IL103051A - Molecules binding to the p75 tnf receptor, their preparation and pharmaceutical compositions containing them - Google Patents

Description

10305 1/2 Molecules binding to the p75 TNF receptor, their preparation and pharmaceutical compositions containing them iruDn ,TNF ^ p75 "jrt n nx mnunpn m^i ^ia ]T))x n^m mnpn ^Dm Applicant: Yeda Research and Development Co. Ltd. Ref: Y/51/92 FIELD OF THE INVENTION The present invention relates to ligands to the p75 tumor necrosis factor receptor (p75TNF-R) which inhibit the effects of TNF but not the binding of the TNF to the receptor. Ligands in the context of this specification are molecules capable of binding to the TNF-R.

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 TNF effects are deleterious to the organism, it is of great interest to find the 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 extracellular 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. l 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 signalled 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.

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 p55 receptor. However, this p55 receptor activity can be assisted by the p75 receptor, through an yet unknown mechanism.

Israel patent specification No. 91229 discloses antibodies to the soluble TNF-Rs. These antibodies were found to recognize the soluble TNF-Rs and some of them inhibited 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.

SUMMARY OF THE INVENTION It was now surprisingly found in accordance with the present invention that ligands exist which interact with a certain region of the p75 TNF-R, the region becoming more exposed in response to TNF, and when interacting with this region, the ligands inhibit the effects of TNF. However, they do not inhibit the binding of TNF to the receptor but rather somewhat increase it.

The present invention therefore provides ligands to the p75 TNF-R, inhibiting the effects of TNF but not its binding to the TNF-Rs.

The ligands may comprise 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 ligandprotein 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. The same holds true for the term "peptide".

The molecules may be produced either by conventional nethods or by recombinant DNA methods.

The invention also provides a method for the isolation of a ligand according to the invention.

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.

DESCRIPTION OF TH FIGURES Fig 1 shows the binding of radiolabeled TNF to wild and transfected HeLa cells; Fig 2 shows a FACS analysis and Western blotting confirming high expression of the p75 receptor Fig 3 shows the extent of shedding of the soluble form of the p75 TNF-R, thus confirming high expression of the receptor in transfected HeLa cells; Fig 4 shows the sensitivity to TNF of wild and transfected HeLa cells; Fig 5 shows the pattern of protection of HeLa p75 cells from TNF cytotoxicity by different monoclonal antibodies directed against TNF-Rs ; Fig 6 shows how different monoclonal antibodies affect binding of TNF to TNF-Rs on HeLa cells; Fig 7 shows monoclonal antibody protection of U937 cells from TNF cytotoxicity; Fig 8 shows how the different monoclonal antibodies affect binding of TNF to TNF-Rs on U937 cells; Figure 9 shows a comparison of the mimicking and non-mimicking anti p75 TNF-R antibodies; Figure 10 demonstrates that the region recognized by the non-mimicking antibodies against the p75 TNF-R becomes more exposed in response to TNF; Figure 11 shows the Western blot illustrating the region recognized by one of the non-mimicking monoclonal antibodies (no. 32).

DETAILED DESCRIPTION OF THE INVENTION We have identified a region in the extracellular domain of the p75 TNF-R, which contains an epitope recognized by certain anti-p75 TNF-R monoclonal antibodies. Surprisingly, these antibodies do not inhibit TNF binding to the receptor, but rather increase the binding of TNF to the receptor, even though protecting from TNF effects.

Another surprising finding, in accordance with the present invention, consists in the fact that the monoclonal antibodies recognizing the particular region bind to the TNF-R to a much lesser extent in the absence of TNF than they do in response to TNF. This indicates that the recognized region on the TNF-R may be partially masked in the absence of TNF and is completely exposed only in response to TNF.

The invention therefore relates to ligands which bind to that region, which consists of a stretch of amino acids in the extracellular domain of the TNF-R. The amino acid stretch to which the molecules bind extends downstream to amino acid 157 (numbering according to Dembick et al., Cytokine J2:231 (1990)). Part or all of it is contained in the region 157 to 182. In the case of antibodies, it may well be shorter, since it is known that these recognize and bind to sequence epitopes of a size of about seven amino acids .

Since the region in the extracellular domain to which the ligands according to the invention bind is at least partially masked in the absence of TNF, it is envisaged that these ligands may be employed as latent agents. If they are injected into the body in a form in which they may be retained for long periods, they will have no effect in the absence of TNF. However, with the advent of TNF production by the body, i.e. during septic shock or the like, or when an excess of TNF is administered, the above region will be exposed and the ligands will bind thereto. In this manner, although binding of TNF to its receptors is not inhibited but rather increased, TNF does not exert its effects and damage to the body is thus avoided.

