Cut off soluble tumour necrosis factor receptors of i and ii type

The invention also relates to a truncated sTNFR which is a recombinant expression product of a prokaryotic or eukaryotic host cell as defined above.

The invention also includes a process for the preparation of a pharmaceutical composition containing an active ingredient and a carrier and optional excipients, which comprises using a truncated sTNFR as defined above and / or a multivalent TNF binding protein (TNFbp) as defined above and a therapeutically effective amount of the active ingredient indicated. mixed with one or more pharmaceutically acceptable carriers.

189 309

The pharmaceutical composition according to the invention, comprising the active ingredient and the carrier and optional excipients, is characterized in that it comprises a truncated sTNFR as defined above as active ingredient in association with a pharmaceutically acceptable carrier.

Alternatively, a pharmaceutical composition comprising the active ingredient and a carrier or optional excipients according to the invention is characterized in that it comprises as active ingredient the multivalent TNF binding protein (TNFbp) as defined above in combination with a pharmaceutically acceptable carrier.

Preferably, the composition according to the invention comprises a composition ensuring the sustained release of the active ingredient. Preferably, the composition according to the invention is also lyophilisovun.

The invention also encompasses the use of a truncated sTNFR as defined above or the polyvalent TNF binding protein (TNFbp) as defined above for the manufacture of a medicament for the treatment of tumor necrosis factor mediated diseases - TNF.

According to the invention, a truncated sTNFR as defined above, or a polyurethral TNF binding protein (TNFbp) as defined above, or a pharmaceutical composition as defined above, find use in the preparation of a medicament for the treatment of diabetes, hyperalgesia, inflammatory bowel disease, ischemic injury, reperfusion injury or disease. rheumatic. The rheumatic disease is selected from the group consisting of rheumatoid arthritis, osteoarthritis, juvenile (rheumatoid) arthritis, ankylosing spondylitis, dermatomyositis, psoriatic arthritis, scleroderma, Sjogren's syndrome or vasculitis.

According to the use of the invention, the medicament prepared mu is in a form suitable for intravenous, intramuscular, intradermal, subcutaneous, intra-articular or infusion administration. Preferably, the prepared composition is in a form suitable for administration before, after or simultaneously with the administration of an anti-inflammatory agent.

In the use of the invention, the anti-inflammatory agent is selected from the group consisting of non-steroidal anti-inflammatory drugs (NSAIDs), corticosteroids, slow-acting anti-rheumatic drugs (SAARDs), or disease-modifying drugs (DMs). The anti-inflammatory agent may also be an interleukin-1 (IL-1) inhibitor selected from the group consisting of an IL-1 receptor antagonist (IL-1ru) and a soluble IL-1 receptor. The preferred anti-inflammatory agent is methotrexate.

According to the invention, the pharmaceutical composition may additionally contain an anti-inflammatory agent. Preferably, the anti-inflammatory agent is selected from the group consisting of non-steroidal anti-inflammatory drugs (NSAIDs), corticosteroids, slow-acting anti-rheumatic drugs (SAARDs) or disease modifying drugs (DMs), especially methotrexate.

The anti-inflammatory agent may also be an interleukin-1 (IL-1) inhibitor selected from the group consisting of an IL-1 receptor antagonist (IL-1ru) and a soluble IL-1 receptor. Preferably, the ŻL-1ra comprises the sequence of human IL-1ra.

The present invention relates to functionally active truncated forms of sTNFR-I, which are referred to herein as "truncated sTNFR" or "truncated sTNFRs". Truncated sTNFRs are modified forms of sTNFR-I that lack the fourth domain (Thr¹²⁷-Asn¹⁶¹ in sTNFR-I); portions of the third domain (Asn¹¹¹-Cys * 2⁶ in sTNFR-I) oruz possibly lack part of the first domain (Asp1-Cys1 amino acid residues in sTNFR-I). These new TNF inhibitors (for example TNF-α and / or TNF-β) have general applicability.

Truncated sTNFRs according to the invention include proteins represented by the formula R1- [Cys-Cys1 ° 3] -R₂. These proteins are truncated forms of sTNFR-I.

By the term "R1- [Cys-Cys^1<)3] -R2 "is meant one or more proteins in which [Cys19-Cys¹⁽⁾3] is residues 19 to 103 in sTNFR-I; an amino residue numbering scheme is given in Figure 1 (SEQ ID NO: 2) to facilitate comparison, where R1 and R₂have the meaning defined above.

In one aspect of the present invention, truncated sTNFRs can be made in glycosylated or non-glycosylated forms. Truncated sTNFRs are produced

189 309 recombinant genetic engineering techniques. In an alternative embodiment, truncated sTNFRs are synthesized by chemical or mixed methods: eecombinant and chemical.

In another aspect of the present invention, truncated sTNFRs can be derivatized by attaching truncated sTNFRs to a water-soluble polymer. For example, truncated sTNFRs can be conjugated to one or more polyethylene glycol molecules to improve pharmacological properties by increasing the apparent molecular weight of that molecule.

Yet another aspect of the present invention includes various polynucleotides encoding truncated sTNFRs. Suitable nucleic acid sequences include, for example, those specifically depicted in the accompanying drawings, as well as degenerate sequences and naturally occurring allelic variants thereof. Such nucleic acid sequences can be used to express truncated sTNFRs in eukaryotic or prokaryotic host cells, the expression products or their derivatives having the ability to modulate TNF activity.

A further aspect of the present invention includes vectors containing uolactylides encoding truncated sTNFRs operably linked to amplification and / or expression control sequences. Both peokaryotic and eukaryotic host cells can be stably transformed or transformed with such vectors for the expression of truncated sTNFRs. The present invention further comprises the fetal production of truncated sTNFRs, whereby host cells containing such pclincclcctides are grown in a suitable culture medium and the truncated sTNFRs expressed in these cells are, optionally, isolated from the host cells and / luC from the culture medium.

Another aspect of the present invention includes pharmaceutical compositions containing truncated sTNFRs or derivatives thereof. Typically truncated sTNFRs or derivatives thereof can be formulated in conjunction with pharmaceutically acceptable excipients. A variety of other excipients can be used to facilitate the manufacture, storage, handling, delivery and i / luC efficacy of truncated sTNFRs or their derivatives.

The medicaments produced in accordance with the invention serve to modulate TNF activity, in particular TNF-mediated diseases (for example TNF-α and / or TNF-β mediated diseases) can be treated by administering to the patient therapeutically effective amounts of truncated sTNFRs or derivatives thereof.

Polinclossotides encoding truncated sTNFRs can also be used in cell therapy or in gene therapy.

Truncated sTNFRs according to the invention are particularly suitable for large-scale production of protein amounts. For example, sTNFR-I deamidation takes place at the oCreC of the amino acid sequence 111 to 126 (amino acids Asn^at -Gly¹²⁶). It can be expected that the inactivity of this site will increase the chirophilic stability of the purified protein, reducing possible degradation products and leading to more shelf-stable proteins. Truncated sTNFRs have fewer bilayer bridges than previously disclosed TNF inhibitor proteins. For example, sTNFR-I has two double-sided bridges in the α-amino acid sequence 111 through 126, and three double-sided bridges in the α-acid sequence 127 through 161. The reduced number of two-way bridges is important because larger numbers of these junctions can complicate the protein refolding process. Surprisingly, truncated sTNFRs have fewer sites for antigenic epitopes than other TNF inhibitor proteins previously disclosed (for example, a truncated form of sTNFRI having the first three domains, has an eo-sleeping cluster caused by the display of certain residues; see Example III), leading to relatively reduced antigenicity and does not give a significant reduction in kewikkCisgu removal rate with repeated administration. It can be expected that the reduced immknkgenicity of truncated sTNFRs is suitable for the treatment of TNF mediated diseases, including in particular chronic inflammatory diseases.

Further aspects and advantages of this invention will be apparent to those skilled in the art from reading the following description, which details the practice of the present invention.

i6 i89 309

Many aspects and advantages of the present invention will be provided by an overview of the drawings in which:

Figure 1 shows a nucleic acid sequence (SEQ ID NO: 1) encoding Asp¹-Asni6i, full-length recombinant human sTNFR-I. The amino acid sequence (SEQ ID NO: 2) for Asp -Asn61 is also shown.

Figure 2 shows the nucleic acid sequence (SEQ ID NO: 3) encoding NH2 -MDSVCPQGKYIHPQNNSIC- [Cys-Cysi °³] -FC-COOH (also referred to as sTNFR-I 2.6D / C105). Also shown is the amino acid sequence (SEQ ID NO: 4) NH2-MDSVCPQGKYIHPQNNSIC- [Cysⁱ⁹-Cys¹⁽⁾3] -FC-COOH.

Figure 3 shows the nucleic acid sequence (SEQ ID NO: 5) encoding NH₂-MDSVCPQGKYIHPQNNSIC- [Cys-Cys^AND03] -FNCSL-COOH (also referred to as sTNFR-I 2.6D / C106). Also shown is the amino acid sequence (SEQ ID NO: 6) NH2-MDSVCPQGKYIHPQNNSIC- [Cysi⁹-Cys1⁽⁾3] -FNCSL-COOH.

Figure 4 shows the nucleic acid sequence (SEQ ID NO: 7) encoding NH2 MDSVCPQGKYTHPQNNSIC- [Cys-Cysi ° 3] -FN-COOH (also referred to as sTNFR-I 2.6D / N105). Also shown is the amino acid sequence (SEQ ID NO: 8) NH2-MDSVCPQGKYIHPQNNSIC- [Cysi9-Cysi ° 3] -FN-COOH.

Figure 5 shows the nucleic acid sequence (SEQ ID NO: 11) encoding NH₂ MYIHPQNNSIC- [Cysi9-Cysi ° 3] - ^ CSL-COOH (also referred to as sTNFR-l 2.3D / d8). The amino acid sequence (SEQ ID NO: 12) of NH is also shown₂- NH₂-MYIHPQNNSIC- [Cysi9-Cysi °³] -FNCSL-COOH.

Figure 6 shows the nucleic acid sequence (SEQ ID NO: 9) encoding NH₂-M- [Cysi9-Cysi ° 3] -FNCSL-COOH (also referred to as sTNFR-I 2.3D / d18). Also shown is the amino acid sequence (SEQ ID NO: 10) NHrMjCys ^ -Cys ^ J-FNCSL-COOH.

Figure 7 shows the nucleic acid sequence (SEQ ID NO: 13) encoding NH₂-MSIS- [Cysi9-Cys^W3] -FNCSL-COOH (also referred to as sTNFR-I 2.3D / d15). Also shown is the amino acid sequence (SEQ ID NO: 14) NH2-MSIS- [Cys-Cys^l° 3] -FNCSL-COOH.

Figure 8 shows the nucleic acid sequence (SEQ ID NO: 34) encoding Leu1-Thβ⁷⁹, mature recombinant human sTNFR-II. The amino acid sequence (SEQ iD NO: 35) of Leu1-Thr is also shown^r79.

Figure 9 shows the amount of swelling induced in the streptococcal cell wall-induced reactivation model described in Example II.

Figure 10 shows the plasma profiles of sTNFR-I 4D / C105db in healthy baboons after a two-minute intravenous infusion of 0.2 mg / kg as described in Example IlI.

Figure 11 shows the plasma profiles of sTNFR-I 3D / C105db in healthy baboons after a two-minute intravenous infusion of 0.2 mg / kg as described in Example III.

Figure 12 shows the plasma profiles of sTNFR-Ι 2.6D / C105db in healthy baboons after a two-minute intravenous infusion of 0.2 mg / kg as described in Example III.

Figure 13 shows the dose-clearance relationship of various dimeric sTNFR-1 constructs as described in Example III.

The present invention is based on the unexpected discovery that sTNFR-1 (and also sTNFR-II) can be reduced in size so as to exclude not only the fourth domain but also part of the third domain and possibly part of the first domain while retaining biological activity and achieving reduced antigenicity. For at least the following reasons, it is considered advantageous to produce biologically active truncated sTNFRs or derivatives thereof. First, these molecules may have one less destabilizing deamidation site. Second, these molecules have fewer disulfide bridges, potentially making reassembly and purification easier. Third, these molecules have reduced sites for potential antigenic epitopes.

The term "truncated sTNFR (s)" as used herein includes one or more biologically active synthetic or recombinant molecules of the formula R1- [Cys19-Cysi⁰³] -R₂ and variants thereof (including variants resulting from insertion, substitution, and deletion), as described below.

189 309

The term "biologically active" as used in this application means that a truncated sTNFR exhibits similar TNF inhibitory properties, but not necessarily all of these properties, and not necessarily to the same extent as sTNFR-I. In general, truncated sTNFRs and their derivatives have the ability to inhibit TNF. Biotests for truncated sTNFRs are further described in Example II, below. The choice of the specific TNF inhibition properties considered depends on the desired application of the truncated sTNFR.

In one aspect of the present invention, truncated sTNFRs can advantageously be produced by recombinant techniques in bacterial, mammalian or insect cell systems and can be in the form of either glycosylated or non-glycosylated proteins. Alternatively, truncated sTNFRs can be produced by chemical synthesis. The production methods currently considered to be preferred are described in more detail below.

Each of the truncated sTNFRs can typically be isolated and purified to be substantially free of the presence of other protein-like materials (i.e., uncut sTNFRs). Preferably, the truncated sTNFR is about 8% devoid of other proteins which may be present due to the production technique used to produce a truncated sTNFR. More preferably, the truncated sTNFR is about 90% free from other proteins, particularly about 95% free from other proteins and most preferably about> 98% free from other proteins. It should be understood, however, that the protein may be combined with other active ingredients, chemical compositions and / or suitable pharmaceutical substances in galenical forms prior to its administration, as described in more detail below.

Truncated sTNFRs

In one basic embodiment, truncated sTNFRs of the invention may be one or more proteins represented by the following formula

MCyS1 -Cys6 where [Cys -Cys 3 represents residues 19 to 103 in TNFR-1, an amino acid residue numbering scheme is given in Figure 1 (SEQ ID NO: 2) for ease of comparison; and wherein R1 is NH₂-MdSvCPQGKyIHPQNNSIC, on R.₂ represents the group -FN-COOH or variants and derivatives thereof

Another aspect of the present invention includes one or more variants of R1- [Cys1⁹-Cys¹⁰3] -R.2, in the methionyl or non-methionyl form. The term "truncated sTNFR (s)" thus includes one or more naturally occurring allelic species R1- [Cys-Cys^I03 ] -R₂ and one or more variant proteins in which the amino acids have been removed ("deletion variants") into which ("addition variants") have been inserted ("addition variants") or in which residues within the amino acid sequences of R1- [Cys¹9-Cys103] -R2.

Amino acid sequence deletions typically include about 20 amino acid residues, especially from about 1 to 10, most typically from about 1 to 5 residues, so as not to disrupt protein folding. Deletions include N-terminal, C-terminal or intra-sequence deletions. The total number of deletions and / or uninterrupted deletions are chosen so as to maintain the tertiary structure of the protein in the respective domain, for example cysteine ​​cross-linking.

Deletions within the R1- uminoquus sequence [Cys1⁹-Cys1 ° 3] -R2 can be made in regions of low homology with the sequences of other members of the NGF / TNF receptor family in the group of cell surface proteins. Deletions within the Rl-lCys amino acid sequence¹9-Cys-⁰3] -R2 can be made in regions of substantial homology with the sequences of other members of the NGF / TNF receptor family and is likely to significantly modify the biological activity. In particular, sequence similarity among members of the NGF / TNF receptor family is particularly high in the region corresponding to the first two disulfide loops of domain 1, the entire domain 2, and the first loop of disulfide domain 3 (Banner et al. (1993), Cell, 73: 431-445. ). For example, two copies of the R1- [Cys'9-Cys1 deletion variants^<)3 *] - R2 are of the form Rl-lCysl8 (ΔThr20-Cys ^^ oruz R ^^ s ^^ Cys ^ Lys²1) -Cys1⁰3] -R₂wherein R1 and R2 are as defined above.

Amino acid sequence additions can include amino and / or carboxyl terminus fusions ranging in length from one to one hundred or more residues, including

189 309 internal intra-sequence insertions of single or amino skim mareswaves. Internal additions can typically be from about 1 to 10 amido-amide residues, more typically from about 1 to 5 amido-amido residues, and most typically from about 1 to 3 amido macromacin residues.

The amide-addition variants include addition of methionine (e.g., as an out-of-control effect of direct protein oxidation in bacterial cell culture) or additional cost or amino acid sequence. A further example of amino-terminal insertion includes the fusion of a signal sequence as well as / or other pre-pro sequences to facilitate protein secretion from tecombidation host cells. In the case of prokaryotic host cells that do not disassemble and process the native sTNFR-I signal sequences, the signal sequence may be replaced by a ptoparyotic signal sequence selected, for example, from the group of alkaline phosphatase, ppnicylidary or thermally stable pdtprotoxin-II leaders. In the case of yeast cells, the signal sequence may be selected, for example, from the group of yeast invertase leader, alpha factor or maca phosphatase. When expressed in mammalian cells, the native sTNFR-I signaling modalities (EP 393 438 and EP 422 339) are satisfactory, although there may be other mammalian signal sequences, respectively (for example, sequences derived from other members of the NGF / TNF receptor family.

Carboxy terminally aadition variations involve the addition of one or more amino acid residues that would lead to sTNFR-I reconstruction. It should be understood that the variant produced by the addition to the carboxy terminus will not include the addition of one or more carboxylic acid residues that would result in the reconstruction of the third or fourth sTNFR-I domain. An example of variants resulting from addition to the carboxy terminus includes chimeric proteins containing the R1-Cys19-Cys fusion.^ropes] -R2 with part or all of the human immunoglobulin heavy or light chain constant domain. TaPie chimeric proteins are preferred when the immudeglobulid portion of each protein comprises all domains except the first heavy chain constant region domain of a human immunoglobulin such as IgG, IgA, IgM or IgE, especially IgG, for example IgG1 or IgG3. It is understood by one of skill in the art that any amino acid of any portion of the immunoglobulide may be deleted or pushed with one or more amino acids, or one or more amino acids may be added as long as the TNF binding portion continues to bind TNF and the immudoallobulin portion will exhibit one or more of its characteristics.

Another group of variants are variants resulting from a subctemion of amino acids. Each of these variations has at least one amino acid residue in R 1 - [Cys19-Cysl ° 3] -R2 removed and a Ϊ)) residue inserted into it. Substitution variants include allelic variants that have changes to the nucleotide sequence naturally occurring in the species population, which may or may not result in an amino acid change. One skilled in the art can use any information known about binding or active site of polippptyau in selecting possible mutation sites.

One method of identifying amino acid residues or regions for a mutaaedpry protein is called "alanine scanning mutagenesis" (Cudningham and Wolls (1989), Science, 244: 1081-1085, which disclosure is incorporated herein by reference). In this method, the amino acid residue or group of protein target residues is iaenti-mada (for example, such as charged residues such as Arg, Asp, His, Lys, and Glu) and replaced with a neutral or negatively charged amino acid (most preferably alanine or polyalanine) to obtain interaction of the amino acids with the surrounding amino acid moadym environment inside or outside the cell. These residues showing functional sensitivity to substitutions are then corrected by introducing additional or alternative costs at the substitution sites. Thus, the site for introducing the modification of the acid amide sequence is predetermined, and alanine scanning or random mutagenesis may be performed to optimize the mutation results at a given site, and the resulting variant polipeatide is subjected to sPrididaomi to the optimal combination of the desired activity and degree of activity.

189 309

Sites of interest for muSagephesis by substitution include sites where the amino acids found in R1- [Cys¹⁹-Cys^l[0039] -R2 are substantially different in terms of side chain mass, charge, and / or hydrophobicity from sTNFR-like proteins such as the sT ^ Rs of other species, or other members of the NGF / TNF receptor family.

Other sites of interest include those where particular residues are similar or identical to those found in sTNbR-I-γ-like proteins. Such positions are generally important for the biological activity of the protein. For example, one skilled in the art knows that prior to the present invention, the effect of truncating sTNFR-I and sTNFR-II on their respective three-dimensional structures would not be predictable. However, in view of the results currently disclosed, the skilled artisan will recognize that the first principles for developing a variation making strategy could be based in part on prior information pertaining to full-length sTNFR-I. Accordingly, the following explanatory information regarding sTNFR-I is indicated (Banner et al. (1993), supra, oroz Fu et al. (1995), Protein Engineering, 8 (12): 1233-1241). Residues Tyr9, Th ^⁹, His⁵⁵ in domain 1, residues Phe ^, Ser⁶3, Asp⁸² in domain 2 and the residues Tyr ^ and Ser ⁷ in domain 3 were identified as potentially important for stabilizing the structure of domains 1, 2, and 3, respectively. Residues Proⁿand His * identified as aako ΡοΟ) ^!) ^ derived from Ser⁸⁶-Tyr7⁷ poąeednostk 7 C TNTa. The rest of the Glu⁴5-Pho⁴⁹ were identified as being in a loop that potentially interacts with Leu2 residues⁹-Arg³² drive up a hundred A TNFa. Gly48 residues were identified as potentially interacting with Asn-Pro20 at the A substituent of TNF ?. Remnants of His58-Leu⁶) was potentially identified as having an extended pici structure and interacting via a side chain with Arg31 -Ala residues³3 yodone A TNFa, residual His⁵8 sTNFR-I specifically interacting with the residue of Arg3. Rest of Lyn6⁴-Arg6 "was identified as having an extended strand structure and interacting through the side chain and through the main chain with the residues Ala1 -Glu1 and the residue Glu⁵ drive up a hundred A TNFa. The remainder of Mot69 was identified as potentially interacting with the rest of Tyr^H.5 podjedpatSki A TNFa. Residues of His94-Phe^Kn has been identified as forming a loop that interacts with the Thr ^ -Leu * and Asn residues 137 of the TNFa subunit, with the sTNbR-I residue Trp96 specifically interacting with the Ser7'-Thr72 residues of the TNFa C subunit, Lou ^ sTNFR-I is in close proximity to adjacent to the remainder of Asn ^ substitute ^ one hundred C TNFa, and the remainder Gln^w2 sT ^ R interacts specifically with the rest of Pro^H.3 drive up a hundred A TNFa. Accordingly, one skilled in the art can judge that the sites initially should be modified by substitution in a relatively conservative manner.

Such conservative substitutions are listed in Table 1 under the "Preferred Substitutions" column. If such substitutions lead to a change in biological activity, then more substantial changes (indicated in the "Exemplary Substitutions" column) can be made and / or other additions / deletions made and the resulting products screened.

Table 1

Amino acid substitutions

<td>Original rests</td><td>Preferred substitutions</td><td>Exemplary substituents</td>

<td> 1</td><td> 2</td><td> 3</td>

<td>Ala (A)</td><td>Val</td><td>Val, Leu; How much</td>

<td>Arg (R)</td><td>Lys</td><td>Lys; GI), Asp</td>

<td>Asp (N)</td><td>Gl)</td><td>Gl); His; Lys, Arg</td>

<td>Asp (D)</td><td>Glu</td><td>Glu</td>

<td>Cys (C)</td><td>Cheese</td><td>Cheese</td>

<td>Gl) (Q)</td><td>Asp</td><td>As)</td>

<td>Glu (E)</td><td>Asp</td><td>Asp</td>

i89 309 continued from table 1

<td> 1</td><td> 2</td><td> 3</td>

<td>Gly (G)</td><td>Pro</td><td>Pro</td>

<td>His (H)</td><td>Arg</td><td>Asn; Gln, Lys, Arg</td>

<td>How much (I)</td><td>Leu</td><td>Leu, Val; Met, Ala; Phe, norleucine</td>

<td>Leu (L)</td><td>How much</td><td>Norleucine; How much; Val; Underworld; Ala; Phe</td>

<td>Lys (K)</td><td>Arg</td><td>Arg; Gln; Asn</td>

<td>Met (M)</td><td>Leu</td><td>Leu; Phe; How much</td>

<td>Phe (F)</td><td>Leu</td><td>Leu; Val; How much; Ala</td>

<td>Pro (P)</td><td>Gly</td><td>Gly</td>

<td>Cheese (S)</td><td>Thr</td><td>Thr</td>

<td>T hr (T)</td><td>Cheese</td><td>Cheese</td>

<td>Trp (W)</td><td>Tyr</td><td>^T.y^r</td>

<td>Tyr (Y)</td><td>Phe</td><td>Trp, Phe, Thr; Cheese</td>

<td>Val (V)</td><td>Leu</td><td>How much; Leu; Underworld; Phe; Ala; norleucine</td>

When making such changes of an equivalent nature, the hydropathic index of amino acids may be considered. Each amino acid has been assigned a hydropathic index based on their hydrophobicity and charge as follows: isoleucine (+4.5); Valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine ​​/ cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9) and arginine (-4.5).

The importance of the hydropathic index of amino acids in imparting a biological interactivity function to a protein is well established in the art (Kyte and Doolittle (1982), J Mol Biol., 157: 105-131, the disclosure of which is incorporated herein by reference). It is known that certain amino acids can be substituted for other amino acids with a similar hydropathic index or value while still retaining a similar biological activity. When making changes based on the hydropathic index, it is preferable to substitute amino acids whose hydropathic indexes are within ± 2, and particularly preferably those that are within ± 1, especially those within ± 0.5.

It is also recognized in the art that substitution of similar amino acids can be made efficiently on the basis of hydrophilicity particularly where a protein or peptide so formed having an equivalent biological activity is intended to be used in immunological embodiments as is the case in the present invention. case.

U.S. Patent No. 4,554,101, the disclosure of which is hereby incorporated by reference in its entirety, states that the highest average local hydrophilicity of a protein, resulting from the hydrophilicity of its adjacent amino acids, correlates with its immunogenicity and antigenicity, that is, with the biological properties of the protein.

As detailed in US Patent No. 4,554,101, the following hydrophilicity values ​​have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0 ± 1); glutamate (3.0 ± 1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5 ± 1); alanine (-0.5); histidine (-0.5); cysteine ​​(-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4).

When making changes based on the similarity of the hydrophilicity value, it is preferable to substitute amino acids whose hydrophilicity indices are in the range of ± 2, and particularly preferably those in the range of ± 1, especially those in the range of ± 0.5.

199 099

US Patent No. 4,554,101 also discloses the identification and generation of epitopes from primary amino acid sequences on the basis of hydrophilicity. By the methods disclosed in US Patent No. 4,554,101, one skilled in the art could identify epitopes of the amino acid sequence such as the sTNFRs sequences currently disclosed. The tz regions are also called "epitope core regions".

Numerous scientific publications have been devoted to predicting sub-order structure and epitope identification from α-amino acid sequence analysis (Chou and Fasman (1974), Biochemistry, 13 (2) 222-245; Chou and Fasman (1978), Biochemistry, 113 (2): 211-222; Chou and Fasman (1978), Adv. Enzymol Relat Areas Mol. Biol, 47: 45-148; Chou and Fasman, Ann. Rev Biochem., 47: 251-276 and Chou and Fasman, (1979), Biophys J, 26: 367-384, the disclosure of which is incorporated herein by reference). In addition, computer programs are now available to help predict the antigenic parts and epitope core regions of proteins. Examples include programs based on the Jameson-Wolf analysis (Jameson and Wolf (1998), Comput. Appl Biosci, 4 (1): 181-186 and Wolf et al. (1998), Comput. Appl. Biosi, (4) 1: 187-191, disclosed of which are incorporated herein by reference), the PepPlot® program (Brutlag et al. (1990), CABS, 6: 237-245 and Wziabzrger et al. (1985), Science, 228: 740-742, disclosed herein by reference) and other new programs for predicting the tertiary structure of proteins (Fetrow and Bryant (1993), BIOTECHNOLOGY, 11: 479-483, the disclosure of which is included here. here as a reference).

