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MULTIMERS OF THE SOLUBLE FORMS OF TNF RECEPTORS, THEIR PREPARATION AND PHARMACEUTICAL COMPOSITIONS CONTAINING THEM
FIELD OF THE INVENTION
The present invention relates to multimers of the soluble forms of the tumor necrosis factor receptors, their preparation and pharmaceutical compositions containing them.
BACKGROUND OF THE INVENTION 15
Tumor necrosis factor (TNF) is a cytokine produced by a number of cell types, primarily by mononuclear phagocytes. At present, two different TNFs have been identified: TNF-a and TNF-P (lymphotoxin). Both TNF-a and TNF-P initiate their effects by binding to specific cell receptors. 20
TNF-a and TNF-p (hereinafter called 'TNF') are known to exert both beneficial as well as deleterious effects on a number of different target cells involved in the inflammatory response. Among its many effects, TNF, for example, stimulates the growth of fibroblasts and induces in these cells the 25 synthesis of collagenase, prostaglandin E2 and IL-6. TNF also decreases in adipocytes the activity of lipoprotein lipase, activates osteoclasts and increases in endothelial cells adhesivity for blood leukocytes. 3Q
However, TNF has also extremely deleterious effects: over-production of TNF can play a major pathogenic role in several diseases, for example, TNF-a is known to be a major cause for the symptoms of septic shock. In some diseases, TNF may cause excessive loss of weight (cachexia) by 35 suppressing activities of adipocytes and by causing anorexia (TNF-a was therefore called cachectin). See, e.g. Beutler et al., Annu. Rev. Biochem., 57, pp. 507-518 (1988) and Old, Sci. Am. 258, pp. 41-49 (1988). Excessive TNF production has also been demonstrated in patients with AIDS. 40
In order to counteract the cytotoxic effects of TNF, ways were sought to antagonize or eliminate endogenously formed or exogenously administered TNF. Furthermore, ways are being sought to induce specifically only some of the many effects of TNF or restrict its action to a specific 45 kind of target cells. The first attempt in this direction was the development of monoclonal antibodies which neutralize the TNF-a cytotoxic activity. Such monoclonal antibodies are described in EP 186 833 and in Israel Patent No. 73883.
As stated above, TNF initiates its function by binding to 50 specific cell surface receptors. Two such TNF receptors (hereinafter 'TNF-R") which are expressed differentially in cells of different kinds are known, the p55-TNF receptor and the p75-TNF receptor (p55-TNF-R and p75-TNF-R). Two proteins called TBP-I and TBP-II which bind specifically to 55 TNF have been shown to cross-react immunologically with the two receptors. Both proteins provide protection against the in vitro cytocidal effect of TNF and both bind TNF-P less effectively than TNF-a. It was found that the formation of the TBPs occurs by proteolytic cleavage of the cell surface 60 TNF-Rs, resulting in release of a major part of their extracellular domain (see EP 308 378, 398 327 and 444,900). Indeed, the sequences of the amino acids in TBP-I and TBP-II were found to be fully identical to sequences found in the extra-cellular domains of the cell-surface receptors, 65 but do not contain any part of the intracellular domain of the receptors.
These findings imply that the inhibition of TNF function by TBP-I and TBP-II reflects the conservation, in TBP-I and TBP-II, of part of the structural features of the cell surface TNF-Rs, which are important for binding of TNF by the receptors and the initiation of cell response to TNF thereby. Due to this conservation of structure, TBP-I and TBP-II have the ability to compete with the cell surface TNF-Rs for TNF and thus block its function.
It is known that TNF, in its natural state, exists as a multimer (trimer) consisting of three identical polypeptide chains, each with a molecular size of about 17,000 D.
To elicit its effects, TNF must bind to the TNF Receptors in its trimeric form. Although the TNF monomer also binds to cells (but at a lower affinity when compared with the TNF trimer), it has no effect.
SUMMARY OF THE INVENTION
The present invention now provides multimers of the soluble forms of the TNF-Rs, and salts for functional derivatives thereof. These multimers, effectively interfere with the binding of TNF to the cell-surface receptors and thus do not allow TNF to exert its deleterious effect.
The term "multimers" as used herein refers to any combination of monomers held together, for example, by covalent bonding, liposome formation, the incorporation of monomers of the soluble form of TNF-R into a single recombinant soluble, or any other combination of monomers.