As stated above, the ligands according to the invention may comprise proteins, peptides, immunoadhesins , antibodies or other organic compounds .

Proteins may be isolated from cellular extracts, e.g. by ligand affinity purification employing a molecule having an amino acid sequence substantially corresponding to the above-mentioned stretch as ligand.

Peptides may be prepared by synthetizing first target peptides which correspond to the amino acid stretch of the TNF-R found in accordance with the invention to bind the ligands which inhibit the effects of TNF. Thereafter, peptide libraries are screened for other ligands which bind thereto. The peptides which bind to these regions are further screened for those which also bind to TNF-R. Finally, the peptides capable of high affinity binding with both the target peptides and the TNF-R, are screened for the ability of the peptide to perform the desired biological activity.

In a similar manner, a variety of organic molecules, including drugs known for other indications, are screened for their ability to bind to the amino acid stretch found to be critical for inhibiting the effects of TNF.

In addition to the organic molecules, also broth of biological matter, such as bacteria culture products, fungi culture products, eukaryotic culture products and crude cytokine preparations are screened with the amino acid target peptides described above. Molecules obtained by this screening are then further screened for their ability to perform the desired biological function.

Alternatively, molecules are designed which spatially fit the quaternary structure of the amino acid stretch in the receptor.

The active molecules obtained by the above procedures, inasfar as they are biological substances, can also be prepared by biotechnological approaches. In this way, massive production of these molecules will be made possible. Peptides may either be produced by known peptide synthesis methods or using expression vectors containing DNA sequences encoding them. Other molecules, if produced in an enzymatic way, can be made by producing the enzymes involved in the appropriate cultured cells.

Pharmaceutical compositions containing the ligands of the present invention may be employed for antagonizing the effects of TNF in mammals .

Such compositions comprise the ligands according to the invention as their active ingredient. The pharmaceutical compositions are indicated for conditions such as septic shock, cachexia, graft-versus-host reactions, autoimmune diseases such as rheumatoid arthritis, and the like. They are also indicated for counteracting e.g. an overdose of exogenously administered TNF.

The pharmaceutical compositions according to the invention are administered depending on the condition to be treated, via the accepted ways of administration. For example, in the case of septic shock, intravenous administration will be preferred. The pharmaceutical compositions may also be administered continuously, i.e. by way of infusion, or orally. The formulation and dose will depend on the condition to be treated, the route of administration and the condition and the body weight of the patient to be treated. The exact dose will be determined by the attending physician.

The pharmaceutical compositions according to the invention are prepared in the usual manner, for example by mixing the active ingredient with pharmaceutically and physiologically acceptable carriers and/or stabilizers and/or excipients, as the case may be, and are prepared in dosage form, e.g. by lyophilization in dosage vials.

As used herein the term "muteins" refers to analogs of the proteins, peptides and the like in which one or more of 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 site-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 of 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 triethanola ine, 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.

"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 do not 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.

The invention is illustrated by the following non-limiting examples : EXAMPLE 1; Expression of the human p75 TNF-R 1.1 Human epithelial HeLa cells expressing prevalently the p55 TNF-R were transfected with the cDNA of the human p75 receptor introduced in the pMPSVHE eukaryotic expression vector. Clones expressing the transfected p75 receptor were isolated by culturing under selection with geneticin. 1.2 In order to estimate the extent of p75 TNF-R in the neomycin-resistant clones of HeLa cells, the binding assay with radiolabelled ligand - TNF, was performed. The wild- type cells and cells of three different clones were seeded at the same density onto plastic 24-well plates. On the next day, binding assay was performed. To each well iodinated TNF was applied in a concentration of 0.1 nM in PBS buffer containing 0.5% BSA and 0.05% sodium azide (binding buffer). To determine the nonspecific binding, to the control wells a 100-fold excess of non-labeled TNF was added. Following 2 hrs incubation on ice, the cells were washed twice with the binding buffer and detached with PBS + 5 mM EDTA, and the bound radioactibvty was courted in a gamma counter. Figure 1 shows that the extent of TNF binding of all three HeLa clones transfected with p75 TNF-R exceeded markedly the level of binding characteristic of HeLa wild-type cells. 1.3 To confirm the high level of expression of transfected p75 TNF-R in HeLa cells, the analysis of surface expression of the receptor was performed by means of the fluorescent activated cells analyzer (FACS). The cells to be stained were detached from culture dishes with PBS + EDTA and washed twice in the binding buffer. Then the mixtures of monoclonal antibodies against p55 (18 & 20 & 34) or p75 (13 & 32 & 70) TNF-R were applied. Following 1 hr. incubation on ice, the cells were washed again with the binding buffer and finally incubated with the FITC-conjugated rabbit antibody directed to mouse immunoglobulins for 30 min. on ice in the dark. After the washing of non-bound antibody, the fluorescence of cells was determined by analysis on FACSCA cytometer. Figure 2 confirms the high level of expression of the transfected p75 TNF-R in HeLa cells. The left panel shows similar expression of the p55 receptor on the wild-type as well as on transfected HeLa clones. The right panel shows the level of expression of the p75 receptor, which is slight on the HeLa wild-type, while very high on the two transfectant clones presented. The expression was then confirmed by Western blot analysis: the detergent extracts prepared from 5 millions of cells were subjected to SDS-PAGE electrophoresis in the reducing conditions. The proteins were then transferred onto nitrocellulose sheet and processed for Western blot analysis with the rabbit antiserum recognizing the extracellular part of the p75 TNF-R and iodinated sheep anti-rabbit antibodies. In the lane marked W, the reaction with the extract of HeLa wild-type is weak, while in the lane T, where the extract obtained from HeLa transfected with p75 TNF-R was analyzed, the intensity of reaction is very high. 1.4 Figure 3 shows the extent of shedding of the soluble form of the p75 TNF-R induced by 4 hrs incubation with a phorbol ester - TPA, by HeLa wild-type and three different clones of HeLa cells transfected with p75 TNF-R. The cells were seeded at the same density in 24-well plates. On the next day, the medium was exchanged with fresh medium containing 32 nM TPA. The medium was harvested and the amount of the soluble p75 TNF-R was determined in a sandwich ELISA. The levels of the soluble p75 receptor were barely detectable in HeLa wild- type, while reaching 2 ng/ml in transfectant clones. This confirms high expression of the p75 TNF-R in the transfected HeLa cells.