Conservative modifications to the amino acid sequences (and corresponding modifications to the coding nucleic acid sequences) of R1- [Cys19-Cys^ll)3] -R2 and R4- [Cys32-Cysⁿ⁵] -R5 will produce proteins with a functional and chemical hybridization similar to that of the modified protein.

Conversely, substantial modifications to the functional and / or chemical characteristics of R1- [Cys19-Cy_S.1°³] -R₂ can be made by selecting substitutions that differ significantly in their effect on the preservation of (a) the structure of the polypsypeptide backbone in the region of substitution, for example as a sheet or hzlisa conformation, (b) the charge or electrophobicity of the protein at the target site, (c) the mass (volume) of the side chain. Naturally occurring residues are divided into groups based on common side chain properties:

1) hydrophobic: nzrijeuine, Met, Ala, Vs1, Leu, Ilz;

2) neutral hydrophilic: Cys, Ser, Thr;

3) acidic: Asp, Glu;

4) basic: Asn, Gln, His, Lys, Arg;

5) residues that influence chain orientation: Gly, Pro; and

6) aromatic: Trp, Tyr, Phe.

Non-behavioral substitutions can replace one of these groups with another. Such substituted residues can be introduced into the regions of Rl- [Cys19-Cyall⁾3] -R_₂which are homologous or unfamiliar to other members of the NGF / TNF receptor family.

Specific mutations in the R &amp; lt; 3 &amp; gt; Cys &amp; lt; -Cys &amp; lt; R &amp; gt; JR sequence may involve substitution of a non-native amino acid at the N-terminus, C-terminus, or at any site of the modified protein by addition of an N-linked or O-linked carbohydrate. Such modifications may be of particular utility, such as the addition of an amino acid (e.g., cysteine), which is advantageous for binding a water-soluble polymer to form a derivative, as described below. See, for example, the drawing of Fig. 5, where naturally occurring Asn from sTNFR-1 is changed to "Cys" to facilitate attachment of the polyethylene glycol molecule (Example 1).

In addition, the sequences R1- [Cys-Cys1 ° 3] -R₂ can be modified to add glyconylaval sites or to remove N-linked or O-linked glycosylation sites. Such an asparagine-linked glycsylation recognition site includes a triptych sequence that is specifically recognized by appropriate cellular glycsylation enzymes. These tripeptide sequences are either Asa-Xaa-Thr or Asa-Xaa-Ser, where Xss can be any amino acid other than Pro. Proven or Predicted Residuals

189 The aspartic sTNFR-I are found at positions 14, 105 and 111. A series of amino acid substitutions or deletions can be made to modify or add an N-linked or O-linked glycosylated site leading to an altered glycosylation protein.

In a particular embodiment, the variants are substantially homologous to the amino acid R1- [Cys19-Cys¹⁰3] -R2. The term "substantially homologous" as used herein means a degree of homology which is preferably greater than 70%, more preferably greater than 80%, even more preferably greater than 90%, most preferably 95%. The percentage of homology, as described in the present application, is calculated as the percentage of the amino acid residues found in the smaller of the two sequences aligned with identical amino acid residues in the sequence being compared when four gaps of 100 amino acids can be introduced to facilitate this alignment. juk was given by: Dayhoff (1972), in Atlas of Protein Sequence and Structure, 5: 124, National Biochemical Research Foundation, Washington, DC, the disclosure of which is incorporated herein by reference. Also included as substantially homologous are truncated sTNFRs, which can be distinguished by reciprocal reactivity with antibodies to the amino acid sequences SEQ ID NO: 2 and SEQ ID NO: 35, respectively, or whose genes can be isolated by hybridization with the DNA of SEQ ID NO: I or SEQ ID NO: 34 Or with their segments.

Exemplary sTNFRs of the invention include the following molecules: NH₂-MDSVCPQGKYJHPQNNSIC- [Cys-Cys1 ° 3] -FC-COOH (also referred to as sTNFR-I 2.6D / C105); NH₂-MDSVCPQGKYIHPQNNSIC- [Cys19-Cys ^⁰3] -FN-CSL-COOH (also referred to as juko sTNFR-I 2.6D / C106); NH2-MDSVCPQGKYinPQNNSIC- [Cys19-Cys' ° 3] -FN-COOH (also referred to as sTNFR-I 2.6D / N105); NH2-MYIHPQNNSIC- [Cys-Cysⁱⁿ3] -FNCSL-COOH (also referred to as sTNFR-I 2.3D / d8); NH2-M-1Cysl9-Cysl^θ3] -FNCSL-COOH (also referred to as sTNFR-I 2.3D / d18); oruz NH₂-MSIC-lCysI⁹-Cysl⁰3] -FNCSL-COOH (also referred to as sTNFR-I 2.3D / d15), methionyl or non-methionyl, variants and derivatives thereof.

The production of truncated variant sTNFRs is described in more detail below. Such variants can be made by introducing appropriate nucleotide changes into the DNA encoding the truncated sTNFRs or by in vitro chemical synthesis of the desired truncated sTNFRs. Those skilled in the art will recognize that many combinations of deletions, insertions and substitutions can be made provided that the terminal truncated sTNFRs are biologically active.

Mutagenesis techniques for replacing, inserting or removing one or more selected amino acid residues are well known to those skilled in the art (for example, US Patent No. 4,518,584, the disclosure of which is incorporated herein by reference). There are two main factors in constructing any amino acid sequence variant, namely; location of the mutation site and the nature of the mutation. When designing each variant, the location of each mutation site and the nature of the mutation will depend on the biochemical features to be modified. Each mutation site can be modified individually or in series, for example by (1) substituting first with selected conservative amino acids and then using more radical choices depending on the results achieved; (2) removing the target uminoa residue; Or (3) inserting amino acid residues adjacent to the site located.

One skilled in the art can make chemically modified derivatives of truncated sTNFRs based on the data given in the present application. Conjuguts can be made using truncated sTNFRs glycosilune, non-glycosilune, or deglycosilune. Typically, non-glycosylated truncated sTNFRs will be used. Suitable chemical moieties for derivatizing truncated sTNFRs include water-soluble polymers.

Water-soluble polymers are desirable because the protein - to which each is attached - will not precipitate in an aqueous environment such as a physiological environment. Preferably, the polymer is to be pharmaceutically acceptable for the preparation of a therapeutic product or composition. One skilled in the art will be able to select the desired polymer based on such considerations as whether a polymer / protein conjugate will be used.

189 Therapeutically, and if so, the desired dosage, circulation time, and protolysis resistance.

Suitable clinically acceptable water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), polyethylene glycol polyaldehyde, copolymers of ethylene glycol and propylene glycol, mimomethoxy polyethylene glycol, carboxymethyl alcohol, dextrose, polyvinyl alcohol (PVA) polyvinyl alcohol. pkli-1,3-dikxolay, poly-1,3,6-teixa, copolymer of ethylene and maleic anhydride, poly-fe-amino acids (homopolymers or random copolymers), poly (n-wrinyl polyethylene glycol) polyethylene glycol, polypropylene glycol (PpG) homopolymers and other polyalkylene oxides, polypropyl oxide / ethylene oxide copolymers, polykxyethyl, polyethylene glycol, polyoxyethylene glycol and other polyoxyethylene glycol (e.g. polyoxyethylene glycol) alcohols (e.g. polyoxyethylene glycol) and other polyoxyethylene glycol , colonic acids luC other carbohydrate polymers, Ficoll luC dextran and mixtures thereof.

As used in the present application, the term polyethylene glycol encompasses all forms that Cyla is used to privatize other proteins, such as glycol flour (C1-C10) alkoxy or arylkxypolyoxy. Polyethylene glycol propionaldehyde may have manufacturing advantages due to its stability in water.

Each of the water-soluble polymers can be of any molecular weight and can be branched or lowbranched. Each of the water-soluble polymers typically has an average molecular weight ranging from about 2 kDa to about 100 kDa (the term "about" indicates that in water-soluble polymer formulations some molecules will weigh more, some less than the stated molecular weight. ). The average molecular weight of each water-soluble polymer is preferably between about 5 kDa and about 50 kDa, more preferably between about 12 kDa and about 40 kDa, and most preferably between about 20 kDa and about 35 kDa.

In general, the higher the molecular weight or the more branches, the greater the polymer: protein ratio will be. Sizes may be used depending on the desired therapeutic profile (e.g., duration of sustained release; effect, if any, on biological activity; ease of processing; degree of lack of antigenicity, and other known aspects of the effect of the water-soluble polymer on the therapeutic protein) .

Each of the water-soluble polymers should be attached to the protein with regard to its effect on the functional or antigenic domains of the protein. In general, chemical derivatisation can be performed in any suitable weighting used to react the protein with the activated polymer molecule. Activating groups that can be used to combine the water-soluble polymer with one or more proteins are the following: sulfone, maleimide, sulfhydryl, thiol, trrflankwa, tresylaque, azidirine, xylene and 5-pyridyl.

Each of the water-soluble polymers is generally linked to a protein at the a-amino IuC groups ε-amino acid luC groups on the reactive lower group, but it is also contemplated that the water-soluble group may Cyc attached to any reactive group of the protein that is sufficiently reactive Cy to be attached to the water-soluble group in the appropriate reaction partners. Thus, the water-soluble polymer may be covalently linked to the protein through a reactive group such as a free amino or carboxyl group. Amino acid residues with a free amino group may include lysine residues and an N-terminal amino acid residue. Residues having a free carboxyl group may include aspartic acid residues, glutamic acid residues, and a C-terminal amino acid residue. Amino acid residues having a thiol reactive group include cysteine ​​residues.

Methods of making proteins conjugated to water-soluble polymers will, in any event, essentially include the steps of: (a) reacting the protein with a water-soluble polymer under conditions where the protein is attached to one or more water-soluble polymers; and (b) obtaining a reaction product. The reaction conditions for each configuration may be selected from conditions known in the art or conditions later developed, but should be chosen to avoid

Or, limit exposure to reaction conditions such as temperature, solvents, and pH levels that could inactivate the modified protein. In general, the optimal reaction conditions for these reactions will be determined on a case-by-case basis based on known parameters and the effect desired. For example, the higher the ratio of water-soluble polymer: protein conjugate, the greater the percentage of conjugated product. The optimal ratio (in terms of reaction yield in the sense that there is no excess of unreacted protein or polymer) can be determined by factors such as the degree of derivatization desired (e.g. mono-, di-, tri-, etc.), the molecular weight of the selected polymer whether the polymer is branched or unbranched and the reaction conditions used. The ratio of water-soluble polymer (e.g., PEG) to protein will generally be in the range of 1: 1 to 1: 100. One or more purified conjugates can be prepared from each mixture by standard purification techniques including, but not limited to, dialysis, salting-out, ultrafiltration, ion exchange chromatography, gel filtration chromatography, and electrophoresis.

One can target a specific protein, chemically modified at the N-terminus. The water-soluble polymer can be selected in terms of molecular weight, degree of branching, etc., the proportion of water-soluble polymer to protein (or peptide) molecules in the reaction mixture, type of reaction performed, method of obtaining the selected N-terminally chemically modified protein. A method for producing an N-terminally modified protein preparation (i.e., separating these particles from other monoderivatized residues, if necessary) may be to purify the N-terminally chemically modified protein material from a population of chemically modified protein molecules. Selective chemical modification at the N-terminus can be achieved by reductive alkylation taking advantage of the difference in reactivity of the different types of primary amine groups (lysine versus the N-terminus) available for derivatization in a given protein. Under suitable reaction conditions, an essentially selective derivatization of the protein at the N-terminus with a carbonyl-containing polymer is achieved. For example, one can selectively attach a water-soluble polymer to the N-terminus of the protein by conducting a reaction at a pH that allows the difference in pKa between the ε-amino group of the lysine residues and the α-amino group of the N-terminal residue of the protein to be used. By this selective derivatization, the binding of the water-soluble polymer to the protein is controlled: the coupling to the polymer takes place primarily at the N-terminus of the protein and no significant modification of other reactive groups, such as the amino groups on the side chains of the lysine residues, occurs. When using reductive alkylation, the water-soluble polymer may be of the types described above and should have one reactive aldehyde for attachment to the protein. Polyethylene glycol propionaldehyde containing one reactive aldehyde may be used.

The present invention particularly relates to a chemically derivatized protein having one or more (for example 2-4) PEG residues. Pegylation can be carried out using one of the pegylation reactions known in the art. Methods of making a protein pegylation product generally include the steps of (a) reacting the protein product with a polyethylene glycol (such as a reactive ester or aldehyde derivative of PEG) under conditions where the protein attaches to one or more PEG groups, and (b) preparing the product (s) reaction. In general, optimal reaction conditions will be determined on a case-by-case basis based on known parameters and the desired result.

There are many methods of attachment known to those skilled in the art. See, for example, EP 0 401 284, the disclosure of which is hereby incorporated by reference; see also: Malik et al. (1992), Exp. Hematol. 20: 1028-1035; Francis (1992), Focus on Growth Factors, 3 (2): 4-10 (published by Mediscript, Mountain Court, Friem Barnet Lane, London N20 OLD, UK); EP 0 154 316; EP 0 401 384; WO 92/16221; WO 95/34326; and other pegylation publications referenced herein, the disclosure of which is now incorporated by reference.

In particular, the pegylation can be carried out by an acylation reaction, or an alkylation reaction with a reactive polyethylene glycol molecule. Thus, the protein products of the invention include pegylated proteins in which the PEG group (s) is (are) attached via acyl or alkyl groups. Such products may be mono-pegylated or poly-pegylated (for example they may contain 2-6 and preferably 2-5 PEG groups). The PEG groups are generally attached to the protein at the α-amino or ε-amino groups in the amino acids, but it is also contemplated that the PEG groups can be attached to any amino group linked to the protein that is reactive enough to attach to the PEG group. under appropriate reaction conditions.

PEGylation by acylation generally comprises reacting an active ester derivative of polyethylene glycol (PEG) with a protein. For the acylation reaction, the polymer (s) selected should have a single reactive ester group. Any known or subsequently discovered reactive PEG molecule can be used to carry out the PEGylation reaction. A preferred activated PEG ester is PEG esterified to N-hydroxysuccinimide (NHS). As used in the present application, "acylation" is intended to include without limitation the following types of linkages between a therapeutic protein and a water-soluble polymer such as PEG: amide, carbamate, urethane, and the like (see Chamow (1994), Bioconjugate Chem. 5 (2) 133-140, the disclosure of which is incorporated herein by reference). The reaction conditions may be selected from those known in the art for pegylation or subsequently developed, but should avoid temperature, solvent and pH conditions that would reactivate the modified protein.

Pegylation by acylation generally leads to a poly-pegylated protein. Preferably, the bonding bond will be an amide bond. Also preferably, the product obtained will be substantially only (for example> 95%) mono-, di-, or tri-pegylated. However, some species with higher degrees of pegylation may be formed in the amounts depending upon the particular reaction conditions used. If desired, more purified pegylated substances can be isolated from the mixture (particularly with unreacted substances) by standard purification techniques including dialysis, screening, ultrafiltration, ion exchange chromatography, gel filtration chromatography and electrophoresis.

PEGylation by alkylation generally involves reacting the terminal aldehyde derivative of PEG with the protein in the presence of a reducing agent. For the reductive alkylation reaction the selected polymer (s) should (should) have a single, reactive aldehyde group. An example of a reactive PEG aldehyde is polyethylene glycol propionaldehyde, which is water-stable, or its mono-derivatives C1-C10 alkoxy or aryloxy (see US Patent No. 5,252,714, the disclosure of which is incorporated herein by reference).

PEGylation by alkylation can also lead to a polypegylated protein. Moreover, the reaction conditions can be altered to favor pegylation substantially only at the α-amino group of the N-terminus of the protein (i.e., to the monopegylated protein). In any event, both the monopegylation or polypegylation of the PeG groups are preferably attached to the protein via the -CH group₂ -NH-. With particular reference to the -CH group₂-, this type of bond is referred to herein as an "alkyl" linkage.

Reductive alkylation to generate a substantially homogeneous population of monopolymer / protein product will generally include the steps of: (a) reacting the protein with a reactive PEG molecule under reductive alkylation conditions at an appropriate pH to allow for the selective modification of the α-amino group at the amino terminus of said protein, and (b) ) obtaining the reaction product (s). Derivatization by reductive alkylation to form a monopegylated product takes advantage of the pKa differences between the lysine amino groups and the α-amino group at the N-terminus (pKa is the pH at which 50% of the amino groups are protonated and the remaining 50% not).

The reaction is performed at a pH that allows the pKa differences between the ε-amino groups of the lysine residues and the α-amino group of the N-terminal protein residue to be taken advantage of. Generally, if the pH is lower, a greater excess of polymer to protein will be desired (ie, the less reactive α-amino group at the N-terminus, the more polymer is required to achieve optimal conditions). If the pH is higher, the polymer-protein ratio will not

189 309 must be large (ie there are more reactive groups, so fewer polymer molecules are needed). For the purposes of the present invention, the pH will generally be in the range 3-9, preferably 3-6. For reductive alkylation, the reducing agent should be stable in aqueous solution and preferably should be capable of reducing only the Schiff's base formed in the initial reductive alkylation process. Suitable reducing agents may be selected from: sodium borohydride, sodium cyanoborohydride, dimethylamine borohydride, trimethylamine borohydride and pyridine borohydride. A particularly suitable reducing agent is sodium cyanoborohydride. Other reaction parameters such as solvent, reaction times, temperature, and methods for purifying the products can be determined on a case-by-case basis based on published information on protein derivatization using water-soluble polymers.

By such selective derivatization, the attachment of a water-soluble polymer (which contains a reactive group such as an aldehyde) to the protein can be controlled: coupling to the polymer occurs predominantly at the N-terminus of the protein, and there are no significant modifications to other reactive groups such as the amino groups in the chain. side lysine. Typically greater than 90% monopolymer / protein conjugate will be formed, and more typically greater than 95% monopolymer / protein conjugate will be formed, with the remaining observable molecules being unreacted molecules (i.e. protein without polymer moiety).

A particular embodiment of the present invention is an unbranched monomethoxypolyethylene glycol aldehyde with an average molecular weight of about 20 kDa or about 33 kDa (for example between 30 kDa and 35 kDa), or t-butyl polyethylene glycol aldehyde with an average molecular weight of about 33 kDa (for example between 30 kDa) 35 kDa), coupled via reductive alkylation to sTNFR-I 2.6D / N105.

Pegylation can also be specifically performed tuk, with water-soluble polymers having at least one reactive hydroxyl group (for example polyethylene glycol) can be reacted with a reagent having a reactive curbonyl, nitrile or sulfone group to convert the hydroxyl group into a reactive Michael acceptor, thereby forming an activated linker useful for modifying various proteins to obtain conjuguts with better biological activity. "Carbonyl, nitrile or sulfone reactive group" means a curbonyl, nitrile or sulfone group to which is attached a group with two carbon atoms that has a thiol-specific reactive site at the second carbon atom of the curbonyl, nitrile or sulfone group (WO 92/16221 ).

Activated linkers can be monofunctional, dual function, or multifunctional. Useful reagents having a reactive sulfone group that can be used in these methods include, without limitation, chlorosulfone, vinyl sulfone, and divinyl sulfone.

In a specific embodiment, the water-soluble polymer is activated with a Michael acceptor. WO 95/13312 describes, inter alia, sulfone-activated water-soluble polyethylene glycols PEGs which are highly selective for coupling to thiol moieties instead of amine moieties on molecules and on surfaces. These PEG derivatives are stable to hydrolysis for extended periods in aqueous media at pH = about 11 or less, and can form bond with molecules to give conjuguts that are also hydrolytically stable. The link by which PEG and the biologically active molecule are conjugated includes a sulfone residue conjugated to a thiol residue and has the structure PEG-SO₂-CH₂-CH2-SW where W is a biologically active molecule wherein the sulfone moiety is vinylsulfone or active ethylsulfone. Two particularly useful homobifunctional derivatives are PEG-bis-chlorosulfone and PEG-bis-vinylsulfone.

International Application No. PCT / US96 / 19459, the disclosure of which is incorporated herein by reference, teaches methods for making sulfone-activated linkers by making a compound with a reactive hydroxyl group and converting the hydroxyl group into a reactive Michael acceptor to form an activated linker, using tetrahydrofuran (THF). ) as a solvent for conversion. International Application No. PCT / US96 / 19459, the disclosure of which is hereby incorporated by reference, teaches a purification process for activated linkers that employs chromium for the purpose of separating lidkids for size and functionality of the end groups.

In short, the present invention contemplates prokaryote-expressed molecules that are chemically dprimatized with mono- or poly- (for example 2-4) PEG residues: sTNFR-I 2.6D / C105, sTNFR-I 2.6D / C106, sTNFR-I 2.6D / N105, sTNFR-I 2.3D / d8, sTNFR-I 2.3D / d18 and sTNFR-I 2.3D / d15, methyinylated or unmethinylated and their variants and derivatives.

Form (my) mieląmartęściowa (wo)

Polyvalent forms (do), that is, molecules with more than one active moiety, can be constructed. In one mypadium, the molecule may have more tumor necrosis factor binding sites for the TNF ligand (for example, a combination of a truncated sTNFR product). Furthermore, the molecule may have at least one quaternary necrosis factor binding site and, depending on the desired characteristics of the multivalent form, at least one other binding site (for example, a combination of at least one sTNFR truncated product and at least one idterleukid-1 receptor antagonist ( "IL-1ra") as episade below).

In one embodiment, a multi-valued form may be constructed, for example, by chemically coupling at least one sTNFR truncated product and another molecule, preferably another sTNFR truncated product, using any clinically acceptable link (for example, a moase-soluble polymer as shown above). In fact, the linkor is not sure to impart new immudeness, nor is it sure to redefine the amidecmasomp residues - to change the hydraubility and charge in the structure to the detriment of its biodistribution and removal from krmiobCoau (clearance).

Such polymers, when used as letters, may be homopolymers, copelimors, or block and terpolymers based on the above-mentioned monomers, either straight chain or branched, substitute or diepered. The polymer can be of a small length or a small molecular weight, but these characteristics can affect the biological properties. Average molecular weights of pellimers, particularly suitable for lowering the clearanco clearance rate in pharmaceutical applications, range from 2,000 to 35,000 Daltons. Furthermore, the length of the polymers may be varied to optimize or impart a desired whiteness activity.

Activating groups that can be used to link the water-soluble polymer to two or more proteins include the following: sulfone, maleimidomo, sulfhydryl, holowa, trieledomo, tresyladomo, aziradome, or 5-ayridyl.

In a particular embodiment, an activated linkor or amuunctions with at least one reactive Michael acceptor can be prepared, preferably with US Patent Application No. 08 / 473,809, and purified in accordance with United States Patent Application No. 08 / 611,918.

The active particles can be joined using conventional coupling techniques (see PCT Publication No. WO 92/16221 and PCT Publication No. WO 95/34326, the references of which are hereby incorporated by reference). In addition, PCT Publication No. WO 92/16221 describes the preparation of various dimorimatic sTNFR-I inhibitor molecules, for example, an polymerized c105 sTNFR-I. An example of a myelvalent protoin that binds tumor necrosis factor with ϋπτο (sTNFR-I 2.6D / C 106)₂- (20 kDa PEG) is disclosed in Example I.

Alternatively, the size of the molecule may consist of two repeats of sTNFR truncated product elements separated by a region with a polypeptide link. The design of the pear-peptidine liners is reportedly similar to the placement of short sequences of loops between domains in de novo designed proteins (Muttor (1988), TIBS, 13: 260-265 and Regan and DeGrado (1988), Science, 241: 976-978, the disclosure of which has been cited here as references). The single-chain attachment linker has been shown to be effective for the generation of the aimoric form of recombinant human sTNFR-II (Neve et al. (1996), Cytokine, 8 (5): 365-370, the disclosure of which is hereby referenced. ). A variety of linkor constructs have been put together and shown to be useful for antibodies; most functional linkers range in size from 12 to 25 amino acids (amino acids having nioreactim side groups, with 28

189 309 e.g. alanine, serine and glycine) which together form a hydrophilic sequence have a number of oppositely charged residues to increase solubility and are flexible (Whitlow and Filpupa (1991), Methods. A Companion to Methods in Enzymology, 2: 97-105 and Brigido et al. (1993), J Immunol., 150: 469-479, the disclosure of which is incorporated herein by reference).

In another embodiment, truncated sTNbRt can be chemically coupled to biotin, and the biotin / abcioSe sTNFRs conjugated systems are then coupled to avidin to form avidin / biotin / truncated sTNbR truncated molecules. The truncated nTNbRt can also be covalently coupled to dinitrophenol (DNP) or trinitrophenol (TNP) and the resulting copjugaSy precipitated with apty-DNP or anti-TNP-IgM to produce decamer canjugots with a valency of 10 for TNF binding sites.

In yet another embodiment, recombinant truncated sTNFR fusion proteins can also be made, wherein each rocomuninative chimeric molecule has the sequence of a sTNFR as described above, substituted with the variable domains of one or both heavy and light chains of the immunoglobulin molecule and has all or part of the constant domains, but at least one human immunoglobulin heavy or light chain constant domain. For example, each such chimeric sTNbR / IgG1 truncated fusion protein can be made from two chimeric genes: tTNbR / human gamma-1 truncated light chimeras (αUsTNFR / Ck) and heavy chain chimeras - tTNFR / human gamma-1 truncated ( truncated sTNbR / Cg-1). After transcription and translation of the two chimeric genes as described below, the gene products can be fused into a single chimeric molecule with a truncated nTNbR situated in the bivalent position. Further details on the construction of such chimeric molecules are disclosed in US Patent No. 5,116,964; PCT Publication No. WO 89/09622; PCT Publication No. WO 91/16437 and EP 315062, the disclosure of which is hereby incorporated by reference.

In yet another embodiment, recombinant truncated sTNFR fusion proteins can also be made in which each recombinant chimeric molecule has the tTNbR sequence as described above and at least one portion of the osteoyroSegerin (OPG) 186-401 region described in European Patent Application No. 96309363.8.

Palynuclootids

The present invention further provides polynucleosides encoding truncated sTNFRs. Based on the present description and using a universal codon table, one of ordinary skill in the art can easily determine all nucleic acid sequences that encode truncated tTNbRs amino acid sequences. Currently preferred nucleic acid sequences include the polynucleotides encoding tTNbR-I 2.6D / C105, sTNFR-I 2.6D / C106, sTNFR-I 2.6D / N105, tTNbR-I 2.3D / d8, sTNFR-I 2.3D / d18, and sTNFR- I 2.3D / d15. Examples of various polynucleotides are shown in Figures 2, 3, 4, 5, 6, and 7.

Recombinant expression techniques performed in accordance with the descriptions provided below can be followed to generate these polynucleotides and express the encoded proteins. For example, by inserting a nucleic acid sequence encoding a truncated sTNFR into a suitable vector, one skilled in the art can easily generate large amounts of the desired nucleoside sequence. These sequences can then be used to generate detection probes or amplification primers. Alternatively, a polynucleotide encoding a truncated sTNFR can be inserted into an expression vector. By introducing an expression vector into an appropriate host, the desired tTNbR truncated can be produced in large amounts.

As described below herein, there are numerous host / vector systems available for propagating the desired nucleic acid sequences and / or producing truncated tTNbRt. These include, but are not limited to, plasmid, viral and ipsertial vectors, and yrakaryotic and eukaryotic hosts. One skilled in the art can select a host / vector system capable of propagating and expressing hoSerologa DNA to generate or express the sequences of the present invention.

