CA2075358C - Multimers of the soluble forms of tnf receptors, their preparation and pharmaceutical compositions containing them - Google Patents

Multimers of the soluble forms of tnf receptors, their preparation and pharmaceutical compositions containing them Download PDF

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CA2075358C
CA2075358C CA002075358A CA2075358A CA2075358C CA 2075358 C CA2075358 C CA 2075358C CA 002075358 A CA002075358 A CA 002075358A CA 2075358 A CA2075358 A CA 2075358A CA 2075358 C CA2075358 C CA 2075358C
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tnf
multimer
receptors
cells
soluble
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CA2075358A1 (en
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David Wallach
Cord Brakebusch
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Yeda Research and Development Co Ltd
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    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K16/00Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2866Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for cytokines, lymphokines, interferons
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00Drugs for disorders of the alimentary tract or the digestive system
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00Drugs for immunological or allergic disorders
    • A61P37/02Immunomodulators
    • A61P37/06Immunosuppressants, e.g. drugs for graft rejection
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00Drugs for disorders of the cardiovascular system
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    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K14/00Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52Cytokines; Lymphokines; Interferons
    • C07K14/525Tumour necrosis factor [TNF]
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K14/00Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/715Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
    • C07K14/7151Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons for tumor necrosis factor [TNF], for lymphotoxin [LT]
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00Medicinal preparations containing peptides
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    • C07KPEPTIDES
    • C07K2319/00Fusion polypeptide

Abstract

Multimers of the soluble forms of the tumor necrosis factor receptors (TNF-Rs) are provided. These multimers are produced either by chemical or by recombinant methods. The multimers of the soluble forms of TNF-Rs are useful for protecting mammals (including humans) from the deleterious effects of TNF.

