptors, especially those of TNF, have the ability to block the function of TNF by binding it in competition with the surface receptors. Application of a ligand according to the invention together with a soluble receptor is, therefore, expected to increase the inhibitory effect of the soluble receptor. 
    
    
     BRIEF DESCRIPTION OF THE FIGURES 
     FIG. 1 shows the results of the test by which epitope 67 was mapped. 
     FIGS. 2A-C shows the nucleotide (SEQ ID NO:1) and deduced amino acid (SEQ ID NO:2) sequences of the p 75  receptor. TBP-II and transmembranal domains are boxed and shaded. The region recognized by the group 67 antibodies is underlined, and the region recognized by the anti-stalk antibodies is underlined by a broken line. 
     FIG. 3 shows the inhibitory effect of the 67 and anti-stalk antibodies on TNF function in HeLa cells. 
     FIG. 4 shows that antibodies against the upper part of extracellular domain of the p 75  TNF-R are signaling in the HeLa cells. 
     FIG. 5 shows that antibodies against the upper part of the extracellular domain of the p 75  TNF-R do not signal in A9 cells which express the human p 75  TNF-R. Antibodies of the 67 group do have, though, an inhibitory effect on TNF function in them (FIG.  6 ). 
     FIG. 6 shows that antibodies against the upper part of the extracellular domain of the p 75  TNF-R inhibit TNF function in A9 cells. 
     FIG. 7 shows that antibodies against the upper part of the extracellular domain of the p 75  TNF-R do not signal in A9 cells which express the cytoplasmically truncated p 75  TNF-R. Antibodies of the 67 group do have, though, an inhibitory effect on TNF function in them (not shown). 
     FIG. 8 shows that antibodies against the 67 epitope impede TNF dissociation from p 75  TNF-R. 
     FIG. 9 shows the sequence homology between several members of the TNF/NGF receptor family (residues 3-155 of hu p 55  TNF-R (SEQ ID NO:3); residues 39-201 of hu p 75  TNF-R (SEQ ID NO:4); residues 31-149 of hu FAS (SEQ ID NO:5); residues 3-161 of hu NGF-R (SEQ ID NO:6); residues 25-187 of hu CDw40 (SEQ ID NO:7); and residues 25-164 of rat Ox40 (SEQ ID NO:8)). 
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     TNF, as stated above, is a cytokine which initiates its effect on cell function by binding to two specific cell surface receptors: the p 55  and p 75  receptors. Binding of antibodies to the extracellular domain of these receptors can interfere with its effect. However, as shown in a number of studies, antibodies binding to the extracellular domain of the receptors can also trigger the effects of TNF by inducing aggregation of the p 55  receptors, as well as by inducing aggregation of the p 75  receptors. (Engelmann, et al. J. Biol. Chem., Vol. 265, No. 24, pp. 14497-14504, 1990; and unpublished data). 
     As disclosed in patent application no. 103051, antibodies binding to one particular region in the p 75  receptor are not mimetic but rather inhibitory to the signaling for the cytocidal effect by this receptor. This, in spite of the fact that when binding to this region, these antibodies do not block TNF binding, but rather increase it to some extent. In application no. 106271 this region is more particularly identified as extending between cys-163 and thr-179, in the fourth cysteine rich domain of the receptor. The present invention reveals that the region recognized by certain other antibodies is the region extending downstream of thr-181 and upstream of cys-163 to about cys-142 in the extracellular domain-of the p 75  receptor. 
     The present invention also reveals that the so-called “stalk-antibody” recognizes a region downstream of the fourth cysteine rich domain, more particularly the region extending from about amino acid 181 to about amino acid 235. 
     It was also found in accordance with the present invention that, in case of the “67 epitope” antibodies, the divalent antibodies have an effect which mimics TNF action, while the monovalent fragments, such as F(ab), inhibit the cytotoxic effect of TNF. 
     Based on these findings, small molecular weight compounds, such as peptides or mimetic compounds, which will either inhibit the function of the p 75  receptor, or enhance it, can be defined. 
     In view of these findings, as well as the close similarity of the receptors in this particular family, this invention relates also to ligands which bind to the same regions in the extracellular domain of the various other members of the TNF/NGF receptor family and modulate the function of the other receptors, similarly to the modulation of the function of TNF. In this receptor family, the localization of cysteines in the extracellular domain and the spacing is highly conserved. Certain members of this family, e.g., CDw40, exhibit particularly high similarity to the p 75  receptor. Particularly in such receptors, ligands binding to these regions are expected to have effects similar to the effect of the ligands according to the present invention on the p 75  receptor. 
     Recombinant production of the ligands is carried out by known methods commonly employed in the art. 
     The invention is illustrated by the following non-limiting examples: 
     EXAMPLE 1 
     Monoclonal Antibodies to TBP-II 
     Production of the Monoclonal Antibodies 
     Female Balb/C mice (8 weeks old) were injected with 1 μg purified TBP-II in an emulsion of complete Freund&#39;s adjuvant into the hind foot pads, and three weeks later, subcutaneously into the back in incomplete Freund&#39;s adjuvant. The other injections were given in weekly intervals, subcutaneously in PBS. Final boosts were given 4 days (i.p.) and 3 days (i.v.) before the fusion with 9.0 μg of TBP-I in PBS. Fusion was performed using NSO/Mr cells and lymphocytes prepared from both the spleen and the local lymphocytes of the hind legs as fusion partners. The hybridomas were selected in DMEM. supplemented with HAT, 15% horse serum and gentamycin 2 μg/ml. Hybridomas that were found to produce antibodies to TBP-1 were subcloned by the limiting dilution method and injected into Balb/C mice that had been primed with pristane for the production of ascites. Immunoglobulins were isolated from the ascites by ammonium sulfate precipitation (50% saturation) and then dialyzed against PBS containing 0.02% azide. Purity was approximately 60% as estimated by analysis on SDS-PAGE and staining with Coomassie blue. The isotypes of the antibodies were defined with the use of a commercially available ELISA kit (Amersham, U.K.). 
     Several positive clones were obtained, subcloned for further studies and characterized. Some of the isolated subclones with their isotype and binding of TBP-II in inverted RIA are listed in Table I. 
     
       
         
               
             
               
               
               
               
             
               
               
               
               
             
           
               
                 TABLE I 
               
             
             
               
                   
               
               
                 Subclones Producing Monoclonal Antibodies to TBP-II 
               
             
          
           
               
                   
                 Screening with 
                 Screening of subclone 
                   
               
               
                 Clone Number 
                 iRIA [CPM] 
                 with iRIA [CPM] 
                 Isotype 
               
               
                   
               
             
          
           