Furthermore, those skilled in the art know that, in view of the present disclosure, the novel nucleic acid sequences include degenerate nucleic acid sequences encoding

199 099 truncated sTNFRs with the sequences shown in the figure, and those nucleic acid sequences that hybridize (preferably under stringent hybridization conditions) to complement these sequences (creating complementary sequences to these sequences) nucleic acid nucleic acid (Maniatis et al. (1982), Molecular Cloning (A Labo ^ ory Manual), Cold Spring Harbor Labzratory, pp. 387-389). An exemplary stringent hybridization condition is hybridization in 4 x SSC at 62-67 ° C followed by a wash in 0.1 x SSC at 62-67 ° C for about 1 hour. Alternatively, exemplary stringent hybridization conditions are hybridization in 45-55% formamide, 4 x SSC at 40-45 ° C. Also included are DNA sequences that hybridize to the nucleic acid sequences shown in Figures 1 and 9, under relaxed hybridization conditions, and that encode truncated sTNFRs. Examples of such strictly relaxed hybridization conditions are 4 x SSC at 45-55 ° C or hybridization in 30-40% formamide at 40-45 ° C.

The present invention also provides trans-combination DNA constructs comprising vector DNA with DNA sequences encoding truncated sTNFRs. In each such DNA quastro, a nucleic acid sequence encoding a truncated sTNFR (-with or -without signal peptides) is operably linked to an appropriate expression control or regulatory sequence capable of replication of the expression of the truncated sTNFR in the host of choice.

Transcombinative expression

Preparation of pzliankleotides

Anklzinzwzgo acid sequences encoding truncated sTNFRs can be readily obtained in a number of ways including without restriction: chemical synthesis, scanning of cDNA or gnomom libraries, screening of expression libraries and / or PCR amplification of cDNAs. These and others that are useful for the isolation of such nucleic acid sequences are set out in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, (1989); Ausubel et al., Ed. Current Protocols in Molecular Biology, Current Protzcols Press, (1994); and from: Bzrger iKimmel, Methods in Enzymology: Guide to Molecular Cloning Techniques, Vol. 152, Azαdemiz Press, Inc., San Diego, CA, (1987), the disclosure of which is hereby incorporated by reference.

Chemical synthesis of the auxleinzwzgo acid which encode the truncated sTNFRs can be performed using methods well known in the art such as those described by Engels et al. (1989), Angew. Chem. Intl. Ed. 28: 716-734; and Wzlls et al. (1985), Gene, 34: 315, the disclosures of which are hereby incorporated by reference. These methods include, but are not limited to, the phosphotriester, phosphoramidite, and H-phosphoaidine methods for the synthesis of nucleic acid sequences. Large nucleic acid sequences, for example, greater than 1 ϋϋ auklzeptides in length, may be synthesized as multiple fragments. These fragments can then be ligated together to form a nucleic acid sequence encoding truncated sTNFRs. The preferred method is polymer synthesis using standard phosphoramidite chemistry reactions.

Alternatively, a suitable nucleic acid sequence can be obtained by screening a suitable cDNA library (i.e. a library made from one or more tissue sources believed to express the protein) or a gene library (a library made up of all genomic cDNAs). The source of the cDNA library is typically tissue from any species believed to express the desired protein in reasonable amounts. The source of the gzaome library may be any tissue or tissues from any mammalian or other species believed to contain the gene encoding a truncated sTNFR.

Hybridization media can be screened for DNA encoding a truncated sTNFR using one or more nucleic acid probes (oligonucleotides, cDNA fragments, or gnomes DNA fragments that have an acceptable degree of homo ^ n to the cDNA or gene to be cloned) that will selectively hybridize with cDNA (s) or gene (s) present in the library. Probes typically used for such scriagnoses encode a small region of DNA sequence from the same or similar species as the species from which the library is produced. Alternatively, the probes may be generated as discussed in the present application.

189 309

Hybridization is typically performed by recombining an oligo-claccidic or cDNA probe with clones to form a bilateral form under well-defined stringent conditions that prevent non-specific binding but allow binding of those clones that show significant levels of homology with the probe or primer. Typical stringent hybridization and washing conditions will depend, in part, on size (i.e. the number of nucleotides in the oligonuclectide probe cDNA IuC chain and whether the probe is degenerate. The likelihood of a clone being identified when designing a hybridization medium is also considered (for example, whether it is being truncated with a CiClikteka cDNA or genomic library).

When a DNA fragment (such as cDNA) is used as a probe, typical hybridization conditions include those given by AusuCela et al. (1994), supra ed. After hybridization, the hybridization medium is washed under strictly stringent conditions depending on a number of factors such as: probe size, expected probe homology to the clone, screened hybridization medium, number of clones screened, etc. Examples of strict washing solutions, which are typically low ionic strength and used at relatively high temperatures, are as follows: one such tight washing solution is 0.015 M NaCl, 0.005 M sodium citrate, and 0.1% SDS at 55-65 ° C; another such strict washing solution is 1 mM Na₂EDTA, 40 mM NsHPO4, pH = 7.2, and 1% SDS at about 40-50 ° C; and yet another tight wash solution is 0.2 x SSC and 0.1% SDS at about 50-65 ° C.

There are also exemplary protocols for stringent washing conditions where oligknkclectid probes are used to screen the hybridization media. For example, the first protocol uses 6 x SSC with 0.05% sodium pyrophosphate at a temperature between about 35 ° C and 63 ° C depending on the length of the probe. For example, 14 base probes are washed at 35-40 ° C, 17 base probes at 45-50 ° C, 20 base probes at 52-57 ° C, and 23 base probes at 57-63 ° C. The temperature can be increased by 2-3 ° C where non-specific background binding is high. The second protocol uses quaternary ammonium chloride solution (TMAC) for washing. One such tight wash solution is 3M TMAC, 50mM Tris-HCl, pH 8.0 and 0.2% SDS.

Another suitable method of obtaining a suitable nucleic acid sequence is the pclimerase chain reaction (PCR). In this method, cDNA is produced zpoly (A) + RNA luC total RNA using the enzyme reverse transcriptase. The two primers, typically complementary to two separate cDNA regions (clusters of glucectides) encoding a truncated sTNFR, are then added to the cDNA along with a pnlimease such as a polyme after Taq, and uolymscraza amplifies the cDNA region between the two probes.

Cligoncleotide sequences selected for probes or primers should be of appropriate length and should be sufficiently uniform to minimize the amount of non-specific binding that may occur during screening or PCR amplification. The actual sequence of the probes or primers is usually based on conserved or highly homologous sequences or regions. Alternatively, the luC primer probes may be fully or partially degenerate, that is, they may contain a mixture of probes / primers all encoding the same amino acid sequence but using different codons. An alternative to generating degenerate probes is to place izyz at certain luC at all positions in the code, which vary from species to species. Oligclectide probes or primers can be made by chemical synthesis methods for DNA as described above.

As described above, the variant sequence is a natural sequence (e.g., an allelic variant) or a synthetic sequence, a ^^ and a ^ c ^^ one or more substitutions, deletions and / or insertions of macleotides as compared to the sequence in Figures 2, 3, 4. 5, 6 and 7, and this results in the expression of variations in the amino acid sequence compared to the wild-type amino acid sequence. The preparation of synthetic variant sequences is also well known in the art as described in, for example, Sambrook et al. (1989) supra; and Wells et al. (1985), Geee, 34: 315, the disclosure of which is hereby incorporated by reference.

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3i

Vectors

DNA encoding truncated sTNFRs can be inserted into vectors for further cloning (DNA amplification) or for expression. Suitable vectors are commercially available or the vector may be specifically constructed. The choice or construction of an appropriate vector will depend on: (1) whether it is to be used for DNA amplification or for DNA expression; (2) the size of the inserted DNA into the vector; and (3) from the intended host cell transformed with the vector.

Each of the vectors contains a nucleic acid sequence encoding a desired protein in operable association with one or more of the following expression control or regulatory sequences capable of targeting, controlling or otherwise affecting the expression of the desired protein by the host cell of choice. Each vector contains different components depending on their function (DNA amplification or DNA expression) and their compatibility with the intended host cell. Vector components generally include, but are not limited to, one or more of the following positions: signal sequence, origin of replication, one or more selection or marker genes, promoters, enhancer elements, transcription termination sequence, etc. These components may be obtained from natural sources or may be obtained by known methods by synthesis.

Examples of suitable prokaryotic cloning vectors include bacteriophages such as lambda derivatives or E. coli plasmids (for example: pBR322, col EI, pUC, F-factor and Bluescript® plasmid derivatives (Stratagene, LaJolla, CA)). Other suitable expression vectors, many types of which are known in the art and for the host cells described below, may also be used for this purpose.

Signal sequence

Nucleic acid encoding a signal sequence may be inserted at the 5 'position of the sequence encoding a truncated sTNFR, for example it may be a component of a vector, or it may be part of a nucleic acid encoding a truncated sTNFR. Nucleic acids encoding native sTNFR-I and sTNFR-II signal sequences are known (documents EP 393 438 and EP 422 339). '

Start of replication

Each of the expression and cloning vectors generally contains a nucleic acid sequence enabling the vector to replicate in one or more host cells. In a cloning vector, this sequence is typically one that allows the vector to replicate independently of the host chromosomal DNA and includes origins of replication or autonomously replicating sequences. Such sequences are well known. The origin of replication from plasmid pBR322 is suitable for most gram-negative bacteria, and various origins (e.g., SV40, polyoma, adenovirus, VSV, or BPV) are suitable for cloning vectors in mammalian cells. In general, an origin of replication is not needed for mammalian expression vectors (for example, the SV40 origin is often only used because it contains an early promoter.

Selection gene

Each of the expression and cloning vectors typically contains a selection gene. This gene codes for a "marker" protein necessary for the survival or growth of transformed host cells when grown in a selective culture medium. Host cells that are not transformed with the vector will not contain the selection gene and therefore will not survive in the culture medium. Typical selection genes encode proteins that: (a) confer resistance to antibiotics and other toxins, for example ampicillin, neomycin, methotrexate or tetracycline, (b) supplement auxotrophic deficiencies, or (c) provide critical nutrients not available from the culture medium .

Other selection genes can be used to amplify the genes to be expressed. Amplification is the process by which genes with a greater demand for growth-critical proteins are duplicated one after the other within a chromosome during the successive formation of recombinant cells. Examples of suitable selectable markers for mammalian cells include dihydrofolate reductase (DHFR) and thymidine kinase. Cell transformants are placed under the selected pressure to which only the transformants are adapted to survive due to the markers present in the vector. The selected pressure is

I89 309 is applied by culturing the transformed cells under conditions in which the concentration of the selected agent in the medium is sequentially varied, thereby amplifying the selection genes and the DNA encoding the truncated sTNFRs. As a result, increased amounts of truncated sTNFRs are synthesized from the amplified DNA.

For example, cells transformed with the DHFR selection gene are first identified by culturing all transformants in a culture medium containing methotrexate, a competing DHFR antagonist. A suitable host cell when wild-type DHFR is used is the Chinese hamster ovary cell line deficient in DHFR activity (Urlaub and Chasin (1980), Proc. Natl. Acad Sci, USA, 77 (7): 4216-4220, the disclosure of which is provided here for reference). The transformed cells are then exposed to elevated levels of methotrexate. This leads to the synthesis of multiple copies of the DHFR gene and, along with this, to multiple copies of other DNAs present in the expression vector, such as DNA encoding the truncated sTNFR.

Promoter

Each of the expression and cloning vectors will typically contain a promoter that is recognized by the host organism and is operably linked to the nucleic acid sequence encoding the truncated sTNFR. A promoter is an untranslated sequence located upstream (5 ') of the start codon of a structural gene (generally ranging from about 100 to 1000 bp) that controls the transcription and translation of a particular nucleic acid sequence, such as that encoding a truncated sTNFR. The promoter may - in a typical case - be grouped into one of two classes; inducible promoters and constitutive promoters. An inducible promoter initiates increased levels of transcription from DNA under its control in response to certain changes in the conditions of the medium, such as the presence or absence of a nutrient, or a change in temperature. A number of promoters recognized by many potential host cells are well known. A promoter can be operably linked to the DNA encoding the truncated sTNFR by removing the promoter from the source DNA by restriction enzyme digestion and inserting the desired promoter sequence. The native sTNFR-I promoter sequence can be used to direct the amplification and / or expression of DNA encoding the truncated sTNFR. A heterologous promoter is preferred, however, if it allows for greater transcription and higher yields of the expressed protein compared to the native promoter, and if it is compatible with the host cell system selected for use. For example, any of the native promoter sequences of other members of the NGF / TNF family may be used to direct amplification and / or expression of DNA encoding a truncated sTNFR.

Promoters suitable for use with prokaryotic hosts include the beta-lactamase and lactose promoter systems: alkaline phosphatase, tryptophan (trp) promoter system; the bacterial luminescence gene system (luxR); and hybrid promoters such as the "tac" promoter. Other bacterial promoters are also suitable. Their nucleotide sequences have been published, thereby allowing one of skill in the art to ligate to the desired DNA sequences using linkers or adapters as needed to provide any desired restriction sites.

Suitable promoter sequences for use in yeast hosts are also well known in the art. Suitable promoters for use in mammalian host cells are well known and include those derived from viral genomes such as: polyoma virus, avipox virus, adenovirus (such as Adenovirus 2), bovine papillomavirus, avian sarcoma virus, cytomegalovirus, retrovirus, virus hepatitis-B and - especially - Simian Virus 40 (SV40). Other suitable mammalian promoters include heterologous mammalian promoters, for example, heat shock promoters and the actin promoter.

The enhancer element

Each of the cloning expression vectors will typically contain an enhancer sequence to enhance transcription by higher eukaryotes of the DNA sequence encoding the truncated sTNFR. Enhancers are cis-acting elements of DNA, usually about 10 to 300 base pairs in length, that act on a promoter to increase its transcription. Enhancers are relatively orientation and position independent. Their presence was confirmed

189 309 at positions 5 'and 3' to the transcription unit. Yeast enhancers are preferably used with yeast promoters. A number of enhancer sequences are known available from the mammalian genes (for example: globin, elastase, albumin, alpha-feto-protein, and insulin). In addition, viral enhancers such as the SV40 enhancer, cytomegulovirus early promoter enhancer, polyoma enhancer, and the udenovirus enhancer are exemplary enhancer elements for activating eukaryotic promoters. While the enhancer may be fused in the vector 5 'or 3' to the DNA encoding the truncated sTNFR, it is typically located 5 'to the promoter.

Complete transcription

Each of the expression vectors used in eukaryotic host cells will typically contain a sequence necessary for terminating transcription and for stabilizing the mRNA. Such sequences are commonly available from the 5 'and sometimes 3' non-translocating regions of eukaryotic DNAs or cDNAs. These regions contain transcription nucleotide segments as polyudenyl fragments in the untranslated portion of the mRNA encoding a truncated sTNFR.

Vector construction

Construction of an appropriate vector containing one or more of the components listed above (along with the coding sequence for the truncated sTNFR) is performed using standard ligation techniques. Isolated plasmids or DNA fragments are cleaved, cut, and re ligated in the desired order to generate the desired vector. To confirm that the correct sequence was built, a ligation mix can be used to transform E. coli, and successful transformants can be selected by known techniques, as described above. Some amounts of vector are then generated from the trunsformats, analyzed with restriction endonucleases, and / or sequenced to confirm the presence of the desired construct.

A vector may also be used which provides for the transient expression of the DNA encoding the truncated sTNFR in mammalian cells. In general, transient expression involves the use of an expression vector that is capable of replication efficiently in the tuk host cell, that the host cell accumulates multiple copies of the expression vector, and in turn synthesizes high levels of the desired protein encoded by the expression vector. Any transient expression system including an appropriate expression vector and a host cell allows convenient, positive identification of the proteins encoded by the cloned DNA, as well as the rapid screening of such proteins for the biological and physiological properties desired, i.e. identifying an active biologically truncated sTNFR.

Host cells

Any of a plurality of recombinant host cells, each of which contain a nucleic acid sequence for use in expressing the desired protein, is also provided by the present invention. Exemplary prokaryotic and eukaryotic host cells include bacterial, mammalian, fungal, insect, yeast, or plant cells.

Prokaryotic host cells include, but are not limited to, eubuctivity such as Gram-negative or Grum-dodutically organisms (e.g., E coli (HB 101, DH5a, DH10, and MC 1061); Bacilli such as: B subtilis; Pseudomonas species, such as such as P. aeruginosa, Streptomyces spp., Salmonella typhimurium, or Serratia marcescans In a particular embodiment, the desired protein may be expressed in E. coli.

In addition to prokaryotic host cells, eukaryotic microorganisms such as yucca filamentous fungi or yeast may be suitable hosts for the expression of truncated sTNFRs. Saccharomyces cerevisiae, or common baker's yeast, is the most widely used of the eukaryotic host microorganisms, but a variety of other genera, species and strains are well known and widely available.

A truncated sTNFR may be expressed in glycosylated form by one of a number of suitable host cells derived from multicellular organisms. Such host cells are capable of complex processing and glycosylating activities. In principle, any higher eukuriotic cell culture could be used, regardless of whether the tUka culture involves vertebrate or invertebrate cells34

189 309 heads including plant and insect cells. In a particular embodiment, the desired protein can be expressed in baculovirus cells.

Vertebrate cells can be used because propagation of vertebrate cells in culture (tissue culture) is a well known procedure. Examples of useful mammalian host cell lines include, but are not limited to, monkey kidney CV1 transfoating through SV40 (COS-7) lines, human embryo kidney lines (293 cells or 293 cells cloned for growth for suspension culture), baby hamster kidney cells, and Chinese hamster ovary cells. Other suitable mammalian cell lines include, but are not limited to, HeLa, monkey L-929 cells, derivatives of the 3T3 Swiss line, BalC-c luC nIh mice, and BHK or Hamster HaK cell lines. In a particular embodiment, the desired protein may be exponential in COS cells.

The host cell may be transfused and preferably transformed with the desired nucleic acid under appropriate conditions allowing expression of the nucleic acid. The selection of suitable host cells and methods for transformation, culture, amplification, screening, and product production and purification are well known in the art (Gething and Sambrook (1981), Nature, 293: 620-625 or - alternatively Kaufnan et al. (1985), Mol . Cell Biol., 5 (7) 1750-1759, or US Patent No. 4,419,446, the disclosure of which is hereby incorporated by reference). For example, for mammalian cells without cell walls, the calcium phosphate precipitation method can be used. Slekteopracration, mikekinjekcjp, and other known techniques can also be used.

It is also possible that truncated sTNFRs can be produced by homologous luC recombination using recombinant production methods using container elements introduced into cells already containing DNA encoding kCiptc sTNFRs. Homologous recombination is an original technique for the case where Cy targeting genes induced or corrected mutations in the active genes of teaaypcyjaiś (Kucherlapati (1989), Prog WNucl Acid Res. And Mol. Biol., 36: 301, the disclosure of which is incorporated herein by reference). The principal technique was developed as a method for introducing specific mutations into specific regions of the mammalian genome (Thomas et al. (1986), Cell, 44: 419-428; Thomas and Capecchi (1987), Cell, 51: 503-512, and Dketschmaa et al. . (1988), Proc. Natl. Acad Sci., 85: 8583-8587, the disclosure of which is incorporated herein by reference), or to correct specific mutations in defective genes (Dketschman et al. (1987), Nature, 330: 576-578, the disclosure of which is hereby incorporated by reference). Exemplary techniques are provided in: US Patent No. 5,272,071; WO 92/01069; WO 93/03183; WO 94/12650 and WO 94/31560, the disclosure of which is hereby incorporated by reference.

By homologous recombination, the DNA sequence to be inserted into the genome can be directed to a specific region of the gene of interest by attaching it to the targeting DNA. A targeting DNA is DNA which is complementary (homologous) to the geometry of the DNA. Small fragments of targeting DNA, complementary to a specific region of the genome, are brought into contact with the parental strand during the DNA replication process. A general property of DNA inserted into a cell is that it hybridizes - and hence recombines - with other fragments of endogenous DNA through common homologous regions. If this complementary strand is attached to an oligknucleotide that contains the luC mutation by a different DNA sequence, it is also incorporated into the newly synthesized strand by recombination. As a result of the correction function, it is possible for the new DNA sequence to serve as a template. Therefore, the transferred DNA is integrated into the genome.

Where the sequence of a particular gene is known, such as the sTNFR nucleic acid sequence, an expression control sequence (a DNA fragment complementary to a selected region of the gene) may be synthesized or otherwise obtained, such as by appropriate restriction of native DNA at specific recognition sites binding the region of interest. This fragment serves as a targeting sequence when inserted into a cell and will hybridize to its homologous region within the genome. If this hybridization occurs during DNA replication, this DNA fragment, and any sequences linked to it, will function as an Okazaki fragment and will be "sewn" into the newly synthesized "daughter" strand of DNA.

Attached to these targeting DNA fragments are regions of DNA that can interact with the expression of the truncated sTNFR. For example, a promoter / enhancer element, suppressor or exogenous transcription modulation element is inserted into the genome of the intended host cell in proximity and orientation sufficient to affect transcription of the DNA encoding the desired truncated sTNFR. A control element does not encode the truncated sTNFR, but instead controls a portion of the DNA of the host cell genome present. Thus, expression of a truncated sTNFR can be achieved not by transfection of the DNA encoding the truncated sTNFR, but rather by using targeting DNA (containing regions of homology with the endogenous gene in question) linked to DNA regulatory segments providing the endogenous gene sequence with recognizable transcription signals of the truncated sTNFR.

Host cell culture

The method of culturing each of the one or more recombinant host cells to produce a desired protein will vary depending on many factors and circumstances; the optimal manufacturing procedure for a given situation will be apparent to those skilled in the art after carrying out a minimum number of experiments. Such recombinant host cells are grown in a suitable medium and the truncated sTNFR expressed is then optionally recovered, isolated, and purified from the culture medium (or from the cell if expressed intracellularly) by appropriate methods known to those skilled in the art.

In particular, each of the recombinant cells used to produce the desired truncated sTNFR can be cultured in media suitable for inducing promoters by selecting the appropriate recombinant host cells or by amplifying the gene encoding the desired truncated sTNFR. Nutrients can be supplemented as needed with hormones and / or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (such as sodium, calcium, magnesium, and phosphate chloride), buffers (such as HEPES), nucleosides (such as adenosine). and thymidine), antibiotics (such as gentamicin), trace elements (defined as inorganic compounds usually present in final concentrations in the micromolar range), glucose or other energy sources. Other supplements may also be included, at appropriate concentrations, as will be appreciated by those skilled in the art. Suitable culture conditions, such as temperature, pH, etc., are also well known to those skilled in the art for use in selected host cells.

The expression product obtained can then be purified almost homogeneously using procedures known in the art. Exemplary purification techniques are given in EP 393 438 and EP 422 339, the disclosures of which are incorporated herein by reference.

Pharmaceutical compositions

Each of the pharmaceutical compositions will generally contain a therapeutically effective amount of truncated sTNFRs and chemically modified truncated sTNFRs derivatives (collectively, "truncated sTNFR product (s)") mixed with an excipient. The excipient preferably comprises one or more pharmaceutically and physiologically acceptable substances to form a formulation in admixture with the truncated sTNPR product (s) and the controlled release substance.

The primary solvent in the excipient can be aqueous or non-aqueous. In addition, the excipient may contain other pharmaceutically acceptable excipients to modify or maintain the pH preferably between 5-6.5 and especially between 5.5-6.0 (for example buffers such as citrates, phosphates and amino acids such as glycine); swelling agents for a lyophilized preparation (for example, mannitol and glycine); for maintaining osmolarity (e.g., mannitol and sodium chloride); surfactants (e.g., polysorbate 20, polysorbate 80, triton, and pluzonix); means for maintaining viscosity; clarity; colors; sterility; stability (e.g., sucrose and sorbitol); antioxidants (e.g., sodium sulfite and sodium bisulfite); preservatives (for example, benzoic acid and salicylic acid); the smell of the preparation; flavoring and thinning agents; means for

189 Dissolution rate assurance (for example, salubilizators or solubilizing agents such as alcohols, polyethylene glycols, and sodium chloride); means for providing a rate of release; emulsifying agents; suspending agents; solvents; fillers; delivery earnings; thinners; pharmaceutical excipients and / or adjuvants. Other effective forms of administration are also contemplated such as slow release parenteral formulations, inhalation sprays, orally active formulations, or suppositories. The composition may also include particulate preparations of polymeric compounds such as bulk-degradable polymers (e.g., lactic-coglycolic acid (PLGA) copolymers, PLGA polymer blends, PEG and lactic and glycolic acid block copolymers, poly (cyanacrylates) ); surface degradable polymers (for example, yol (anhydrides) and poly (artotter)); hydrogel esters (for example, pluronic polyhydric alcohols, polyvinyl alcohol), polyvinylpyrrolidone), copolymers of maleic anhydride and alkyvinyl ether, cellulose, hyaluronic acid derivatives, alginate, collagen, gelatin, albumin and starches and dextran systems thereof; Iub preparations of lipatams or microspheres. Such compositions can affect the physical state, stability, rate of in vivo release, and rate of in vivo removal of the proteins and derivatives present. The optimal pharmaceutical formulation for the desired protein will be determined by one of skill in the art depending upon the route of administration and the desired dosage. Exemplary pharmaceutical compositions are disclosed in Remington's Pharmaceutical Sciences, 18th ed. (1990), Mack Publinhipg Co., Eosto) PA 18092, pp. 1435-1712; Gombotz and Pettit (1995), Blasonjugate Chem., 6: 332- 351; Leone-Bay, and ip. (1995), Journal of Medicinal Chemistry, 38: 4263-4269; Haas, et al. (1995), Clinical Immunology and Immunopathology, 76 (1:93: WO 94/06457; WO 94/21275; FR 2706772 and WO 94/21235, the disclosures of which are incorporated herein by reference.