EXAMPLE 2; Cytocidal effect of TNF on the cells and protection therefrom by certain monoclonal antibodies In order to test the sensitivity of the HeLa wild type cells and the transfectant clones, TNF was applied to the HeLa p75 wild-type HeLa cells and to transfectants 75.1 and 75.3. The cytotoxic assay was performed in 96-well plates by applying various dilutions of TNF in the presence of cycloheximide (25 ug/ml). The number of viable cells was estimated by the neutral red uptake test after 10 hours of incubation with TNF. Figure 4 shows that the clones of HeLa cells transfected with the p75 TNF-R are more sensitive to the cytotoxic effect of TNF.

The protection afforded by different monoclonal antibodies from TNF cytotoxicity was evaluated. The cells were incubated for 10 hours with 100 U/ml of TNF in the presence of cycloheximide (25 ug/ml) and different dilutions of the antibodies in order to examine their protective capabilities. Monoclonal antibody no. 32, even though having no inhibitory effect on TNF binding to the p75 TNF-R, inhibited the cytocidal effect on HeLa p75 cells just as effectively as monoclonal anti-p55 TNF-R antibody no. 18, which blocks the binding of TNF to the p55 receptor. In contrast thereto, the monoclonal anti-p75 TNF-R antibody no. 13, which blocked TNF binding to the p75 TNF-R, did not inhibit the cytotoxicity of TNF (see Fig. 5).

EXAMPLE 3: Effect of different antibodies on binding of TNF to receptors and on the cytocidal effect of TNF 3.1 Table I summarizes the behaviour of a number of monoclonal antibodies. 3.2 The manner in which selected antibodies affect binding of TNF to the receptors is shown also in figure 6. HeLa wild-type and HeLa p75.3 cells were seeded at the same density in 24-well plastic plates. On the next day, binding assay was performed. The cells were washed and then the antibodies were applied in a concentration of 10 ug/ml in the binding buffer and kept on ice for 2 hrs . The cells were then washed twice with the binding buffer and then iodinated TNF was applied in a concentration of 0. InM in the binding buffer to each well. To determine the nonspecific binding to the control wells, a 100-fold excess of non-labeled TNF was added. Following 2 hrs incubation on ice, the cells were washed twice with the binding buffer and detached with PBS + TABLE I Subclones were chosen according to results in iRIA and frozen in four different batche Assay was performed with the hybridoma supernatant. The result represents the best sub between subclones were less than 5Z.

Western blots were performed with ammonium sulfate precipitated ascites fluids. Blots diluted in 10Z milk in phosphate buffered saline (PBS) at concentrations between 1.1 a SDS PAGE was performed in the presence of (3-mercaptoethanol (+|1ME) SDS PAGE was performed in the absence of β-mercaptoethanol (-f)ME) mM EDTA. Then the bound radioactivity was counted in a gamma counter- In the HeLa wild-type cells, most of the TNF binding was inhibited with anti-p55 TNF-R monoclonal antibody no. 18, while the effect of the anti-p75 TNF-R antibody no. 13 was much weaker. This shows that although the HeLa wild-type cells expressed prevalently p55 receptors, considerable amounts of p75 receptors were also expressed. Anti-p75 TNF-R antibodies nos . 32, 57 and 70 did not significantly affect the binding of TNF to the HeLa wild-type cells. In the HeLa p75.3, most of the TNF binding was inhibited by anti-p75 TNF-R monoclonal antibody no. 13, while the effect of the anti-p55 TNF-R antibody no. 18 was much weaker. This shows that the HeLa p75.3 cells expressed prevalently p75 receptors, as well as considerable amounts of p55 receptors. Anti-p75 TNF-R antibodies nos. 32, 57 and 70 strongly upregulated the binding of TNF to the HeLa p75.3 cells .