               
                 13.11 
                 31800 
                 31000 
                 IgG 1   
               
               
                 .12 
                   
                 31500 
                 IgG 1   
               
               
                 .13 
                   
                 31100 
                 IgG 1   
               
               
                 14.1 
                 15300 
                 15400 
                 IgG 2a   
               
               
                 .6 
                   
                 16200 
                 IgG 2a   
               
               
                 .7 
                   
                 15300 
                 IgG 2a   
               
               
                 20.2 
                 12800 
                 14200 
                 IgG 2b   
               
               
                 .5 
                   
                 14300 
                 IgG 2b   
               
               
                 .6 
                   
                 14800 
                 IgG 2b   
               
               
                 22.7 
                 20400 
                 20000 
                 IgG 1   
               
               
                 .8 
                   
                 19300 
                 IgG 1   
               
               
                 27.1 
                  1800 
                 27000 
                 IgG 2a   
               
               
                 .3 
                   
                 25000 
                 IgG 2a   
               
               
                 .9 
                   
                 28000 
                 IgG 2a   
               
               
                 32.4 
                 11315 
                 10900 
                 IgG 2b   
               
               
                 .5 
                   
                 10700 
                 IgG 2b   
               
               
                 .6 
                   
                 11200 
                 IgG 2b   
               
               
                 33.1 
                 18400 
                 11400 
                 IgG 1   
               
               
                 .3 
                   
                 10500 
                 IgG 1   
               
               
                 .4 
                   
                 14800 
                 IgG 1   
               
               
                 36.1 
                 27500 
                 26600 
                 IgG 2a   
               
               
                 .5 
                   
                 24900 
                 IgG 2a   
               
               
                 .6 
                   
                 24900 
                 IgG 2a   
               
               
                 41.3 
                 13800 
                 18100 
                 IgG 1   
               
               
                 .7 
                   
                 18100 
                 IgG 1   
               
               
                 .10 
                   
                 18800 
                 IgG 1   
               
               
                 67.1 
                 16800 
                 10900 
                 IgG 2a   
               
               
                 .16 
                   
                 10800 
                 IgG 2a   
               
               
                 .17 
                   
                 10900 
                 IgG 2a   
               
               
                 70.2 
                 15100 
                  5100 
                 IgG 2a   
               
               
                 .3 
                   
                  5200 
                 IgG 2a   
               
               
                 .4 
                   
                  5300 
                 IgG 2a   
               
               
                 77.2 
                 15300 
                 11800 
                 IgG 2b   
               
               
                 78.9 
                 25300 
                 21400 
                 IgG 2a   
               
               
                 82.1 
                 17600 
                 25900 
                 IgG 1   
               
               
                 .4 
                   
                 25700 
                 IgG 1   
               
               
                 .10 
                   
                 26400 
                 IgG 1   
               
               
                 86.2 
                  8800 
                 12200 
                 IgG 2b   
               
               
                 .5 
                   
                 12600 
                 IgG 2b   
               
               
                 .11 
                   
                 12800 
                 IgG 2b   
               
               
                 19.6 
                   
                 29700 
                 IgG 2a   
               
               
                 .9 
                   
                 28900 
                 IgG 2a   
               
               
                   
               
             
          
         
       
     
     Hybridomas TBP-II 13-12 and TBP-II 70-2 were deposited with the Collection Nationale de Cultures de Microorganismes (CNCM), Institut Pasteur, 25, rue du Docteur Roux, 75724 Paris CEDEX 15, France on Mar. 12, 1990, and were assigned No. I-929 and No. I-928. respectively. 
     EXAMPLE 2 
     Inverted Radioimmunoassay (iRIA) for the Detection of the Monoclonal Antibodies to TBP-II 
     This assay was used for estimating the level of the anti-TBP antibodies in the sera of the immunized mice and for screening for the production of the antibodies by hybridomas. PVC 96 -well microtiter plates (Dynatech 1-220-25) were coated for 12 hr at 4° C. with affinity purified goat anti mouse F(ab) immunoglobulins (Biomakor, Israel 10 μg/ml in PBS containing 0.02: NaN 3 ), then blocked for 2 hr at 37° C. with 0.52 BSA in PBS supplemented with 0.05% Tween 20 (Sigma) and 0.02% NaN 3  (blocking buffer) and washed 3 times with PBS containing 0.05% Tween 20 and 0.02% NaN 3  (washing buffer). Serum samples, in serial dilutions, or samples of hybridoma growth media (50 μl) were applied into the wells for 2 hr at 37° C. The plates were rinsed with washing buffer and  125 I-labelled TBP-I (10,000 cpm, in blocking buffer) was applied into the wells. After further incubation of 2 hr at 37° C., the plates were washed and the amount of label which bound to individual wells was determined in the gamma-counter. 
     EXAMPLE 3 
     The Use of Anti-TBP-II Antibodies for Affinity Chromatography 
     Antibodies against TBP-II can be utilized for the purification of TBP-II by affinity chromatography, according to the following procedure. The monoclonal antibodies for affinity chromatography were selected by testing their binding capacity for the radiolabeled antigen in a solid phase radio immunoassay. Ascites from all hybridomas was purified by ammonium sulfate precipitation at 50% saturation followed by extensive dialysis against PBS. PVC 96-well plates were coated with the purified McAbs, and after blocking the plates with PBS containing 0.5% BSA, 0.05% Tween 20 (Sigma) and 0.02% NaN 3 , the wells were incubated with 50,000 cpm  125 I-TNF for 2 hr at 37° C., then washed and the radioactivity which had bound to each well was quantitated in the gamma-counter. The antibodies with the highest binding capacity were examined for their performance in immunoaffinity chromatography. 
     Polyacryl hydrazide agarose was used as resin to immobilize the antibodies. The semipurified immunoglobulins were concentrated and coupled to the resin as specified by Wilchek and Miron,  Methods in Enzymology  34:72-76, 1979. Three monoclonal antibodies against TBP-I, clones 16, 20, and 34 were tested in these experiments. Antibody columns of 1 ml bed were constructed. Before use, all columns were subjected to 10 washes with the elusion buffer, each wash followed by neutralization with PBS. Then the columns were loaded with 120 ml of concentrated urinary proteins in PBS with 0.02% NaN 3 . The flow rate of the columns was adjusted to 0.2 to 0.3 ml per minute. After loading, the columns were washed with 50 ml PBS and then eluted with a solution containing 50 mM citric acid, pH 2.5, 100 mM NaCl and 0.02% NaN 3 . Fractions of 1 ml were collected. Samples of the applied urinary proteins, the last portion of the wash (1 ml) and of each elusion fraction (8 fractions of 1 ml per column) were taken and tested for protein concentration and activity in the bioassay for T3P-II. According to the protein measurements before and after coupling of the antibodies to hydrazide agarose, the amounts of immunoglobulin bound to the columns ranged from 7 to 10 mg/ml agarose. All protein measurements were done according to a micro-flurescamin method in comparison to a standard solution containing 100 μg BSA/ml (Stein, S. and Moschera. J.,  Methods Enzymol.  79:7-16, 1981). 
     EXAMPLE 4 
     Determination of TBP-II Using Anti-TBP-II Antibodies 
     The levels of TBP-II in the sera of healthy individuals, patients with cancer or systemic lupus erythematosus (SLE) and of pregnant women at term were determined by an ELISA method employing a monoclonal antibody to TBP-IO coating the plates. 50 μl of each sample was added and after a 2.5 hr incubation at 37° C. the wells were washed with a solution of PBS, Tween 0.05% and sodium azide 0.02%, after which a rabbit anti-TBP-II polyclonal antibody was added for 2.5 hr at 37° C. Then the wells were washed again (no azide) and goat anti-rabbit horseradish peroxidase-coupled antibody was added for 2 hr. Following this incubation, and washing, an ABTS buffer was added and optical density (O.D.) read 30 min. later at 600 nm. 
     The normal levels of TBP-II in human serum of healthy individuals as determined by the ELISA method are 1.48±0.46 ng/ml. 
     EXAMPLE 5 
     Epitope Mapping of TBP-II by Cross Competition Analysis with Monoclonal Antibodies (mAbs) to TBP-II 
     PVC 96-well microtiter plates were coated as described above, with purified mAbs to TBP-II (25 μg/ml). Following rinsing and blocking, samples of  125 I-labelled T3P-II (100,000 cpm per well) which had been preincubated for 2 hr, at 37° C. with the same or a different monoclonal antibody to TBP-II (at 1 μg/ml) were put into the wells; the plates were incubated overnight at 4° C., washed and the radioactivity bound to each well was determined by gamma counting. The results are expressed as percent of the control values (TBP-II binding in the absence of competing mAbs). 
     The results are depicted in Table II. The monoclonal antibodies are indicated by the clone numbers in the first row and ˜n left column. Low percent binding values indicate that the two antibodies compete for each other&#39;s epitope on TBP-II, while higher values indicate that they bind to different epitopes. Non-competitive antibodies are suitable for use in double-sandwich ELISA, e.g., clones 13 and 70. 
     