Particular sustained release compositions are available from the following suppliers: Doyotosh (Depofoam ™, multi-vesicle liposome); Alkermes (PraLeaso ™, PLGA microsphere). In the present application, hyaluronan is intended to include hyaluronan, hyaluropic acid, its (such as sodium hyaluronate), esters, ethers, enzymatic derivatives, and hlaluronic acid gels strained and chemically modified hyaluronic acid derivatives (such as hylan). Exemplary forms of hyaluronan are disclosed in Peyrop and Balazs (1974), Path. Biol., 22 (8): 731-736: Isdalo et al. (1991), J Drug Dev., 4 (2): 93-99; Larsep and ip. (1993), Journal of Biomedical Materials Research, 27: 1129-1134; Namiki et al. (1982), International Journal of Clinical Pharmacology, Therapy and Toxicology, 20 (11): 501-507; Meyer et al. (1995), Journal of Controlled Release. 35: 67-72; Kikuchi et al. (1996), Osteoarthritis and Cartilage, 4: 99-110; Soka ^ am et al. (1994), Clinical Orthopedics and Related Research, 299: 282-292: Meyors and Brandt (1995), 22 (9): 1732-1739; Laurent et al. (1995), Acta Orthop Scand, 66 (266): 116-120; Cascone and ip. (1995), Biomatericds, 16 (7): 569-574; Yerashalmi et al. (1994), Arch. Of Biochemistry and Biophysics, 313 (2): 267-273; Bomatchez et al. (1993), Journal of Biomedical Materials Research, 27 (5): 677-681: Tan et al. (1990), Australian Journal of Blotechnology, 4 (1): 38-43; Gombotz and Pettit (1995), Blasanjugote Chem., 6: 332-351; U.S. Patent Nos. 4 582 865, 4 605 691, 4 636 524, 4 713 448, 4 716 154, 4 716 224, 4 772 419, 4 851 521, 4 957 774, 4 863 907, 5 128 326, 5 202 431, 5 336 767,5356,883; European Patent Applications Nos. 0 507 604 A2 and 0 718 312 a2; and WO 96/05845, the disclosures of which are incorporated by reference in the present application. Specific hyaluronan compositions are available from the following suppliers: BioMatrix, Inc. Ridgefield, NJ (Synyisc ™, 90:10 mixture of hylap fluid and hylan gel); Fidia SpA, Abano Terme, Italy (Hyalgan ™, sodium salt of halopurous acid derived from rooster comb (MW -500,000 to -700,000)); Kaken Pharmaseutlcal Co., Ltd., Tokyo, Japan (Artz ™, 1% Rooster Comb Hyaluropic Acid Solution, C. Part. -700,000 MW); Pharmacia AB, Stockholm, Sweden (Healop ™, rooster comb hyaluronic acid, molecular weight ~ 4 x 106 MW); Genzyme Corporation, Cambridge, MA (Surgicoot ™, a recombinant hyaluropic acid); Rights of Biopolymer, Inc. Parttmoush, NH (FCH hyaluronic acid, high molecular weight (e.g., -1.5-2.2 x 106 MW) hyoluropic acid produced from Streptococcus zooepidemicus cultures; Sodium hyaluronate MV, -1.0-1.6 x 10 MW and sodium hyaluronate LY, -1.5-2.2 x 106 MW); Calbiochem-Novabiachem AB, Lautelfmgen,

189 309

Switzerland (hCaluronomy acid, sodium salt (Company Catalog 1997 No. 385908) prepared from Streptococcus sp.); Interfon Company, Purcha.se, NY (Rooster comb derived healuronic acid> 1x10 ° MW); Diosynth Inc., Chicago, IL; Amorchol Corp., Edison, NJ and Kyoma Hakko Koaya Co., Ltd., Tokyo, Japan.

After the pharmaceutical composition is formulated, it can be stored in sterile vials as a solution, suspension, gel, emulsion, solid or dehydrated powder or liquid liquid. Such preparations may be stored in a ready-to-use form, or in a form (e.g.

In a particular embodiment, the present invention is directed to kits for producing a single dose administration unit. Each of the kits may contain both a dry protein first container and a second aqueous container. Kits falling within the scope of the present invention are single and ground brown pre-filled syringes; examples of pre-filled syringes (for example, liquid syringes and syringes such as Lyo-Joct®, a pre-filled twin-chamber syringe) are available from Vetter GmbH, Ravensburg, Germany].

Applicant

Truncated sTNFR products may be useful as research reagents, and as therapeutic and diagnostic agents. Thus, truncated sTNFRs can be used in in vitro and / or in vivo diagnostic assays to quantify native sTNFR-I in a tissue or organ sample or to identify and / or isolate cells that express TNF (Scallon et al. (1995), ^ upra). In tissue or organ samples, there will be less radioactivity from m-truncated sTNFRs binding to TNF as compared to the standardized binding curve of 12 [alpha] -truncated sTNFRs due to the significant amount of native sTNFR-I or sTNFR-I binding to TNF. Similarly, 1 can be used²⁵I-truncated sTNFRs to detect the presence of TNF in various cell types.

The present invention also contemplates the use of truncated sTNFR products in the formation of antibodies and reactive antibodies (more generally, those that also bind to native sTNFR-I or sTNFR-II). Antibodies that bind to truncated sTNFRs can be obtained, such as epitopes within the amino acid sequence of the formula Rι- [Cycl⁹-Cys1 ° 3] -R2 Iub within the sequence aminePmasomaj Ri- [Cys32-CyS^H5] -R5. One of ordinary skill in the art can use well-known published procedures to obtain modoplonaldo and polycyclic antibodies, or polyclinic antibodies that specifically recognize and bind to a variety of proteins targeted by the emino acid sequence of the present invention. Such antibodies can then be used to purify and characterize the half-length, full-length, 30 kDa mature TNF inhibitor, the 40 kDa mature TNF inhibitor.

The present invention also relates to methods of treating certain diseases and health conditions (many of which may be characterized as inflammatory diseases) mediated by TNF. A disease Iub is considered a "TNF mediated disease" if the disease (spontaneous Iub associated with experiments) is associated with current levels of TNF in body fluids or tissue adjacent to the outbreak or within the body's indications. TNF mediated diseases can also be diagnosed by the following two conditions. (1) Pathological disease-related findings can be mimicked experimentally in animals by administration of TNF, and (2) the pathological condition induced in experimental animal models of disease can be inhibited or abolished by treatment with agents. which inhibit the action of TNF. Many TNF-mediated diseases fulfill two of these three merunks and others fulfill all three marunpi. A non-exhaustive list of TNF-mediated diseases and related sequelae and associated symptoms, each of which may be treated by the methods of the present invention, include: acute respiratory distress syndrome; Pachexia / adorexia; cancer (for example leukemia); chronic fatigue syndrome; graft versus host reaction; hyperalgesia; inflammatory bowel disease; neurozapaldehyde; non-invasive / roperfusion-related trauma, including dipauPrmiedio of the brain (trauma to the brain as a result of epilepsy, haemorrhage, or stroke, any of which may lead to neurodegedwork); diabetes (for example, juvenile onset

I89 309 diabetes type i); multiple sclerosis; eye diseases; ache; inflammation of the pancreas; pulmonary fibrosis; rheumatic diseases (for example rheumatoid arthritis, osteoarthritis, juvenile (rheumatoid) arthritis, seronegative polyarthritis, ankylosing spondylitis, Reiter's syndrome and reactive arthritis, psoriatic arthritis, enteropathic arthritis, polymyositis, dermatitis- muscle, scleroderma, systemic sclerosis, vasculitis, vasculitis, Sjogren's kit, acute rheumatoid arthritis, polychondritis and high ESR polymuscular pain syndrome and giant cell arteritis); septic shock; side effects of radiation therapy; systemic lupus erythematosus; temporomandibular joint disease; thyroiditis and tissue transplantation.

Each of the truncated sTNFR products can be administered to a patient in therapeutically effective amounts for the treatment of TNF mediated diseases as defined above, including diseases such as rheumatic diseases (e.g., Lyme disease, juvenile (rheumatoid) arthritis, osteoarthritis, psoriatic arthritis, and psoriatic arthritis. arthritis, rheumatoid arthritis and staphylococcal ('septic') arthritis). The term "patient" is intended to include animals (for example cats, dogs, and horses) as well as humans.

The truncated sTNFR product can be administered by topical, enteral, or parenteral administration including, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardial, intradermal, intraperitoneal, transtracheal, subcutaneous, subcutaneous, subcutaneous, subcutaneous, subcutaneous, subcutaneous, subcutaneous, intra-intra-arterial, intrathecal, intra-intracapsular, intra-intra-arterial, intra-epidural, , intramedullary, intraventricular and intrasternal. The truncated sTNFR product may also be administered orally or be administered through the mucosa, i.e., nasally, sublingually, buccally or rectally for systemic administration.

It is preferred that the truncated sTNFR products are administered by intra-articular, subcutaneous, intramuscular or intravenous injection. In addition, the truncated sTNFR product may be administered by continuous infusion (for example, modulating the flow of an implanted or external continuous or intermittent infusion device) so as to continuously provide a desired level of truncated sTNFR product in the blood at the time of administration. This is preferably done by continuous infusion through, for example, a mini-pump such as an osmotic mini-pump. By these methods, it can be ensured that the amount of drug is kept at the desired level, and blood samples can be taken and the amount of drug in the blood stream can be monitored. A variety of pumps are commercially available from suppliers such as MiniMed Inc, Sylmar, CA (for example MT507) and Alza Corp., Palo Alto, CA (for example Alzet osmotic pump, model 2MLI).

It is also contemplated that other continuous or near-continuous dosing methods may be used. For example, chemical derivatization can lead to slow release forms of the protein resulting in a continuous presence in the blood stream in predictable amounts based on the designated dosing regimen.

Methods of using truncated sTNFR products to treat TNF mediated diseases, including arthritis (e.g., osteoarthritis, psoriatic arthritis, and rheumatoid arthritis), are given in European Patent Application 567566, the disclosure of which is hereby incorporated by reference. . By way of example and not limitation, in one particular embodiment truncated sTNFR products can be administered intraarticularly to treat rheumatoid arthritis and osteoarthritis. By way of example - but not limitation - in another specific embodiment, truncated sTNFR products may be administered subcutaneously or intramuscularly to treat rheumatoid arthritis, inflammatory bowel disease, cachexia / anorexia, or multiple sclerosis. By way of example - but not limitation - in yet another particular embodiment truncated sTNFR products may be administered intravenously to treat trauma, epilepsy, hemorrhage or stroke trauma; or administered intraventricularly to treat traumatic brain injury. A preferred method of treating arthritis includes: (i) a single intra-articular injection of a truncated sTNFR product administered periodically as needed to prevent or treat reactivation of arthritis, and (2) periodic subcutaneous injections of a truncated sTNFR product. Initiation

199 399 treatment of septic shock should begin as soon as possible after sepsis or diagnosing the possibility of sepsis. For example, treatment may be initiated immediately following surgery or an accident, or any event that may lower the risk of initiating septic shock. The preferred methods of treating adult respiratory distress syndrome include: (1) single or multiple intra-ophthalmic administration of a truncated sTNFR product, and (2) single bolus administration or continuous intravenous infusion of a truncated sTNFR product.

In another embodiment, cell therapy is also contemplated, for example implantation of cells producing a truncated sTNFR. This embodiment of the present invention may involve implanting into patient cells that are capable of synthesizing and secreting the bizlogizzniz of an active form of the truncated sTNFR. Such cells that produce a truncated sTNFR may be cells that do not normally produce a truncated sTNFR, but which have been modified to produce a truncated sTNFR, or which may be cells whose ability to produce a truncated sTNFR has been increased by conversion with a pzlinucleotide suitable for expression and secretion of the truncated sTNFR . To minimize a potential immune response in patients by administering a truncated sTNFR of a foreign species, it is preferable that the cells are of the same species as the patient (e.g., human) or that the cells are encapsulated with a material that provides a barrier against the recognition of the immnnologizzaemn, or that the cells are inserted. at an immunologically privileged anatomical site such as in the testes, eye, or central nervous system.

Human or animal cells can be implanted in patients with immune, semi-transmissive polymer sheaths or membranes to allow the release of the truncated sTNFR but prevent the destruction of the cells by the patient's immune system or other harmful agents from the surrounding tissue. Alternatively, the patient's own cells, transformed ex vivo to produce a truncated sTNFR, can be imputed directly into the patient's body from such an envelope. The encapsulated membrane methodology of living cells is known to those of ordinary skill in the art, and the production of encapsulated cells and their implantation into the body of patients can be performed.

In yet another embodiment, in vivo gene therapy is also contemplated in which a nucleic acid sequence encoding a truncated sTNFR is introduced directly into the patient. For example, a nucleic acid sequence encoding a truncated sTNFR is introduced into target cells by local coaster injection of the nucleic acid, with or without a suitable delivery vector, such as an adeno-associated virus vector. Alternative viral vectors include (but are not limited to): retrovirus, adeaovirn, herpes simplex virus, and papillomavirus vectors. Physical transfer can be achieved in vivo by local injection of the desired nucleic acid or other appropriate delivery vector containing the desired nucleic acid sequence, liposm-mediated transfer, direct injection of ("naked" DNA), receptor mediated transfer (ligand-DNA complex) or bombardment microparticles (gene cannon).

Exemplary gene and cell therapy techniques are shown in the following entries: US Patent No. 4,892,538; U.S. Patent 5,011,472; U.S. Patent 5,106,627; DE 4219626, WO 94/20517 and 96/22793, the disclosures of which are incorporated by reference in the present application.

Regardless of the mode of administration, treatment of TNF mediated disease requires a dose or total regimen of truncated sTNFR effective to reduce or treat symptoms of the disease. Other factors in determining an appropriate dosage may include: the disease or condition being treated or prevented, the severity of the disease, the route of administration, and the age, sex and medical condition of the patient. Further refinement of the calculations needed to determine an appropriate dosage for treatment is routinely made by those skilled in the art, especially in view of the dosing information in the experiments herein. Dosages can also be determined by the use of known trials to determine the dosages used in conjunction with the respective ones

189 309 dose-response data. The specific dose is calculated according to the approximate body weight or body surface area of ​​the patient.

The frequency of dosing depends on the pharmacokinetic parameters of the truncated sTNFR in the formulation used. A truncated sTNFR can be administered once, or for acute and chronic disorders, given daily in less frequent doses, or given as a high initial dose followed by a continuous dose or slow delivery. In the case of parenteral administration, parenteral unit doses, for example, may be up to 10 mg each, generally up to 15 mg, especially up to 20 mg. For intra-articular administration, the pharmaceutical composition is preferably administered as a single injection, for example in the form of a 3 to 10 ml syringe containing a dose, for example between about 5 mg / ml to 10 mg / ml of truncated sTNFR dissolved in isotonic phosphate buffered saline. The preparation may be administered into the joint cavity with a frequency of, for example, once every 7 to 10 days. In such a manner, administration is carried out continuously, for example 4 to 5 times, with dose changed as necessary.

In some cases, truncated sTNFR products can be administered as an adjunct to other therapy and also with other pharmaceutical preparations suitable for the indication in question. The truncated sTNFR product and any one or more of the traditional or novel anti-inflammatory drugs may be administered separately or in combination.

Products of a truncated sTNFR (for example proteins of the formula R1- [Cys19-Cys1 °³] -R2) and any one or more of the additional anti-inflammatory drugs may be administered separately or in combination. Information on the following compounds can be found in The Merck Manual of Diagnosis und Therupy, 16th Ed., Merck, Sharp &amp; Dohme Research Laboratories, Merck &amp; Co., Ruhway, NJ (1992) and Pharmaprojects, PJB Publications Ltd.

Current treatments for TNF mediated diseases as defined above include acute and chronic inflammation such as rheumatic diseases (e.g., Lyme disease, juvenile (rheumatoid) arthritis, osteoarthritis, psoriatic arthritis, rheumatoid arthritis, and staphylococcal-induced ( "Septic") arthritis includes first-line drugs for pain and inflammation control classified as non-steroidal anti-inflammatory drugs (NSAIDs). Secondary treatments include corticosteroids, slow-acting anti-rheumatic drugs (SAARDs), or disease-modifying drugs (DMs).

In a particular embodiment, the present invention is directed to the use of a truncated sTNFR product (e.g. protein R | -lCysI⁹-CysI^θ3] -R2) and any of one or more NSAIDs for the treatment of TNF mediated diseases as defined above including acute and chronic inflammation such as rheumatic diseases (e.g. Lyme disease, juvenile (rheumatoid) arthritis, osteoarthritis , psoriatic arthritis, rheumatoid arthritis and staphylococcal ("septic") arthritis); and graft-to-host reaction disease. NSAIDs owe their anti-inflammatory effects, at least in part, to inhibition of prostaglandin synthesis (Goodman and Gilman in "The Pharmucological Basis of Therapeutics," MacMillan, 7th ed. (1985)). NSAIDs can be classified into nine groups: (1) salicylic acid derivatives; (2) propionic acid derivatives; (3) acetic acid derivatives; (4) phenamic acid derivatives; (5) carboxylic acid derivatives; (6) butyric acid derivatives; (7) oxicams; (8) pyrazoles and (9) pyrazolones.

In a particular embodiment, the present invention is directed to the use of a truncated sTNFR product (for example R1- [Cys19-Cys1 ° 3] -R2 protein) in combination (pretreatment, follow-up or concurrent treatment) with any one or more salicylic acid derivatives, esters a prodrug or a pharmaceutically acceptable salt thereof. Such salicylic acid derivatives, prodrug esters, and pharmaceutically acceptable salts thereof include: acetaminosalol, aloxyprin, aspirin, benorylate, bromosaligenin, calcium acetylsalicylate, choline magnesium trisulicylate, diflusinal, etersalate, fendosal, gentisic acid, glycol salicylate, imidazole salicylate, lysine acetylsalicylate, mesalaminol, olicylsalicylparsilin, olicylsalicylparsilin phenyl, phenyl salicylate, salacetumid, O-acetic acid salicylamide, salsalate, and sulfasalazine. Structurally related derivative I89 309

4 and ne of salicylic acid with similar analgesic and anti-inflammatory properties are also intended to be encompassed by this group.

In a particular embodiment, the present invention is directed to the use of a truncated sTNFR product (e.g. protein R1- [Cysi9-Cysi0³] -R.2) in combination (pretreatment, follow-up or concurrent treatment) with any one or more propionic acid derivatives, prodrug esters or pharmaceutically acceptable salts thereof. Propionic acid derivatives, prodrug esters, and pharmaceutically acceptable salts thereof include: alminoprofen, benoxaprofen, bucloxic acid, carprofen, dexindoprofen, fenoprofen, flunoxaprofen, fluprofen, flurbiprofen, furcloprofen, ibuprofen, ibuprofen, isoprofen, ibuprofen, isoprofen, ibuprofen, isoprofen, iboprofen, ibuprofen, ketoprofen , naproxen, oxaprozin, piketoprofen, pimeprofen, pirprofen, pranoprofen, prothizic acid, pyridoxyprofen, suprofen, thiaprofenic acid and thioxaprofen. Structurally related propionic acid derivatives with similar analgesic and anti-inflammatory properties are also intended to be encompassed by this group.

In a particular embodiment, the present invention is directed to the use of a truncated sTNFR product (e.g. protein R1- [Cysi9-Cysi °³] -R2) in combination (pretreatment, follow-up or concurrent treatment) with any one or more acetic acid derivatives, prodrug esters or pharmaceutically acceptable salts thereof. Acetic acid derivatives, prodrug esters, and pharmaceutically acceptable salts thereof include: acemetacin, alklofenac, ampenac, bufexamac, cinmetacin, clopyrac, delmetacin, diclofenac sodium, etodolac, felbinac, fenclofenac, fenclorac, fenclosic acid, fentiazac, fentiazac, furlofenac , isofezolac, isoxepac, lonazolac, methiazic acid, oxamethacin, oxspinac, pimethacin, proglumetacin, sulindac, talmetacin, thiaramide, thiopinac, tolmetin, zidomethacin and zomepirac. Structurally related acetic acid derivatives with similar analgesic and anti-inflammatory properties are also intended to be encompassed by this group.

In a particular embodiment, the present invention is directed to the use of a truncated sTNFR product (e.g., R <1> Cys ^ -Cys ^] ^ protein) in combination (pretreatment, aftertreatment or concurrent therapy) with any one or more phenamic acid derivatives, prodrug esters or their pharmaceutically acceptable salts. Phenamic acid derivatives, prodrug esters, and pharmaceutically acceptable salts thereof include: enphenamic acid, etofenamate, flufenamic acid, isonicixin, meclofenamic acid, meclofenamate sodium, medophenamic acid, mefanamic acid, niflumic acid, talnifluman, terofenamate, tolfenamic acid and tolfenamic acid. Structurally related phenamic acid derivatives with similar analgesic and anti-inflammatory properties are also intended to be encompassed by this group.

In a particular embodiment, the present invention is directed to the use of a truncated sTNFR product (e.g. protein R1- [Cysi9-Cysi °³] -R2) in combination (pretreatment, follow-up or concurrent treatment) with any one or more carboxylic acid derivatives, prodrug esters, or pharmaceutically acceptable salts thereof. Carboxylic acid derivatives, prodrug esters, and pharmaceutically acceptable salts thereof that can be used include: clidanac, diflunizal, flufenizal, inoridine, ketorolac, and tinoridine. Structurally related carboxylic acid derivatives with similar analgesic and anti-inflammatory properties are also intended to be encompassed by this group.

In a particular embodiment, the present invention is directed to the use of a truncated sTNFR product (e.g. protein R1- [Cysi9-Cysi °³] -R2) in combination (pretreatment, follow-up or concurrent treatment) with any one or more butyric acid derivatives, prodrug esters or pharmaceutically acceptable salts thereof. Butyric acid derivatives, prodrug esters, and pharmaceutically acceptable salts thereof include: bumadizone, butibufen, fenbufen, and xenbucin. Structurally related butyric acid derivatives with similar analgesic and anti-inflammatory properties are also intended to be encompassed by this group.

In a particular embodiment, the present invention is directed to the use of a truncated sTNFR product (e.g. Rr protein [Cys^I9-Cysi °³] -R2) in combination (le42

189 Pre-treatment, follow-up treatment or concurrent treatment) with any one or more oxicams, washing machine esters, or pharmaceutically acceptable salts thereof. Oxicams, prodrug esters, and pharmaceutically acceptable salts thereof include: droxicam, eolixm, isoxicam, phoxicam, sudoxicam, tenoxicam, and 4- (N-phenyl) -carboxamide 4-hydroxy-1,2-Csazothiazide-1,1-dioxide. Structurally related oxicams with similar analgesic and anti-inflammatory properties are also intended to be encompassed by this group.

In a particular embodiment, the present invention is directed to the use of a truncated sTNFR product (e.g. protein R1- [Cys19-Cys^W3] -R2) in combination (pretreatment, follow-up treatment, or concurrent treatment) with any one or more pyrazoles, washing machine esters, or pharmaceutically acceptable salts thereof. Pyrazoles, cone esters, and pharmaceutically acceptable salts thereof that can be used include: difenamizcil and epirizole. Structurally related pyrazoles with similar analgesic and anti-inflammatory properties are also intended to be encompassed by this group.

In a particular embodiment, the present invention is directed to the use of a truncated sTNFR product (e.g. R? Cys? -Cys?? -Ri protein) in combination (pretreatment, follow-up treatment, or concurrent treatment) with any one IuC more pyrazolones, prodrug esters, or prodrug esters thereof. pharmaceutically acceptable salts. Pyrazole, prodrug esters, and pharmaceutically acceptable salts thereof that can be used include: apase, azapropaoon, piperilone, fepraokn, mkfebutaoo, mkrazka, oxyphena, phenylCutaoo, sucrose, propylphenautase, eamifenase. Some related pyrazolones with similar anti-inflammatory and anti-inflammatory properties are also intended to be included in this group.

In a particular embodiment, the present invention is directed to the use of a truncated sTNFR product (e.g. protein R1- [Cys1⁹-Cys^W3] -R2) in combination (pre-treatment, follow-up luC concurrent treatment) with any one IuC of more of the following NSAIDs: ε-ayetamidocapronquva acid, S-adśąozylmethiaąa, 3-amino-4-hydroxybutyric acid, amixetrin, anitrazafśą, anthrafenin, Cjdaseαk, beadazac lysinate, Cequoydamiąa, bspprosine, broperamoximolacolacolase, dipprosin, broperamoximolacolkamethyl , difenpyramide, difrsalsmine, ditazol, emorphsoo, fanetil mesylate, fenflumizole, flkktaquaqa, flumizole, flunixin, fluprocquoa, fopirtoliaα, fosfozal, guajmezal, g ^ az ^ en, izkaixim, lefetlamine Hefetil lofemizole, ^ fazo) lysine clonicoxylate, meseklαoon, abumśtkn, nikindol, quimesulide, orgotein, oepanoxyya, oxaceprolm, oxapadol, paranyline, perisoxal, ueeisoxal citrate, pifoxime, pyroximolacilakxen, ueeisoxime, uiphoximolacilakxen timegadin, tolektin, tklpadkl, tryptamide and compounds with company code numbers such as 4801565, AA861, AD1590, AFPB02, AFPB6O, AI778, AP504, AU8001, BPPC, BW540C, CHINOIN 127, CN100, EH382, EL508, F1044, FK , GV3658, ITF182, KCNTEI6090, KME4, LA2851, MR714, MR897, MY309, 0N03144, PR823, PV102, PV108, R830, RS2131, SCR152, SH440, SIR133, SPAS510, SQ27239, ST281, SY6001, TAI-Cenz1O acid -1-mdankaeboxy), TVX2706, U60257, UR2301 and WY41770. Structurally related NSAIDs with similar analgesic and anti-inflammatory properties as the above NSAIDs are also intended to be encompassed by this group.

In a particular embodiment, the present invention is directed to the use of a truncated sTNFR product (e.g. protein R 1 - [Cys^|9-Cys1 °³] -R2) in combination (pretreatment, follow-up or concurrent treatment) with any one IuC or more keto keto steroids, prodrug esters, or pharmaceutically acceptable salts thereof for the treatment of TNF mediated diseases as defined above, including acute and chronic inflammation such as rheumatic (e.g. Lyme disease, juvenile (rheumatoid) arthritis, osteoarthritis, psoriatic arthritis, rheumatoid arthritis and staphylococcal ("septic") arthritis); and multiple sclerosis. Kyketic steroids, prodrug esters, and their pharmaceutically acceptable salts include hydrocortisone and compounds which are derived from hydrocrystalline acid, such as 21-acetoxyureaoleolone, alklomseazone, α-estone, amcinonide, beclomethazoin, chloropamethaseolone, benzopropamethase, valentisolone

189 309 clobetasol, clobetusone, clobetasone butyrate, clocortolone, cloprednol, corticosterone, cortisone, cortivasol, deflazacone, desonide, desoxymerazone, dexamethusone, diflorazone, diflucortolone, diflupredetlorone, enoxidin flucinolucetlorone, flucidinum flucinolone, enoxolone, flucinolucinolucinolide, enoxolone fluocortolone acetonide, butyl fluocortin, fluocortolone, fluocortolone hexane, diflucortolone valerate, fluorometolone, pubic fluperol acetate, flupredniden acetate, fluprednisolone, flurandenolide, formocortal, halcinonide, halometasone, halopredone acetate, hydrocortamate, hydrocortisone, hydrocortisone vinegar, hydrocortisone butyrate, hydrocortisone phosphate, hydrocortisone sodium 21-succinate, hydrocortisone tebutate, mazypredomethone, mesilepredinnicornin, mesilepredolonin prednisolone, prednisolone 21-diedryaminoacetate, prednisolone sodium phosphate, prednisolone sodium succinate, 21 - prednisolone sodium m-sulfobenzoate, prednisolone sodium 21-stearoglycolate, prednisolone tebutute, prednisolone 21-trimethylacetone, prednisone, prednisolone, prednilidene, prednilidene-21-diethylacetate, thixocortol, triamcinolone, triamcinolone triamcinolone acetonide, and triamcinolone triamcinolone acetonide. Structurally related corticosteroids with similar analgesic and anti-inflammatory properties are also intended to be encompassed by this group.