Description

<,~ a f7 ''.'.' s~ j~, Ci ~a L~ ~ ar ,9 ;,1 The present invention relates to multimers of the soluble forms of the tumor necrosis factor receptors, their preparation and pharmaceutical compositions containing them.
Tumor necrosis factor (TNF) is a cytokine produced by a number of cell types, primarily by mononuclear phagocyte . At present, two different TNFs have been identified: TNF-a and TNF-D (lymphptoxin). Both TNF-a and TNF-~ initiate their effects by binding to specific cell surface receptors.
TNF-a and TNF-p (hereinafter called "TNF") are known to exert both beneficial as well as deleterious effects on a nutaber of different target cells involved in the inflaamatory response. Among its many effects, TNF, for example, stimulates the growth of fibroblasts and induces in these cells the synthesis of collagenase, prostaglandin B2 and IL-6. TNF also decreases in adipocytes the activity of lipoprotein lipase, activates osteoclasts and increases .in endothelial cells adhesivity for blood leukocytes.
However, TNT' has also extremely deleterious effects: over-production of TNF
can play a major pathogenic role in several diseases, for exampl~, 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 (cachexla) by suppressing activities ~~~J35~
of adipocytes and by causing anorexia (TNF-a was therefore called cash~ctin).
See, e.g. Beutler et al., Annu. Rev. Biochem., S7, pp. 507-518 (1988) and Old, Sci. Am, 258, pp. 4i-49 (1988). Excessive TNF production has also been demonstrated in patients with AIDS.
In order to counteract the cytotoxic effects of TNF, w.ys were sought to antagonize or eliminate endogenously formed or exogenously administered TNF.
Furthermore, ways are being sought to induce specifically only soae of the many effects of TNF or restrict its action to a specific kind of target cells. The first attempt in this direction was the development of monoclonal antibodies which neutralize the TNF-a cytotoxic activity. Such uonoclonal antib0~ies art described in Canadian S.N. 498,017 and in Israel Patent No.
73883.
As stated above, 'fNF initiates its function by binding to specific cell surface receptors. Two such TNF receptors (hereinafter "TNF-R«) which are expressed differentially in cells of different kinds are known, the pSS-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 TNF have bean shown to cross-react i~munologically with the two receptors. Both proteins provide protection against the In vitro cytocidal effect of TNF and~both bind TNF-B
less effectively than TNF-a. It was found that the formation of the TBPs occurs by proteolytic cleavage of the cell surface xNF-Rs, resulting in release of d mayor part of their extracellular domain (see Canadian S.N.
577;176, Canadian $.N.2,017,025 and Canadian S.N.2,032,191:!. 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, 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 TNP 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 a multimer of a soluble form of a tumor necrosis factor receptor (TNF-R), or a salt or functional derivative thereof, having the ability to interfere with the binding of TNF to its receptors and to block the effects of TNF, with the proviso that said multimer is other than two identical TBP-II molecules having amino acids 1-235 in two tandem repeats separated by a linker region. 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 molecule, or any other combination of monomers.
The multimers may either be in dimeric, trimeric or other multimeric form and ~~~'~~~~rV~
may comprise, for example, TBP-I, TBP-II, or etixtures 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 presont invention relates to DNA molecules cotaprising the nucleotide sequences encoding the multimers of the soluble forms of the TNF-Rs, to expression vehicles cotaprising them, to host cells transformed therewith, and to processes for producing the multimers by culturing the transformed cells in a suitable culture medium.
The inv6ntion also relates to DNA Molecules which hybridize to the above DNA
aolecules and which code for a multimer of the soluble forms of the TNF-Rs, or a fvntional derivative thereof. The term "functional derivatives" is defined hereinafter.
The multit~ers according to the invention, and the salts and functional derivatives thereof, naY comprise the active ingredient of pharmaceutical compositions for protecting mammals from the deleterious.effects of TNF.
These co~positions are yet another aspect of the present invention.