       
         
               
             
               
               
             
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
             
           
               
                 TABLE II 
               
             
             
               
                   
               
               
                 Cross Competition Analysis with Monoclonal Antibodies to TBP II 
               
             
          
           
               
                   
                 Solid Phase Antibodies 
               
             
          
           
               
                   
                 13 
                 14 
                 19 
                 20 
                 22 
                 27 
                 32 
                 33 
                 36 
                 41 
                 67 
                 70 
                 77 
                 78 
                 82 
                 86 
               
               
                   
                   
               
             
          
           
               
                 13 
                 4 
                 64 
                 53 
                 73 
                 31 
                 51 
                 161 
                 35 
                 177 
                 72 
                 131 
                 128 
                 77 
                 102 
                 50 
                 101 
               
               
                 14 
                 119 
                 20 
                 90 
                 13 
                 13 
                 84 
                 156 
                 11 
                 132 
                 173 
                 134 
                 113 
                 14 
                 70 
                 89 
                 179 
               
               
                 19 
                 103 
                 28 
                 7 
                 19 
                 11 
                 5 
                 144 
                 11 
                 144 
                 133 
                 179 
                 123 
                 18 
                 5 
                 85 
                 126 
               
               
                 20 
                 119 
                 17 
                 93 
                 14 
                 10 
                 88 
                 149 
                 9 
                 135 
                 170 
                 137 
                 135 
                 16 
                 70 
                 101 
                 181 
               
               
                 22 
                 109 
                 26 
                 94 
                 22 
                 13 
                 82 
                 128 
                 12 
                 115 
                 164 
                 136 
                 114 
                 17 
                 68 
                 98 
                 167 
               
               
                 27 
                 106 
                 23 
                 11 
                 27 
                 14 
                 8 
                 145 
                 17 
                 152 
                 133 
                 196 
                 136 
                 24 
                 8 
                 82 
                 125 
               
               
                 32 
                 150 
                 267 
                 150 
                 291 
                 156 
                 186 
                 14 
                 163 
                 139 
                 200 
                 205 
                 18 
                 294 
                 143 
                 103 
                 226 
               
               
                 33 
                 115 
                 19 
                 98 
                 23 
                 16 
                 86 
                 133 
                 12 
                 118 
                 156 
                 120 
                 114 
                 24 
                 78 
                 90 
                 155 
               
               
                 36 
                 155 
                 262 
                 168 
                 271 
                 144 
                 185 
                 167 
                 158 
                 12 
                 169 
                 223 
                 135 
                 265 
                 158 
                 93 
                 150 
               
               
                 41 
                 117 
                 119 
                 119 
                 118 
                 101 
                 109 
                 118 
                 76 
                 93 
                 9 
                 179 
                 107 
                 106 
                 111 
                 8 
                 9 
               
               
                 67 
                 112 
                 138 
                 125 
                 141 
                 125 
                 157 
                 136 
                 107 
                 138 
                 213 
                 30 
                 117 
                 120 
                 127 
                 106 
                 236 
               
               
                 70 
                 150 
                 246 
                 150 
                 255 
                 145 
                 166 
                 4 
                 162 
                 166 
                 217 
                 204 
                 6 
                 232 
                 132 
                 107 
                 234 
               
               
                 77 
                 121 
                 18 
                 98 
                 15 
                 13 
                 78 
                 148 
                 11 
                 145 
                 184 
                 142 
                 132 
                 18 
                 66 
                 103 
                 184 
               
               
                 78 
                 118 
                 20 
                 9 
                 26 
                 10 
                 6 
                 153 
                 13 
                 157 
                 137 
                 183 
                 131 
                 19 
                 6 
                 94 
                 172 
               
               
                 82 
                 107 
                 110 
                 130 
                 116 
                 112 
                 121 
                 128 
                 89 
                 90 
                 8 
                 162 
                 102 
                 121 
                 113 
                 8 
                 7 
               
               
                 86 
                 122 
                 181 
                 125 
                 166 
                 126 
                 129 
                 131 
                 120 
                 86 
                 18 
                 253 
                 109 
                 152 
                 125 
                 20 
                 17 
               
               
                 100% 
                 31582 
                 3958 
                 2057 
                 5437 
                 2947 
                 17395 
                 25923 
                 3525 
                 6368 
                 8042 
                 4368 
                 24113 
                 5887 
                 22222 
                 11608 
                 9703 
               
               
                 Value 
               
               
                   
               
             
          
         
       
     
     EXAMPLE 6 
     Mapping of the Epitope 67 of the p 75  TNF-R 
     In order to compare the function of the 67 group antibodies, not only to antibodies which bind to the receptor at the 67 epitope region, but also to antibodies that bind to the receptor downstream to that epitope region, we immunized rabbits with a chimeric construct corresponding to the region extending downstream to the 32 epitope (amino acids 181 to 235; the “stalk” region), linked to MED. The rabbits developed antibodies which bound to the chimera with which they were immunized as well as to the intact p 55  TNF receptor. These antibodies were affinity purified by binding to the chimeric protein, linked to an AFFIGEL 10 column (N- hydroxysuccinimide ester of a derivatized cross-linked agarose gel bead support, available from Bio-Rad Laboratories), and tested for effect on TNF function and binding. (The affinity purified antibody preparation was termed “318”). The mapping of epitope 67 was carried out by examining the ability of antibodies number 67 and 13 (an antibody that binds to the upper part of the extracellular domain of the p 75  TNF-R) as well as antiserum 318, to immunoprecipitate the following methionine-labeled soluble p 75  TNF-R mutants: WT- a receptor extending from amino acid 22 to amino acid 234, D4D- a receptor like WT, from which the 4th cysteine-rich domain has been deleted (amino acids 141 to 180). The receptors were produced by in vitro transcription of cDNAs encoding them (from the BLUESCRIPT vector (a phagemid vector derived from pUC19, available from Stratagene), using the T7 promoter) followed by in vitro translation using the PROMEGA TNT kit (an in vitro translation kit available from Promega) The immnunoprecipitated proteins were analyzed by SDS PACE, followed by autoradiography. (A) Immnunoprecipitation of WT. All antibodies were effective. (B) Immunoprecipitation of D4D. Only antibodies 13 and 318 were effective. The findings indicate that epitope 67 is located at the upper part of the 4th cysteine rich domain, within about amino acids 141-180. 
     EXAMPLE 7 
     Titration of the Inhibitory Effect of the Group 67 Antibodies and the Anti-Stalk Antibodies on TNF Function 
     As shown in FIG. 3, the protective effect of the different antibodies studied on the cytocidal effect of TNF on HeLa p75.3 cells was found to vary depending on the particular antibody used: antibodies 32 and antiserum 313 and their Fab monovalent fragments, which protect, antibody 67, which protects as Fab monovalent fragment and enhances TNF cytotoxicity in its divalent form, and antibody 13 (which binds to the upper part of the extracellular domain of the -75-R) which enhances the cytocidal effect of TNF (p75.3 cells are HeLa cells transfected with the full length p 75  TNF-R). 
     EXAMPLE 8 
     The Inhibitory Effect of the Group 67 and Anti-Stalk Antibodies Is Independent of the Expression and Function of the Intracellular Domain of the p 75  TNF-R 
     In HeLa cells which over-express the p 75  TNF-R, antibodies against the upper part of the extracellular domain of the receptor have a cytocidal effect, synergistic with that or antibodies against the p 55 -R (FIG.  4 ). However, these antibodies do not have such an effect in A9 cells which express either tine full-length or cytoplasmically-truncated human p 75  TNF-R (FIGS. 5 and 7, respectively). However, antibodies which bind to the lower part of the receptor did show inhibitory effect on TNF function even in these cells, irrespective of whether the cells expressed the full-length or the cytoplasmically truncated receptor (see FIG. 6 as well as data not shown). 
     EXAMPLE 9 
     Effect of the Various Antibodies on the Dissociation of TNF Form p 75  TNF 
     FIG. 8 shows a comparison of the rate of the dissociation of TNF from the p 55  TNF-R, as assessed by measuring the dissociation of radiolabeled TNF from mouse A9 cells expressing transfected human p55 TNF-R (A9D2 cells, in which over 90% of the cell-bound TNF is associated with the human p 55  TNF-R) and from the HeLa p75.3 cells, in which most of the bound TNF is associated with the over-expressed p 75  TNF-R. As opposed to the very slow dissociation of TNF from the p 55  TNF-R, TNF dissociates rather rapidly from the p 75  TNF-R. 
     EXAMPLE 10 
     FIG. 9 shows the internal cysteine rich repeats in the extracellular domains of the two TNF-Rs and their alignment with the homologous repeats in the extracellular domain of the human FAS, nerve growth factor receptor (NGF) and CDw40, as well as rat Ox40. The amino acid sequences (one letter symbols) are aligned for maximal homology. The positions of the amino acids within the receptors are denoted in the left hand margin. 
     DEPOSIT INFORMATION 
     Hybridomas TBP-II 67 and 81 were deposited with the Collection National de Cultures de Microorganismes (CNCM), Institut Pasteur, 25, rue du Docteur Roux, 75724 Paris Cedex 15, France, on Oct. 11, 1993 and assigned Nos. I-1368 and I-1369, respectively. 
     