The multimers may either be in dimeric, trimeric or other multimeric form and may comprise, for example, TBP-I, TBP-II, or mixtures thereof.
The invention also provides methods of producing these multimers by covalent cross-linking of the soluble forms of the TNF-Rs.
In another aspect, the present invention relates to DNA molecules comprising the nucleotide sequences encodin the multimers of the soluble forms of the TNF-Rs, to expression vehicles comprising them, to host cells transformed therewith, and to processes for producing the multimers by culturing the transformed cells in a suitable culture medium.
The invention further relates to nucleic acid, such as DNA or RNA, which hybridizes to DNA or RNA which encodes a multimer in accordance with the present invention, under stringent conditions. Such nucleic acid is useful as a probe in identification and purification of the desired nucleic acid. Furthermore, such nucleic acid would be a prime candidate to determine whether it encodes a polypeptide which retains the functional activity of the multimers of the present invention. The term "stringent conditions" refers to hybridization and subsequent washing conditions which those of ordinary skill in the art conventionally refer to as "stringent". See Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates and Wiley Interscience, N.Y., §§6.3 and 6.4 (1987,1992), and Sambrook et al., supra. Without limitation, examples of stringent conditions include washing conditions 12°-20° C. below the calculated Tm of the hybrid under study in, e.g., 2xSSC and 0.5% SDS for 5 minutes, 2xSSC and 0.1% SDS for 15 minutes; O.lxSSC and 0.5% SDS at 37° C. for 30-60 minutes and then a O.lxSSC and 0.5% SDS at 68° C. for 30-60 minutes. Those of ordinary skill in this art understand that stringency conditions also depend on the length of the DNA sequences, oligonucleotide probes (such as 10-40 bases) or mixed oligonucleotide probes. If mixed probes are used, it is preferable to use tetramethyl ammonium chloride (TMAC) instead of SSC. See Ausubel, supra.
The multimers according to the invention, and the salts and functional derivatives thereof, may comprise the active ingredient of pharmaceutical compositions for protecting mammals from the deleterious effects of TNF. These compositions are yet another aspect of the present invention.
DETAILED DESCRIPTION OF THE
As stated hereinbefore, TNF exists and exerts its biological action as a trimer. However, nothing has been known so far as to the form of the TNF-Rs to which TNF binds, i.e. whether the TNF trimer binds to individual molecules of the TNF-Rs, or the receptors themselves also exist as multimers or become multimers following TNF binding which better accommodates the TNF trimers.
We have now found that the TNF-Rs exist in aggregated forms in cells exposed to TNF.
This was shown by analysis of full-length and C-terminal truncated forms of the human p55-TNF-Rs tagged by crosslinking to labelled TNF. For this purpose we produced truncated forms of the human p55-TNF-R by site-directed mutagenesis of the cDNA and expressed them in murine A9 cells. Radiolabeled TNF was applied on these cells and cross-linked chemically to the TNF-Rs. The TNF-Rs were solubilized with a detergent, and antibodies specific to the human receptors were applied to immunoprecipitate the human receptors, examining thereby whether murine receptors associate noncovalently with the human receptor as a consequence of aggregation of the receptors.
TBP-I and TBP-II monomers must be administered in very high doses in order to result in effective inhibition of TNF-binding to cells in the human body. The multimers of the soluble forms of TNF-Rs according to the invention, are believed to be more effective in inhibiting TNF activity at lower doses, since they can effectively compete with the TNF trimers for the binding sites on the aggregates of the cell surface TNF-Rs.
The multimers of the soluble forms of the TNF-Rs may be produced chemically by using known methods which will result in the formation of either dimers or higher multimers of the soluble forms of the TNF-Rs.
Another way of producing the multimers of the soluble forms of the TNF-Rs is by recombinant techniques. In this way, massive production of multimers with optimal TNF binding activity will be made possible.
The multimers of the present invention have the ability to interfere with the binding of TNF to its receptors and/or to block the effects of TNF. Each multimer comprises two or more monomers, each comprising the soluble form of a TNF-R or a salt or functional derivative thereof. The upper limit for the number of monomers in a multimer is not important and liposomes having many such monomers thereon may be used. Such multimers preferably have 2-5 monomers and more preferably 2 or 3.