As stated hereinbefore, TNF exists and exerts its biological action as a trimer. However, nothing has been known so far as to the farm of the TNF-Rs to which TNF binds, 1.e. whether the TNF trimer binds to individual molecules of the TNF-Rs, or the receptors themselves also exist as multimers or become atultiners following TNF binding which better accomadates the TNF trimers.
a 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 G-terminal truncated forms of the human p55-TNF-Rs tagged by cross-linking to labelled TNF. Fox this purpose we produced truncated forims of the human p55-TNF-R by site-directed ~utagenesis of the cDNA and expressed them in murine A9 cells. Radiolabelled 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 hur~an receptors, examining thereby whether murine receptors associate noncovalenty with the human receptor as a consequence of aggregation of the receptors.
THP-1 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 hua~an body. The multimers of the soluble forms of TNF-Rs according to the invention, are believed to be more eff~ctive in inhibiting TNF activity at lower doses, since they Can effectively coMpete with the TNF trimers for the binding sftes on the aggregates of the cell surface TNF-Rs.
The ~ultimers 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 produclng the multimers of the soluble forms of the TNFwRs is by recombinant teChni9ues. In this way, massive production of multimers with optimal TNF binding activity will be made possible.

Pharmaceutical compositions containing the multimers of the soluble fvrtas of the TNF-Rs lay be eutployed for antagonizing the deleterious effects of THE 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 phar~aaceutical 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, yr exogenously administered, i.e.
administration of overdoses of TNF.
As used herein the term '°salts" refers to bath 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 niheral ~cids~
such as for example, hydrochloric acid or sulfuric acid, and .salts with organic acids such as, for example, acetic acid or oxalic arid.
"Functional derivatives" as used herein covers derivatives which stay 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, ~J '~
i.e. they do not destroy the activity of the protein and do not confer toxic properties on compositions containing it.
These derivatives ntay, for example, include aliphatic esters of the carboxyl groups, amides of the carboxyl groups by reaction with admonia or with primary or secondary amines, N-aryl derivatives of free amino acid groups of the amino acid residues formed with acyl tuoieties (e.g. alkanoyl or carboxyclic aroyl groups) or 0-aryl derivatives of free hydroxyl group (for example that of seryl or threonyl residues) formed with acyl moieties.
"Functional derivatives" also comprise multimers laade up of soluble farms of TNF-Rs in which changes have been introduced In the sequence of the amino acids making up the soluble TN1~-Rs by any conventional method. These changes may comprise elongation or truncation of the soluble TNF-R molecule or deletion ar replacement of one or more amino acids making up the soluble TNF-R. It is understood that none of the above changes txay affect the biological properties of the TNF-Rs.
The phars~aceutical compositions according to the invention are administered depending on the condition to be treated, via the accepted ways of adminfstratlon. For example, in the case of septic shock, intravenous administration will be preferred, while in the case of arthritis, local inflection stay be indicated. The pharmaceutical compasitions~ 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 ~r~p~n~d~ih the usual manner, for example by mixing the active ingredient with pharmaceutically and physiologically acceptai>le carriers and/ar stabilizers andlor excipients, as the case may be, and are prepared in dosage fore, e.g.
by lyophilization in dosage vials.
When the pharmaceutical composition comprises a liposome composition, the latter is ad3usted so as to assure optimal interaction of the liposom~ with phagocytic cells, 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.
~jg~ga 1A.__ and 1B: show the different receptor sizes after covalent cross-linking with labelled TNF, immunoprecipitation and SDS-PA6g analysis.
The patterns shown are as follows:
~g 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-TNF-R (lane 3), and from extracts of. A9 tails expressing mutants of the human p55-TNF-R:.~the x:310-426 human p55-TNF-R (lane 4), the ~:2G4-426 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 mol~cular weight markers.
shows the same receptor size analysis, however without acidification prior to inmunoprecipltation.