       
         
               
             
           
               
                   
               
             
             
               
                                    
               
               
                 #             SEQUENCE LISTING  
               
               
                   
               
               
                   
               
               
                 (1) GENERAL INFORMATION:  
               
               
                   
               
               
                    (iii) NUMBER OF SEQUENCES: 8  
               
               
                   
               
               
                   
               
               
                 (2) INFORMATION FOR SEQ ID NO:1:  
               
               
                   
               
               
                      (i) SEQUENCE CHARACTERISTICS:  
               
               
                           (A) LENGTH: 2224 base  
               
               
                 #pairs  
               
               
                           (B) TYPE: nucleic acid  
               
               
                           (C) STRANDEDNESS: single  
               
               
                           (D) TOPOLOGY: linear  
               
               
                   
               
               
                     (ii) MOLECULE TYPE: cDNA  
               
               
                   
               
               
                     (ix) FEATURE:  
               
               
                           (A) NAME/KEY: CDS  
               
               
                           (B) LOCATION: 90..1472  
               
               
                   
               
               
                     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:  
               
               
                   
               
               
                 GCGAGCGCAG CGGAGCCTGG AGAGAAGGCG CTGGGCTGCG AGGGCGCGAG GG  
               
               
                 #CGCGAGGG     60  
               
               
                   
               
               
                 CAGGGGGCAA CCGGACCCCG CCCGCACCC ATG GCG CCC GTC GCC  
               
               
                 # GTC TGG GCC      113  
               
               
                                    
               
               
                 #              Met Ala P  
               
               
                 #ro Val Ala Val Trp Ala  
               
               
                                    
               
               
                 #                1   
               
               
                 #             5  
               
               
                   
               
               
                 GCG CTG GCC GTC GGA CTG GAG CTC TGG GCT GC  
               
               
                 #G GCG CAC GCC TTG CCC      161  
               
               
                 Ala Leu Ala Val Gly Leu Glu Leu Trp Ala Al  
               
               
                 #a Ala His Ala Leu Pro  
               
               
                      10              
               
               
                 #     15              
               
               
                 #     20  
               
               
                   
               
               
                 GCC CAG GTG GCA TTT ACA CCC TAC GCC CCG GA  
               
               
                 #G CCC GGG AGC ACA TGC      209  
               
               
                 Ala Gln Val Ala Phe Thr Pro Tyr Ala Pro Gl  
               
               
                 #u Pro Gly Ser Thr Cys  
               
               
                  25                  
               
               
                 # 30                  
               
               
                 # 35                  
               
               
                 # 40  
               
               
                   
               
               
                 CGG CTC AGA GAA TAC TAT GAC CAG ACA GCT CA  
               
               
                 #G ATG TGC TGC AGC AAA      257  
               
               
                 Arg Leu Arg Glu Tyr Tyr Asp Gln Thr Ala Gl  
               
               
                 #n Met Cys Cys Ser Lys  
               
               
                                  45  
               
               
                 #                 50  
               
               
                 #                 55  
               
               
                   
               
               
                 TGC TCG CCG GGC CAA CAT GCA AAA GTC TTC TG  
               
               
                 #T ACC AAG ACC TCG GAC      305  
               
               
                 Cys Ser Pro Gly Gln His Ala Lys Val Phe Cy  
               
               
                 #s Thr Lys Thr Ser Asp  
               
               
                              60      
               
               
                 #             65      
               
               
                 #             70  
               
               
                   
               
               
                 ACC GTG TGT GAC TCC TGT GAG GAC AGC ACA TA  
               
               
                 #C ACC CAG CTC TGG AAC      353  
               
               
                 Thr Val Cys Asp Ser Cys Glu Asp Ser Thr Ty  
               
               
                 #r Thr Gln Leu Trp Asn  
               
               
                          75          
               
               
                 #         80          
               
               
                 #         85  
               
               
                   
               
               
                 TGG GTT CCC GAG TGC TTG AGC TGT GGC TCC CG  
               
               
                 #C TGT AGC TCT GAC CAG      401  
               
               
                 Trp Val Pro Glu Cys Leu Ser Cys Gly Ser Ar  
               
               
                 #g Cys Ser Ser Asp Gln  
               
               
                      90              
               
               
                 #     95              
               
               
                 #    100  
               
               
                   
               
               
                 GTG GAA ACT CAA GCC TGC ACT CGG GAA CAG AA  
               
               
                 #C CGC ATC TGC ACC TGC      449  
               
               
                 Val Glu Thr Gln Ala Cys Thr Arg Glu Gln As  
               
               
                 #n Arg Ile Cys Thr Cys  
               
               
                 105                 1  
               
               
                 #10                 1  
               
               
                 #15                 1  
               
               
                 #20  
               
               
                   
               
               
                 AGG CCC GGC TGG TAC TGC GCG CTG AGC AAG CA  
               
               
                 #G GAG GGG TGC CGG CTG      497  
               
               
                 Arg Pro Gly Trp Tyr Cys Ala Leu Ser Lys Gl  
               
               
                 #n Glu Gly Cys Arg Leu  
               
               
                                 125   
               
               
                 #               130   
               
               
                 #               135  
               
               
                   
               
               
                 TGC GCG CCG CTG CGC AAG TGC CGC CCG GGC TT  
               
               
                 #C GGC GTG GCC AGA CCA      545  
               
               
                 Cys Ala Pro Leu Arg Lys Cys Arg Pro Gly Ph  
               
               
                 #e Gly Val Ala Arg Pro  
               
               
                             140       
               
               
                 #           145       
               
               
                 #           150  
               
               
                   
               
               
                 GGA ACT GAA ACA TCA GAC GTG GTG TGC AAG CC  
               
               
                 #C TGT GCC CCG GGG ACG      593  
               
               
                 Gly Thr Glu Thr Ser Asp Val Val Cys Lys Pr  
               
               
                 #o Cys Ala Pro Gly Thr  
               
               
                         155           
               
               
                 #       160           
               
               
                 #       165  
               
               
                   
               
               
                 TTC TCC AAC ACG ACT TCA TCC ACG GAT ATT TG  
               
               
                 #C AGG CCC CAC CAG ATC      641  
               
               
                 Phe Ser Asn Thr Thr Ser Ser Thr Asp Ile Cy  
               
               
                 #s Arg Pro His Gln Ile  
               
               
                     170               
               
               
                 #   175               
               
               
                 #   180  
               
               
                   
               
               
                 TGT AAC GTG GTG GCC ATC CCT GGG AAT GCA AG  
               
               
                 #C ATG GAT GCA GTC TGC      689  
               
               
                 Cys Asn Val Val Ala Ile Pro Gly Asn Ala Se  
               
               
                 #r Met Asp Ala Val Cys  
               
               
                 185                 1  
               
               
                 #90                 1  
               
               
                 #95                 2  
               
               
                 #00  
               
               
                   
               
               
                 ACG TCC ACG TCC CCC ACC CGG AGT ATG GCC CC  
               
               
                 #A GGG GCA GTA CAC TTA      737  
               
               
                 Thr Ser Thr Ser Pro Thr Arg Ser Met Ala Pr  
               
               
                 #o Gly Ala Val His Leu  
               
               
                                 205   
               
               
                 #               210   
               
               
                 #               215  
               
               
                   
               
               
                 CCC CAG CCA GTG TCC ACA CGA TCC CAA CAC AC  
               
               
                 #G CAG CCA ACT CCA GAA      785  
               
               
                 Pro Gln Pro Val Ser Thr Arg Ser Gln His Th  
               
               
                 #r Gln Pro Thr Pro Glu  
               
               
                             220       
               
               
                 #           225       
               
               
                 #           230  
               
               
                   
               
               
                 CCC AGC ACT GCT CCA AGC ACC TCC TTC CTG CT  
               
               
                 #C CCA ATG GGC CCC AGC      833  
               
               
                 Pro Ser Thr Ala Pro Ser Thr Ser Phe Leu Le  
               
               
                 #u Pro Met Gly Pro Ser  
               
               
                         235           
               
               
                 #       240           
               
               
                 #       245  
               
               
                   
               
               
                 CCC CCA GCT GAA GGG AGC ACT GGC GAC TTC GC  
               
               
                 #T CTT CCA GTT GGA CTG      881  
               
               
                 Pro Pro Ala Glu Gly Ser Thr Gly Asp Phe Al  
               
               
                 #a Leu Pro Val Gly Leu  
               
               
                     250               
               
               
                 #   255               
               
               
                 #   260  
               
               
                   
               
               