Each monomer of the multimer is a soluble form of a TNF-R or a salt or functional derivative thereof. Preferably, the monomers are TBP-I and/or TBP-II or their salts or functional derivatives. More preferably, each monomer is a protein having an amino acid sequence essentially corresponding to that of TBP-I or TBP-II and, most preferably, exactly corresponding to TBP-I and TBP-II. The term "essentially corresponding to" is intended to comprehend proteins with minor changes to the sequence of the natural protein which do not affect the basic characteristics of the natural protein insofar as its ability to bind to TNF is
concerned and to thereby inhibit the binding of TNF to a natural TNF receptor in situ. The type of changes which are generally considered to fall within the "essentially corresponding to" language are those which would result from conventional mutagenesis techniques of the DNA encoding these proteins, resulting in a few minor modifications, and screening for the desired activity.
All of the monomers in the multimer may be TBP-I or a polypeptide essentially corresponding to TBP-I or they may all be TBP-II or a polypeptide essentially corresponding to TPB-II. Alternatively, the monomers of a given multimer may comprise a mixture of TBP-I and TBP-II monomers.
As indicated above, the term "multimer" is intended to include molecules including at least two of the defined monomers, which monomers may be linked by any of various methods. For example, the monomers may be chemically cross-linked by means of known linker molecules. Those of ordinary skill in the art will be able to determine the optimum length of any such linker molecules to produce multimers which best bind to the TNF trimer. Similarly, if the multimer is produced by recombinant techniques, the DNA which encodes each monomer may be linked in the manner well known for the production of fusion proteins so that the entire multimer will be encoded by a single DNA molecule which is present in a replicable expression vehicle in a manner to permit expression of the multimer in a transformant host cell, which host cell may be either prokaryotic or eukaryotic. Again, the nature of the amino acids which link the monomers in the recombinantly produced multimer is not critical and the optimum length of such linkers in such recombinantly produced proteins can also be determined by routine experimentation.
The term "multimer" is also intended to include pharmaceutically administrable aggregates of monomers having the ability to interfere with the binding of TNF to its receptors and to block the effects of TNF such as on the surface of liposomes. Thus, while it is preferable that the monomers be directly linked, they may be indirectly linked such as by being expressed on the surface of liposomes.
Pharmaceutical compositions containing the multimers of the soluble forms of the TNF-Rs may be employed for antagonizing the deleterious effects of TNF in mammals, i.e. they serve for treating conditions where excess of TNF is either endogenously formed or exogenously administered.
Such compositions comprise the multimers of the soluble forms of the TNF-Rs according to the invention, or their salts or functional derivatives as their active ingredient. The pharmaceutical compositions are indicated for any condition of excess TNF, either endogenously produced, such as in septic shock, cachexia, graft-versus-host reactions, autoimmune diseases such as rheumatoid arthritis, and the like, or exogenously administered, i.e. administration of overdoses of TNF.
As used herein the term "salts" refers to both salts of carboxyl groups and to acid addition salts of amino groups of the protein molecule. 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.
"Functional derivatives" as used herein covers derivatives 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 accept- 5 able, i.e. they do not destroy the activity of the protein and do not confer toxic properties on compositions containing it.
These derivatives may, for example, include aliphatic esters of the carboxyl groups, amides of the carboxyl groups by reaction with ammonia or with primary or secondary 10 amines, N-acyl derivatives of free amino acid groups of the amino acid residues formed with acyl moieties (e.g. alkanoyl or carboxylic aroyl groups) or O-acyl derivatives of free hydroxyl group (for example that of seryl or threonyl residues) formed with acyl moieties. 15
"Functional derivatives" also comprise multimers made up of soluble forms of TNF-Rs in which changes have been introduced in the sequence of the amino acids making up the soluble TNF-Rs by any conventional method. The sequence of any such changed soluble form of TNF-R must essentially 20 correspond to the sequence of the soluble form of TNF-R. It is understood that none of the above changes may affect the biological properties of the TNF-Rs.
The pharmaceutical compositions according to the inven- 25 tion 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, while in the case of arthritis, local injection may be indicated. The pharmaceutical composi- 3Q tions 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 3J 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, 40 as the case may be, and are prepared in dosage form, e.g. by lyophilization in dosage vials.
When the pharmaceutical composition comprises a liposome composition, the latter is adjusted so as to assure optimal interaction of the liposome with phagocytic cells, 45 optimal accessibility of the liposomes to the circulation and/or other compartments in the body, and optimal rates of clearance of the liposomes from those compartments.
