CZ499A3 - Shortened soluble receptors of necrotizing tumor type I and type II factor - Google Patents

Abstract

Disclosed are novel proteins, referred to as tumor necrosis factor binding proteins, that modulate the activity of tumor necrosis factor. Also disclosed are processes for obtaining the tumor necrosis binding proteins by recombinant genetic engineering techniques.

Description

The present invention relates to the issue of inflammation. More specifically, the present invention relates to the problem of truncated tumor necrosis factor receptors (sTNFRs).

The truncated sTNFRs have the following formula: R _x - [Cys ¹⁹ -Cys ¹⁰³ ] -R ₂ , wherein [Cys ¹⁹ -Cys ¹⁰³ ] represents residues 19-103 of sTNRF-I, and wherein R 1 is the methionylated or nonmethionylated amino group of Cys ¹⁹ or the amino acid the amino acid tail residue (s) selected from the group:

C

IC

SIC

NSIC (SEQ ID NO: 15) NNSIC (SEQ ID NO: 16) QNNSIC (SEQ ID NO: 17) PQNNSIC (SEQ ID NO: 18) HPQMMSIC (SEQ ID NO: 19) IHPQNNSIC (SEQ ID NO: 0) YIHPQNNSIC (SEQ ID NO: 21) KYIHPQNNSIC (SEQ ID NO: 22) GKYIHPQNNSIC (SEQ ID NO: 23) QGKYIHPQNNSIC (SEQ ID NO: 2 4) PQGKYIHPQNNSIC (SEQ ID NO: 25) CPQGKYIHPQNNSIC (SEQ ID NO: 2 6) VCPQGKYIHPQNNSIC (SEQ ID NO: 27) SVCPQGKYIHPQNNSIC (SEQ ID NO: 28) DSVCPQGKYIHPQNNSIC (SEQ ID NO: 2 9)

and wherein R ₂ is a carboxy group of Cys ¹⁰³ or a carboxy group of a carboxy-terminal amino acid residue selected from the group of:

FC

FCC

FCCS (SEQ ID NO: 30) FCCSL (SEQ ID NO: 31) FCCSLC (SEQ ID NO: 32) FCCSLCL (SEQ ID NO: 33)

and derivatives or derivatives thereof derived therefrom, but provided that when R 1 represents the methionylated or unmetethiolated amino group of the amino acid sequence VCPQGKYIHPQNNSIC or the N terminal truncation of 1 to 15 residues derived therefrom, then R _x - [Cys ¹⁹ -Cys ^10j ] -R2 is not an added variant having the formula R 1 - [Cys ¹⁹ -Cys ¹⁰³ ] -FCCSLCL-R 3, wherein R ₃ represents the carboxy group of amino acid residues Asn ^µl -Asn ¹⁶¹ or truncation at the carboxyl terminus Asn ^µl -Asn ¹⁶¹ .

BACKGROUND OF THE INVENTION

Inflammation is a body's defense response to injuries caused, for example, by mechanical damage, infection, or antigens. An inflammatory reaction can manifest itself pathologically when inflammation is induced by inappropriate stimuli (such as an autoantigen), when it manifests itself prominently, or when it persists even after removal of the original harmful agent. Such an inflammatory response may involve the production of certain cytokines.

While the etiology of inflammation is not yet fully understood, our knowledge of the molecular aspects of inflammation has recently been improved. This research has led to the identification of certain cytokines that are believed to play a significant role in inflammatory processes. Cytokines are extracellular proteins that modify the behavior of cells, particularly those that are in the immediate vicinity of the site of cytokine synthesis and release. Tumor necrosis factors (TNFs) are a class of cytokines produced by a variety of cell types, including monocytes and macrophages.

At least two types of TNF have been previously described, namely TNF alpha (TNF α) and TNF beta (TNF β or lymphotoxin (lymphotoxin)). Both are active as a trimeric molecule and assume

have been shown to initiate the transfer of cellular signals by receptor binding (Engelmann et al. (1990), J. Biol. Chem., 265: 14497-14504).

Some evidence suggests that TNF-α and TNF-β are major cytokines of inflammation. These known TNFs have important physiological effects on a number of different target cells that are involved in the inflammatory response to various stimuli, for example, infection or injury. Proteins stimulate secretion of latent collagenase and prostagladin E _{2 in} both fibroblasts and sinovial cells, and in addition, they act to stimulate bone resorption by osteocyte cells. These proteins enhance the surface adhesion properties of endothelial cells to neutrophils. They also cause endothelial cells to secrete coagulation activity and reduce their ability to clot (lyse). In addition, they redirect adipocyte activity from lipid deposition by inhibiting expression of the enzyme lipoprotein lipase (lipoprotein lipase). TNF also stimulates the synthesis of one class of proteins known as "acute phase reactants" in hepatocytes, which act on the hypothalamus as pyrogens (Selby et al. (1988), Lancet, 1 (8583): 483; Starness, Jr. et al.