                 ATT GTG GGT GTG ACA GCC TTG GGT CTA CTA AT  
               
               
                 #A ATA GGA GTG GTG AAC      929  
               
               
                 Ile Val Gly Val Thr Ala Leu Gly Leu Leu Il  
               
               
                 #e Ile Gly Val Val Asn  
               
               
                 265                 2  
               
               
                 #70                 2  
               
               
                 #75                 2  
               
               
                 #80  
               
               
                   
               
               
                 TGT GTC ATC ATG ACC CAG GTG AAA AAG AAG CC  
               
               
                 #C TTG TGC CTG CAG AGA      977  
               
               
                 Cys Val Ile Met Thr Gln Val Lys Lys Lys Pr  
               
               
                 #o Leu Cys Leu Gln Arg  
               
               
                                 285   
               
               
                 #               290   
               
               
                 #               295  
               
               
                   
               
               
                 GAA GCC AAG GTG CCT CAC TTG CCT GCC GAT AA  
               
               
                 #G GCC CGG GGT ACA CAG     1025  
               
               
                 Glu Ala Lys Val Pro His Leu Pro Ala Asp Ly  
               
               
                 #s Ala Arg Gly Thr Gln  
               
               
                             300       
               
               
                 #           305       
               
               
                 #           310  
               
               
                   
               
               
                 GGC CCC GAG CAG CAG CAC CTG CTG ATC ACA GC  
               
               
                 #G CCG AGC TCC AGC AGC     1073  
               
               
                 Gly Pro Glu Gln Gln His Leu Leu Ile Thr Al  
               
               
                 #a Pro Ser Ser Ser Ser  
               
               
                         315           
               
               
                 #       320           
               
               
                 #       325  
               
               
                   
               
               
                 AGC TCC CTG GAG AGC TCG GCC AGT GCG TTG GA  
               
               
                 #C AGA AGG GCG CCC ACT     1121  
               
               
                 Ser Ser Leu Glu Ser Ser Ala Ser Ala Leu As  
               
               
                 #p Arg Arg Ala Pro Thr  
               
               
                     330               
               
               
                 #   335               
               
               
                 #   340  
               
               
                   
               
               
                 CGG AAC CAG CCA CAG GCA CCA GGC GTG GAG GC  
               
               
                 #C AGT GGG GCC GGG GAG     1169  
               
               
                 Arg Asn Gln Pro Gln Ala Pro Gly Val Glu Al  
               
               
                 #a Ser Gly Ala Gly Glu  
               
               
                 345                 3  
               
               
                 #50                 3  
               
               
                 #55                 3  
               
               
                 #60  
               
               
                   
               
               
                 GCC CGG GCC AGC ACC GGG AGC TCA GAT TCT TC  
               
               
                 #C CCT GGT GGC CAT GGG     1217  
               
               
                 Ala Arg Ala Ser Thr Gly Ser Ser Asp Ser Se  
               
               
                 #r Pro Gly Gly His Gly  
               
               
                                 365   
               
               
                 #               370   
               
               
                 #               375  
               
               
                   
               
               
                 ACC CAG GTC AAT GTC ACC TGC ATC GTG AAC GT  
               
               
                 #C TGT AGC AGC TCT GAC     1265  
               
               
                 Thr Gln Val Asn Val Thr Cys Ile Val Asn Va  
               
               
                 #l Cys Ser Ser Ser Asp  
               
               
                             380       
               
               
                 #           385       
               
               
                 #           390  
               
               
                   
               
               
                 CAC AGC TCA CAG TGC TCC TCC CAA GCC AGC TC  
               
               
                 #C ACA ATG GGA GAC ACA     1313  
               
               
                 His Ser Ser Gln Cys Ser Ser Gln Ala Ser Se  
               
               
                 #r Thr Met Gly Asp Thr  
               
               
                         395           
               
               
                 #       400           
               
               
                 #       405  
               
               
                   
               
               
                 GAT TCC AGC CCC TCG GAG TCC CCG AAG GAC GA  
               
               
                 #G CAG GTC CCC TTC TCC     1361  
               
               
                 Asp Ser Ser Pro Ser Glu Ser Pro Lys Asp Gl  
               
               
                 #u Gln Val Pro Phe Ser  
               
               
                     410               
               
               
                 #   415               
               
               
                 #   420  
               
               
                   
               
               
                 AAG GAG GAA TGT GCC TTT CGG TCA CAG CTG GA  
               
               
                 #G ACG CCA GAG ACC CTG     1409  
               
               
                 Lys Glu Glu Cys Ala Phe Arg Ser Gln Leu Gl  
               
               
                 #u Thr Pro Glu Thr Leu  
               
               
                 425                 4  
               
               
                 #30                 4  
               
               
                 #35                 4  
               
               
                 #40  
               
               
                   
               
               
                 CTG GGG AGC ACC GAA GAG AAG CCC CTG CCC CT  
               
               
                 #T GGA GTG CCT GAT GCT     1457  
               
               
                 Leu Gly Ser Thr Glu Glu Lys Pro Leu Pro Le  
               
               
                 #u Gly Val Pro Asp Ala  
               
               
                                 445   
               
               
                 #               450   
               
               
                 #               455  
               
               
                   
               
               
                 GGG ATG AAG CCC AGT TAACCAGGCC GGTGTGGGCT GTGTCGTAG  
               
               
                 #C CAAGGTGGGC     1512  
               
               
                 Gly Met Lys Pro Ser  
               
               
                             460  
               
               
                   
               
               
                 TGAGCCCTGG CAGGATGACC CTGCGAAGGG GCCCTGGTCC TTCCAGGCCC CC  
               
               
                 #ACCACTAG   1572  
               
               
                   
               
               
                 GACTCTGAGG CTCTTTCTGG GCCAAGTTCC TCTAGTGCCC TCCACAGCCG CA  
               
               
                 #GCCTCCCT   1632  
               
               
                   
               
               
                 CTGACCTGCA GGCCAAGAGC AGAGGCAGCG AGTTGGGGAA AGCCTCTGCT GC  
               
               
                 #CATGGTGT   1692  
               
               
                   
               
               
                 GTCCCTCTCG GAAGGCTGGC TGGGCATGGA CGTTCGGGGC ATGCTGGGGC AA  
               
               
                 #GTCCCTGA   1752  
               
               
                   
               
               
                 CTCTCTGTGA CCTGCCCCGC CCAGCTGCAC CTGCCAGCCT GGCTTCTGGA GC  
               
               
                 #CCTTGGGT   1812  
               
               
                   
               
               
                 TTTTTGTTTG TTTGTTTGTT TGTTTGTTTG TTTCTCCCCC TGGGCTCTGC CC  
               
               
                 #AGCTCTGG   1872  
               
               
                   
               
               
                 CTTCCAGAAA ACCCCAGCAT CCTTTTCTGC AGAGGGGCTT TCTGGAGAGG AG  
               
               
                 #GGATGCTG   1932  
               
               
                   
               
               
                 CCTGAGTCAC CCATGAAGAC AGGACAGTGC TTCAGCCTGA GGCTGAGACT GC  
               
               
                 #GGGATGGT   1992  
               
               
                   
               
               
                 CCTGGGGCTC TGTGTAGGGA GGAGGTGGCA GCCCTGTAGG GAACGGGGTC CT  
               
               
                 #TCAAGTTA   2052  
               
               
                   
               
               
                 GCTCAGGAGG CTTGGAAAGC ATCACCTCAG GCCAGGTGCA GTGGCTCACG CC  
               
               
                 #TATGATCC   2112  
               
               
                   
               
               
                 CAGCACTTTG GGAGGCTGAG GCGGGTGGAT CACCTGAGGT TAGGAGTTCG AG  
               
               
                 #ACCAGCCT   2172  
               
               
                   
               
               
                 GGCCAACATG GTAAAACCCC ATCTCTACTA AAAATACAGA AATTAGCCGG GC  
               
               
                 #           2224  
               
               
                   
               
               
                   
               
               
                 (2) INFORMATION FOR SEQ ID NO:2:  
               
               
                   
               
               
                      (i) SEQUENCE CHARACTERISTICS:  
               
               
                           (A) LENGTH: 461 amino  
               
               
                 #acids  
               
               
                           (B) TYPE: amino acid  
               
               
                           (D) TOPOLOGY: linear  
               
               
                   
               
               
                     (ii) MOLECULE TYPE: protein  
               
               
                   
               
               
                     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:  
               
               
                   
               
               
                 Met Ala Pro Val Ala Val Trp Ala Ala Leu Al  
               
               
                 #a Val Gly Leu Glu Leu  
               
               
                   1               5  
               
               
                 #                 10  
               
               
                 #                 15  
               
               
                   