~,v~~ ~c~
schematically illustrates the structure of the wild type human p55-TNF-R and of three truncated forns thereof, i.e. truncated at amino acid 310 (the 4:310'426 human p55,TNF-R ~autant), at amino acid 244 (the d:244~-426 human p55-TNF-R mutant), and at amino acid 215 (the 4:215-426 human p55-TNF-R
~autant).
~,~,ure 3A illustrates the cytocidal effects of TNF in A9 cells expressing the full length hulttan p55°TNF-R, and in A9 cells expressing the cytoplasatic deletion mutants thereof 0:310-42b, .4:244-426 and A:215:426 human p55-TNF-R).
Fieure ~ illustrates the cytocidal effects of monoclonal antibodies against the human p55-TNF-R fn the same cells as Fig. 3A.
E~,E'~llCi'~~Ph 4.~.~ show that treatment of A9 cells which express a eytoplasnic deletion mutant of the human p55-TNF-R with antibodies to the receptor restores their sensitivity to the cytocidal effect of TNF.
The following examples illustrate the invention without limiting it thereto.
$gILWPLE 1: Ite ec i n of a~a$regat a of heean Tt~IF-Rs in the anal~rsis of their A9 cells, as well as cells expressing the wild type and mutant forges of the huaan p55-TNF-R, and HeLa cells were detached by incubation in P8S containing 5mM ~DTA and, after rinsing with binding buffer, were suspended in aliquots of 5X10' calls in 1 ml binding medium, containing radiolabelled THF. After incubation with occasional shaking far 4 hrs. on ice, the cells were washed once with Dulbecco's balanced salt solution (PHS+) and incubated for 20 min.
~3 in the same buffer containing 1 mM bis(sulfo~succinimidyl)suberate (Pierce).
Cross-linking was stopped by adding Tris-HC1 aind glycine-HC1, pH 7.4 (both to a final concentration of 100nM) followed by 'two washes with PHS+. The cells were then extracted far 1 hr. at 4°C, using 600 u1 of a lysis buffer containing 20 mM Hepes, pH 7.4, 150mM NaCI, 1~ demonised Triton X°100, 1ug/ml ieupeptin and 1 mM-phenylraethylsulfonyl fluoride. After centrifugation for 30 min, at 10.OOOxg the cell extracts were divided into two equal portions.
One (portion A) was acidified by adding 90u1 1 M glycine-HC1 bufer pH 2.5 and, after 1 hr. incubation on ics, neutralized with 30u1 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, 20p1 protein-A Sepharose beads (Pharmacia), equilibrated with PBS+, were added and, following b0 min. incubation at b°C, washed three times with the lysis buffer containing 2M KC1, and two times with PBS. The beads were resuspended in 15 u1 sataple buffer containing 4~ (w/v) SDS and 6%
(v/v) a-mercaptaethanol and boiled for 3 min. The supernatant was analysed by SDS-PAGg (10~ polyacryla~tide) followed by autoradiagraphy.
As shown in Figs. 1A and 1B, 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 forns in Fig. 2) expressed in murine A9 cells and tagged by applying radio-labelled TNF on the cells followed by . , cross-linking. .
The receptors were immunoprecipitated fram 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 "°°