In a particular embodiment, the present invention is directed to the use of a truncated sTNFR product (e.g., R 1 Cys 1 -Cys 1 protein³] -R2) in combination (pretreatment, follow-up, or concurrent therapy) with any one or more slow-acting anti-rheumatic drugs (SAARDs) or disease-modifying anti-rheumatic drugs (DMARDs), prodrug esters, or their pharmaceutically acceptable salts for the treatment of diseases mediated by TNF as defined above, including acute and chronic inflammation such as rheumatic diseases (e.g., Lyme disease, juvenile (rheumatoid) arthritis, osteoarthritis, psoriatic arthritis, rheumatoid arthritis and staphylococcal ("septic") arthritis); and multiple sclerosis. SAARDs or DMARDs, prodrug esters, and pharmaceutically acceptable salts thereof include: sodium allocupreide, auranofma, aurothioglucose, aurothioglycanide, azathioprine, sodium brechinar, bucylamine, 3-aurothio-2-propanol --- calcium sulfoman, chlorambucil, chloroquine, clobusarite, kuproxoline, cyclophosphamide, dioxyacualosporin, -5cyclosperemidine glucosamine, gold salts (for example, cyclochine gold salt, sodium sodium thiosulfate, sodium sodium thiosulfate), hydroxychloroquine, hydroxyurea, kebuzone, levamisole, lobenzite, melittin, 6-mercaptopurine, methotrexate, mizoribine, mycophenolate mofetil, mioral, nitrogen mustard, D-penicillamine, pyridinolimidazoles such as SKNF86002 and SB203580, rupamycin, thiols, thymopoietin and vincristine. Structurally related SAARDs or DMARDs with similar analgesic and anti-inflammatory properties are also intended to be encompassed by this group.

In a particular embodiment, the present invention is directed to the use of a truncated sTNFR product (e.g. protein Rr [Cys19-Cys13-IG) in combination (pretreatment, follow-up or concurrent treatment) with any one or more COX2 inhibitors, prodrug esters or their pharmaceutically acceptable salts for the treatment of TNF mediated diseases as defined above, including acute and chronic inflammation. Examples of COX2 inhibitors, prodrug esters, or pharmaceutically acceptable salts thereof include, for example, celecoxib. Structurally related COX2 inhibitors with similar analgesic and anti-inflammatory properties are also intended to be encompassed by this group.

In a particular embodiment, the present invention is directed to the use of a truncated sTNFR product (e.g. protein R1- [Cys^AND9-CysI^θ3j-R.2) in combination (pretreatment, follow-up or concurrent treatment) with any one or more antibacterial agents, prodrug esters or pharmaceutically acceptable salts thereof for the treatment of TNF mediated diseases as defined above, including acute and chronic inflammation . Antimicrobial agents include, for example, ampicillin, amoxicillin, aureomycin, bacitracin, ceftracin, cefti ^^ ydymium, ceftriaxone, cefotaxime, cefachlor, cephalexin, cefradin, ciprofloxacin, neutricanic acid, cloxacillicin, diclucilicin, diclucilicin, diclucilicin, diclucillin, diclucilicin,

I89 309 oxacylate, penicillin and vancomycin. Structurally related antimicrobial agents with similar analgesic and anti-inflammatory properties are also intended to be encompassed by this group.

In a particular embodiment, the present invention is directed to the use of a truncated sTNFR product (e.g. protein R1- [Cysi⁹-Cysi °³] -R2) in combination (pretreatment, follow-up or concurrent treatment) with any one or more of the following compounds for the treatment of TNF mediated diseases as defined above, including acute and chronic inflammation: granulocyte colony stimulating factor; thalidomide; BN50730; tenidap; E 5531; PCA thiapaphant 4248; nimesulide; panavir; rolipram; RP 73401; T peptide; MDL 201,449A; (13R4, 3S) -cis-1- [9- (2,6-N-diaminopurinyl)] -3-hydroxy-4-cyclopentene hydrochloride; (1R, 3R) -trans-1- [9- (2,6-diamino) purine] -3-acetoxycyclopentane; (1R, 3R) -trans --- [9-adenyl) -3-azidocyclopentane hydrochloride 1 (1 R, 3R) -trans-1 - [6-hydroxy-purin-9-yl) -3-azidocyclopentane.

In a particular embodiment, the present invention is directed to the use of a truncated sTNFR product (for example R ^ Cys ^ -Cys ^ J ^ protein) in combination (pretreatment, follow-up or concurrent therapy) with one or more additional TNF inhibitors for the treatment of diseases mediated by TNF mediated as defined above, including acute and chronic inflammation. TNF inhibitors contain compounds and proteins that block in vivo synthesis or extracellular release of TNF, including the following compounds.

Additional TNF inhibitors include anti-TNF antibodies (e.g. MAK 195F Fab antibody (Holler et al. (1993), 1st International Symposium on Cytokines in Bone Marrow Transplantation, 147; CDP 571 - anti-TNF monoclonal antibody (Rankin et al. 1995), British Journal of Rheumatology, 34: 334-342, the disclosures of which are hereby incorporated by reference); BAY X 1351 murine anti-tumor necrosis factor monoclonal antibody (Kieft et al. (1995), 7th European Congress of Clinical Microbiology and Infectious Diseases, 9, the disclosure of which is hereby incorporated by reference); CenTNF cA2 - anti-TNF monoclonal antibody (Elliott et al. (1994), Lancet, 344: 11251127 and Elliott et al. (1994), Lancet, 344: 1105-1 110, the disclosures of which are hereby incorporated by reference).

In a particular embodiment, the present invention is directed to the use of a truncated sTNFR product (e.g. protein R1- [Cysi⁹-Cysi °³] -R2) in combination (pretreatment, follow-up or concurrent treatment) with soluble recombinant human Fas antigen or recombinant variants thereof (WO 96/20206 and Mountz et al., J. Immunology, 155: 4829-4837; iEP 510 691; ), the disclosures of which are hereby incorporated by reference in the present application. WO 96/20206 discloses a secreted human Fas antigen (native and recombinant, including an Ig fusion protein), methods for isolating the genes responsible for encoding soluble recombinant human Fas antigen, methods for cloning the gene in suitable vectors and cell types, and methods for expressing the gene to produce inhibitors . EP 510 691 teaches DNA encoding a human Fas antigen, including soluble Fas antigen, expression vectors for said DNAs, and vector transfected transformants. When administered parenterally, the dosages of each Fas antigen fusion protein are generally from 1 microgram / kg to 100 microgram / kg.

In a particular embodiment, the present invention is directed to the use of a truncated sTNFR product (e.g. protein R1- [Cysi⁹-Cysi ° 3] -R₂) in combination (pretreatment, follow-up or concurrent treatment) with any one or more interleukin-1 inhibitors for the treatment of TNF mediated diseases as defined above, including acute and chronic inflammation such as rheumatic diseases (e.g. (rheumatoid) arthritis, osteoarthritis, psoriatic arthritis, rheumatoid arthritis and staphylococcal ('septic') arthritis); trauma to the brain as a result of trauma, epilepsy, hemorrhage or stroke; and multiple sclerosis. The classes of interleukin-1 inhibitors include interleukin-i receptor antagonists (any compound capable of specifically preventing activation of cellular receptors to IL-1) such as IL-1ra, as described below; anti-IL-1 - receptor monoclonal antibodies (for example EP 623674, of which

199 099 the disclosure is incorporated herein by reference); IL-1 binding proteins, such as soluble IL-1 receptors (for example, USP 5,492,888, USP 5,488,032, USP 5,464,937, USP 5,319,071, and USP 5,180,812, the disclosures of which are hereby incorporated by reference); anti-IL-1 human antibodies (e.g. WO 9501997, WO 9402627, WO 9006371, USP 4,935,343, EP 364778, EP 267611 and EP 220063, the disclosures of which are incorporated by reference in the present application); additional IL-1 receptor proteins, for example WO 96/23067 (the disclosure of which is hereby incorporated by reference) and other compounds and proteins that block in vivo synthesis or extracellular release of IL-1.

The iatzrleukin-1 receptor antagonist (Il-1ra) is a human protein that acts as a natural iaterlznkSny-1 inhibitor. Preferred receptor antagonists, as well as methods of making and using them, are described in US Patent 5,075,222 (referred to herein as the '222 patent); WO 91/08285; WO 91/17184; AU 9173636; WO 92/16221; WO 93/21946; PCT International Application No. US97 / 02131, which teaches a pharmaceutical composition comprising (a) an effective amount of a controlled release polymer (e.g., hyalnuric acid), and (b) an effective amount of Il-1ra; WO 94/06457; WO 94/21275; FR 2706772; WO 94/21235; DE 4219626, WO 94/20517; and WO 96/22793, the disclosures of which are incorporated by reference in the present application. Proteins will become glycosylated as well as nizglycosylated forms of IL-1 receptor antagonists.

In particular, three preferred forms of Il-1ra (IL-1raa, Il-1raP, and Il-1rax), each derived from the same DNA coding sequence, are disclosed and described in US Patent 5,075,222 by Hannum et al. "Interlzukin-T Inhibitors." The US patent, referred to herein as the '222 patent, is specifically incorporated herein by reference. All three of the iaterleukin-1 inhibitors have similar functional and immune activities. Methods of making inhibitors of IL-1, in particular IL-1ras, are also disclosed in the '222 patent. One method disclosed relates to the isolation of inhibitors of human moaositites (where they are naturally produced). The second method disclosed involves isolating the gene responsible for encoding 11L-1ra, cloning the gene in appropriate vectors and cell types, expressing the gene to produce 11L-1ra, and harvesting IL-1ra. The latter method, which exemplifies DNA restoration methods in general, is the preferred method of the invention. In a particular embodiment, lL-1ra contains an N-terminal metiary group as a consequence of E. coli dispersion. The present invention also includes modified 11ra. Modified IL-1ra contains, for example, muteins of such inhibitors, in which a cysteine ​​residue is substituted for an amino acid at the nucleus or more sites in the amino acid sequence of the naturally occurring inhibitor. Such mzines may then be site-selectively reacted with polyethylene glycol (PEG) units or other thiol-containing polyethers to form 11L-1ra PEG. PCT Publication No. WO 92/16221 discloses a number of modified IL-1ra and methods for making such PEG modified inhibitors.

An additional class of interlenkin-1 inhibitors will include compounds that are able specifically to prevent activation of cellular receptors to IL-1. The compounds also contain IL-1 binding proteins, as well as soluble mzazclonal receptors and antibodies. Such compounds also contain monzclone antibodies to the receptors.

A further class of iaterleukiay-1 inhibitors contains compounds and proteins that block in vivo synthesis and / or extracellular release of IL-1. The compounds also contain agents that affect the transcription of IL-1 genes or the processing of IL-1 preproteins.

The foregoing is given by way of example and does not exclude other therapies that may be used concurrently with those anti-inflammatory compounds that are known to those skilled in the art or that could be accomplished by those skilled in the art using the guidelines set forth herein.

It is especially advantageous to formulate additional anti-inflammatory compounds in unit dosage form for ease of administration and uniformity of dosage. A "unit dosage form" in the present application refers to physically discontinuous units suitable as unitary dosages for a mammalian subject to be treated, each unit containing a predetermined amount of additional compounds.

189 309 anti-inflammatory drugs calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. In the present application, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antimicrobial and anti-fungal agents, isotidic and absorption delaying agents, etc. that are compatible with the active ingredient and the mode of administration and the other ingredients of the formulation and are not harmful to the recipient. The use of such media and agents is well known in the art (see, for example, Remington's Pharmaceutical Sciences, 18th Ed. (1990), Mack Publishing Co., Easton, PA 18042, pp. 1435-1712, the disclosure of which is incorporated in the present application. as a link). Supplementary active ingredients can also be incorporated into the compositions.

For oral therapeutic administration, the additional compound may be incorporated with excipients and formulated in the form of digestible tablets, oral tablets, lozenges, capsules, tinctures, suspensions, syrups, wafers, etc., or may be incorporated directly into the food. Tablets, lozenges, pills, capsules, etc. also may contain the following: a binder such as gum tragacadium, acacia, corn starch, or gelatin; excipients such as aimapdioma phosphate; a disintegrant such as corn starch, alginic acid and the like; a lubricant such as magnesium stearate; a sweetening agent such as sucrose, lactose or saccharin; Or a flavor such as peppermint, winter oil, real or cherry or orange flavor. When the dosage unit form is a capsule, it may contain, in addition to a material of the above type, a liquid carrier. Various other materials may be present as the coating or to otherwise modify the physical form of the dosage unit. For example, tablets, pills, or capsules may be coated with shellac, sugar, or both. Of course, any material used in preparing any unit form of tomato acomania should be pharmaceutically pure and substantially non-toxic in the amounts used. In addition, an additional anti-inflammatory compound may be included in the untimely release formulation. The amount of additional anti-inflammatory compound in such therapeutically useful kemaesis is such as to obtain the desired damomane.

In the case of ara-enterally capable of therapeutic administration, any additional anti-inflammatory compound may be included in a sterile injectable solution. The sterile idiopathic solution can be prepared by incorporating the additional anti-inflammatory compound in the required amount in a pharmaceutically acceptable carrier, with various other ingredients enumerated below (desired), followed by filter sterilization. In the case of dispersions, each can be prepared by including the additional prrecimzapalnpga compound in a sterile vehicle that contains the basic dispersion medium and the desired other ingredients from those enumerated above. In the case of sterile injection solutions, each of them may be prepared by including a powder - an additional anti-inflammatory compound and, optionally, a further desired ingredient from a previously filtered solution, the powder being prepared by any technique (e.g. ).

The specific dose of any desired anti-inflammatory compound is calculated according to the approximate body weight or body surface area of ​​the patient. Other factors involved in determining eaaomipadieae damkomania may include: the disease or condition being treated or prevented, severity of the disease, route of administration, and the age, sex and health condition of the patient. Further refinement of the calculations needed to determine an appropriate dose of treatment dosages is routinely made by those skilled in the art, especially in light of the dosing information in the trials disclosed herein. Dosage can also be determined by using known assays to determine damaskadia used in conjunction with damask behavior data.

Thus, for example, it is within the scope of the invention that doses of additional anti-inflammatory compounds selected for the treatment of particular acute or chronic inflammatory diseases such as rheumatic diseases (e.g. Lymp disease, juvenile (rheumatoid) arthritis, osteoarthritis, psoriatic arthritis, rheumatoid arthritis, arthritis and udder inflammation by arądPomce ('septic') inflammation

189 309 joints) can be varied to achieve the desired therapeutic effect. Where one of the additional anti-inflammatory compounds has side effects, it may be administered to patients during alternating treatment periods in combination therapy. For example, chronic treatment with metaSrexate has been associated with gastrointestinal, hepatic, bone marrow and pulmonary toxicity (Sapdoval et al. (1995), British Journal of Rheumatology, 34: 49-56, the disclosure of which is incorporated by reference in the present application).

Assays for monitoring disease improvement may include specific targeted toast, for example, pa, a systemic response, pa, inflammation, which includes erythrocyte sedimentation rate (ESR) and acute phase reactants (APR). Observations of swelling etc. of the affected body parts are made. Improvement in stiffness and grip is also seen (where applicable) and reduction of pain in the patient. If the patient's foot is stable, he or she is re-treated at the same dosing weekly and assessed weekly. Treatment can be continued as long as the patient's sta) is stable. After six months of treatment, the anatomical changes of the skeleton are determined by means of radiological imaging, for example, by X-ray.

At the end of each period, the patient is reassessed. A comparison of pre-treatment and follow-up using radiographic assessment, ESR and APR indicates treatment efficacy. According to the effectiveness of the treatment and the patient's condition, the dosage may be increased or kept constant for the duration of the treatment.

Preferably, the present invention is directed to a method with, optionally, one of the following combinations for the treatment or prevention of an acute or chronic inflammatory disease and condition as defined above, such as rheumatic diseases (e.g. Lyme disease, juvenile (rheumatoid) arthritis, osteoarthritis and arthritis, psoriatic arthritis, rheumatoid arthritis and staphylococcal (optic) arthritis): a product of a truncated sTNFR (for example protein R: - [Cys1⁹-Cys1⁰³] -R2) and method-oxate; truncated sTNbR product (for example, protein R: - [Cytl⁹-Cytl^θ3] -R2), a metaSrectate and an inhibitor of IL-1, preferably IL-1ra; a product of a truncated sTNFR (for example protein R: - [Cyt: 9-Cytl^θ3i-R2) and any one or more selected from: oxate-method, immunotuyretapt (e.g. cyclaspora), ciproflactasin, Fas antigen and an IL-1 inhibitor, preferably IL-1ra; a product of a truncated tTNbR (for example R ^ Cys ^ -Cys ^^ protein) and metatroctate and immunotupretant (for example cyclotharipe); truncated sTNFR product (for example protein R: - [Cys -Cytl⁰³i-R2) and metaSrectat and ciproflaxacin; and a truncated sTNFR product (for example protein R: - [Cysl⁹-Cytl^θ3] -R2) and a metatrectate and inhibitor of IL-1, preferably IL-1ra; truncated sTNFR product (exemplary protein R1- [Cys1⁹-Cys^W3] -R2) and any one or more of the following: meSotrexate, sulfatazine and hydroxychloroship; a product of a truncated sTNFR (for example the protein R1- [Cysl⁹-Cytl^θ3i-R2). metatrectate and hydroctyhloroquine; and a truncated sTNFR product (for example protein R1- [Cysl⁹-Cytl^θ3] -R2), mothotrexate and sulfasazine.

In a specific preferred embodiment, the method comprises administering (e.g. dasSawe, subcutaneously or intramuscularly) a truncated NFR product (e.g. protein R: - [Cytl9-Cysl⁰³] -R2), optionally in the form of a slow release formulation (e.g. hyaluropate)), optionally in combination (pretreatment, follow-up or concurrent treatment) with metatrexate and / or an IL-1 inhibitor (e.g. IL-1ra) and / or solvent recombinant human Fas antigen for the treatment of rheumatic diseases as defined above (e.g. Lyme disease, juvenile (rheumatoid) arthritis, osteoarthritis, psoriatic arthritis, rheumatoid arthritis and staphylococcal ("septic") arthritis and symptoms associated therewith.

In a particular preferred embodiment, the method comprises administering (e.g. intravenously or intraventricularly) a sT1R truncated product (e.g. protein R1- [Cys^!9-Cit⁰³i-R2. optionally in the form of a slow-release formulation (e.g. hyaluroniap)) optionally in combination (pretreatment, follow-up or concurrent treatment) with a tissue plosminogep activator and / or an IL-1 inhibitor (e.g. IL-1ra) to treat trauma to the brain , epilepsy, haemorrhage or stroke, any of which can lead to neurodegeneration.

I89 309

In a particular preferred embodiment, the method comprises administering (e.g. subcutaneously or intramuscularly) a truncated sTNFR product (e.g. protein - Rr [Cys'9-Cysi °³] -R2, optionally in the form of a slow release formulation (e.g. hyaluronan)), optionally in combination (pretreatment, follow-up or concurrent treatment) with one or more corticosteroids, cyclosporin, FK-506, or an interferon (e.g. alpha interferon, beta-interferon, gamma-interferon or consensus interferon) and / or TL-Ira to treat multiple sclerosis.

In a particular preferred embodiment, the method comprises administering (e.g. subcutaneously or intramuscularly) a truncated sTNFR product (e.g. protein R 1 Cys 1 -CysD³] -R2, optionally in the form of a slow release formulation (for example hyaluronan)), optionally in combination (pretreatment, follow-up or concurrent treatment) with G-CSF and / or IL-1ra to treat an inflammatory disease of the gut.

In a particular preferred embodiment, the method comprises administering (e.g. subcutaneously or intramuscularly) a truncated sTNFR product (e.g. protein R1- [Cys¹⁹-Cysi ° 3] -R2, optionally in the form of a slow release formulation (for example hyaluronan)) optionally in combination (pretreatment, follow-up or concurrent treatment) with leptin, Marinol ™ or Megace ™ to treat cachexia / anorexia.

In a particular preferred embodiment, the method comprises administering (e.g. subcutaneously, intraventricular or intrathecal) a truncated sTNFR product (e.g. protein R1- [Cysi⁹-Cysi °³] -R2), optionally in the form of a slow release preparation (e.g. hyaluronan)), optionally in combination (pretreatment, follow-up or concurrent treatment) with an NSAID (e.g. indomethacin) and / or an IL-1 inhibitor (e.g. IL-1ra) to treat Alzheimer's disease.

In a particular preferred embodiment, the method comprises administering (e.g. subcutaneously, intraventricular or intrathecal) a truncated sTNFR product (e.g. protein R1- [Cysi⁹-Cysi0³] -R2, optionally in the form of a slow release formulation (e.g. hyaluronan)), optionally in combination (pretreatment, follow-up or concurrent treatment) with a soluble recombinant human Fas antigen to treat cancer (e.g. leukemia), diabetes (e.g. juvenile onset) type i) diabetes; graft versus host reaction; hepatitis; ischemia / reperfusion injury, including cerebral ischemia (trauma to the brain as a result of trauma, epilepsy, hemorrhage, or stroke, any of which can lead to neurodegeneration); neuroinflammatory diseases; rheumatic diseases as defined above (for example, Lyme disease, juvenile (rheumatoid) arthritis, osteoarthritis, psoriatic arthritis, rheumatoid arthritis and staphylococcal ("septic") induced and tissue transplantation.

Other aspects and advantages of the present invention will be apparent from consideration of the following illustrative examples.

Standard methods for many of the procedures described in the examples below or suitable alternative procedures are given in widely recognized textbooks of molecular biology such as, for example, Sambroock et al. (1989) supra and Ausubel et al (1990) supra. For the convenience of our readers, 'mL' stands for milliliters and 'L' stands for liter.

Example I.

The following examples teach the preparation of various forms of truncated, soluble recombinant TNFR-I:

NH2-MDSVCPQGKYIHPQNNSIC- [Cysi⁹-Cyys⁰³] -FC-COOH (sTNFR-I 2.6D / C105);

NH2-MDSVCPQGKYTHPQNNSIC- [Cys19-Cys¹⁰³l-FNCSL-COOH (sTNFR-I2.6D / C106);

NH2-MDSVCPQGKYIHPQNNSIC- [Cys19-Cys103] -FN-COOH (sTNFR-12.6D / N105);

NH2-MYIHPQNNSIC- [Cys-Cysi0³] -FNCSL-COOH (sTNFR-I 2.3D / d8);

NH2-M- [Cys19'-Cysi⁰³] -FNCSL-COOH (sTNFR-I 2.3D / d18); and

NH2-MSIS- [Cys^r9-Cysi °³] -FNCSL-COOH (sTNFR-I 2.3D / d15).

A. Production of DNA:

1. sTNFR-I 2.6D / C106

PCR amplification of sTNFR-I 2.6D / C106 is carried out using a derivative of the cloned cDNA from the Iambda-gt107ctnfbp clone (EP 422339) as a template and the following PCR primers:

189 309

5 'OLIGO # 1: (SEQ ID NO: 68)

5 '-GGTTAGCCATATGGACAGCGTTTGCCCCCAA-3'

3'- OLIGO # 2 (SEQ ID NO: 69)

5'-CCCAAGCTTTTACAGAGAGCAATTGAAGCACTG-3 '

Compounds OLIGO11 and OLIGO # 2 encode NdeI and HindIII and stick to the 5 'and 3' ends of the truncated gene, respectively. PCR amplification is performed for 25 cycles; each cycle consists of 30 seconds at 94 ° C for denaturation, 15 seconds at 55 ° C for adhesion and 1 minute at 72 ° C for elongation [Model 2400 thermal cycler (Perkin-Elmer Cetus, Norwalk, CT]. The PCR product is purified using the "QIAquick ™ PCR Purification Kit" (QIAGEN, Chatsworth, CA) according to the manufacturer's instructions. The purified PCR product is cut with NdeI and Hindm and then gel purified using the "QIAquick ™ Gel Extraction Kit" (QIAGEN, Chatsworth, CA) according to the manufacturer's instructions. The gel separated PCR product is ligated into pAMGII (WO 95/26746) and transformed into FM15 E. coli cells (ATCC 55765).

2. sTNFR-I 2.6D / C105

PCR amplification of sTNFR-I 2.6D / C105 is carried out using sTNFR-I 2.6D / C106 plasmid DNA as template and the following PCR primers:

OLIGO # 3: (SEQ ID NO: 70)

5'-ACTCGAGGATCCGCGGATAAATAAGTAACGATCCGGTCCA-3 '

OLIGO # 4: (SEQ ID NO: 71)

5'-CAGGTCGGATCCTATCAGCAGAAGCACTGGAAAAGGTTTTC-3 '

The compounds OLIGO # 3 and OLIGO # 4 encode BamHI and a mutation N (105) C followed by stopcodone. They are (OLIGO # 3 and OLIGO # 4) designed to extend completely around the pattern in order to introduce a new BamHI site for the string. PCR amplification is performed for 35 cycles; 10 cycles each of 10 seconds at 92 ° C for denaturation, 30 seconds at 55 ° for adhesion, and 4 minutes at 68 ° C for elongation, followed by 25 cycles of each composition 10 seconds at 92 ° C for denaturation, 30 seconds at 55 ° C for sticking, and 4 minutes + 20 seconds at 68 ° C for elongation [Model 2400 thermal cycler (Perkin-Elmer Cetus, Norwalk, CT]. The PCR product is gel purified using the "QIAquick ™ Gel Extraction Kit" (QIAGEN, Chatsworth, CA) according to the manufacturer's instructions, cut with BamHI, extracted with phenol / chloroform and ethanol precipitated. After resuspension, it is ligated into pAMG11 and transformed into E. coli Fm cells.

3. sTNFR-I 2.6D / N105

PCR amplification of sTNFR-I 2.6D / N105 is carried out using sTNFR-I 2.6D / C106 plasmid DNA as a template and the following PCR primers

5 'OLIGO # 5: (SEQ ID NO: 72)

5'-GGTTAGCCATATGGACAGCGTTTGCCCCCAA-3 '

3 'OLIGO # 6: (SEQ ID NO: 73)' -CGCGGATCCCTATTA.TTGAAGCACTGGAAAAGG-3 '

Compounds OLIGO # 5 and OLIGO # 6 encode NdeI and BamHI, respectively, and stick to the 5 'and 3' ends of the truncated gene. PCR amplification is performed for 35 cycles; each cycle consists of 45 seconds at 95 ° C for denaturation, one minute at 65 ° C for adhesion and 2 minutes at 72 ° C for elongation [Model 2400 thermal cycler (Perkin-Elmer Cetus, Norwalk, CT] .

The PCR product is purified using the "Wizard ™ DNA Clean-Up System (Promega, Madison, WI) according to the manufacturer's instructions. The purified PCR product is cut with NdeI and BamHI, extracted with phenol / chloroform and precipitated with ethanol. After resuspension, it is ligated into pA-MG11 and transformed into FM15 E. coli cells.

Based on the description of the present invention, those of ordinary skill in the art will appreciate that a variety of materials and methods can be readily used or adapted for appropriate expression in a host cell (e.g., E. coli and other bacteria).

4. sTNFR 2.3D / d18; sTNFR-I 2.3D / d8 and sTNFR-I 2.3D / d15

189 309

Each of the sTNFR 2.3D / d18 PCR amplification; sTNFR-I 2.3D / d8 and sTNFR-I 2.3D / d15 were run using plasmid DNA 2.6D / C106 as template and the following primers:

sTNFR-I 2.3D / d8 PCR primers:

5 'OLIGO # 7: (SEQ ID NO: 74)

5'-CCCCATATGTATATCCACCCTCAAAATAAT-3 '

3 'OLIGO # 9: (SEQ ID NO.75)

5 '-CCCAAGCTTTTACAG AGAGCAATTGAAGCACTG-3' sTNFR-I 2.3D / d15 PCR primers:

5 'OLIGO # 9: (SEQ ID NO: 76)

5'-CCCCATATGTCGATTAGCTGTACCAAGTGCCACAAAGG-3 '

3 'OLIGO # 10: (SEQ ID NO: 77)

5'-CCCAAGCTTTTACAGAGAGCAATTGAAGCACTG-3 'sTNFR-I 2.3D / d18 PCR primers

3 'OLIGO # 11: (SEQ ID NO: 78)

5'-CCCCATATGTGTACCAAGTGCCACAAAGGA-3 '

3 'OLIGO # 12: (SEQ ID NO: 79)

5'-CCCAAGCTTTTACAGAGAGCAATTGAAGCACTG-3 '

The OLIGO # 7, OLIGO # 9 and OLIGO # 11 each encode Ndel and each zOLIGO # 8, OLIGO # 10 and OLIGO # 12 encode Hindlia. PCR amplification is performed in 25 cycles: each cycle consists of 45 seconds at 95 ° C for denaturation) one minute at 65 ° C for sticking and 2 minutes at 72 ° C for extension [tśrmkcykler Model 2400 (Peekią-Elmśr Cetus, Ndrwalk, CT]. PCR products are purified using the "Wizard ™ DNA Clean-Up System (Promega, Madison, WI) according to the manufacturer's instructions. Purified PCR products are cut with NdeI and HindIII, extracted with ffnK1 / chlorophyll chloride and ethanol precipitated. After resuspension, it is ligated into pAMG11 and transformed into E coli FM cells.