               
               
                 Trp Ala Ala Ala His Ala Leu Pro Ala Gln Va  
               
               
                 #l Ala Phe Thr Pro Tyr  
               
               
                              20      
               
               
                 #             25      
               
               
                 #             30  
               
               
                   
               
               
                 Ala Pro Glu Pro Gly Ser Thr Cys Arg Leu Ar  
               
               
                 #g Glu Tyr Tyr Asp Gln  
               
               
                          35          
               
               
                 #         40          
               
               
                 #         45  
               
               
                   
               
               
                 Thr Ala Gln Met Cys Cys Ser Lys Cys Ser Pr  
               
               
                 #o Gly Gln His Ala Lys  
               
               
                      50              
               
               
                 #     55              
               
               
                 #     60  
               
               
                   
               
               
                 Val Phe Cys Thr Lys Thr Ser Asp Thr Val Cy  
               
               
                 #s Asp Ser Cys Glu Asp  
               
               
                  65                  
               
               
                 # 70                  
               
               
                 # 75                  
               
               
                 # 80  
               
               
                   
               
               
                 Ser Thr Tyr Thr Gln Leu Trp Asn Trp Val Pr  
               
               
                 #o Glu Cys Leu Ser Cys  
               
               
                                  85  
               
               
                 #                 90  
               
               
                 #                 95  
               
               
                   
               
               
                 Gly Ser Arg Cys Ser Ser Asp Gln Val Glu Th  
               
               
                 #r Gln Ala Cys Thr Arg  
               
               
                             100       
               
               
                 #           105       
               
               
                 #           110  
               
               
                   
               
               
                 Glu Gln Asn Arg Ile Cys Thr Cys Arg Pro Gl  
               
               
                 #y Trp Tyr Cys Ala Leu  
               
               
                         115           
               
               
                 #       120           
               
               
                 #       125  
               
               
                   
               
               
                 Ser Lys Gln Glu Gly Cys Arg Leu Cys Ala Pr  
               
               
                 #o Leu Arg Lys Cys Arg  
               
               
                     130               
               
               
                 #   135               
               
               
                 #   140  
               
               
                   
               
               
                 Pro Gly Phe Gly Val Ala Arg Pro Gly Thr Gl  
               
               
                 #u Thr Ser Asp Val Val  
               
               
                 145                 1  
               
               
                 #50                 1  
               
               
                 #55                 1  
               
               
                 #60  
               
               
                   
               
               
                 Cys Lys Pro Cys Ala Pro Gly Thr Phe Ser As  
               
               
                 #n Thr Thr Ser Ser Thr  
               
               
                                 165   
               
               
                 #               170   
               
               
                 #               175  
               
               
                   
               
               
                 Asp Ile Cys Arg Pro His Gln Ile Cys Asn Va  
               
               
                 #l Val Ala Ile Pro Gly  
               
               
                             180       
               
               
                 #           185       
               
               
                 #           190  
               
               
                   
               
               
                 Asn Ala Ser Met Asp Ala Val Cys Thr Ser Th  
               
               
                 #r Ser Pro Thr Arg Ser  
               
               
                         195           
               
               
                 #       200           
               
               
                 #       205  
               
               
                   
               
               
                 Met Ala Pro Gly Ala Val His Leu Pro Gln Pr  
               
               
                 #o Val Ser Thr Arg Ser  
               
               
                     210               
               
               
                 #   215               
               
               
                 #   220  
               
               
                   
               
               
                 Gln His Thr Gln Pro Thr Pro Glu Pro Ser Th  
               
               
                 #r Ala Pro Ser Thr Ser  
               
               
                 225                 2  
               
               
                 #30                 2  
               
               
                 #35                 2  
               
               
                 #40  
               
               
                   
               
               
                 Phe Leu Leu Pro Met Gly Pro Ser Pro Pro Al  
               
               
                 #a Glu Gly Ser Thr Gly  
               
               
                                 245   
               
               
                 #               250   
               
               
                 #               255  
               
               
                   
               
               
                 Asp Phe Ala Leu Pro Val Gly Leu Ile Val Gl  
               
               
                 #y Val Thr Ala Leu Gly  
               
               
                             260       
               
               
                 #           265       
               
               
                 #           270  
               
               
                   
               
               
                 Leu Leu Ile Ile Gly Val Val Asn Cys Val Il  
               
               
                 #e Met Thr Gln Val Lys  
               
               
                         275           
               
               
                 #       280           
               
               
                 #       285  
               
               
                   
               
               
                 Lys Lys Pro Leu Cys Leu Gln Arg Glu Ala Ly  
               
               
                 #s Val Pro His Leu Pro  
               
               
                     290               
               
               
                 #   295               
               
               
                 #   300  
               
               
                   
               
               
                 Ala Asp Lys Ala Arg Gly Thr Gln Gly Pro Gl  
               
               
                 #u Gln Gln His Leu Leu  
               
               
                 305                 3  
               
               
                 #10                 3  
               
               
                 #15                 3  
               
               
                 #20  
               
               
                   
               
               
                 Ile Thr Ala Pro Ser Ser Ser Ser Ser Ser Le  
               
               
                 #u Glu Ser Ser Ala Ser  
               
               
                                 325   
               
               
                 #               330   
               
               
                 #               335  
               
               
                   
               
               
                 Ala Leu Asp Arg Arg Ala Pro Thr Arg Asn Gl  
               
               
                 #n Pro Gln Ala Pro Gly  
               
               
                             340       
               
               
                 #           345       
               
               
                 #           350  
               
               
                   
               
               
                 Val Glu Ala Ser Gly Ala Gly Glu Ala Arg Al  
               
               
                 #a Ser Thr Gly Ser Ser  
               
               
                         355           
               
               
                 #       360           
               
               
                 #       365  
               
               
                   
               
               
                 Asp Ser Ser Pro Gly Gly His Gly Thr Gln Va  
               
               
                 #l Asn Val Thr Cys Ile  
               
               
                     370               
               
               
                 #   375               
               
               
                 #   380  
               
               
                   
               
               
                 Val Asn Val Cys Ser Ser Ser Asp His Ser Se  
               
               
                 #r Gln Cys Ser Ser Gln  
               
               
                 385                 3  
               
               
                 #90                 3  
               
               
                 #95                 4  
               
               
                 #00  
               
               
                   
               
               
                 Ala Ser Ser Thr Met Gly Asp Thr Asp Ser Se  
               
               
                 #r Pro Ser Glu Ser Pro  
               
               
                                 405   
               
               
                 #               410   
               
               
                 #               415  
               
               
                   
               
               
                 Lys Asp Glu Gln Val Pro Phe Ser Lys Glu Gl  
               
               
                 #u Cys Ala Phe Arg Ser  
               
               
                             420       
               
               
                 #           425       
               
               
                 #           430  
               
               
                   
               
               
                 Gln Leu Glu Thr Pro Glu Thr Leu Leu Gly Se  
               
               
                 #r Thr Glu Glu Lys Pro  
               
               
                         435           
               
               
                 #       440           
               
               
                 #       445  
               
               
                   
               
               
                 Leu Pro Leu Gly Val Pro Asp Ala Gly Met Ly  
               
               
                 #s Pro Ser  
               
               
                     450               
               
               
                 #   455               
               
               
                 #   460  
               
               
                   
               
               
                   
               
               
                 (2) INFORMATION FOR SEQ ID NO:3:  
               
               
                   
               
               
                      (i) SEQUENCE CHARACTERISTICS:  
               
               
                           (A) LENGTH: 153 amino  
               
               
                 #acids  
               
               
                           (B) TYPE: amino acid  
               
               
                           (C) STRANDEDNESS: single  
               
               
                           (D) TOPOLOGY: linear  
               
               
                   
               
               
                     (ii) MOLECULE TYPE: peptide  
               
               
                   
               
               
                     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:  
               
               
                   
               
               
                 Val Cys Pro Gln Gly Lys Tyr Ile His Pro Gl  
               
               
                 #n Asn Asn Ser Ile Cys  
               
               
                 1               5    
               
               
                 #                10   
               
               
                 #                15  
               
               
                   
               
               
                 Cys Thr Lys Cys His Lys Gly Thr Tyr Leu Ty  
               
               
                 #r Asn Asp Cys Pro Gly  
               
               
                             20       
               
               
                 #            25       
               
               
                 #            30  
               
               
                   