In order to ascertain the influence of selected antibodies on the cytocidal effect, U937 human monocytoid cells, which are sensitive to TNF-mediated cytocidal effect were examined. It is known that these cells express prevalently the p75 TNF-R as well as, to a lesser extent, p55 receptors. The cells were incubated for 12 hours with 1000 U/ml of TNF in the presence of cycloheximide (5 ug/ml) and different dilutions of the antibodies in order to examine their protective capabilities. As can be seen from Figure 7, anti-p75 TNF-R monoclonal antibody no. 32 was able to protect U937 cells from TNF cytotoxicity. Anti-p55 TNF-R monoclonal antibody no. 18 also exerted some protective activity on this type of cells, unlike the anti p75 monoclonal antibody no. 13, even though it blocks the binding of TNF to the p75 receptor.

The effect of different monoclonal antibodies on the binding of TNF to TNF receptors on U937 cells was also investigated. The cells were washed twice and the antibodies were applied in a concentration of lOug/ml in the binding buffer and kept on ice for 2 hrs . Then the cells were washed again and iodinated TNF was applied to each sample, in a concentration of O.lnM in the binding buffer. To determine the nonspecific binding, 100-fold excess of non-labeled TNF was added to the control samples. Following 2 hrs incubation on ice the cells were washed twice with the binding buffer and the cell-bound radioactivity was measured with a gamma counter. As shown in figure 8 , most of the TNF binding could be inhibited by the anti-p75 monoclonal antibody no. 13, while the effect of the anti-p55 TNF-R antibody no. 18 was much weaker. Thus the U937 cells expressed prevalently p75 receptors as well as some p55 receptors. Anti-p75 antibodies nos. 32 and 57 strongly upregulated the binding of TNF* to the U937 cells.

Under certain conditions, it is possible to mimic the cytotoxic effect of TNF by applying anti-p75 TNF-R antibodies. HeLa cells transfected with the human p75 receptor show susceptibility to agonistic antibodies after pretreatment with TNF or anti p55 TNF-R antibodies. Applying p75 receptor agonists together with cycloheximide in such pretreated cells induces strong cell killing. TNF (100 U/ml) was applied to HeLa p75.3 cells and kept for 4 hrs . Then the cells were washed three times and various dilutions of anti- p75 TNF-R monoclonal antibodies were added to the medium supplemented with cycloheximide (25 ug/ml). Following 10 hrs incubation, the neutral red uptake assay was performed in order to measure the number of viable cells. Figure 9 shows that anti-p75 TNF-R monoclonal antibodies nos . 32, 57 and 70 did not exhibit the mimicking activity against HeLa p75.1 and p75.3 cells, while the antibodies recognizing epitopes involved in TNF binding (13, 31 and 36) kill HeLa p75 cells clone 1 or 3 effectively.

EXAMPLE 4; Determination of the extent of exposure of the region of the TNF-R recognized by the non-mimicking antibodies Nearly confluent HeLa p75.3 cells were incubated with and without TNF (10* U/ml) for 15 min at 37°C. The medium was removed and the cells were washed with cold PBS containing 0.5% BSA. The cells were then incubated for 1.5 hrs on ice in the same buffer containing the indicated anti-p75 TNF-R monoclonal antibodies at a concentration of 10 ug/ml. Thereafter, the cells were washed, lysed and extracted with RIPA buffer (10 itiM Tris-HCl, pH 7.5, 150 mM NaCl, 1% of NP-40, 1% deoxycholate, 0.1% SDS and 1 mM EDTA) , supplemented with 1 mM PMSF and 10 mM benzamidine HC1. Insoluble material was pelleted by centrifugation for 30 min at ,000 g. The antibody-receptor complexes were then precipitated with Protein G Sepharose beads. The beads were washed extensively, resuspended in SDS sample buffer containing mercaptoethanol , and boiled. After SDS-PAGE, Western blotting analysis was performed using rabbit polyclonal antibodies against the soluble p75 TNF-R. For each antibody, immunoprecipitation was performed both with extracts of cells pretreated (+) and not treated (-) with TNF. As can be seen in Fig. 10, in contrast to antibodies 14, 19 and 78, which effectively immunoprecipitated the p75 TNF-R both from TNF-treated and untreated cells, antibody no. 70 (and also 32, 57) precipitated the TNF-R effectively only from TNF-treated cells. This implies that the epitope which antibodies 32, 57 and 70 recognize is partially masked under normal growth conditions and becomes exposed only in response to TNF.