A. Production in E. coli:

At the outset, one small fresh culture was initiated of the strain of the desired E. coli female Cinα fetal clone containing the desired construct for sTNFR-I 2.6D / N105, sTNFR-I 2.6D / C105, sTNFR-I 2.6D / C106, sTNFR 2.3D / d18, sTNFR-I 2.3D / d8 and sTNFR-I 2.3D / d15 by transferring the entire contents of the ampoule (approximately 1.5 ml) of frozen spare embryos in a 2 liter spike containing 500 ml Luna broth. Cultivate and crawl in a vortex chaser at 37 ° C and 350 rpm. The culture density is determined by measuring the abcence at 660 nm (OD660). The embryo culture is grown to a density> 2.0 (OD660) and at this point 125 ml is aseptically transferred to a 15 liter production fermentation vessel containing 10 liters of sterile growth medium.

The type of medium and fermentation conditions in the production fermentation vessel are complex fermentation conditions in the medium, some described by Sniff (1993) in the doctoral dissertation "A Chemically-Defmśd Medium for the Overproduction of a Reckmbiąaąat Protein in E coli". Bodies in E. coli), Colorado State University. Generally, reference discloses the use of a complex medium containing casein hydrolyzate, salts, glycerin and a diluting agent which is sterilized in a fermentation vessel. After the container has cooled to below 40 ° C, filter-sterilized trace minerals and thiamine hydrochloride are added.

When the medium temperature is fixed at 37 ° C, the medium is inoculated with the embryo culture. The growth of the culture is monitored by measuring the OD660. The culture is kept at pH = 6.0 by the automatic addition of 5M sodium hydroxide and 5M hydrochloric acid. When the OD660 is between 9.5 and 10.5, the culture is induced by aseptic addition of sterile isopropyl (IPTG) to a final concentration of 0.50 mM. The culture is harvested after cessation of growth.

The medium and growth conditions are as described by Sniff (1993) - supra, with the following exceptions: ammonium sulfate (2.0 g / L) and L-cystsine hydrochloride monohydrate (1.0 g / L) are added to the medium; tetracycline hydrochloride is omitted; The pH is maintained at 89 309

5i at 6.0 with sodium hydroxide and hydrochloric acid rather than at 7.0 with only sodium hydroxide; the growth temperature is increased to 37 ° C; the inducer concentration is increased from 0.15 mM to 0.50 mM IPTG; the harvest criterion is based on the cessation of growth rather than the lapse of time after induction.

After fermentation is complete, cells are harvested by centrifugation in 500 ml bottles. Cells are pelleted by centrifugation at 10,000 rpm. for 30 minutes. The recovered cell paste is diluted to 15% solids in a breaking buffer consisting of 50 mM Tris and 5 mM EDTA at pH 8.0. The cell suspension is then lysed by passing the solution three times through a homogenizer (APV Gaulin, Inc., Everett, MA) operating at a pressure of 5.52 x 10⁷ Pa (8,000 psi). The resulting homogeneous material is then centrifuged at 10,000 rpm. for 30 minutes to separate inclusion bodies (IBs). IBs bodies are washed by resuspending in stop buffer and centrifuging the solution a third time at 10,000 rpm for 30 minutes. IBs bodies are resuspended in deionized water (1: 1 ratio) and centrifuged one last time at 10,000 rpm for 30 minutes for a second wash. The separated, washed inclusion bodies for each protein are ready to dissolve, reassemble and cleanse. Each flush produces approximately 200-250 grams of IBs bodies.

In an alternative embodiment, a truncated sTNFR-I may be fermented as follows:

Initially, one small fresh culture of the desired recombinant E. coli clone strain that stores the desired construct for sTNFR-I 2.6D / N105 or sTNFR-I 2.6D / C106 is started by transferring the entire contents of the ampoule (approximately 1.5 ml) frozen in glycerol of spare embryos to a 2-liter flask containing 500 ml of BBL yeast extract at a concentration of 10 g / l, opH = 7.0. The culture is incubated in a vortex shaker at 37 ° C and 300 rpm. The density of the culture is determined by measuring the absorbance at 600 nm (OD 600). The embryo culture is grown to a density> 2.0 OD600, at which point it is aseptically transferred (80 ml) to a 15 liter production fermentation vessel containing 7 liters of sterile growth medium.

The production fermentation is carried out by the reagent feed method. The medium is a complex medium containing yeast extract, salts and an anti-foaming agent which is sterilized in the fermentation vessel. After the container has cooled to below 40 ° C, filter-sterilized trace minerals, glucose, magnesium sulfate and hexametaphosphate are added. A feed is used with two media, the first, Feed 1, is a carbon (glucose / magnesium sulfate) feed, and the other, Feed 2, is a nitrogen feed, based on yeast extract.

When the medium temperature is stabilized at 33 ° C, the medium is inoculated with the embryo culture. The growth of the culture is monitored by measuring the OD 600. The culture is kept at pH = 7.0 by automatic addition of ammonia and 48.78% citric acid solution. When the OD600 is between 8.0 and 12.0, Feed 1 is started using an exponential feed rate. When the OD600 is between 30.0 and 40.0, Feed 2 is started using a constant feed rate. When the OD 600 reaches 67-83, the culture is induced by aseptic addition of a sterile auto-inducer (homoserine lactone) to a final concentration of 0.6 mg / L. Feed 1 and Feed 2 feed speed becomes constant when induction starts. The culture is harvested after 16 ± 2 hours.

After fermentation is complete, cells are harvested by centrifugation in 500 ml bottles. Cells are pelleted by centrifugation at 10,000 rpm. for 30 minutes. The recovered cell paste is diluted to 15% solids in a breaking buffer consisting of 50 mM Tris and 5 mM EDTA at pH 8.0. The cell suspension is then lysed by passing the solution three times through a homogenizer (APV Gaulin, Inc., Everett, mA) operating at 5.52 x 107 Pa (8000 psi). The resulting homogeneous material is then centrifuged at 10,000 rpm. for 30 minutes to separate inclusion bodies (IBs). IBs bodies are washed by resuspending in stop buffer and centrifuging the solution a third time at 10,000 rpm for 30 minutes. IBs bodies are resuspended in deionized water (1: 1 ratio) and centrifuged one last time at 10,000 rpm for 30 minutes for a second wash. Separated, washed

189 309 the bodies that contain each protein are ready to be lubricated and can be assembled and cleaned.

3. Selubilization / Seeking assembly:

The washed bodies and inclusions (IBs) with each total of 10 liters of fermentation are dissolved in 800 ml of solubilization buffer (50 mM Tris, 8M urea, 160 mM cysteine, pH = 9.5). The pH of the mixture is adjusted to 9.5 with 10N NaOH and stirred at room temperature for 2-3 hours. Each roll gives about 200-250 grams of IBs bodies.

Each mixture is or is preferably diluted 1:20 with cold renaturation buffers (50 mM Tris, 1.1 M urea). The final volume of each myocadine will be approximately 16 liters. Then each mixture is adjusted to pH = 9.7 with 6N HCl and slowly stirred at 4 ° C for 2-3 days.

The pH of each stirrer is then adjusted to pH 5.0 with glacial acetic acid and 6N HCl. Each mioszadid precipitates which is removed by purge at 10,000 g in a Beckman Model J2-HS centrifuge. Each material is then filtered through a 5 [mu] m and 0.22 [mu] m filter.

D. Cleansing:

The composite materials are ready for column purification on an IX-1 SP-Sepharose Big Boad ™ column (Pharmacia Biotech, Inc., Piscatemay, NJ).

Column Type IX-1 SP-Sepharaso Big Boad ™ (4.4 cm x 20 cm)

Buffer A Buffer B mM acetate 25 mM ocaan mM NaCl 375 mM NaCl pH = 5.0

The column is equilibrated with 4-5 column volumes of Buffer A before distributing the charges of each refolding material. The reassembled materials are separately fed to the column for purification. Each time the column is loaded with less than 12 grams of protein per liter of resin. After each aliquot applied to the column, the column is washed with 3-4 column volumes of buffer A (until the UV recovers to background deadness). Each time the column is loaded, the protein is eluted from the column using 8 column volumes of a buffer with a linearly increasing salt concentration gradient between 50-375 NaCl. The entire protein peak is collected as one aliquot. Harvesting of each protein peak begins as the UV absorbance increases to about 20% of the peak maximum. Harvesting stops as soon as the UV absorption reaches about 50% of the peak peak or when the abrasion stops degrading whatever comes first.

Flow rate - 7.5 cm3 / aoaz. During balancing and washing

- 15 cm3 / h for rałaaąmadie

- 6 cn ^ / gomz go yyamiu

Each column purification is performed at 4 ° C.

Each IX-1 harvest solution is ready for purification on a Toyo Pearl ™ Butyl 650M HIC column (Toso Haas, Philadelphia, PA).

300 ml column - Toyo Pearl Butyl ™ 650M (4.4 cm x 20 cm)

Buffer A buffer diluting buffer B mM NaPO₄ 40 mM N NaPO₄ Milli Q H2O

1.8 M NaCl, pH = 6.0 4 M NaCl, pH = 6.0

The column is equilibrated with 4-5 column volumes of Buffer A before separating the charges of each material collected from the column IX-1. Each aliquot collected from column IX-1 was diluted 1: 1 with dilution buffers and the pH was adjusted to 6.0. In either case, the diluted material is introduced on

199 009 column. Each time the column is loaded with less than 10 grams of protein per liter of resin. After each aliquot applied to the column, the column is washed with 3 column volumes of buffer. Each time the column is loaded, the protein is eluted from the column using 8 column volumes of buffer with a linearly decreasing salt concentration gradient from 1.8 M NaCl to pure water. Harvesting of each protein peak begins as the UV absorbance increases to about 15-20% of the peak maximum. Harvesting is terminated as soon as UV abaorbaasia is about 50% at peak maximum or when absorbαnzta ceases to decline, whichever comes first.

Flow rate - 6 cm3 / hour during balancing, loading and washing

- 3 cm / hour for leaching

Each column purification is carried out in room temperature. Each solution collected from the HIC column is ready for bilge / diafiltration.

Zαtezaaie / Diαfiltration (C / D)

0.093 m2 (one square foot) PLCC ™ 5,000 MW regenerated cellulose membrane (MilliPzre, Bedford, MA) is used for the C / D step for each HlC harvest solution. Each HlC harvest solution is concentrated to approximately 200 mL, then diafiltered against 6-7 column volumes of 20 mM NaPCU at pH = 6.0 until the conductivity is &lt;4 mm / hour.

Each concentration step / filtration step is performed at room temperature.

Each batch of material from step C / D is then ready for purification on a 1Χ-2-365 mL SP-Sepharose HP ™ column (Phyrmyclα Biotzch, Inc., Piszataway, NJ).

1Χ-2 - 365 ml SP-Sepharose HP ™ column (5 cm x 18.5 cm)

Equilibration buffer Buffer A mM Na NaPO4 20 mM NsPO4 pH = 6.0 pH-6.3 50 mM NaCl

Buffer B 20 mM NaPO4 pH-6.8

The column is equilibrated with 4 column volumes of equilibration buffer prior to distributing the charges for each aliquot of C / D material. Each batch of C / D material is loaded onto the column using no more than 8 grams of protein per liter of resin. After each aliquot applied to the column, the column is washed with 3 kLnm of buffer to equilibrate, and then with 3 column volumes of buffer A. After each loading of the column, the protein is eluted from the column using 8 column volumes of buffer with an increasing pH gradient between 6.3 and 6.8 and a linear increasing salt concentration gradient from 0 to 50 mM NaCl (buffer B). Harvesting begins at 1.0 OD and continues on the increasing froat area of ​​the peak and stops at 50% of the maximum peak height on the descending part of the peak.

In an alternate embodiment, a truncated sTNFR-1 may be septicized, reassembled and refined as follows:

C. l Subletting / Reassembly:

Washed iacclusal bodies (1Bs) are solubilized with 8M canSk, 60mM Tris, 100mM cysteine ​​to a final concentration of 6.5M urea, 50mM Tris and 80mM cysteine, 0 pH = 9.5 and 5-10 mg / mL truncated sTNFR-1. (The latter result is based on counting the amount of truncated sTNFR-1 in washed IBs bodies in g / L. The material is allowed to stir at room temperature for 90 minutes and then reassembled by diluting 1:10 in cold (4 - 8 ° C) 0.85 M urea, 50 mM Tris opER ^^ (pH measured at 4 - 8 ° C) C).

The refolded material solution is allowed to stir for 24-72 hours at 4-8 ° C. At the end of this time, glacial acetic acid (about 20 mM) is added and the pH is adjusted to 5.0. The resulting precipitate is removed by centrifugation and the supernatant is retained until it is loaded onto the first column.

D. 1 Purification

The acid-clarified precipitate solution is loaded onto a SP-Sepharose Big Bead ™ column (Pharmyzia Biotech, Inc., Piscataway, NJ) adjusted to

189 309 equilibrium with 20 mM sodium acetate, 75 mM NaCl, pH = 5.0. The column is filled with no more than 15 g of truncated sTNFR-1 per one liter of column bed volume. After loading, the column is washed with 3 column volumes of 20 mM sodium acetate, 75 mM NaCl, pH = 5.0 and eluted with 9 column volumes with a linear concentration gradient from 75 mM to 450 mM NaCl in 20 mM sodium acetate, pH = 5 , 0. The entire purification process on the SP-Sepharose Big Beud ™ column (SP-BB) was carried out at 4 - 8 ° C.

The SP-BB harvest solution is diluted 1: 1 in 2 M NaCl, 60 mM acetate, pH 4.5, and adjusted to pH 4.5 if necessary. The diluted SP-BB collection solution is placed on a Toyopearl ™ Butyl 650M column (Toso Haas, Philadelphia, PA) that has been equilibrated with a 1 M NaCl solution, 30 mM acetate, pH 4.5. About 10-13 grams of truncated sTNFR-I are loaded onto the column per liter of bed volume. After loading, the column is washed with 3 column volumes of 1 M NaCl, 30 mM acetate, pH 4.5, and eluted with 8 column volumes with a linear concentration gradient of 1 M - 0 M NuCl in 30 mM acetate, pH = 4. 5.

The purified truncated sTNFR-I fractions from the Butyl 650M column were pooled, diluted 1: 5 with water and loaded onto a SP-Sepharose High Performance ™ column (Sp-HP) (Pharmacia Biotech, Inc., Piscataway, NJ) equilibrated with a 30 mM acetate solution of pH = 4.5 (will introduce no more than about 15 g / L bed volume). The column is then washed with 3 column volumes of a 30 mM acetate solution, pH 4.5, and eluted with 12 column volumes with a linear concentration gradient from 100 mM to 400 mM NaCl in 30 mM acetate, pH 4.5. The purified fractions of truncated sTNFR-I were pooled and adjusted to pH = 5.0 with NaOH.

C. PEGylation:

1. Production of sTNFR-I 2.6D / N105-t-BuPEG (33 kDa)

To a chilled (4 ° C) mixed solution of sTOFR-2.6D / N105 (3.5 mg / ml) in 50 mM sodium acetate, pH = 4, a 3-fold molar excess of t-BuPEG (monot-butoxypolyethylene glycol, average molecular weight MW = 33 kDa, Shearwater Polymers, Inc.). NaCNBH3 is added to a final concentration of 20 mM and the reaction mixture is stirred at 7 ° C for 18-24 hours.

The degree of protein modification during the course of the reaction is monitored by SEC HPLC using a TSKG3000swx column_L. (Toso Haas, Montgomeryville, PA) and elution with 0.1 M sodium phosphate buffer, pH = 6.9, 0.5 M NaCl and 10% ethanol at a rate of 0.7 ml / min (Toso Haas, Montgomeryville , PA).

The reaction mixture is adjusted to about pH = 3.5 with 1 M HCl and the reaction mixture is diluted with water to a final protein concentration of 1.5 mg / ml. sTNFR-I 2.6D / N105-t-BuPEG (33 kDa) is separated from excess t-BupEG and other reaction by-products using SP Sepharose HP 161/10 ™ ion exchange chromatography (Pharmacia Biotech, Inc., Piscataway, NJ).

The reaction mixture is loaded onto the column and unreacted BuPEG is eluted with three column volumes of the starting buffer A (20 mM sodium vinegar, pH 4.0). sTNFR-I 2.6D / N105-t-BuPEG (33 kDa) was eluted using 20 column volumes of a solution with a linear gradient of 0-30% buffer B (1 M NaCl in 20 mM acetate, pH 4.0). The leakage is monitored at 280 nm. Each fraction containing sTNFR-I 2.6D / N- ° 5-t-BuPEG (33 kDa) is analyzed by SDS-PAGE using 4-20% preformed gradient gels (Novex, Sun Diego, CA). Based on SDS-PAGE analysis, fractions were pooled, concentrated, and sterile filtered. Each final crop of purified sTNFR-I 2.6D / N-05-t-BuPEG (33 kDa) is reanalyzed by SDS-PAGE and SEC HPLC. The protein is formulated in 10 mM sodium sulfate, pH 6.5, and 20 mM NaCl.

2. Production of sTNFR-I 2.6D / N 105-33 kDa (MePEG)

To the cooled (7 ° C) sTNFR-2.6D / N- ° 5 mixed solution (4 mg / ml) 10% acetic acid is added until pH = 5.0. To this solution is added 15 mM NaCNBH3 and a 2 fold molar excess of t-butoxy PEG (t-butoxypolyethylene glycol, average molecular weight MW = 33 kDa, Shearwater Polymers, Inc.). The reaction mixture is briefly stirred at the same temperature and then incubated for approximately 18 hours.

189 309

After 18 hours, the protein concentration in the reaction mixture is adjusted to pH = 3.0 with citric acid.

The sTNFR-2.6D / N105-MśPEG (33 kDa) is separated from excess MSPEG and other reaction products by ion exchange chromatography using a SP Sepharose HP ™ column (Pharmacia Biotech, Inc., Piscataway, NJ).

The reaction mixture is placed (no more than 8 mg / ml resin) on the column and the converted MSGPEG is eluted with 3 kb column startin buffer A (20 mM sodium citrate, pH = 3.0). The sTNFR-2.6D / N105-MśPEG (33 kDa) was eluted using 16 column volumes of a solution with a concentration gradient from 0.1 to 0.5 M NaCl in 20 mM citrate pH 3.0. The leakage is monitored at 280 nm. Each fraction containing sTNFR-2.6D / N105-Mś-PEG (33 kDa) is analyzed by SDS-PAGE using 4-20% preformed gradient gels (NKVx, San Diego, CA). Based on the results of SDS-PAGE analyzes, fractions were pooled, concentrated and sterile filtered. Each final crop of purified sTNFR-I 2.6D / N105-MśPEg (33 kDa) is reanalyzed by SDSPaGe. Purified sTNFR-I 2.6D / N105-MśPEG (33 kDa) is concentrated to 5 - 20 mg / ml and formulated either in PBS kpH = 6.5 (10 mM sodium phosphate, 35-100 mM NaCl) alCo in 20 mM acetate solution and 100 mM NaCl, pH 5.0.

3. Production of sTNFR-I 2.6D / N105-MśPEG (20 kDa)

The procedure described in point "A" is generally repeated for the production of sTNFR-I 2.6D / N 105-MŚPEG (33 kDa), with the difference that MESPeG (monomethoxyphyloxide glycol, average molecular weight MW = 20 kDa, Shearwater P ^^ ers, Inc.) is replaced with MŚPEG (mca-methoxypolyethylene glycol, average molecular weight MW = 33 kDa, S ^ water Polymers, Inc.). This protein is formulated in 10 mM sodium phosphate, pH 6.5, and 20 mM NaCl.

4. Producing additional kojugats

Additional sTNFR-I 2.6D / N105 kknjugates are prepared essentially as sTNFR-I 2.6D / N105-MśPEG (33 kDa) except that the following types of PEG aldehydes (Shearwater Polymers, Inc.) are used:

linear single function - MW 5 kDa, 6 kDa and 57 kDa;

branched monofunctional - MW 10 kDa, 20 kDa and 40 kDa;

linear double loops - MW 8 kDa and 20 kUa;

branched tayfkakcyjaj - M ^ W10kD ^ £ i.

These proteins are formulated in 10 mM sodium phosphate, pH = 6.5, and 20 mM NaCl.

5. Alternative PEGylation Methods

In an alternative embodiment, truncated sTNFR-1 molecules can Cyc PEGylkwαj and purified using the following techniques:

The effluent SP-HP (3 - mg / ml adjusted to pH = 5.0) is reacted with 2 moles of polyethylene glycol (e.g. MśPEG luC t-BuPEG) per 1 mole of sTNFR-I 2.6D / N105 (about 5 grams t -BuPEG per 1 gram of sTNFR-I 2.6D / N105). After the polyethylene glycol has dissolved, 10-20 mM sodium cyanobnohydride is added and the solution is allowed to incubate overnight at 7-15 ° C. After completion of the pegylation reaction (approximately 18 hours), the reaction is quenched by adding 10 mM glycine.

The pegylation mixture is diluted with 4 volumes of 50 mM acetate, pH 4.0 or adjusted to pH 4.0 if necessary, and loaded onto an SP-HP column equilibrated with 50 mM acetate, pH 4.0. The column is loaded with no more than about 8 grams of sTNFR-2.6D / N105 per liter kb volume of bed. After loading, the column is washed with 3 kC column volumes of the equilibration buffer and eluted with a linear gradient solution of 0 - 0.3 M NaCl in 50 mM acetate, pH 4.0. The fractions of mkpegylkvαne-goSTNFR-2.6D / N105-30kDa are collected, adjusted to pH = 5.0, concentrated and diafiltered into a mkpegylkvαne-goSTNFR-2.6D / N105-30kDa buffer. All purification steps are performed at room temperature. The protein is formulated in either PBS pH 6.5 (10 mM sodium phosphate, 35-100 mM NaCl) alCo in 20 mM acetate and 100 mM NaCl at pH 5.0.

I89 309

6. Production of sTNFR-I 2.6D / C105 db and sTNFR-I 2.6D / C106 db

Sulfone-activated polyethylene glycol (made and purified substantially in accordance with U.S. Patent Application No. 08 / 473.809, filed June 7, 1995 and U.S. Patent Application No. 08 / 611.918, filed March 6, 1996) [PEG 20,000-eight-vinyl sulfone ] is used for protein dimerization essentially according to the method described in PCT Publication No. WO 95/34326, except for the reduction and reaction conditions. Proteins are reduced prior to the addition of polyethylene glycol with 4 moles of DTT per one mole of protein at 5-6 ° C at pH 7.6. All reactions are performed in the presence of 30% glycerol. The dimerized proteins are called sTNFR-I 2.6D / C105db and sTNFR-I 2.6D / C106db. Each protein is formed in either PBS pH 6.5b (10 mM sodium phosphate, 35-100 mM NaCl) or 20 mM acetate, 100 mM NaCl, pH 5.0.

7. Production of comparative sTNFR-I molecules (i). sTNFR-I 4D / N105 is prepared as described in EP 422339. sTNFR-I 4D / N105-t-BuPEG (33 kDa) is prepared by pegylating sTNFR-I 4D / N105 essentially following the procedure outlined above for pegylating sTNFR-I 2.6D / N105-t-BuPEG (33 kDa). sTNFR-I 4D / N105-t-MePEG (33 kDa) is prepared by pegylating sTNFR-I 4D / N105 essentially following the procedures outlined above for pegylating sTNFR-I 2.6D / N105MePEG (33 kDa). sTNFR-I 4D / C105 and sTNFR-I 4D / C105db are prepared as described in PCT Publication No. WO 95/34326. This protein is formulated in 10 mM sodium phosphate, pH 6.5, and 20 mM NaCl.

(ii). sTNFR-I 4D / C105-33 kDa (MePEG) is prepared by 4D / C105 pegylation essentially following the procedures outlined above for sTNFR-I 2.6D / C 105-33 kDa (MePEG) pegylation except that the reaction takes place at pH = 7.5 using 1.3 moles DTT per one mole of sTNFR-I for about 5-6 hours, then by removing DTT on a SPSepharose ™ fF column and pegylating with 1.5-3 moles PEG per one mole of protein for at least 15 hours at room temperature. This protein is formed in either PBS pH 6.5 (10 mM sodium phosphate, 35-100 mM NaCl) or 20 mM acetate, 100 mM NaCl pH 5.0.

(iii). sTNFR-I 3D / N105 truncated C-terminus 34 amino acids in sTNFR-I 4D / N104) is prepared as follows. PCR amplification is performed using sTNFR-I 4D / N105 as a template and OLIGO # 13 and OLIGO 14 which encode MM and HindII respectively, and annealing to the 5 'and 3' ends of the truncated gene, respectively. PCR amplification is performed for 25 cycles; each cycle consists of 30 seconds at 94 ° C for denaturation, 15 seconds at 60 ° for annealing and 1 minute at 72 ° C for extension [Model 2400 thermocycler (Perkin-Elmer Cetus, Norwalk, CT)]. The PCR product is purified using the QIAquick ™ PCR Purification Kit (QIAGEN, Chatsworth, CA). The purified PCR product was cut with NdeI and Hindill and the gel was purified using the QIAquick ™ Gel Extraction Kit (QIAGEN, Chatsworth, CA). The gel-isolated PCR product is ligated into pAMG11 and transformed into FM15 E. coli cells.

5 'OLIGO # 13: (SEQ ID NO: 80)

5'-GGTTAGCCATATGGACAGCGTTTGCCCCCAA-3 '

3'-OLIGO # 14: (SEQ ID NO: 81) '-CCC / AAGCTTTTAGGTGCACACOGTGTTCTGTTT-This protein is formulated in 10mM sodium phosphate pH = 6.5 and 20mM NaCl.

(iv). sTNFR-I 3D / C105 (sTNFR-I 4D / C105 C-terminal 34 amino truncation) is produced essentially as sTNFR-I 3D / N105 with the difference that the template is sTNFR-I 4D / C105. sTNPR-I 3D / C105 is formed in either PBS pH 6.5 (10 mM sodium phosphate, 35-100 mM NaCl) or 20 mM acetate, 100 mM NaCl, pH 5.0.

(v). sTNFR-I 3D / C105db is produced essentially as sTNFR-I 4D / C105db with the difference that the starting material is sTNFR-I 3D / C105 instead of sTNFR-I 4D / C105. sTNFR-I 3D / N105db is developed either in PBS pH- 6.5 (10 mM sodium phosphate, 35-100 mM NaCl) or in 20 mM acetate, 100 mM NaCl, pH 5.0.

Example II

Various forms of truncated, recombinant soluble TNFR-I were evaluated for their ability to inhibit TNF activity.