               
               
                 Pro Gly Gln Asp Thr Asp Cys Arg Glu Cys Gl  
               
               
                 #u Ser Gly Ser Phe Thr  
               
               
                         35           
               
               
                 #        40           
               
               
                 #        45  
               
               
                   
               
               
                 Ala Ser Glu Asn His Leu Arg His Cys Leu Se  
               
               
                 #r Cys Ser Lys Cys Arg  
               
               
                     50               
               
               
                 #    55               
               
               
                 #    60  
               
               
                   
               
               
                 Lys Glu Met Gly Gln Val Glu Ile Ser Ser Cy  
               
               
                 #s Thr Val Asp Arg Asp  
               
               
                 65                   
               
               
                 #70                   
               
               
                 #75                   
               
               
                 #80  
               
               
                   
               
               
                 Thr Val Cys Gly Cys Arg Lys Asn Gln Tyr Ar  
               
               
                 #g His Tyr Trp Ser Glu  
               
               
                                 85   
               
               
                 #                90   
               
               
                 #                95  
               
               
                   
               
               
                 Asn Leu Phe Gln Cys Phe Asn Cys Ser Leu Cy  
               
               
                 #s Leu Asn Gly Thr Val  
               
               
                             100       
               
               
                 #           105       
               
               
                 #           110  
               
               
                   
               
               
                 His Leu Ser Cys Gln Glu Lys Gln Asn Thr Va  
               
               
                 #l Cys Thr Cys His Ala  
               
               
                         115           
               
               
                 #       120           
               
               
                 #       125  
               
               
                   
               
               
                 Gly Phe Phe Leu Arg Glu Asn Glu Cys Val Se  
               
               
                 #r Cys Ser Asn Cys Lys  
               
               
                     130               
               
               
                 #   135               
               
               
                 #   140  
               
               
                   
               
               
                 Lys Ser Leu Glu Cys Thr Lys Leu Cys  
               
               
                 145                 1  
               
               
                 #50  
               
               
                   
               
               
                   
               
               
                 (2) INFORMATION FOR SEQ ID NO:4:  
               
               
                   
               
               
                      (i) SEQUENCE CHARACTERISTICS:  
               
               
                           (A) LENGTH: 163 amino  
               
               
                 #acids  
               
               
                           (B) TYPE: amino acid  
               
               
                           (C) STRANDEDNESS: single  
               
               
                           (D) TOPOLOGY: linear  
               
               
                   
               
               
                     (ii) MOLECULE TYPE: peptide  
               
               
                   
               
               
                     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:  
               
               
                   
               
               
                 Thr Cys Arg Leu Arg Glu Tyr Tyr Asp Gln Th  
               
               
                 #r Ala Gln Met Cys Cys  
               
               
                 1               5    
               
               
                 #                10   
               
               
                 #                15  
               
               
                   
               
               
                 Ser Lys Cys Ser Pro Gly Gln His Ala Lys Va  
               
               
                 #l Phe Cys Thr Lys Thr  
               
               
                             20       
               
               
                 #            25       
               
               
                 #            30  
               
               
                   
               
               
                 Ser Asp Thr Val Cys Asp Ser Cys Glu Asp Se  
               
               
                 #r Thr Tyr Thr Gln Leu  
               
               
                         35           
               
               
                 #        40           
               
               
                 #        45  
               
               
                   
               
               
                 Trp Asn Trp Val Pro Glu Cys Leu Ser Cys Gl  
               
               
                 #y Ser Arg Cys Ser Asp  
               
               
                     50               
               
               
                 #    55               
               
               
                 #    60  
               
               
                   
               
               
                 Asp Gln Val Glu Thr Gln Ala Cys Thr Arg Gl  
               
               
                 #u Gln Asn Arg Ile Cys  
               
               
                 65                   
               
               
                 #70                   
               
               
                 #75                   
               
               
                 #80  
               
               
                   
               
               
                 Thr Cys Arg Pro Gly Trp Tyr Cys Ala Leu Se  
               
               
                 #r Lys Gln Glu Gly Cys  
               
               
                                 85   
               
               
                 #                90   
               
               
                 #                95  
               
               
                   
               
               
                 Arg Leu Cys Ala Pro Leu Arg Lys Cys Arg Pr  
               
               
                 #o Gly Phe Gly Val Ala  
               
               
                             100       
               
               
                 #           105       
               
               
                 #           110  
               
               
                   
               
               
                 Arg Pro Gly Thr Glu Thr Ser Asp Val Val Cy  
               
               
                 #s Lys Pro Cys Ala Pro  
               
               
                         115           
               
               
                 #       120           
               
               
                 #       125  
               
               
                   
               
               
                 Gly Thr Phe Ser Asn Thr Thr Ser Ser Thr As  
               
               
                 #p Ile Cys Arg Pro His  
               
               
                     130               
               
               
                 #   135               
               
               
                 #   140  
               
               
                   
               
               
                 Gln Ile Cys Asn Val Val Ala Ile Pro Gly As  
               
               
                 #n Ala Ser Met Asp Ala  
               
               
                 145                 1  
               
               
                 #50                 1  
               
               
                 #55                 1  
               
               
                 #60  
               
               
                   
               
               
                 Val Cys Thr  
               
               
                   
               
               
                   
               
               
                 (2) INFORMATION FOR SEQ ID NO:5:  
               
               
                   
               
               
                      (i) SEQUENCE CHARACTERISTICS:  
               
               
                           (A) LENGTH: 119 amino  
               
               
                 #acids  
               
               
                           (B) TYPE: amino acid  
               
               
                           (C) STRANDEDNESS: single  
               
               
                           (D) TOPOLOGY: linear  
               
               
                   
               
               
                     (ii) MOLECULE TYPE: peptide  
               
               
                   
               
               
                     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:  
               
               
                   
               
               
                 Gln Asn Leu Glu Gly Leu His His Asp Gly Gl  
               
               
                 #n Phe Cys His Lys Pro  
               
               
                 1               5    
               
               
                 #                10   
               
               
                 #                15  
               
               
                   
               
               
                 Cys Pro Pro Gly Glu Arg Lys Ala Arg Asp Cy  
               
               
                 #s Thr Val Asn Gly Asp  
               
               
                             20       
               
               
                 #            25       
               
               
                 #            30  
               
               
                   
               
               
                 Glu Pro Asp Cys Val Pro Cys Gln Glu Gly Ly  
               
               
                 #s Glu Tyr Thr Asp Lys  
               
               
                         35           
               
               
                 #        40           
               
               
                 #        45  
               
               
                   
               
               
                 Ala His Phe Ser Ser Lys Cys Arg Arg Cys Ar  
               
               
                 #g Leu Cys Asp Glu Gly  
               
               
                     50               
               
               
                 #    55               
               
               
                 #    60  
               
               
                   
               
               
                 His Gly Leu Glu Val Glu Ile Asn Cys Thr Ar  
               
               
                 #g Thr Gln Asn Thr Lys  
               
               
                 65                   
               
               
                 #70                   
               
               
                 #75                   
               
               
                 #80  
               
               
                   
               
               
                 Cys Arg Cys Lys Pro Asn Phe Phe Cys Asn Se  
               
               
                 #r Thr Val Cys Glu His  
               
               
                                 85   
               
               
                 #                90   
               
               
                 #                95  
               
               
                   
               
               
                 Cys Asp Pro Cys Thr Lys Cys Glu His Gly Il  
               
               
                 #e Ile Lys Glu Cys Thr  
               
               
                             100       
               
               
                 #           105       
               
               
                 #           110  
               
               
                   
               
               
                 Leu Thr Ser Asn Thr Lys Cys  
               
               
                         115  
               
               
                   
               
               
                   
               
               
                 (2) INFORMATION FOR SEQ ID NO:6:  
               
               
                   
               
               
                      (i) SEQUENCE CHARACTERISTICS:  
               
               
                           (A) LENGTH: 159 amino  
               
               
                 #acids  
               
               
                           (B) TYPE: amino acid  
               
               
                           (C) STRANDEDNESS: single  
               
               
                           (D) TOPOLOGY: linear  
               
               
                   
               
               
                     (ii) MOLECULE TYPE: peptide  
               
               
                   
               
               
                     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:  
               
               
                   
               
               
                 Ala Cys Pro Thr Gly Leu Tyr Thr His Ser Gl  
               
               
                 #y Glu Cys Cys Lys Ala  
               
               
                 1               5    
               
               
                 #                10   
               
               
                 #                15  
               
               
                   