All three monoclonal antibodies exhibiting this activity were distinguished by their ability to bind to the p75 TNF-R following its complete denaturation in SDS and β-mercaptoethanol indicating that the epitope(s) is (are) not conformational but rather sequence epitope(s).

EXAMPLE 5: Determination of the region of the p75 receptor which is recognized bv the selected group of antibodies In order to determine the region of p75 receptor recognized by antibodies 32, 57 and 70, four different constructs of the extracellular part of the p75 TNF-R were expressed in E. coli by cloning into bacterial expression plasmid pET8c the PCR-amplified DNA segments coding for the truncated forms of the extracellular part of the p75 TNF receptor, as follows : Amino acids Molecular weight 1 Ala 3 to Thr 132 about 13kDa 2 Ala 3 to Ser 182 about 20kDa 3 Ala 3 to Val 192 about 20kDa 4 Ala 3 to Asp 235 about 25kDa The results are shown in Figure 11. The proteins were blotted onto two separate nitrocellulose sheets and incubated with polyclonal antibodies against the soluble p75 TNF-R (lower part) and with the no. 32 monoclonal antibody (upper part of the autoradiograph) . All four bacterial products were recognized by the polyclonal antibodies while only the three longer ones were recogized by monoclonal antibody no. 32. It can therefore be concluded that the epitope recognized by antibody no. 32 maps between C-terminal ends of the two shortest constructs prepared; i.e. between amino acids 125-182.

This conclusion was verified in a further experiment where this region of 57 amino acids (Phe 125 to Ser 182) was expressed in E. coli as a fusion protein with maltose binding protein in the pMalCRI expression system. The resulting product (construct 8) was recognized by both monoclonal no. 32 and polyclonal antibodies against p75 receptor thus confirming the localization of the epitope inside this polypeptide. Two additional truncated versions of the extracellular part of the p75 receptor which terminated inside the presumed epitope region were also prepared in order to determine more precisely the protein sequence recognized by the antibodies : Amino acids Molecular weight : Ala 3 to Thr 146 about 16kDa 6: Ala 3 to Asn 164 about 18kDa Both constructs were not recognized by the monoclonal antibody no. 32, thus suggesting that the region recognized by this antibody lies either behind the C-terminal end of construct 6 or within the region of about seven amino acids upstream to the C-terminal end that is approximately between amino acids 157-182 of the mature protein. The set of other constructs which contained (constructs 9, 10) or did not contain (construct 7) the presumed epitope region expressed in pMAlCRI bacterial expression system confirmed this conclusion. Involvement of amino acids located downstream to amino acid 182 cannot be excluded.

Table II summarizes the results obtained from the experiments carried out in accordance with this example.

TABLE II Recognition by Construct Amino acids Polyclonal Ab Monoclonal # 32 1 :pET Ala 3 to Thr 132 + - 5:pET Ala 3 to Thr 146 + - 6:pET Ala 3 to Asn 164 + - 2:pET Ala 3 to Ser 182 + + 3:pET Ala 3 to Val 192 + + 4 :pET Ala 3 to Asp 235 + + 7:pMal Phe 125 to Thr 146 + - 8:pMal Phe 125 to Ser 182 + + 9:pMal Phe 125 to Val 192 + + :pMal Phe 125 to Asp 235 + + EXAMPLE 6: Preparation of a peptide capable of binding to the region of the TNF-R which inhibits TNF cytotoxicity A. Synthesis of target peptides The target peptides having an amino acid sequence corresponding to one of the amino acid stretch found in accordance with the invention are synthesized using Fmoc protected amino acid derivatives , according to the usual procedures, e.g. according to Chang, CD., and Meienhoffer, J., (1978) Int. J. of Peptide Protein Res., 11:246-249; Grandas, A., et. al . (1989) Tetrahedron, 45:4637-4648; Fournier, A., et. al . (1989) Int. J. Peptide Protein Res., .3_3: 133-139 ; Stewart M.J. and Young J.D., Solid phase peptide synthesis (1984), Pierce Chemical Company; or similar methods .

B. Synthesis of combinatorial peptide library Beads and linkers The combinatorial peptide library is synthesized on suitable bead carriers, e.g. beads made of polystyrene crossed with divinyl benzene (1% DVB), diameter about 200 microns, containing about 1 mili-equivalent of binding site, e.g. primary amines, or acid labile chloromethyl group. Linkers, such as cystamine, can be coupled to the beads by using a suitable cross linker e.g. glutardialdehyde. Peptide synthesis is performed on the free amine of the cystamine. At the termination of the synthesis, the cystamine bridge is cleaved by mild reduction, e.g. by use of dithiotreitol , resulting in a peptide having a free C terminal SH group.