BRIEF DESCRIPTION OF THE FIGURES 50
FIGS. 1A and IB: show the different receptor sizes after covalent cross-linking with labelled TNF, immunoprecipitation and SDS-PAGE analysis. The patterns shown are as follows: 55
FIG. 1A: precipitation with prior acidification so as to disrupt non-covalent association between receptors. Precipitation of receptors from HeLa cells (lane 1), precipitation from extracts of non-transfected A9 cells (lane 2), from extracts of A9 cells expressing the wild type human p55- go TNF-R (lane 3), and from extracts of A9 cells expressing mutants of the human p55-TNF-R: the A:310-426 human p55-TNF-R (lane 4), the A:244-^26 human p55-TNF-R (lane 5) and the A:215-426 human p55-TNF-R (lane 6). The lane marked Mr is the one of the molecular weight markers. 55
FIG. IB: shows the same receptor size analysis, however without acidification prior to immunoprecipitation.
FIG. 2 schematically illustrates the structure of the wild type human p55-TNF-R and of three truncated forms thereof, i.e. truncated at amino acid 310 (the A:310-426 human p55-TNF-R mutant), at amino acid 244 (the A:244-426 human p55-TNF-R mutant), and at amino acid 215 (the A:215-426 human p55-TNF-R mutant).
FIG. 3 A illustrates the cytocidal effects of TNF in A9 cells expressing the full length human p55-TNF-R, and in A9 cells expressing the cytoplasmic deletion mutants thereof (A:310-426, A:244-426 and A:215:426 human p55-TNF-R).
FIG. 3B illustrates the cytocidal effects of monoclonal antibodies against the human p55-TNF-R in the same cells as FIG. 3A.
FIGS. 4A, 4B and 4C show that treatment of A9 cells which express a cytoplasmic deletion mutant of the human p55-TNF-R antibodies to the receptor restores their sensitivity to the cytocidal effect of TNF.
The following examples illustrate the invention without limiting it thereto.
Detection of Aggregates of Human TNF-Rs in the
Analysis of their Sizes
A9 cells, as well as cells expressing the wild type and mutant forms of the human p55-TNF-R, and HeLa cells were detached by incubation in PBS containing 5 mM EDTA and, after rinsing with binding buffer, were suspended in aliquots of 5x10 cells in 1 ml binding medium, containing radiolabelled TNF. After incubation with occasional shaking for 4 hrs. on ice, the cells were washed once with Dulbecco's balanced salt solution (PBS+) and incubated for 20 min. in the same buffer containing 1 mM bis(sulfosuccinimidyl)suberate (Pierce). Cross-linking was stopped by adding TrisHC1 and glycine-HCl, pH 7.4 (both to a final concentration of 100 nM) followed by two washes with PBS+. The cells were then extracted for 1 hr. at 4° C, using 600 ul of a lysis buffer containing 20 mM Hepes, pH 7.4,150 mM NaCl, 1% deionised Triton X-100 1 ug/ml leupeptin and 1 mMphenylmethylsulfonyl fluoride. After centrifugation for 30 min at lO.OOOxg the cell extracts were divided into two equal portions. One (portion A) was acidified by adding 90 ul 1M glycine-HCl buffer pH 2.5 and, after 1 hr. incubation on ice, neutralized with 30 ul 1M NaOH. To this portion of the extracts as well as to the other one (B), monoclonal antibodies against the human p55-TNF-R were added. After 12 hrs further incubation at 4° C, 20 ul protein-A Sepharose beads (Pharmacia), equilibrated with PBS+. were added and, following 60 min incubation at 4° C, washed three times with the lysis buffer containing 2M KC1, and two times with PBS. The beads were resuspended in 15 ul sample buffer containing 4% (w/v) SDS and 6% (v/v) p-mercaptoethanol and boiled for 3 min. The superantant was analysed by SDS-PAGE (10% polyacrylamide) followed by autoradiography.
As shown in FIGS. 1A and IB, the receptors for TNF exist in aggregated forms in cells exposed to TNF. These figures present the SDS-PAGE analysis of the full-length and truncated forms of the human p55-TNF-Rs (see the schematic representation of the different forms in FIG. 2) expressed in murine A9 cells and tagged by applying the radio-labelled TNF on the cells followed by cross-linking.
The receptors were immunoprecipitated from detergent extracts of the cells following acidification of the extracts, in order to dissociate non-covalent aggregates of proteins (A) or without such acidification (B). The patterns of the labelled proteins, precipitated from extracts of HeLa cells (lane 1), from extracts of non-transfected A9 cells (lane 2), from