r'~~.~'~~j'~
the labelled proteins, precipitated from extracts of HeLa cells (lane 1), from extracts of non-transfected A9 cells (lane 2), from extracts of A9 cells expressing the wild type human p55-TNF-R (3)u and from extracts of A9 cells expressing the ,4:310-426 human p55-TNF-R (4), the,d:244-426 human p55-TNF-R
(5) and the Q:215:426 human p55-TNF-R (6) mutants are shown in comparison to the migration of molecular weight markers (Mr). Labelled bands whose sizes correspond to the expressed human receptors, tagged by cross-linking to wither one or to two labelled TNF molecules, are denoted with solid arrows (sizes of 72 and 89 kD for the full length receptor, 59 and 76 kD for the 4:310-426 human p55-TNF-R, 51 and 68 kD for the b:244-426 human p55-TNF-R and 48 and 65 kD for the e:215:426 human p55-TPtF-R). The labelled bands whose sizes correspond to the full-length marine receptors, cross-linked either to one or to two TNF molecules t72 and 89 kD) are denoted with empty arrows, and the bands which correspond to cross-linked monomers, divers and tri~xers of TNF (17, 34 and 51 kiladaltons) - by stippled lines.
The antibodies applied for immunoprecipitation in this analysis of the receptor size, recognized specifically the receptors of human origin (solid arrows). These antibodies did not precipitate TNF receptors from extracts of non-transfected A9 cells (compare lanes 1 and 2). However, in application of these antibodies to extracts of the A9 cells which express the human p55-TNB-R, it was found that, together with the human receptors, tho antibodies precipitated also some of the murlne receptors, which~were easily distinguishable from the truncated human r~ceptors by their full length size (empty arrows) (lanes 3~6 fn Fig. 1B), implying that the TNF receptors exist in the cells as aggregates, containing both receptors of hupan and of marine origin. Consistently with this notion, it was found that if, prior to immunoprecipitation, the cell extracts were exposed to low pH, in order to )nyt ~"r 'fsrupt non-covalent association between the receptors, the human~~chf~d~s could still be precipitated. however, the precipitation of the murine receptors was abolished (coa~pare Fig. 1A to Fig. 18).
$BA~L~...~:
~ Construction of autant PAS--TNF-R~
The cDNA of the human TNF-RI (see EP application 90 12 4133.1) was cut with BanII(at nucleotides 218-222) and Nhel(at nucleotides 1723 and 1728), resulting in removal of large parts of the non-coding regions including an ATG in the S' non-coding region and a multiple GTn(n=4-S) in the 3' non-codfng region. Site directed mutagenesis of this shortened form of the cDNA
was carried out using the "Altered Sites" (ututagenesis) kit of Promega. Stop codons wera introduced in th~ foliowing points: After leucine 309 (mutant 4:310-426) using the oligonucleotide:
S'-CCC CAA CCC CCT CTA GAA GTG GGA GG-3' and after leucine 214 (mutant X1:215-426) using the oligonucleotide:
5'-AGT CCA AGC TCT AGA CCA TTG TTT GTG G-3' (Fig. 1). The wild type and mutated cDNAs were introduced to an sukaryotic expression vector. For the generation of the 4:244-426 mutant, the expression vector containing the wild type cDNA was cut with HindIII. The 3.9 Kb fragment was isolated and then, after fill-in of the protruding ends, religated, thus replacing amino acid 244 by a stop codon.
Cells o~ murine A9, 1,929, NIH3T3 and the hamster BHK lines were cultured with Dulbecco's Minimal Essential Medium (DMEM), containing 10~ fetal calf serum, 100 units/ml penicillin and 100ug/ml streptomycin. The expression constructs encoding the wild type and the mutant receptors were cotransfected together ~~,~~~~ f.i:~>~3 with the neomycin resistance conferring plamsid pSU2neo into these cells, using the calciu~t phosphata precipitation method. After 10 to 14 days selection in growth medium containing 540ug/ml 6418, resistant colonies were isolated and checked for expression of the human p55 TNF-R by ~aeasuring TNF
binding to the cells.
Figure 2 illustrates the structure of the wild type huatan p55-TNF-receptor.
It consists of icons depicting the full length human p55-TNF-R and the truncated forms of this receptor created by site-directed nutagenesis. Using these oligonucleotides, stop colons were introduced into the intracellular domain of the receptor at amino acids 310 (the Q:310-426 hurian p55-TNF-R
mutant), 244 (the 4:244-426 human p55-TNF-R mutant), and 21S (the A:215:426 hu~an p55-TNF-R mutant).
Figs. 3 and 4 provide further evidence for the existence of the cell-surface TNF-R as aggregates as well as fox two other points, namely that aggregation of functional receptors is necessary for the activity of these receptors, as well as that fnvolveaent of non-functional receptors in this aggregation results fn effective inhibition of TNF function.
Fig. 3 illustrates the cytocidal effects of TNF (A) and of teonoclbnal antibodies against the hunan pS5-TNF-R (E) in A9 cells, in A9 cells expressing the full length human p55-TNF-R, and in A9 cells expressing the cytoplaslnic deletion mutants of the human p55-TNF-R (the :310-426 human p55-TNF-R, the :244-42b human p55-TNF-R, and the :215:426 human p55-TNF-R.
Cells were seeded into 96-well plates, 24 hrs. before the assay, at a density of 30,000 cells/well. TNF and the monoclonal antibodies, were applied simultaneously with CHl (50 ug/rol). After further 1i hrs, incubation at 37°C, viability of the cells was assessed in a neutral red uptake assay. The two monoclonal antibodies were applied at equal axounts which summed up to the concentration specified in the figurR.
Turning now to Fig. 4, the cytocidal effect of TNF, applied together with CHI
(50 ug/nl) to A9 cells (A), A9 cells expressing the X1:910-426 hudan p55-TNF-R
(H) and A9 cells expressing the 4:215:426 human p55-TNF-R (C), was examined in the presence and absence of antibodies to the human p55-TNF-R. Similar sensitization by the antibodies to the cytocidal effect of TNF was observed in A9 cells expressing the A:244-426 human p55-TNF-R mutant.
Thus, while having a pronounced cytocidal effect in the A9 cells which express the full--length human p55-TNF-R, the antibodies had no effect at all in cells expressing any of the three truneated forms of the receptor, suggesting that these truncated forms are not functional. Furthermore, testing the effect of TNF itself on the cells revealed that, in contrast to the full-length human p55-TNF-R receptors, whose expression results in increased sensitivity of the A9 Ce115 to TNF, the truncated forms of the receptors conveyed a decreased reponsiveness to the cytocidal effect of TNF.
This decrease Could be observed in clones of A9 cells, expressing either one of the truncated forms of the receptors, at extents which seemed roughly , , proportional to the extent of receptor expression. Sensitivity to TNF could be recovered by applying on these cells antibodies to the h,p55-fiNF-R, confirming that the decrease in response to TNF reflects an inhibitory effect of the truncated human receptors on the function of the full length rodent receptors.
14 .--,.