               
               
                 Cys Asn Leu Gly Glu Gly Val Ala Gln Pro Cy  
               
               
                 #s Gly Ala Asn Gln Thr  
               
               
                             20       
               
               
                 #            25       
               
               
                 #            30  
               
               
                   
               
               
                 Val Cys Glu Pro Cys Leu Asp Ser Val Thr Se  
               
               
                 #r Ser Asp Val Val Ser  
               
               
                         35           
               
               
                 #        40           
               
               
                 #        45  
               
               
                   
               
               
                 Ala Thr Glu Pro Cys Lys Pro Cys Thr Glu Cy  
               
               
                 #s Val Gly Leu Gln Ser  
               
               
                     50               
               
               
                 #    55               
               
               
                 #    60  
               
               
                   
               
               
                 His Ser Ala Pro Cys Val Glu Ala Asp Asp Al  
               
               
                 #a Val Cys Arg Cys Ala  
               
               
                 65                   
               
               
                 #70                   
               
               
                 #75                   
               
               
                 #80  
               
               
                   
               
               
                 Tyr Gly Tyr Tyr Gln Asp Glu Thr Thr Gly Ar  
               
               
                 #g Cys Glu Ala Cys Arg  
               
               
                                 85   
               
               
                 #                90   
               
               
                 #                95  
               
               
                   
               
               
                 Val Cys Glu Ala Gly Ser Gly Leu Val Phe Se  
               
               
                 #r Cys Gln Asp Lys Gln  
               
               
                             100       
               
               
                 #           105       
               
               
                 #           110  
               
               
                   
               
               
                 Asn Thr Val Cys Glu Glu Cys Pro Asp Gly Th  
               
               
                 #r Tyr Ser Asp Glu Ala  
               
               
                         115           
               
               
                 #       120           
               
               
                 #       125  
               
               
                   
               
               
                 Asn His Val Asp Pro Cys Leu Pro Cys Thr Va  
               
               
                 #l Cys Glu Asp Thr Glu  
               
               
                     130               
               
               
                 #   135               
               
               
                 #   140  
               
               
                   
               
               
                 Arg Gln Leu Arg Glu Cys Thr Arg Trp Ala As  
               
               
                 #p Ala Glu Cys Glu  
               
               
                 145                 1  
               
               
                 #50                 1  
               
               
                 #55  
               
               
                   
               
               
                   
               
               
                 (2) INFORMATION FOR SEQ ID NO:7:  
               
               
                   
               
               
                      (i) SEQUENCE CHARACTERISTICS:  
               
               
                           (A) LENGTH: 162 amino  
               
               
                 #acids  
               
               
                           (B) TYPE: amino acid  
               
               
                           (C) STRANDEDNESS: single  
               
               
                           (D) TOPOLOGY: linear  
               
               
                   
               
               
                     (ii) MOLECULE TYPE: peptide  
               
               
                   
               
               
                     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:  
               
               
                   
               
               
                 Ala Cys Arg Glu Lys Gln Tyr Leu Ile Asn Se  
               
               
                 #r Gln Cys Cys Ser Leu  
               
               
                 1               5    
               
               
                 #                10   
               
               
                 #                15  
               
               
                   
               
               
                 Cys Gln Pro Gly Gln Lys Leu Val Ser Asp Cy  
               
               
                 #s Thr Glu Phe Thr Glu  
               
               
                             20       
               
               
                 #            25       
               
               
                 #            30  
               
               
                   
               
               
                 Thr Glu Cys Leu Pro Cys Gly Glu Ser Glu Ph  
               
               
                 #e Leu Asp Thr Trp Asn  
               
               
                         35           
               
               
                 #        40           
               
               
                 #        45  
               
               
                   
               
               
                 Arg Glu Thr His Cys His Gln His Lys Tyr Cy  
               
               
                 #s Asp Pro Asn Leu Gly  
               
               
                     50               
               
               
                 #    55               
               
               
                 #    60  
               
               
                   
               
               
                 Leu Arg Val Gln Gln Lys Gly Thr Ser Glu Th  
               
               
                 #r Asp Thr Ile Cys Thr  
               
               
                 65                   
               
               
                 #70                   
               
               
                 #75                   
               
               
                 #80  
               
               
                   
               
               
                 Cys Glu Glu Gly Trp His Cys Thr Ser Glu Al  
               
               
                 #a Cys Glu Ser Cys Val  
               
               
                                 85   
               
               
                 #                90   
               
               
                 #                95  
               
               
                   
               
               
                 Leu His Arg Ser Cys Ser Pro Gly Phe Gly Va  
               
               
                 #l Lys Gln Ile Ala Thr  
               
               
                             100       
               
               
                 #           105       
               
               
                 #           110  
               
               
                   
               
               
                 Gly Val Ser Asp Thr Ile Cys Glu Pro Cys Pr  
               
               
                 #o Val Gly Phe Phe Ser  
               
               
                         115           
               
               
                 #       120           
               
               
                 #       125  
               
               
                   
               
               
                 Asn Val Ser Ser Ala Phe Glu Lys Cys His Pr  
               
               
                 #o Thr Ser Cys Glu Thr  
               
               
                     130               
               
               
                 #   135               
               
               
                 #   140  
               
               
                   
               
               
                 Lys Asp Leu Val Val Gln Gln Ala Gly Thr As  
               
               
                 #n Lys Thr Asp Val Val  
               
               
                 145                 1  
               
               
                 #50                 1  
               
               
                 #55                 1  
               
               
                 #60  
               
               
                   
               
               
                 Cys Gly  
               
               
                   
               
               
                   
               
               
                 (2) INFORMATION FOR SEQ ID NO:8:  
               
               
                   
               
               
                      (i) SEQUENCE CHARACTERISTICS:  
               
               
                           (A) LENGTH: 140 amino  
               
               
                 #acids  
               
               
                           (B) TYPE: amino acid  
               
               
                           (C) STRANDEDNESS: single  
               
               
                           (D) TOPOLOGY: linear  
               
               
                   
               
               
                     (ii) MOLECULE TYPE: peptide  
               
               
                   
               
               
                     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:  
               
               
                   
               
               
                 Asn Cys Val Lys Asp Thr Tyr Pro Ser Gly Hi  
               
               
                 #s Lys Cys Cys Arg Glu  
               
               
                 1               5    
               
               
                 #                10   
               
               
                 #                15  
               
               
                   
               
               
                 Cys Gln Pro Gly His Gly Met Val Ser Arg Cy  
               
               
                 #s Asp His Thr Arg Asp  
               
               
                             20       
               
               
                 #            25       
               
               
                 #            30  
               
               
                   
               
               
                 Thr Val Cys His Pro Cys Glu Pro Gly Phe Ty  
               
               
                 #r Asn Glu Ala Val Asn  
               
               
                         35           
               
               
                 #        40           
               
               
                 #        45  
               
               
                   
               
               
                 Tyr Asp Thr Cys Lys Gln Cys Thr Gln Cys As  
               
               
                 #n His Arg Ser Gly Ser  
               
               
                     50               
               
               
                 #    55               
               
               
                 #    60  
               
               
                   
               
               
                 Glu Leu Lys Gln Asn Cys Thr Pro Thr Glu As  
               
               
                 #p Thr Val Cys Gln Cys  
               
               
                 65                   
               
               
                 #70                   
               
               
                 #75                   
               
               
                 #80  
               
               
                   
               
               
                 Arg Pro Gly Thr Gln Pro Arg Gln Asp Ser Se  
               
               
                 #r His Lys Leu Gly Val  
               
               
                                 85   
               
               
                 #                90   
               
               
                 #                95  
               
               
                   
               
               
                 Asp Cys Val Pro Cys Pro Pro Gly His Phe Se  
               
               
                 #r Pro Gly Ser Asn Gln  
               
               
                             100       
               
               
                 #           105       
               
               
                 #           110  
               
               
                   
               
               
                 Ala Cys Lys Pro Trp Thr Asn Cys Thr Leu Se  
               
               
                 #r Gly Lys Gln Ile Arg  
               
               
                         115           
               
               
                 #       120           
               
               
                 #       125  
               
               
                   
               
               
                 His Pro Ala Ser Asn Ser Leu Asp Thr Val Cy  
               
               
                 #s Glu  
               
               
                     130               
               
               
                 #   135               
               
               
                 #   140