This group, which in many peptides is unique, serves as a specific modification site for binding of a recognition molecule, e.g. by reaction with iminobiotin-maleimide.

Library Synthesis strategy Primary library The library synthesis strategy is designed to allow screening of peptides of sizes of 9 or more residues, and composed of up to about 100 different amino acid derivatives. The first 6 amino acids are randomly synthesized by incorporation of all the different amino acid derivatives into each synthesis cycle. In the last 3 synthesis cycles , a unique sequence of amino acids per bead is synthesized, as follows: Following the incorporation of amino acid no. 6, the beads are aliquoted. The number of aliquots is identical to the number of different amino acid derivatives. A single amino acid derivative is then incorporated into the 7th position of growing polypeptide chain of all the beads in a single aliquot. The beads are then mixed together, and redistributed as before. This procedure is repeated 3 times, resulting in incorporation of a total of 3 unique amino acid residues per bead. This format of synthesis is called "structured synthesis" to differentiate from the random synthesis format used to synthesize the peptide in positions 1-6.

The primary library is then screened as described below. The results of the screening allow selection of specific tripeptide sequences capable of binding to the target sequences .

Secondary library Synthesis of the secondary library is performed after the appropriate tripeptide sequence(s) are identified by screening of the primary library. The synthesis of the secondary library is carried out similarly to the synthesis of the primary library in concept. The first 3 amino acid residues are randomly synthesized, the next 3 amino acid residues are synthesized by structured synthesis, and the last 3 amino acids are synthesized according to the sequence(s) derived from screening of the primary library.

Tertiary library The tertiary library is synthesized similarly to the primary and secondary libraries in concept. The first 3 amino acid residues are synthesized by structured synthesis, and the final 6 amino acid residues are incorporated according to the sequence(s) derived from screening of the primary and secondary libraries.

Extended synthes s/screening process The process described above results in identification of peptides composed of 9 amino acid residues, which are capable of binding specifically to the target ligands. It is possible to continue the synthesis/screening process in order to prepare longer peptides, which might perform better. It may also be advantageous to further modify the peptide, e.g. stabilize the peptide conformation by cross-linking of side chains, protect against proteolytic cleavage by modification of the peptide bond, increase potential for transmembrane transport by conjugation of hydrophobic residues to side chains, etc., in order to improve its activity. It is then possible to carry out the modifications according to the above strategy. This approach allows fast screening of many modified peptides and greatly reduces the time needed in order to identify the desired peptide.

Screening of the library The screening of the library (peptides) is divided into 2 phases : Phase A: Screening for ligand binding i.e. binding to the target peptide and to the native protein.

Phase B: Screening for the capability of the peptides to diminish or abolish TNF-R shedding in response to TNF, or to block signal transduction by the TNF-R.

Phase A: Screening for ligand binding potential The libraries are exposed to the ligand, e.g. any of the target peptides indicated above or the purified TNF-R. Detection of the bound ligand is possible following direct labeling of the ligand, e.g. by labeling with a fluorescent dye, such as tetramethyl rhodamine, or by using specific polyclonal antibodies and an appropriate second antibody coupled to a suitable marker, e.g. the enzyme alkaline phosphatase or horseradish peroxidase. The bound enzyme is detected using an appropriate substrate e.g. for alkaline phosphatase BCIP coupled with a tetrazolium salt such as nitroblue tetrazolium, or for horseradish peroxidase chloronaphtol or tetramethyl benzidine.

Detection of beads which bind the ligand is effected either by monitoring bead fluorescence e.g. by using a FACS machine, or by monitoring bead color by visual inspection. The labeled beads are retrieved from the mixture and the peptides of individual beads are sequenced by automatic peptide sequencer. The sequence data obtained enable designing of further libraries.

After detection of beads which bind to the target peptide, their binding to the protein carrying the target peptide is confirmed. This is carried out using a process similar to the one used for determination of the binding of the target peptide, but using highly purified TNF-R as ligand.

Phase B: screening for bioactivit After identification of peptides capable of high affinity binding with the target peptides and to the TNF-R through the library screening cycles described above has been effected, screening for the ability of the peptide to perform the desired biological activity is carried out.

EXAMPLE 7; Creation of recombinant DNA molecules comprising nucleotide sequences coding for the active peptides and other molecules and their expression The peptides and other molecules can also be prepared by genetic engineering techniques and their preparation encompasses all the tools used in these techniques. Thus DNA molecules are provided which comprise the nucleotide sequence coding for such peptides and other biological molecules. These DNA molecules can be genomic DNA, cDNA, synthetic DNA and a combination thereof.