a) Introduction of cystelne or, alternatively, of Biotin, to the C-termini of the soluble receptars_ P~rS,~~ure I: Samples of the soluble forms of either the human p55- or the human p75-TNh-R are incubated with cysteine amide, or alternatively, with Biotin auide V I
Biotin - C - NH(CHa).~ - CH - CONHz and with carboxypeptfdase X. In the presence of excoss of the cysteine amide or the Biotfn amide the enzya~atic reaction leads primarily to incorporation to these compounds to the C-termini of the soluble receptors.
Procedure II: The reactions ara carried out as above, except that the amino acid. within the receptor chosen to be the C-tera~inal for~the soluble receptors is lysine and that lysine endopeptidase is applied instead of carboxypeptidase. Use of this enzy~te assures that, beside rearoval of a single amino acid frost the C-ternihus of the soluble receptor, no further truncation of the soluble receptor takes place.

~~r~5~~~
b) Use of soluble receptors, to whose C-teraini cysteine was introduced, for foraation of diners of the soluble receptor.c:
Receptors to whose C-termini cysteins were introduced as described above, are cross-linked to form dimers, using activated linkers of either one of the following two fornulae;
,~,0 0 0 I ~ - NH - CHz - CHs - CO[NH-CH-C]., - NH - N

~0 R 0°' l1 ti II Br - CHm - C [NH-CH-C)" - NH ~ C - CHs - Br R
Linkers of different lengths can be employed. The function of the products will be conpared to each other and thus the optional linker length will b~
defined. In case free cysteines turn out to ba present within the soluble receptors, they will be blocked before the introduction of cystelne to'the C-termini, by way of alkylation.
c) Use of soluble receptors, to whose C-teraini Biotin was introduced, for forsation of tetraaers of the soluble receptors, as ~aell as wore coaplex structures of the receptors:
The soluble receptors to whose C-tera~ini Biotin was introduced, as described above, are cross-linked wfth Avidin. Each Avidin contains four is ,~""

f ~., i Biotin binding sites. Tn that way tetra~ers of the receptors ~f~~
S~~f9t°e~.
Alternatively, part of the four binding sites in the Avidfn serve for bihdihg other proteins to which Biotin was linked. Tn that way, several Avidity molecules are linked to each other, resulting in formation of higher aggregates of the soluble forms of the human TNF--Rs. This approach allaws also the binding of additional molecules to the soluble forms of the TNF--Rs (e. g. Fc portions of antibodies).
Alternatively, Avidity may be replaced with Streptavidin, the procedure itself remaining the same.
d) Foraation of tultit~ers of the soluble receptors by cross-linking cysteins located within the receptors' sequence:
Sueh multimers of 'fNF receptors are formed by linking the molecules of soluble forms of the TNg-Rs to each other using cross-linking. reagents specific to amine groups or to thiols. (The latter following partial reduction of the cysteins in the soluble receptors with a-mercapthoethanol or dithiothreitol).
e) Foraation of liposot~es containing wultiple molecules of the truncated receptors: . -.
Procedure I: Liposomes expressing any Biotin-containing protein on their surface can be applied fpr that purpose. Soluble forms of the TPdF-Rs to whose C-tera~ini Biotin was linked, are finked to these liposoaes with the use of (the tetravalent) Avidity.
p~~g~.: Reco~abinant TNF receptors, truncated in the intracellular domains, but containing the transmembrane dot~ains, are produced as described in Rxampie 2. They are then incorporated into l~p~s~~e ~ y'a dissociation in detergents, fo3lowed by their reconstitution in the presence of lipids by removal of the detergs~nts.
f) Isolation of soluble receptor multitrers c~it:h highest inhibitory effect on 1°HF functions Preparations of soluble receptor diners or taultiaers are fractionated chromatographically, and fractions showing highest inhibitory effects on TNF function are further analyzed, to define the structures optimal for TNF inhibition. Alternatively, affinity purification of the optimal inhibitors is perforated, by applying preparations of soluble receptor dimt3rs or multimers to TNF-affinity columns.
RH~MPi R 4' Creation of recombinant TiriA ,oj~e~ules crn risine nuc eotide The diners and higher muititners of the soluble receptors 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 diners or oligomers. These DNA molecules can be genonic DNA, cDNA, synthetic DNA and a cotabfnation thereof.
Creation of DNA molecules coding for a diner of soluble receptors is carried ,.
out by . ligating the cDNA sequence encoding the soluble form of a TNF-R to a sequence encoding a linker peptide and then further to a synthetic polynucleotide encoding the translation of the soluble receptor in the reverse orientation - from the original C-terminus to the N-terminus and lacking the sequence of the leader peptide.
18 """'"
.. . - , .