(1988) J. Clin. Invest., 82: 1321; Oliff et al. (1987) Cell 50:

555 and Wage et al. (1987), Lancet, 1 (8529): 355). Also, preclinical results in various animal models of inflammation (including rheumatoid arthritis) suggest that TNF inhibition has a significant impact on disease development and severity (Dayer et al. (1994), European Cytokine Network, 5 (6): 563-571 and Feldman et al. (1995), Annals of the New York Academy of Sciences, 66: 272-278). Recent preliminary clinical trials in humans with rheumatoid arthritis with TNF inhibitors have also shown promising results (Rankin et al. (1995), British Journal Of

Rheumatology, 3 (4): 4334-4342. Elliot et al. (1995), Lancet 344: 1105-1110; Tak et al. (1996) Arthritis and Rheumatism 39: 10771081; and Paleolog et al. (1996), Arthritis and Rheumatism, 39: 1082,1091.

TNF protein inhibitors are mentioned in EP 308 378, proteins isolated from the urine of patients with fever showing TNF inhibitory activity. The effect of these proteins is probably due to a competitive mechanism at the receptor level. EP 308 378 discloses proteins sufficiently pure to be characterized by their own

N-end. However, the reference does not say anything about any DNA sequence or recombinantly produced TNF inhibitor.

However, there are also data on recombinantly prepared TNF inhibitors. For example, EP 393 438 and EP 422 339 disclose the amino acid and nucleotide sequences of a mature (recombinant) human 30 kDa inhibitor TNF (also known as the p55 receptor and as a sTNFR-I) and a mature recombinant human 40 kDa inhibitor (also known as the p75 receptor). sTNFR-II); also discloses modified forms thereof, such as fragments, functional derivatives, and variants thereof. EP 393 438 and EP 422 339 also disclose methods for isolating the genes encoding these inhibitors, cloning the respective genes in suitable vectors and cells, and expressing the genes leading to the production of inhibitors. The finished recombinant human 30 kDA TNF inhibitor and the finished recombinant human 40 kDa TNF inhibitor are capable of inhibiting TNF (EP 393 438, EP 422 339, PCT Publication No. WO 92/16221 and PCT Publication No. WO 95/34326.

sTNFR-I and sTNFR-II are members of the nerve growth factor / TNF receptor superfamily, receptors including the nerve growth factor receptor (NGF), CD40 B cell antigen, Fas antigen and CD27 and CD30 antigens (Smith et al (1990) Science 248: 1019-1023).

The most conservative feature in this family of cell surface receptors is the cysteine-rich, extracellular ligand-binding domain, which can be divided into four repeating motifs of about 40 amino acids and containing 4-6 cysteine residues at extensively conserved positions (Smith et al. (1990) ), see above).

EP 393 438 further discusses 40 kDa TNF inhibitor Δ51 and 40 kDa TNF inhibitor Δ53, which are truncated versions of the full-length recombinant 40 kDa protein of the TNF inhibitor, where amino acid residues 51 or 53 at the carboxy terminus of the finished protein are removed. One of skill in the art will appreciate that the fourth domain of both the 30 kDa inhibitor TNF and the 40 kDa inhibitor is not required for TNF inhibition. This fact has been confirmed by more groups. The variants of the 30 kDa and 40 kDa TNF inhibitors with the deleted domains were prepared and these variants without the fourth domain retain full TNF binding activity; in contrast, variants lacking the first, second, or third domains do not retain TNF binding activity (Corcoran et al. (1994), Eur. J, 1994). ··. Biochem.,

223: 831-840; Chih-Hsueh et al. (1995) The Journal of Biological Chemistry, 270 (6): 2874-2878, and Scallon et al. (1995), Cytokine, (8): 759-770.

Because of the relatively low inhibition of cytotoxicity exhibited by the 30 kDa TNF inhibitor and the 40 kDa TNF inhibitor (Butler et al. (1994),

Cytokine, 6 (6): 616-623), various groups prepared protein dimers of TNF inhibitors (Butler et al. 1994, supra and Martin et al. (1995), Exp. Neurol., 131: 221-228). However, dimers may stimulate an antibody response (Martin et al. (1995), supra and Fisher et al. (1996), The

New England Journal of Medicine, 334 (26): 1697-1702.

It is an object of the present invention to provide a functionally active truncated sTNFR. This and other objects of the present invention will become apparent from the following description.

SUMMARY OF THE INVENTION

The present invention is directed to functionally active truncated forms of sTNFR-Ι and sTNFR-II, hereinafter referred to as truncated (truncated) sTNRF. Truncated sTNRFs are modified forms of sTNFR-Ι and sTNFR-II that do not contain the fourth domain (amino acid residues Thr ¹²⁷ -Asp ¹⁶¹ in sTNFR-Ι and amino acid residues Pro ¹⁴¹ -Thr ¹⁷⁹ in sTNFR-II); portion of the third domain (amino acid residues Asn Cys ^{lu 12b} with sTNFR-Ι and Pro ¹²³ -Lys ¹⁴⁰ of sTNFR-II in); and which optionally do not contain part of the first domain (amino acid residues Asp ^x -Cys ¹⁹ for sTNFR-Ι and Leu ¹ Cys ³² for sTNFR-II). These novel TNF inhibitors (eg, TNF-α and / or TNF-β) have general utility.