189 309

A. WEHI sytotoxicity test:

The WEHI assay is an in vitro cell proliferation assay (Edwards et al. (1991), Endocrinology, 128: 989-996). Cell lines are sensitive to TNF-α (that is, TNF-α is cytotoxic). In the presence of a TNF-α inhibitor, cells are protected against the cySatoxic effect and are therefore capable of proliferation.

Protocol:

TNF-sensitive cells of clone WEHI-164 (ATCC, Rockvillo, MD) were suspended at a concentration of 20 x 10⁴ cells / mL in RPMI medium (Gibco, Grand Island, NY) supplemented with 5% Fotal Calf Serum (Hyclone, Ogdon, UT) and penicillin 50 U / mL: streptomycin 50 mg / mL. One hundred microliters of this cell suspension was placed in each well of a 96-well flat bottom microanalysis plate and the cells were allowed to adhere for 4-6 hours at 37 ° C in a 5% CO 2 atmosphere. To each well was added 10 μΐ 0.0060 mg / mL actinysin-D (Sigma Chemical Co., St. Louis, MO). To each well was added 10 microliters of human TNF? Rocombustion at a concentration of 50 ng / mL (final concentration 5 ng / ml). Serially two different forms of sTNFR (sTNbR-I 2.6D / C106, sTNFR-I 4D / C105 and sTNbR-I 4D / C105db) were serially two-fold diluted with PBS, then added to duplicate wells (10 μL / well) adherent WEHI-164 cells after addition of recombinant human TNF-α. WEHI-164 clone 13 cells are incubated for 18 hours at 37 ° C and 5% CO 2. After the incubation was complete, 10 mL of a 2 mg / mL solution of the TeSrazalium MTT organic dye (3- [4.5 dlmethylatiose-2-yl] bromide) 2.5-diphenyltetrazolium; Sigma Chemical Co., St. Louis, MO) was added, and the cells were incubated for an additional 4-6 hours. Cells were salubilized by adding 50 µL of DMF / SDS solution (20% SDS and 50% N, N dimotylaformamide, pH = 4.7). The DMF / SDS solution was pipetted up and down several times until all MTT crystals had dissolved, and the cells were incubated for a further 2-22 hours. Ab (abs) are ready to be read pa on the "Vmax leader" device at 570. Percentage content Specific toxicity was calculated from the optical densities using the formula:

_{n /} _. ,. 100% x [abs (cells + medium) - abs (cells + sample)]% specific satotaxicity = -^Ł s - + -— ż a -) - tabs (cells + ροΐΑ-ΑνΤη) - abs (cells + TX -100)

The amount of TNF units in each sample was determined using the percentage quotas of the appropriate mouse standards as previously described.

The WEHI test results are listed in Table 2 below:

Table 2 Activity in vitro in the WEHI assay

<td>Relationship</td><td>IC₅0 () g / mL)</td>

<td>sTNFR-I 2.6D / C106</td><td> 208</td>

<td>sTNFR-I 4D / C105</td><td> 238</td>

<td>sTNFR-I 4D / C105db</td><td>ON</td>

Based on the results of the WEHI trial, it can be seen that there are no significant differences between sTNFR-I 2.6D / C106 and sTNFR-I 4D / C105 in terms of in vitro bioofactivity.

B. Attempt of Cytatoxicity L929:

The satitoxicity assay with L929 is an in vitro cell proliferation assay (Parmely et al. (1993), J, Immunol, 151: 389-396). which also assesses the "cytotoxicity of killing sensitive to TNF-α. Cell lines are sensitive to TNF-α (for example, TNF-α is cytotaxic). In the presence of a soluble TNF-α inhibitor, cells are protected against the cyto-toxic effect and are thus able to proliferate.

Protocol:

The L929 cell line was obtained from the American Typo Culturo Callestian collection (CCL pumor catalog 1, NCTC 929 clone, L strain clones, connective tissue, mouse). The propagation medium was RPMI Medium 1640, supplemented with 10% FBS solutions, + 1% L-glutamine solutions, + 1% yeplcyhna-stroytomysin solutions.

i89 309

96-well microanalysis plates (Corning) were used in the trial and only 60 internal wells were used. The standard sample and the test sample were tested in triplicate on the same plate.

The TNFα used in the assay was obtained from R&amp;D Systems (Minneapolis, MN). The final concentration of TNFα used in the assay is 1 ng / mL in all assay wells.

The assay diluent was L929 growth medium, TNF? At 10pg / mL, and actinomycin D at 10ng / mL (Sigma Chemical Co., St. Louis, MO).

Plates were harvested using an XTT / MEN solution (1.5 mg / mL XTT + 75 mM MEN).

On the first day, cells were seeded in the assay plates. Cells were suspended by trypsinizing and resuspended at 3.33 x 10⁴cells / mL. 180 mL of this cell suspension was seeded into each of the 60 inner wells of the assay plates. 200 mL of growth medium was spread in 36 outer wells of the plate to prevent evaporation in the assay. The plates covered with foil were left at room temperature in an unventilated place for about 1 hour. The test plates were placed in a 37 2 ° C incubator with high humidity and 5 1% CO 2. Plates were incubated for approximately 20-22 hours before adding serial dilutions of sTNFR-I.

On the second day, standard sTNFR-I 4D / N105 and test samples were prepared as follows: standard sTNFR-I 4D / N105 and test samples were diluted to approximately 2.0 mg / mL (or other appropriate concentration). Serial dilutions of this concentration were prepared to plot a 10-point dilution curve from approximately 1.0 x 106 ng / mL to 1.0 x 10'3 ng / mL, including the 0 ng / mL point (for the dilution test only). If other concentrations are appropriate, they can be used. To each plate, 1000 pL of each dilution was added in triplicate. Incubate the plates in an incubator at 37 ± 2 ° C, high humidity and 5 ± 1% CO2 for 20 ± 1 hours after transferring the serial dilution samples to the sample plates.

On day 3, 50 µL / well of the XTT / MEN solution was added to the 60 inner wells of the assay plates. Plates were incubated in an incubator (Falcon, New York, New York) at 37 ± 2 ° C, high humidity and 5 ± 1% CO2 for 24 ± 0.5 hours.

On the fourth day, the optical density (OD) of the test plates was read at 450 nm minus 650 nm on an ELISA plate reader (SpectraMAx, Beckman Instruments, Inc., Fullerton, CA). If values ​​of 4,000 OD are obtained for the wells in the plate at these wavelengths, the plate should be reread directly at 490 nm minus 650 nm and the data so read should be used for the calculation.

A standard dose-response curve against a logarithmic scale is plotted using a four parameter curve fit. The original concentrations of the unknown samples are calculated from the standard curve and the ED50 for the standard and the correlation coefficient for the standard curve are determined.

Results: The results of the L929 cytotoxicity assay are summarized in Table 3 below:

Table 3:

In vitro activity in the L929 cytotoxicity assay

<td>Relationship</td><td>Concentration</td><td>ED50 (ng / ml)</td>

<td>sTNFR-I 4D / C105db</td><td> 7,8</td><td> 1,0±0,1</td>

<td>sTNFR-l 2 6D / C105db</td><td> 2,6</td><td> 1,1+0,0</td>

<td>sTNFR-l 2.6D / C106db</td><td> 2,2</td><td> 1,0±0,1</td>

<td>sTNFR-I 4D / N105-t-BuPEG (33kDa)</td><td> 2,0</td><td> 229,2±18</td>

<td>sTNFR-1 4D / C105-t-BuPEG (33 kDa)</td><td> 1,1</td><td> 325,5±147</td>

<td>sTNFR-I 2 6D / C105-t-BuPEG (33kDa)</td><td> 1,7</td><td> 210,2±9</td>

<td>Internal Pattern</td><td></td><td></td>

<td>sTNFR-1 4D / C105</td><td> 3,5</td><td> 210,2+671881</td>

189 309

The above data indicate that sTNFR-I 4D / C105db and sTNFR-I 2.6D / C105db and sTNFR-I 2.6D / C106db are active and show comparable dose responses when compared to the reference. These results also indicate that sTNFR-I and sTNFR-I 4D / N105-t-BuPEG (33 kDa) and sTNFR-I 2.6D / C105-t-BuPEG (33 kDa) are nearly 100 times less active, but are active in this test, however, when compared with sTNFR-I 4D / C105db.

Throw # 2

<td>sTNFR-I 3D / C105db</td><td> 0,2</td><td> 2,27±0,3</td>

<td>sTNFR-I 3D / C105db</td><td> 0,,2</td><td> 2,0 *;</td>

<td>sTNFR-I 3D / C105db</td><td> 1,,9</td><td> 1,8 *</td>

<td>sTNFR-I 3D / N105</td><td> 2,,4</td><td> 413,3</td>

<td>Internal standard: sTNFR-I 4D / C105</td><td> 3,5</td><td>Π5.9 ± 42.1</td>

* 'Single result

The above data indicate that sTNFR-I 3D / C105db is active and the area of ​​ED50 values ​​is within the range for sTNFR-I 4D / C105db (projection # 1), sTNFR-I 2.6D / C105db (projection # 1) and sTNFR-I 2.6D / C106db (throw # 1). These data also indicate that sTNFR-I 3D / N105 is less active compared to the sTNFR-I 4D / C105 internal standard.

C. Model of reactivation induced by the streptococcal cell wall:

The reactivation model induced by the streptococcal arthritis cell wall when tested with rats is performed using known protocols (Esser et al. (1985), Arthritis AndRheumatism, 28: 1402-1411, and Makarov et al. (1996), Proc Natl. Acad. Sci USA, 93: 402-406).

Protocol:

Female Lewis rats (Charles River Laboratories, Inc., Wilmington, MA), each weighing 175 to 185 grams, were intraarticularly injected into the right ankle joint with a sterile suspension of streptococcal cell wall products containing peptidoglycan polysuccharide (SCW) (Lee Laboratory, Grayson, GA) at doses containing 1.5 mg / 10 mg per joint. Saline solution was injected into the contralateral leg to ensure control. Intra-articular injection of SCW causes acute arthritis of a relatively short duration, with joint swelling reaching a maximum one to two days after injection. After 20 days of an acute inflammatory reaction, SCW was re-administered by intravenous injection at doses of 200 mg / 200 mL per rat. The second dose of SCW is sufficient to reactivate inflammation in the previously injected SCW ankle and has little effect on the saline injected ankle joint. To assess the degree of inflammation after a 72 hour period after SCW injection, posterior ankle dimensions were measured at 0; 24; 36; 48 and 72 hours after reactivating arthritis, then a portion of the posterior ankle joint was collected for histology (for example: inflammation, corneal pannus formation, cartilage damage, and bone damage).

Results:

The effect of sTNFR-I 2.6D / C106db when administered on the development of joint swelling during reactivation of arthritis was investigated. Inhibitors and vehicle were administered as single intravenous injections 24 hours prior to SCW reactivation.

sTNFR-I 2.6D / C106db has statistically significant efficacy in reducing joint swelling by analysis of variance (ANOVA) and Fisher's "post-hoc" test (Stutview®) at all four doses, on the second and third days after reactivation and at all except j one - dose (1.5 mg / kg) on ​​the first day. This reduction in edema is comparable to a positive sTNFR-I 4D / C105db control sample administered at a dose of 0.5 mg / kg per day (i.e. 8.8 nM) from the first day before reactivation to three days after reactivation. sTNFR (s) also show significant efficacy when considering the total amount of edema after three days. The area under the curve (AUC) shows the dose-response relationship at all doses (see Figure 9, where sTNFR-I 2.6D / C106db was defined as "sTNFR-I 2.6D" and sTNFR-I 4D / C106db was defined as "sTNFR-I). -I 4D ").

189 309 sTNFR-I 2.6D / N105-t-BuPEG (33 kDa) shows a significant reduction in the ankle width and a mismatch rate compared to the disease control group in the model.

D. Model of D-aalaPtozoamina / hpope> liseoharyau:

D-galactizoamide (D-GalNH2) / lipepąlicacharyau (LPS) model (Parmoly et al. (1993), supra), in vivo, highly dependent on TTF-α to measure the totality model. In addition, it has been shown that auteimmtmizomano MRL-lpr / lpr mice are remarkably sensitive to TNF-α induced by LPS or SEB. (Mountz et al. (1995), J. Immunol, 155: 4829-4837).

Protocol:

After fasting, 6-8th daytime adult MRL-lpr / lpr self-mice (Jackson Laboratery, Bar Harbor, ME) received an IP dose of the following pharmacological reagents: 25 mg D-GalNH2 (Sigma Chemical Co., St. Louis, MO) suspended in Hank's solution (HankN Balanoed Salt Slutiod (Gibco Laboratories, Inc., Grand Island, NY) at a concentration of 50 mg / mL; and lippolysaccharide (LPS) from E. coli Serotype 0127: B8 (Sigma Chemical Co., St. Louis, MO) in sterile, endant-Psynl-free phosphate buffered saline (PBS) (25 mg / mouse), or SEB (Toxin Technologies, Sarasota, FL) in normal saline (50 mg / mouse). The various forms of sTNFR were given in duplicate runs (mg / kg doses) to obtain ED50 curves generated using statistical programs for Macintosh (Statview®, Mountain Viem, CA). Total obsermąmadą through 48 gassies after dosing.

Results:

As shown in Table 4, when sTNFR-I 2.6D / C106db is administered as described above, 1 hour prior to LPS / DGalNH2, ED50 dosing (i.e. sTNFR-I 2.6D / C106ab dose required for 50% protection) after 48 hours, yield 50 pg / kg (N = 8 mice). Compared to sTNFR-I 4D / C105db nip raobsermom there are significant differences (P> 0.05) in the ability of this form to prevent lethality (ED50 = ~ 50 pg / kg; N = 8 mice).

Table 4:

Comparison of sTNFR and optimal forms of truncated sTNFR in the LPS / D-GaINH2 model

<td>Center</td><td>ED 100</td><td>ED50</td>

<td>sNFR-I 4D / C105db</td><td>~ 100 pg / kg</td><td>~ 50 pg / kg</td>

<td>sTNFR-I 2.6D / C106db</td><td>~ 100 pg / kg</td><td>~ 50 pg / kg</td>

<td>sTNFR-I 2 6D / N105-t-BuPEG (33kDa)</td><td>~ 2 pg / kg</td><td>~ 400 pg / kg</td>

<td>sTNFR-I 2.6D / N 105-MePEG (20 kDa)</td><td>~ 800-1000 pg / kg</td><td>~ 1 pg / kg</td>

<td>sTNFR-I 2.6D / N105-MePEG (20 kDa, branched)</td><td>2 mg / kg</td><td>-111.5 pg / kg</td>

<td>sTNFR-I 2.6D / N105-MePEG (40 kDa, branched)</td><td>1.5 mg / kg</td><td>~ 1 pg / kg</td>

The above data indicate that sTNFR-I 2.6D / C106ab shows equivalent activity compared to sTNFR-I 4D / C105db, but with sTNFR-I 2.6D / N105-t-BuPEG (33 kDa) it is less active in this model with ED50 about 400pg / kg (n = 5 individual mice). Moreover, the sTNFR-I activity of 2.6D / N105-Me PEG (20 kDa, branched) and 2.6D / N105-MePEG (40 kDa, branched) are less active in this model.

E. Modd of phlegmonous arthritis (uartaclcbzmt pemoaoigzγn) and: Rgumatojgajne arthritis induced in rats by pharmaceutical pomadeonic substances has many features similar to human rheumatoid arthritis. The colom of the present experiment is to demonstrate that systemic administration of truncated sTNFRs has a mitigating effect on excipient-induced arthritis in mice.

Protocol:

Male Lewis rats (5-7 per group) (Charles River Laboratories Inc., Wllmingted, MA), each weighing at least 200 g, are cannulated with SQ catheters and are allowed to recover for several days. Then they were placed

189 309 in infection cages, where they acclimated for a week before starting the infusion with saline solution.

On day "zero", all rats were injected with 100 µl of Freunds complete adjuvant (Sigma Chemical Co., St. Louis, MO) to which was added 50 mg / ml of the synthetic excipient N, N-dictyldicyclic-N ', N-bis. (2-hydroxy-etylk) propa-diamine. On the eighth day, different groups of rats were infused continuously with Sq, sTNFR-I 4D / C105 and sTNFR-I 2.6D / N105.

The results are summarized in Table 5.

Table 5:

arthritis caused by excipients

<td>Relationship</td><td>Dose mg / kg / h</td><td>% AUC (% mh.)</td><td>Foot weight (% inh)</td><td colspan="2">Inflammation of the bone Histopathology (% inh.) (% mh.)</td>

<td>Study # 1</td><td></td><td></td><td></td><td></td><td></td>

<td>sTNFR-I 4D / C105</td><td> 5</td><td> 61</td><td> 46</td><td> 37</td><td> 89</td>

<td></td><td> 1</td><td> 49</td><td> 45</td><td> 26</td><td> 855</td>

<td></td><td> 0,2</td><td> 33</td><td> 40</td><td> 14</td><td> 34</td>

<td>sTNFR-I 2 6D / N105</td><td> 1</td><td> 55</td><td> 53</td><td> 33</td><td> 51</td>

<td>Study # 2</td><td></td><td></td><td></td><td></td><td></td>

<td>sTNFR-I 2.6D / N105</td><td> 5</td><td> 42</td><td>ND</td><td> 19</td><td> 67</td>

<td></td><td> 1</td><td> 38</td><td>ND</td><td> 13</td><td> 49</td>

<td>Study # 3</td><td></td><td></td><td></td><td></td><td></td>

<td>sTNFR-I 2.6D / N105-</td><td> 9</td><td> 50</td><td> 40</td><td> 13</td><td> 27</td>

<td>-MePEG (20 kDa)</td><td> 3</td><td> 35</td><td> 34</td><td> 9</td><td> 22</td>

<td></td><td> 1</td><td> 36</td><td> 30</td><td> 0</td><td> 0</td>

<td>sTNFR-I 2.6D / C105-</td><td> 9</td><td> 43</td><td> 37</td><td></td><td></td>

<td>-MŚPEG (33 kDa)</td><td> 3</td><td> 38</td><td> 33</td><td></td><td></td>

<td></td><td> 1</td><td> 24</td><td> 20</td><td></td><td></td>

Surprisingly, it was found that sTNFR-I 2.6D / N 105-t-BuPEG (33kDa) and sTNFR-I 4D / C105db had a comparative anti-inflammatory effect in excipient-induced arthritis in Lświs rats, although sTNFR-I 4D / C105db is more effective in the in vitro cytotoxicity assays of WEHI-164 and L929 as well as in the LPS / GalN model.

F. Model of Collagen Induced Arthritis:

Collagen-induced arthritis type II has many features similar to human rheumatoid arthritis. The aim of the present experiment is to demonstrate that systemic administration of truncated sTNFRs has a mitigating effect on the pathogenesis of collagen-induced arthritis type II in rats and mice.

Cadan protocol on rats:

Female Lewis rats (Charles Rwer Laboratories, Inc., Wilmiągtoą, MA), had SQ catheters implanted and were accustomed to catheterization through continuous infectious disease. They were then immunized with complete bovine type II collagen

199 099 αdjuwynciz Frenads. On days 13, 14, or 15 after immunization, arthritic animals were randomized into groups of eight each. The animals in the experimental groups were infused with vehicle alone or vehicle with different doses of sTNFR-1 as described in Table 6 for 7 days. Inflammation in the paws was assessed by daily caliper measurement of the ankle joints. On the seventh day, the animals were anesthetized and their paws were removed in order to determine the weight of the paws as an indicator of inflammation. The ankle and knee joints were dissected for histopathological evaluation of the parameters of arthritis.

The results are summarized in Table 6A below.

Interestingly, in the rats in the collagen model, all treatment groups had almost the same performance (for example, the shape of the curves, percentage and area under the curve (AUC), ranging from 30-59%, and paw mass inhibition, ranging from 40-64%. None. the treatment groups was not statistically different from the others in this arthritis model.

Study protocol in mice:

Male DBA / 1 family (Jackson Laboratories, 1nc., Bar Harbor, ME), are immunized with bovine type 11 collagen (Sigma Chemical Co., St. Louis, MO) in incomplete adjnwαnzle Freunds. On days 24, 25, and 26 after immunity, arthritic animals were randomized into groups of eight each. The animals in the experimental groups were dosed twice daily by intraperitoneal infusion with saline alone or with sTNFR-1 2.6D / N105-MePEG (33 kDa) vehicle for 3 consecutive days (days +27, +28, +29). Inflammation in the paws was assessed by daily caliper measurement of the ankle joints. On day +34, the animals were anesthetized and their paws were removed in order to determine the weight of the paws as an indicator of inflammation. The ankle and knee joints were dissected for histopathological evaluation of the parameters of arthritis.

The results are summarized in Table 6B below.

Table 6A:

Collagen-induced arthritis

<td rowspan="2">Relationship</td><td rowspan="2">Dose mg / kg / h</td><td rowspan="2">% AUC (% inh.)</td><td rowspan="2">Paw weight. (% inh)</td><td colspan="2">Inflammation of the Resorpzsy bones Histopathology</td>

<td>(% inh.)</td><td>(% inh.)</td>

<td> 1</td><td> 2</td><td> 3</td><td> 4</td><td> 5</td><td> 6</td>

<td>Study # 1</td><td> 5</td><td> 65</td><td> 81</td><td>ND</td><td>ND</td>

<td>STNFR-1 4D / C105</td><td> 1</td><td> 35</td><td> 34</td><td>ND</td><td>ND</td>

<td></td><td> 0,2</td><td> 19</td><td> 22</td><td>ND</td><td>ND</td>

<td>STNFR-1 2 6D / N105</td><td>1 mg / kg / day</td><td> 39</td><td> 41</td><td>ND</td><td>ND</td>

<td>Study # 2</td><td></td><td></td><td></td><td></td><td></td>

<td>STNFR-1 2.6D / N105-MePEG (33 kDa)</td><td> 3</td><td> 50</td><td> 60</td><td> 76</td><td> 46</td>

<td>STNFR-I 4D / N105- MePEG</td><td> 3</td><td> 47</td><td> 50</td><td>ND</td><td>ND</td>

<td>(33 kDa)</td><td>mg / kg / day</td><td></td><td></td><td></td><td></td>

<td>Study # 3</td><td></td><td></td><td></td><td></td><td></td>

<td>STNFR-I 2 6D / N105-MePEG (33 kDa)</td><td> 9</td><td> 25</td><td> 44</td><td>ND</td><td>ND</td>

i89 309 Continued Table 6 A.

<td> 1</td><td> 2</td><td> 3</td><td> 4</td><td> 5</td><td> 6</td>

<td>STNFR-I 2 6D / N 105-HePEG (33 kDa)</td><td> 3</td><td> 25</td><td> 37</td><td>ND</td><td>ND</td>

<td>STNFR-1 2 6D / N105-MePEG (20 kDa)</td><td> 9</td><td> 35</td><td> 52</td><td>ND</td><td>ND</td>

<td>STNFR-I 2 6D / N 105-HePEG (20 kDa)</td><td> 3</td><td> 35</td><td> 37</td><td>ND</td><td>ND</td>

Table 6B Collagen-induced arthritis

<td>Relationship</td><td>Dose mg / kg / 2D</td><td>% AUC (% inh)</td><td>Histopathology (% inh)</td>

<td>Study # 1 STNFR-I 4D / C105db</td><td> 5</td><td> 61</td><td> 39</td>

<td>sTNFR-I 4D / C105-t-BuPEG (33 kDa)</td><td> 1</td><td> 55</td><td> 55</td>

<td>Study # 2 sTNFR-I 2.6D / N 105-HePEG (33 kDa)</td><td> 9</td><td> 73</td><td>ND</td>

<td>sTNFR-I 2 6D / N 105-HePEG (33 kDa)</td><td> 3</td><td> 75</td><td>ND</td>

G. Rat model of TNF-α production induced by continuous LPS infusion: sTNFR-I 2.6D / C105db and sTNFR-I 2.6D / C106db, sTNFR-I 2.6D / N105 and sTNFR-I

4D / N105 is implanted into the jugular vein (IV) using Alzet ™ mini-pumps (Alza Corp., Palo Alto, CA) according to the manufacturer's instructions for 48 hours continuous infusion (1 mg / kg). Serum TNF-α levels, as determined using an ELISA kit (Genzyme, Cambridge, MA), are significantly lowered compared to the level in control animals at +2 hours after high dose LPS administration.

Example III: Immunogenicity Studies

Various forms of truncated, recombinant soluble TNFR-I have been assessed for immunogenicity in a number of animal models.

A. Rodents:

sTNFR-I 2.6D / N105-t-BuPEG (33 kDa); and sTNFR-I 4D / C105db (control) were administered subcutaneously in this study on days 1 and 5 to female Sprague Dawley rats (Charles Rivers Labs, Wilmington, MA) at a dose of 4 mg / kg, (n = 6-8 animals per group) . Behind the sockets blood samples were collected weekly through day 21 after the initial administration. The samples were evaluated for the production of IgM and IgG antibodies.

As can be seen from Table 7, in the case of sTNFR-I 4D / C105db administered subcutaneously (SC) on days +1 and +5, higher IgG, anti-sTNFR-I antibody titers were observed in rats up to day +21 than in the case of sTNFR- I 2.6D / N105-t-BuPEG (33 kDa), which corresponds to very low, if any, antibody titers. Similar trends in immunogenicity were also observed in rats producing IgM anti-TNFR-I antibodies for +21 days. sTNFR-I 2.6D / N105-t-BuPEG (33 kDa) did not produce rat IgM anti-sTNFR-I antibodies for +21 days.

B. Papio anubis:

The aim of phase A of part 1 of this study was to determine the pharmacokinetics and immunogenicity of sTNFR-I 4D / C105db (0.2 mg / kg body weight [BW]), sTNFR-I 3D / C105db, respectively.

I89 309 sTNFR-I 3D / C105db (0.2 mg / kg BW); or sTNFR-I 2.6D / C105db (0.2 mg / kg BW), when IV is administered twice +21 days apart to a healthy baboon.

The research in Part 1 was divided into two phases. Part 1, Phase A, was designed to determine the pharmacokinetics and immunogenicity of various sTNF-R1 constructs in a healthy baboon in response to two injections. The twelve baboons were divided into three groups. After anesthesia, each group received a dose of 0.2 mg / kg BW sTNFR-I 4D / C105db, sTNFR-I 3D / C105db, or sTNFR-I 2.6D / C105db. Three baboons were examined during each session. The animals were observed for 21 days after which they received a second identical IV injection of protein and were tested for a further 21 days. The pharmacokinetics and immunogenicity were then determined at intervals.