lP v a Polymeric forns of the soluble receptors can be created, as mentioned above, by fortaing recombinant truncated receptors, deficient of intrac~llular domains but containing transmembrane domains, and incorporating theft, by membrane reconstitution techniques, into liposomes.
lixpression of the rgcotabinant proteins can be effected in eukaryotic cells, bacteria or yeasts, using the appropriate expression vectors. Any method known in the art ~aay be employed.
For ~xample, the DNA Molecules coding for the multimers of the soluble forms of the TNF-Rs obtained by the above methods are inserted into appropriately constructed expression vectors by techniques well known in the art (sse Maniatis et al., ). Double-stranded cDNA is linked to plasnid vectors by homapolyMgric 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 treatsaent with alkaline phosphatase.
In order to be capable of expressing a desired protein, an expression vector should comprise also specific nucleotide sequences containing .I
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 transariptinn prncesx. There are a variety of such promoters in use, which work with different efficiencies (strong and weak promoters). They are Y

different for prokaryotic and eukaryatic cells.
~~~~~a~~
The promoters that can be used in the present invention may be either constitutive, for example, the i1~ promoter of bacteriophage ~, the bla promoter of the m~lactamase gene of pBR322, and the CAT promoter of the chlora~nphenicol acetyl transferase gene of pPR325, etc., or inducible, such as the prokaryotic promoters including the mayor right and left promoters of bacteriophage ~ (P~ and Pse), the gyp, recA, l,~s~Z.. l~s.~., omnF
and g~ promoters of E.E, coil, or the tro~lac hybrid promoter, etc. (Glick, B.R. (l9By) J. Ind. Microbiol. 1:277-282).
Fesides the use of strong praraoters 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 sequence (5D
sequence) appropriately positioned from the initiation codon and complementary to the 3'-terminal sequence of I6S RNA.
For eukaryotic hosts, different transcriptional and translational regulatory sequences may be employed, depending an the nature of the host.
They may be derived fram 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 ANA molecule comprising the nucleotide sequence codi ~ or the TNF
multimera of the invention tend the operably linked transcriptional and translational regulatory signals is inserted into a vector which is capable of integrating the desired gone sequences iota the host cell chromosome. The cells which have stably integrated the introduced DNA into their chromosoz~es can ba eoloctad by also introducing; one or yore ae~rker~ which bllow For selection of host calls which contain the expression vector. The ~arker may provide for prototrophy to an auxotropic host, bioctde resistance, e.g., antibiotics, or heavy d~etals, such as copper, or the like. The selectable narker gene can either be directly linked to th~ DNA gene sequences to be expressed, or introduced into the same cell by co~transfection. Additional elenents nay al.rc ha naA~ta~l for cptin°al syntho~i~ 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 Okayana, H., (1983) ilol. Cel. Bfol. x:280.
In a preferred embodiment, the introduced DNA molecule will be incorporated into a plasmid or viral vector Capable of autonoa~ous replication in the recipient host. Factors of importance in selecting a particular plesmid or viral vgctor~ 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 wh~ther it is desirable to be able to °"shuttle°' the vector between host cells of different spacies.
Preferred prokaryotic vectors include plasmids such as those capable of replication in ~ cell, for exa~ple, pBR322, ColBl, pSC101, pACYC 18h, etc.

b (see Maniati& et al., Molecular -Craning: ~ j,~~gratorv Manual, op.cit.);
Bacillus plasmids such as pCl9G, pC221, pT127, etc. (Gryczan, T., Thg IN,olecular Biolog,K of t4~,0 ~C~ 1111., Acadanic Press, NY (1982), pp. 307-329);
Streptonyces plas>ittds including pIJ101 (Kendall, K.J. et al., (19$7) J.
Bacterioi. ]~Q:4177-4183); Streptomyces bacteriophages such as ~C31 (Chater, K.F. et al., in: Sixth LI1W patio ~1 Svia~osiuiQ on nt~;.~inotnycetales BioloEV, Akademiai Kaido, Budapest, Hungary (1986), pp. 45-54), and Pseudomonas piasmids (John, J.F., et al. (1986) Rev. Infect. Dis. x:693-704), and Izaki, R. (1978) Jpn. J. Bacteriol. x.:729-742).