Creation of DNA molecules coding for such peptides and molecules is carried out by conventional means, once the amino acid sequence of these peptides and other molecules has been determined .

Expression of the recombinant proteins can be effected in eukaryotic cells, bacteria or yeasts, using the appropriate expression vectors. Any method known in the art may be employed.

For example, the DNA molecules coding for the peptides or other molecules obtained by the above methods are inserted into appropriately constructed expression vectors by techniques well known in the art (see Maniatis, T. et al., Molecular Cloning; A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor (1982)). Double-stranded cDNA is linked to plasmid vectors by homopolymeric tailing or by restriction linking involving the use of synthetic DNA linkers or blunt-ended ligation techniques. DNA ligases are used to ligate the DNA molecules and undesirable joining is avoided by treatment with alkaline phosphatase.

In order to be capable of expressing a desired biological substance, i.e. a peptide or protein (hereinafter "protein", for simplicity's sake), an expression vector should comprise also specific nucleotide sequences containing transcriptional and translational regulatory information linked to the DNA coding for the desired protein in such a way as to permit gene expression and production of the protein. First, in order for the gene to be transcribed, it must be preceded by a promoter recognizable by RNA polymerase, to which the polymerase binds and thus initiates the transcription process. There are a variety of such promoters in use, which work with different efficiencies (strong and weak promoters). They are different for prokaryotic and eukaryotic cells .

The promoters that can be used in the present invention may be either constitutive, for example, the int promoter of bacteriophage lambda, the bla promoter of the β-lactamase gene of pBR322, and the CAT promoter of the chloramphenicol acetyl transferase gene of pPR325, etc., or inducible, such as the prokaryotic promoters including the major right and left promoters of bacteriophage lambda (PL and PR), the trp, recA, lacZ , lacl , ompF and gal promoters of E. coli, or the trp-lac hybrid promoter, etc. (Glick, B.R. (1987) J . Ind. icrobiol . , :277-282) .

Besides the use of strong promoters to generate large quantities of mRNA, in order to achieve high levels of gene expression in prokaryotic cells, it is necessary to use also ribosome-binding sites to ensure that the mRNA is efficiently translated. One example is the Shine-Dalgarno (SD) sequence appropriately positioned from the initiation codon and complementary to the 3 '-terminal sequence of 16S RNA.

For eukaryotic hosts, different transcriptional and translational regulatory sequences may be employed, depending on the nature of the host. They may be derived from viral sources, such as adenovirus, bovine papilloma virus, Simian virus, or the like, where the regulatory signals are associated with a particular gene which has a high level of expression. Examples are the TK promoter of Herpes virus, the SV40 early promoter, the yeast gal4 gene promoter, etc. Transcriptional initiation regulatory signals may be selected which allow for repression and activation, so that expression of the genes can be modulated.

The DNA molecule comprising the nucleotide sequence coding for the peptides or other molecules of the invention and the operably linked transcriptional and translational 'regulatory signals is inserted into a vector which is capable of integrating the desired gene sequences into the host cell chromosome. The cells which have stably integrated the introduced DNA into their chromosomes can be selected by also introducing one or more markers which allow for selection of host cells which contain the expression vector. The marker may provide for prototrophy to an auxotropic host, biocide resistance, e.g., antibiotics, or heavy metals, such as copper, or the like. The selectable marker gene can either be directly linked to the DNA gene sequences to be expressed, or introduced into the same cell by co-transfection. Additional elements may also be needed for optimal synthesis of single chain binding protein mRNA. These elements may include splice signals, as well as transcription promoters, enhancers, and termination signals. cDNA expression vectors incorporating such elements include those described by Okayama, H. , (1983) Mol. Cell Biol. , 3.: 280.

In a preferred embodiment, the introduced DNA molecule will be incorporated into a plasmid or viral vector capable of autonomous replication in the recipient host. Factors of importance in selecting a particular plasmid or viral vector include: the ease with which recipient cells that contain the vector may be recognized and selected from those recipient cells which do not contain the vector; the number of copies of the vector which are desired in a particular host; and whether it is desirable to be able to "shuttle" the vector between host cells of different species .

Preferred prokaryotic vectors include plasmids such as those capable of replication in E. coli, for example, pBR322, ColEl, pSClOl, pACYC 184, etc. (see Maniatis et al., (1982) op. cit.); Bacillus plasmids such as pC194, pC221, pT127, etc. (Gryczan, T. , The Molecular Biology of the Bacilli, Academic Press, NY (1982)); Streptomyces plasmids including pIJlOl (Kendall, K.J. et al . , (1987) J. Bacterid . 169:4177-83 ) : Streptomyces bacteriophages such as (}>C31 (Chater, K.F. et al . , in: Sixth International Symposium on Actinomycetales Biology, (1986)), and Pseudomonas plasmids (John, J.F., et al. (1986) Rev. Infect . Dis . 8.:693-704; and Izaki, K. (1978) Jpn. J. Bacteriol . , 33:729-742) .