Table 7:

Rodent immunogenicity

<td>Time (days)</td><td> 0,01</td><td> 7</td><td> 14</td><td> 21</td>

<td>Group + number of pets</td><td>Igm titer</td><td>Igm titer</td><td>Igm titer</td><td>Igm titer</td>

<td>sTNFR-I 2 6D / N105-33 kDaPEG</td><td>NEG</td><td> 0</td><td> 0</td><td> 0</td>

<td> 1</td><td>NEG</td><td> 0</td><td> 0</td><td> 0</td>

<td> 2</td><td>NEG</td><td> 0</td><td> 0</td><td> 0</td>

<td> 3</td><td>NEG</td><td> 0</td><td> 0</td><td> 0</td>

<td> 4</td><td>NEG</td><td> 0</td><td> 0</td><td> 0</td>

<td> 6</td><td>NEG</td><td> 0</td><td> 0</td><td> 0</td>

<td>sTNFR-I 2.6D / N105-t-BuPEG33 kDa</td><td> 0</td><td> 0,00</td><td> 0,00</td><td> 0,00</td>

<td>SEM</td><td> 0</td><td> 0,00</td><td> 0,00</td><td> 0,00</td>

<td>Control</td><td></td><td></td><td></td><td></td>

<td> 7</td><td> 0</td><td> 0</td><td> 50</td><td> 0</td>

<td> 8</td><td> 0</td><td> 0</td><td> 50</td><td> 0</td>

<td> 3</td><td> 0</td><td> 0</td><td> 100</td><td> 0</td>

<td> 9</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td>

<td> 10</td><td> 0</td><td> 0</td><td> 100</td><td> 50</td>

<td> 11</td><td> 0</td><td> 0</td><td> 100</td><td> 0</td>

<td> 12</td><td> 0</td><td> 0</td><td> 100</td><td> 0</td>

<td> 13</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td>

<td>Control</td><td> 0</td><td> 0</td><td> 62,5</td><td> 6,3</td>

<td>SEM</td><td> 0</td><td> 0</td><td> 15,7</td><td> 6,3</td>

189 309

<td>Time (days)</td><td> 0,01</td><td> 7</td><td> 14</td><td> 21</td>

<td>Group + animals #</td><td>IgG titer</td><td>IgG titer</td><td>IgG titer</td><td>IgG titer</td>

<td>sTNFR-I 2 6D / N105-t-BuPEG 33 kDa</td><td>NEG</td><td>NEG</td><td> 0</td><td> 0</td>

<td> 1</td><td>NEG</td><td>NEG</td><td> 0</td><td> 0</td>

<td> 2</td><td>NEG</td><td>NEG</td><td> 0</td><td> 0</td>

<td> 3</td><td>NEG</td><td>NEG</td><td> 0</td><td> 0</td>

<td> 4</td><td>NEG</td><td>NEG</td><td> 0</td><td> 0</td>

<td> 6</td><td>NEG</td><td>NEG</td><td> 0</td><td> 0</td>

<td>sTNFR-I 2.6D / N105-t-BuPEG33 kDu</td><td> 0,00</td><td> 0,00</td><td> 0,00</td><td> 0,00</td>

<td>SEM</td><td> 0,00</td><td> 0,00</td><td> 0,00</td><td> 0,00</td>

<td>Control</td><td></td><td></td><td></td><td></td>

<td> 7</td><td>NEG</td><td>NEG</td><td> 0</td><td> 200</td>

<td> 8</td><td>NEG</td><td>NEG</td><td> 0</td><td> 200</td>

<td> 9</td><td>NEG</td><td>NEG</td><td> 0</td><td> 0</td>

<td> 10</td><td>NEG</td><td>NEG</td><td> 200</td><td> 400</td>

<td> 11</td><td>NEG</td><td>NEG</td><td> 0</td><td> 200</td>

<td> 12</td><td>NEG</td><td>NEG</td><td> 200</td><td> 800</td>

<td> 13</td><td>NEG</td><td>NEG</td><td> 0</td><td> 50</td>

<td>Control</td><td> 0</td><td> 0</td><td> 50</td><td> 231,3</td>

<td>SEM</td><td> 0</td><td> 0</td><td> 32,7</td><td> 94,0</td>

Phase B of Part 1 of the study is designed to evaluate the efficacy of these formulations in a well established TNFu-mediated lethality model (Espat et al., J Surq Res., 59: 153-158, 1995). Lethal E. coli bacteraemia is induced in 16 animals in groups of 4 by the administration of 5-10 x WOciu / kg (colony forming units / kg) of live E. coli. Placebo is compared to IV pretreated baboons with sTNFR-I 4D / C105db (0.2 mg / kg bw), sTNFR-I 3D / C105db (0.2 mg / kg bw), or sTNFR-I 2.6 D / C105db given at 1 mg / kg body weight.

In both phases of Part 1, adult Young baboons, male and female Papio anubis (6-11 kg) (Biomedical Research Foundation, San Antonio, TX) were fasted overnight. The animals were anesthetized with ketumin (10 mg / kg, intramuscular - im), and a needle was inserted subcutaneously into a vein. Anesthesia was maintained with an initial administration of up to 35 mg / kg sodium pentobarbital, followed by repeated injections with 3-5 mg / kg / h sodium pentobarbital. The upper airway was secured by placing a cuffed endotracheal tube (endotracheotomy) and the animals maintained spontaneous breathing. A femoral artery catheter was inserted through the skin allowing repeated arterial blood sampling as well as continuous monitoring of heart rate and mean blood pressure using the "Datascope 2000 anesthesiu monitor" (Datuscope, San Antonio, TX). Arterial blood samples were collected at intervals, anticoagulated with EDTA or heparin, and chilled on ice immediately after collection. Plasma fractions were separated by centrifugation at 4 ° C and stored at -70 ° C until assayed. Core temperature was monitored with a rectal probe. A Foley urinary catheter was placed to allow urine collection and monitoring of urine output and creatinine removal. Hemodynamic parameters

189 309 manitors every 15 minutes. All animals received intravenous injection of 0.9% sodium chloride (4 ml / kg) as a maintenance fluid (/'.ν). In the phase B studies, animals received additional fluid (10 ml / kg every 15 minutes) if two of the following physiological criteria were met: 1) mean arterial sleepiness decreased by more than 30%; 2) there was an increase in heart rate by more than 30%; 3) decrease in urine output to &lt;1 ml / kg / h. After taking a blood sample at the baseline and waiting for a period of at least 1 hour to allow equilibration, the protein infusion was started.

In Phase A of Part One of the study, reclamation proteins were infused through the head vein and the animals were observed for a period of 8 hours, by which time all catheters were removed and the animals returned to their cages for 21 days. After 24 and 48 hours and on days 3, 5, 8, 11, 16, and 21, animals were briefly anesthetized with 1M ketamine (10 mg / kg) and venous blood samples were collected. On day 21, the animals were re-anesthetized, they received a second injection of the protein and the entire procedure performed on day "0" was repeated for the next 21 days by which time the animals were sacrificed.

In Phase B of Part 1 of the study, one hour before E. coli infusion, four animals were randomly assigned to receive either a placebo or one of the previously mentioned co-constructs. The animals were observed for a period of 8 hours, by which time all catheters were removed, the animals returned to their cages, and after survival of the lethal bacterium. Animals subject to undue discomfort were sacrificed. Excessive discomfort is defined by IACUC as: 1) inability to sit or upright for the previous 12 hours), 2) inability to take food or water for the last 12 hours, 3) uncontrolled bleeding from catheter sites, or 4) unresponsive stimuli Outside. Venous blood samples were taken in: -1; 0; 0.5; 1; 1.5; 2; 2.5; 3; 4; 5; 6; 7; 8; 24 and 48 hours and on: 3, 5, 8, 11, 16 and 21. On the twenty-first day, the surviving animals were sacrificed.

The presence of Papio antibodies against the administered rocombypsy proteins was determined using the ELIS sandwich kit. Very briefly, the sTNFR-1 constructs nanietiopa on ELISA plates (1 μg / ml0 and added (100 μΐ) diluted baboon blood plasma (1:50 to 1: 100,000). After washing the samples, protein A (0.5 μg / ml) was added ) conjugated horseradish peroxidase (HRP), and the trials were visualized using TMB.

Results (Part I):

Plasma durations differed significantly for the three constructs. The disappearance times were determined using a model independent method and the apparent half-lives were generally estimated to be between 8 and 172 hours. In naïve animals, the plasma half-life is longest in baboons treated with the 4.0 domain construct (29 hours) and the melt temperature is decreased in baboons treated with sTNFR-I 3D / C105dU (24.7 hours) and sTNbR-I 2.6D / C105dU (21.5 hours). The difference - although statistically significant - is only 26%.

Surprisingly, after the second administration of the proteins to the respective baboons, the plasma half-lives tended to be shortened, indicating a faster clearance. This reduction in half-life is most pronounced in baboons receiving tTNbR-I 4D / C105dU where it is reduced by 48% (p &lt;0.01) [Figure 10]. The half-life reduction is indirect in baboons treated with tTNbR-I 3D / C105db (31%) [Fig. 11] and at least in animals treated with sTNbR-I 2.6D / C105dU (14%) [Figure 12]. The reduction in half-life is not statistically significant in baboons treated with tTNFR-1 2 ^ / Ο05όΚ

All preparations are immunogenic in baboons. However, the frequency of immunogenicity is higher in sTNFR-I 4D / C105db-treated baboons, intermediate in sTNFR-I 3D / C105dU-treated baboons, and lowest in sTNFR-I 2.6D / C105dU-treated animals (Table 8). ).

189 309

Table 8:

Maximum Antibody Responses θ

<td rowspan="2"></td><td colspan="2">The first 21 days</td><td colspan="2">The second 21 days</td>

<td>median</td><td> 25%-27%</td><td>median</td><td> 25%-75%</td>

<td>sTNFR-I 4D / C105dC (n = 4)</td><td> 3,20</td><td> 3,20 3,20</td><td> 3,95</td><td> 3,50 4,40</td>

<td>sTNFR-I 3D / C105dC (n = 4)</td><td> 1,60</td><td> 0,00 3,65</td><td> 3,50</td><td> 1,30 4,75</td>

<td>sTNFR-I 2.6D / C105db (n = 4)</td><td> 0,00*)</td><td> 0,00 1,75</td><td> 1,45</td><td> 0,00 3,50</td>

logarithmic scale (plasma detachment necessary to obtain a semi-maximum concentration on the saądwicok ELISA; see Experimental Methods) ⁺⁾ p = 0.056, ANOVA - two-tailed analysis, according to Kruskal-Wallis (log transformed values ​​did not meet the ąkr'maląkśyi test)

Antibody responses generally develop around day 8 after administration of the co-particles and are present throughout the 21 days of the study. Moreover, the responses to antibodies tend to be stronger during the response to the second occurrence of the protein compounds.

All four baboons receiving sTNFR-I 4D / C105db developed antibodies, two of four animals receiving sTNFR-I 3D / C105dC developed antibodies, and one of four animals receiving sTNFR-I 2.6D / C105dC developed antibodies. According to Keuskall-Wallis weighting analysis (ANOVA), the magnitude of the antibody response (log transformed) is significantly different for the three groups as a function of time (p &lt;0.05). "Pkst-hoy" analysis suggests that a significant difference in antibody responses generally exists between animals receiving sTNFR-I 4D / C105db and sTNFR-I 2.6D / C105db for intermediate (and non-snapping) responses in sTNFR-treated animals- And 3D / C105dC.

A correlation was observed between antibody development and change in clearance between the two 21-day studies (p &lt;0.01). It is not unexpected that in those animals which develop a strong antibody response after the first administration of the construct, the protein is cleared more rapidly than after the second administration. The change in clearance between the first and second injection is compared between animals that developed an antibody response (n = 7) and those that did not (n = 5) (Figure Fig. 13).

Antibodies detected in the plasma of baboons are assessed in a selected number of animals for direct cytotoxicity to the ME-180 cell line and for neutralization capacity in assay L-929. No cytotoxicity or neutralization was observed with the antibodies produced against any of these factors.

In Part A of the Phase I baboon study, the animals that developed the strongest antibody responses also showed the fastest increase in co-particle clearance after their second administration. Thus, such findings suggest that antibody responses may reduce the biological half-life and thus may be the selection of therapeutic efficacy of the co-particles and dose adjustment. However, no uncertain clinical response to the presence of antibodies was observed when Cila co-constructs were administered a second time. Thus, therapeutic efforts for Cy to modify such contours to reduce immunogenicity without significantly affecting the half-life or efficacy are directed, primarily, at reducing the need for incremental dose adjustments rather than at the risk of adverse reactions.

Part 1 Phase B - Results:

Ultimately, for unstimulated baboons, all three tartlets are almost equally effective in preventing yytokia-mediated E. coli injury when administered at 1.0 mg / kg SW. One of the four placebo-treated baboons survived; 4 of 4 sTNFR-I 4D / C105dC- and sTNFR-I 3D / C105dC- treated baboons survived; and 3 of 4 sTNFR-I 2.6D / C105db treated baboons survived, respectively. All three co-molecules prevent TNF? Cicactivity and provide excess neutralizing capacity.

189 309

Part II:

A particular goal of part II of the studies in baboons was to determine whether the streaks of repeated exposure to Cp on the action (i.e. 3 separate injections) of animals on different angles of sTNF-RI led to further immunity and reduced half-trimmunity. In addition, this study aimed to compare the immense validity and pharmacokinetics of a number of sTNF-R1 substructures, including sTNFR-I 2.6D / CI05ab; and sTNFR-I 4D / C105db; sTNFR-I 2.6D / N105-tBuPEG (33 kDa) and sTNFR-I 4D / N105-t-BuPEG (33 kDa). Ultimately, this study was designed to evaluate the importance of the PlCnioste antibody response, and the alteration of clearance as a result of subsequent reaction to TNFα mediated injury (E. coli bacteria).

On days 0, 21 and 42, aamadam was administered intravenously with 0.2 mg / kg of various cedstructures (sTNFR-I 4D / C105db; sTNFR-I 2.6D / C105db; sTNFR-I 2.6D / N105-t-BuPEG (33 kDa) ; Or sTNFR-I 4D / N105-t-BuPEG (33 kDa), odpomipddie). On day 63, the baboons received 2.0 mg / kg BW of their stomach acid. On day 65 (i.e. 48 hours later), baboons were given a lethal dose of E. coli as eaisade above in Part I. The principal findings of Part II are as follows:

Results (part II):

Essentially, sTNFR-I 4D / N105-t-BuPEG (33 kDa); and sTNFR-I 2.6D / N105-t-BuPEG (33 kDa) have longer half-lives than TNFR-I 4D / C105db and sTNFR-I 2.6D / C105db in non-stimulated baboons for domon, half-lives are up to from 30-35 hours for sTNFR-I medepegylated forms compared to 10-20 hours for dimeromyoh peylated forms. In addition, sTNFR-I 4D / N105-t-BuPEG (33 kDa) and TNFR-I 4D / C105db have longer half lives than 2.6D / N105-t-BuPEG (33 kDa) and sTNFR-I 2.6D / C105db in baboons untreated.

sTNFR-I 4D / C105db and sTNFR-I 2.6D / CI05ab are also immunocompatible, with a moderate trend towards reduced immunity at sTNFR-I 2.6D / C105db. However, only TNFR-I 4D / C105db shows reduced clearance with repeated administrations. sTNFR-I 4D / N105-t-BuPEG (33 kDa) and 2.6D / N 105-t-BuPEG (33 kDa) are neither antigenic nor do they significantly alter their clearance rates with repeated administration.

The serum obtained from each baboon (N = 3) treated with the different compounds on days 21, 42, and 61 was assessed in vitro for immunoreactivity (by sadamic ELISA capture) against other codstructures by using different codstructures as the uptake antigen. For example, the serum obtained with pamiados given 2.6D / N105-t-BuPEG (33 kDa) on day 21 (Table 9) did not "react" to sTNFR-I 4D / C105db or to sTNFR-I 4D / N105, when rt compounds were used on the ELISA plate as the uptake antigen.

A positive reaction is that of an antibody with a titer> 1: 400. The data of days 42 and 61 are given in Tables 10 and 11. It is important that there is a positive in vitro response with sera obtained from one baboon previously treated with 2.6D / N105-t-BuPEG (33 kDa) when tested for antigen sTNFR-I 4D / C105db capture (Table 11).

For baboons previously exposed to kedstructures three times, the efficacy of the TNFα-mediated response to injury is greatest in: (1) sTNFR-I 4D / C105db; (2) sTNFR-I 2.6D / C105db; (3) sTNFR-I 4D / N105-t-BuPEG (33 kDa); and (4) 2.6D / N105-t-BuPEG (33 kDa) (as determined by survival, multisystem organ immobility (MSOF), serum IL-6 and WBC reaction). The "warning" is that this n and p study examines the differences in the ability to neutralize TNF from different codegroups.

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Table 9

IgG baboon antibody response (titer> 1: 400) day 21

<td>Quantity animals- n = 3</td><td>sTNFR-I 2 6D / C105db</td><td>sTNFR-I 2 6D / N105- t-BuPEG (33 kDa)</td><td>sTNFR-I 4D / N105-1- BuPEG (33 kDa)</td><td>sTNFR-I 4D / C105-tBuPEG (33 kDa)</td><td>sTNFR-I 4D / C105db</td><td>sTNFR-1 4D / N3105</td>

<td>sTNFR-I 2 6D'N105-1BuPEG (33 kDa)</td><td></td><td>3/3 neg</td><td></td><td></td><td>3/3 neg</td><td>3/3 neg</td>

<td>sTNFR-I 2.6D / C105db</td><td>3/3 neg</td><td></td><td></td><td></td><td>3/3 neg</td><td>3/3 neg</td>

<td>sTNFR-I 4D / N105-tBuPEG (33 kDa)</td><td></td><td></td><td>3/3 neg</td><td></td><td>3/3 neg</td><td>3/3 neg</td>

<td>sTNFR-I 4D / C105-tBuPEG (33 kDa)</td><td></td><td></td><td></td><td></td><td></td><td></td>

<td>sTNFR-1 4D / C105db</td><td></td><td></td><td></td><td></td><td>1/3 react (400)</td><td>3/3 neg</td>

Table 1

IgG baboon antibody response (titer> 1,400) day 42

<td>Quantity animals- n = 3</td><td>sTNFR-I 2.6D / C105db</td><td>sTNFR-1 2 6D / N105- t-BuPEG (33 kDa)</td><td>sTNFR-I 4D / N105-t- BuPEG (33 kDa)</td><td>sTNFR-I 4D / C105-tBuPEG (33 kDa)</td><td>sTNFR-I 4D / C105db</td><td>sTNFR-I 4D / N3105</td>

<td>sTNFR-1 2.6D / N105-tBuPEG (33 kDa)</td><td></td><td>3/3 neg</td><td></td><td></td><td>3/3 neg</td><td>3/3 neg</td>

<td>sTNFR-1 26D / C105db</td><td>1/3 react (1600)</td><td></td><td></td><td></td><td>3/3 neg</td><td>3/3 neg</td>

<td>sTNFR-I 4D / N105-t- BuPEG (33 kDa)</td><td></td><td></td><td>3/3 neg</td><td></td><td>3/3 neg</td><td>3/3 neg</td>

<td>sTNFR-I 4D / C105-tBuPEG (33 kDa)</td><td></td><td></td><td></td><td></td><td></td><td></td>

<td>sTNFR-I 4D / C105db</td><td></td><td></td><td></td><td></td><td>2/3 react (3200)</td><td>2/3 react (800)</td>

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Table 11:

IgG baboon antibody response (titer> 1: 400) day 61

<td>Quantity animals' n = 3</td><td>sTNFR-I 2.6D / C105db</td><td>sTNFR-I 2.6D / N105t-BuPEG (33 kDa)</td><td>sTNFR-I 4D / N105-t- BuPEG (33 kDa)</td><td>sTNFR-I 4D / C105-tBuPEG (33 kDa)</td><td>sTNFR-I 4D / C105db</td><td>sTNFR-I 4D / N3105</td>

<td>sTNFR-I 2 6D / N105-tBuPEG (33 kDa)</td><td></td><td>3/3 neg</td><td></td><td></td><td>1/3 react (1600)</td><td>3/3 neg</td>

<td>sTNFR-I 2 6D / C105db</td><td>2/3 react (204800)</td><td></td><td></td><td></td><td>1/3 react (1600)</td><td>1/3 react (3200)</td>

<td>sTNFR-I 4D / N105-tBuPEG (33 kDa)</td><td></td><td></td><td>3/3 neg</td><td></td><td>1/3 react (6400)</td><td>1/3 react (6400)</td>

<td>sTNFR-I 4D / C105-tBuPEG (33 kDa)</td><td></td><td></td><td></td><td></td><td></td><td></td>

<td>sTNFR-I 4D / C105db</td><td></td><td></td><td></td><td></td><td>1/3 react (3200)</td><td>3/3 react (800)</td>

C. Chimpanzees:

The purpose of this study is to evaluate the immunogenicity of various forms of sTNF-R1 that are repeatedly injected intravenously into chimpanzees over a period of 1 month. The forms of sTNF-R1 tested in these studies are: sTNFR-I 2.6D / C105db; sTNFR-I 4D / C105db; sTNFR-I 4D / C105-t-BuPEG (33 kDa); sTNFR-I 2.6D / N105-t-BuPET (33 kDa); and sTNFR-I 4D / N105-t-BuPEG (33 kDa). There are a total of 3 chimpanzees in each study group.

The dosing regimen and parameters in this study are as follows: Each chimpanzee receives the test substance by intravenous injection at 0.1 mg / kg twice a week on Mondays and Fridays for 4 weeks (8 doses in total). The dose volume is variable depending on the concentration of the test substance delivered. A 5 ml serum sample is obtained from each animal on day "0" prior to testing. Additional serum samples are collected immediately prior to dosing on days 7, 14, 21, and 28.

Chimpanzee immunogenicity data are presented in Table 12.

By day 28, all animals (N = 3) treated with either sTNFR-I 4D / C105db or sTNFR-I 2.6D / C105db showed a positive response (as measured by ELISA) with the highest titer observed being 1 : 12,800 Or 1: 3200 (table 12).

(Note: in this part of the experiment, all "immunizing" antigens are used as respective uptake antigens immobilized on the ELISA plate).

One of the animals treated with sTNFR-I 4D / Cl05-t-BuPET (33 kDa) or with sTNFR-I 2.6D / N105-t-BuPEG (33 kDa) showed a positive antibody response on days 21 and 28 (Table 12) . It is important that none of the animals treated with sTNFR-I 4D / C105-tBuBEG (33 kDa) or sTNFR-I 2.6D / N105-t-BuPET (33 kDa) developed anti-sTNFR antibodies- And during the experiment (Table 12).

As described in the baboon experiments, serum obtained from each chimpanzee (N = 3) treated with different forms of sTNF-R1 on days 28 was assessed in vitro for immunoreactivity (by ELISA) against other constructs by using the sTNF-R1 species. as an antigen uptake. A positive reaction is an antigen reaction with a titer> 1: 400. Importantly, the serum obtained from chimpanzees administered sTNFR-I 2.6D / 105N-t-BuPEG (33 kDa) did not "react" with any of the sTNFR-I 4D / C105db; sTNFR-I 4D / N105, with sTNFR-I 4D / C105db; sTNFR-I 4D / N105 when these compounds were used on ELISA plates as the capture antigen (Table 13).

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This was also observed in animals treated with sTNFR-I 4D / C105db; sTNFR-1 4D / C105-t-BnPEG (33 kDa), or sTNFR-1 4D / N105db-t-BnPEG (33 kDa) (Table 13).

Table 12:

Antibody results IgG titer (number of animals)

<td></td><td>Day "0" (initial dose)</td><td>Day 7 (2 doses)</td><td>Day 14. (4 doses)</td><td>Day 21 (6 doses)</td><td>Day 28 (8 doses)</td>

<td>sTNFR-I 2 6D / C106db</td><td></td><td></td><td> 100(1)</td><td> 400(1) 1600 (1) 3200(1)</td><td> 800 (1) 3200 (2)</td>

<td>sTNFR-1 4D / C105db</td><td></td><td></td><td> 3200(1)</td><td> 400(1) 16^0(1)</td><td> 800 (2) 12800 (1)</td>

<td>sTNFR-I 4D / N105-t- BuPEG (33 kDa)</td><td></td><td></td><td></td><td></td><td></td>

<td>sTNFR-I 2.6D / N105-tBuPEG (33 kDa)</td><td></td><td></td><td></td><td> 200 (1)* 1600(1)</td><td></td>

<td>sTNFR-1 4D / N105-tBuPEG (33 kDa)</td><td></td><td></td><td></td><td></td><td> 100(1)* 400(1)</td>

♦ Note Titers obtained using sTNFR-1 4D / C105db as the uptake antigen

Table 13:

Antibody Findings in Chimpanzees I._gG (Titer> 1: 400)

<td>Number of animals: n = 3</td><td>sTNFR-1 2 6D / C105db</td><td>sTNFR-1 2 6D / N105-tBuPEG (33 kDa)</td><td>sTNFR-1 4D / N105-t- BuPEG (33 kDa)</td><td>sTNFR-1 4D / C105-tBuPEG (33 kDa)</td><td>sTNFR-1 4D / C105db</td><td>sTNFR-1 4D / N105</td>

<td>sTNFR-I 2 6D / N1054BuPEG (33 kDa)</td><td></td><td>3/3 neg</td><td></td><td></td><td>3/3 neg</td><td>3/3 neg</td>

<td>sTNFR-I 2.6D / C105db</td><td>3/3 react (3200)</td><td></td><td></td><td></td><td>2/3 react (3200)</td><td>1/3 react (400)</td>

<td>sTNFR-I 4D / N105-t- BuPEG (33 kDa)</td><td></td><td></td><td>3/3 neg</td><td></td><td>2/3 react (1600)</td><td>2/3 react (3200)</td>

<td>sTNFR-I 4D / C105-tBuPEG (33 kDa)</td><td></td><td></td><td></td><td>3/3 neg</td><td>1/3 react (400)</td><td>3/3 neg</td>

<td>sTNFR-I 4 D / Cl 05 db</td><td></td><td></td><td></td><td></td><td>3/3 react (12800)</td><td>3/3 react (6400)</td>

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Example IV

EAE is an acute or chronic inflammatory demyelinating disease of the CNS (CNS) resulting from the sensitization of genetically susceptible animals to neuroantigens such as myelin essential protein (MBP). EAE is an established and widely used animal model for acute human MS.

Female Lewis rats (Jackson Laboratories, Bar Harbor, ME) were anesthetized and immunized on day "0" in the left hindpaw with 0.1 mL of an emulsion containing essential myelin protein in complete Freunds adjuvant dissolved in phosphate buffered saline (PBS) with an equal volume of Complete Freunds Adjuvant (CFA) containing 5 mg / mL of Mycobacterium tuberculosis H37Ru (Difco Lab MI). Control rats received 0.1 mL of PBS / CFA emulsion without MBP in the foot of the left hind paw.

Clinical disease assessment is based on the conventional 0-5 score scale. The range is as follows: 0 - normal; 0.5 - partial loss of tail tone; 1 - complete loss of tail tension; 2 - pulling one hind paw; 3 - paralysis of both hind legs; 4 - state of failure; 5 - death. All injections of sTNFR-I constructs or vehicle were given at 1 mg / kg SC every other day starting on day 9 after immunization. All animals were completed on day 21. The results are presented in two forms - the clinical severity scale as a function of time and the integrated clinical score for each rat throughout the disease period, calculated as the area under the daily clinical score versus time curve. The values ​​of the studied groups for the integrated clinical outcome were statistically compared to the results obtained for the control group using the Mann-Whitney test.

Vehicle-treated animals had an onset of disease around day 10, a maximum of disease on day 16, and then subsided in disease state. sTNFR-I 4D / C106db attenuated clinical signs by approximately 73% compared to vehicle treated animals. sTNFR-I 4D / C-05-t-BuPEG (33 kDa) also reduces clinical symptoms by approximately 85%. sTNFR-I 4D / C105-t-BuPEG (33 kDa) and sTNFR-I 2.6DN105-t-BuPEG (33 kDa) are of equal potency in reducing clinical symptoms (64 and 57%, respectively).

In conclusion, truncated sTNFRs appear to be effective in mediating some clinical sequelae in this animal model of MS.

While the present invention has been described above both generally and in terms of the preferred embodiments, it should be understood that other variations and modifications may be envisaged by those skilled in the art from the foregoing description.