Preferred eukaryptic piasmfds include BPV, vaccinia, SV40, 2-micron circle, etc., or th9ir derivatives. Such plasmids are well known in the art (Botstein, DY, et al. (1982) tRiaai Wint. Symp. x:265-274; Broach, J.R., in:
Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, pp. 445-a70 (1981);
Broach, J.R., (1982) Cell 2~:2p3-204; Hollon, D.P., et al. (1980) J. Clip.
Hematol. Oncol. ~.Q,:39-48; Maniatis, T,, in:.Cei1 Biology: A Cp,~pre ,give Treati;~0~, Vol. 3: Gene $~grQ~gion, Academic Press, NY, pp. 563-608 (1980)).
Once the vector or DNA sequence containing the constructs) has been prepared for' expression, the DNA constructs) may be introduced into an appropriate host cell by any of a variety of suitable neaps: transformation, transfection, conJuBation, protoplast fusion, electroporation, calciun . , phosphate-precipitation, direct microin,iecti~n, etc.
Host cells to be used in this invention may be either prokaryotic or eukaryotic. Preferred prokaryotic hosts include bacteria such as K. cola, Ea~ctllua, DtrC~hrW y~G~rs, Ps~d~vrras, 3a3artrrrelW , Serratla, etc. Ttte most 22 '"'-Y

~,~'~ i~=~r preferred prokaryotic host is 1(, coil, Bacterial hosts of particular interest include l(12 strain 294 (ATCC 31446), g177g (ATCC
3153'7), ~ w3110 (F-, larabda-, prototroplc (ATCC 27325)), and other enterobacteriun~ such as Salmonella typhimurtum or Serratia ararcescens and various Pseudotronas species. Under such conditions, the protein will not be glycosylated. The prokaryotic host must be cot~patible with the replicon and control sequences in the expression plasmid.
Preferred eukaryotic hosts are ntamrtalian cells, e.g., human, monkey, house and Chinese har~ster ovary (CHO) sells, 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 nub~ber of recombinant Dt3A strategies exist which utilize strong promoter sequences and high copy tlu~lber of plasa~ids which can be utilized fox 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, fixpression of the cloned gene sequences) results in the production of the desired sultinters~of the soluble forms of the TNF-Rs.
Purification of the recombinant proteins is carried out using monoclonal antibodies to the soluble forms of the TNF-Rs, or by affinity purification on ligand (TNF) columns.
P~

Claims (16)

1. A multimer of a soluble form of a tumor necrosis factor receptor (TNF-R), or a salt or functional derivative thereof, having the ability to interfere with the binding of TNF to its receptors and to block the effects of TNF, with the proviso that said multimer is other than two identical TBP-II molecules having amino acids 1-235 in two tandem repeats separated by a linker region.
2. A multimer according to claim 1 in dimeric form.
3. A multimer according to claim 1 in trimeric form.
4. A multimer according to any one of claims 1 to 3, wherein the multimer is comprised by a liposome.
5. A multimer according to any one of claims 1 to 4 comprising TBP-I.
6. A multimer according to any one of claims 1 to 4 comprising TBP-II.
7. A multimer according to any one of clams 1 to 4 comprising a mixture of TBP-I and TBP-II.
8. A process for the production of a multimer of a soluble form of a tumor necrosis factor receptor (TNF-R) comprising chemically cross-linking the C-termini of the soluble forms of TNF-R.
9. A process according to claim 8, wherein the cross-linking is effected by introduction of cysteine amide to the C-terminus of the soluble form of the TNF-R, followed by cross-linking with an activated linker.
10. A process according to claim 9, wherein Biotin amide is employed instead of cysteine amide.
11. A process according to claim 9 or claim 10; wherein the activated cross-linker is of the formula
12. A process according to claim 9 or claim 10, wherein the activated cross-linker is of the formula:
13. A process according to claim 8, wherein cross-linking is effected by introduction of Biotin to the C-terminus followed by cross-linking with Avidin.
14. A process according to claim 13, wherein Streptavidin is employed instead of Avidin.
15. A pharmaceutical composition comprising a multimer of a soluble form of a tumor necrosis factor receptor (TNF-R), a salt or a functional derivative thereof, together with a pharmaceutically acceptable carrier or excipient or stabilizer, with the proviso that said multimer is other then two identical TBP-II molecules having amino acids 1-235 in two tandem repeats separated by a linker region.
16. A pharmaceutical composition according to claim 15, comprising liposomes containing multiple molecules of soluble TNF-R.
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