Preferred eukaryotic plasmids include BPV, vaccinia, SV40, 2-micron circle, etc., or their derivatives. Such plasmids are well known in the art (Botstein, D. , et al. (1982) Miami Wint. Symp. .19, pp. 265-274; Broach, J.R., in: The Molecular Biology of the Yeast Saccharomyces : Life Cycle and Inheritance, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, pp. 445-470 (1981); Broach, J.R., (1982) Cell, 28.: 203-204 ; Bollon, D.P., et al. (1980) J. Clin. Hematol. Oncol., 10.: 39-48; Maniatis, T., in: Cell Biology: A Comprehensive Treatise, Vol. 3: Gene Expression, Academic Press, NY, pp. 563-608 (1980)).

Once the vector or DNA sequence containing the construct(s) has been prepared for expression, the DNA construct(s) may be introduced into an appropriate host cell by any of a variety of suitable means: transformation, transfection, conjugation, protoplast fusion, electroporation, calcium phosphate-precipitation, direct microinjection, etc.

Host cells to be used in this invention may be either prokaryotic or eukaryotic. Preferred prokaryotic hosts include bacteria such as E. coli. Bacillus, Streptomyces, Pseudomonas, Salmonella, Serratia, etc. The most preferred prokaryotic host is E. coli. Bacterial hosts of particular interest include E. coli K12 strain 294 (ATCC 31446), E. coli X1776 (ATCC 31537), E. coli W3110 (F~, lambda-, prototropic (ATCC 27325)), and other enterobacterium such as Salmonella typhimurium or Serratia marcescens and various Pseudomonas species. Under such conditions, the protein will not be glycosylated. The prokaryotic host must be compatible with the replicon and control sequences in the expression plasmid.

Preferred eukaryotic hosts are mammalian cells, e.g. human, monkey, mouse and Chinese hamster ovary (CHO) cells, because they provide post-translational modifications to protein molecules including correct folding or glycosylation at correct sites. Also yeast cells can carry out post-translational peptide modifications including glycosylation. A number of recombinant DNA strategies exist which utilize strong promoter sequences and high copy number of plasmids which can be utilized for production of the desired proteins in yeast. Yeast recognizes leader sequences on cloned mammalian gene products and secretes peptides bearing leader sequences (i.e. pre-peptides ) .

After the introduction of the vector, the host cells are grown in a selective medium, which selects for the growth of vector-containing cells. Expression of the cloned gene sequence(s) results in the production of the desired proteins .

Purification of the recombinant proteins is carried out by any one of the methods known for this purpose. 103051/4

Claims (10)

1. A method for the isolation of a ligand (except antibodies 32,57 and 70 herein) which specifically binds to the region of amino acids 157 to 182 of the p75 TNF-R, the ligand inhibiting the effects of TNF, but not the binding of TNF to the p75 TNF-R, comprising : a) if the ligand is a protein, ligand affinity purification from cellular extracts employing a molecule having an amino acid sequence corresponding to amino acids 157 to 182 of the p75 TNF-R; b) if the ligand is a peptide, (i) synthesis of target peptides which correspond to amino acids 157 to 182 of the p75 TNF-R (ii) screening of a peptide library for peptides binding thereto; c) if the ligand is another organic molecule, including a drug known for other indications, screening for the ability of the molecule to bind to amino acids 157 to 182 of the p75 TNF-R; or d) screening with a target peptide as obtained under b(i) above a broth of biological matter, and testing the ability of the ligands obtained to inhibit TNF function.

2. A method according to claim 1, wherein the broth of biological matter is selected from bacteria culture products, fungi culture products, eukaryotic culture products and crude cytokine preparations.

3. A ligand obtainable by the method claimed in claims 1 or 2, except antibodies 32, 57 and 70 herein.

4. A ligand according to claim 3, being a protein.

5. A ligand according to claim 3, being a peptide.

6. A ligand according to claim 3, being an immunoadhesin.

7. A ligand according to claim 3, being an antibody, except antibodies 32, 57 and 70 herein. 103051/4

8. A ligand according to claim 3, being another organic compound.

9. The use of an antibody according to claim 7 for the preparation of a pharmaceutical composition which inhibits the effects of TNF, but not the binding of TNF to the p75 TNF-R, substantially as described in the specification.

10. A pharmaceutical composition which inhibits the effects of TNF, but not the binding of TNF to the p75 TNF-R, comprising a ligand obtainable by the method of claim 1 or 2.