The truncated sTNFRs of the present invention include proteins that correspond to the formula R 1 - [Cys ¹⁹ -Cys ¹⁰³ ] -R 2 and R 4 - [Cys ³² -Cys ¹¹³ ] -R 5. These proteins are truncated forms of sTNFR-Ι and sTNFR-II.

The formula R _x - [Cys ¹⁹ -Cys ¹⁰³ ] -R ₂ means one or more proteins wherein [Cys ¹⁹ -Cys ¹⁰³ ] correspond to TNFR-idu residues 19-103 when numbering the amino acid residues according to the scheme in Figure 1 (SEQ ID NO : 2); wherein R 1 represents a methionylated or unmethionylated amino group Cys ¹⁹ or the amino terminus of any amino acid residue (s) of the following group:

· · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · «· · * ··

IC SIC NSIC (SEQ ID NO 15) NNSIC (SEQ ID NO 16) QNNSIC (SEQ ID NO 17) PQNNSIC (SEQ ID NO 18) HPQNNSIC (SEQ ID NO 19) IHPQNNSIC (SEQ ID NO 20) YIHPQNNSIC (SEQ ID NO 21) KYIHPQNNSIC (SEQ ID NO 22) GKYIHPQNNSIC (SEQ ID NO 23) QGKYIHPQNNSIC (SEQ ID NO 29) PQGKYIHPQNNSIC (SEQ ID NO 25) CPQGKYIHPQNNSIC (SEQ ID NO 26) VCPQGKYIH PQNNSIC (SEQ ID NO 27) SVC PQGKYIH PQNN SIC (SEQ ID NO 28) DSVCPQGKYIHPQNNSIC (SEQ ID NO 29)

and wherein R ₂ represents a carboxy group of Cys ¹⁰³ or a carboxy terminus of an amino acid residue selected from the group of:

F

FC

FCC

FCCS (SEQ ID NO: 30) FCCSL (SEQ NO ID: 31) FCCSLC (SEQ ID NO: 32) FCCSLCL (SEQ ID NO: 33)

and variants thereof, but provided that when Rx represents a methionylated or non-methylated amino group of the amino acid sequence VCPQGKYIHPQNNSIC or a N-terminal truncation of 1-15 residues, then the R _x - [Cys ¹⁹ -Cys ¹⁰³ ] -R ₂ protein is not a variant of R ₂ - [Cys ¹⁹ -Cys ¹⁰³ ] -FCCSLCL-R 3, wherein R 3 represents the carboxy group of the amino acid sequence Asn ¹¹¹ Asn ¹⁶¹ of Figure 1, or a truncation thereof at the carboxy terminus.

• «« · ·

The following molecules may serve as examples of truncated sTNFR-I of the present invention: NH ₂ -MDSVCPQGKYIHPQNNSIC- [Cys ¹⁹ Cys ¹⁰³ ] -FC-COOH (also referred to as sTNFR-I 2.6D / C105); NH2MDSVCPQGKYIHPQNNSIC- [Cys ¹⁹ -Cys ¹⁰³ ] -FNCSL-COOH (also referred to as sTNFR-I 2.6D / C106); NH2-MDSVCPQGKYIHPQNNSIC- [Cys ¹⁹ -Cys ¹⁰³ ] -FN-COOH (also referred to as sTNFR-I 2.6D / N105); NH2-MYIHPQNNSIC- [Cys ¹⁹ Cys ¹⁰³ ] -FNCSL-COOH (also referred to as sTNFR-I 2.3D / d8); NH 2 -M [Cys ¹⁹ -Cys ¹⁰³ ] -FNCSL-COOH (also referred to as sTNFR-I 2.3D / dl8) and NH ₂ -MSIS- [Cys ¹⁹ -Cys ¹⁰³ ] -FNCSL-COOH (also referred to as sTNFR-I 2.3D / dl5) either methionylated or non-methionylated and variants and derivatives thereof.

"R ₄ - [Cys ³² -Cys ¹¹⁵ ] -R ₅ means one or more proteins wherein [Cys ³² -Cys ¹¹⁵ ] represent residues of Cys ³² through Cys ¹¹⁵ sTNFR-I according to the scheme used in Figure 8 (SEQ ID NO: (35) for ease of comparison and wherein R 4 represents a methionylated or non-methylethylated Cys ³² amino acid residue

residues) at the amino terminus selected from groups: C MC QMC AQMC (SEQ ID NO: 37) TAQMC (SEQ ID NO: 38) QTAQMC (SEQ ID NO: 37) DQTAQMC (SEQ ID NO: 38) YDQTAQMC (SEQ ID NO: 41) YYDQTAQMC (SEQ ID NO: 40) EYYDQTAQMC (SEQ ID NO: 43) REYYDQTAQMC (SEQ ID NO: 42) LREYYDQTAQMC (SEQ ID NO: 43) RLREYYDQTAQMC (SEQ ID NO: 44) CRLREYYDQTAQMC (SEQ ID NO: 47) TCRLREYYDQTAQMC (SEQ ID NO: 48) STCRLREYYDQTAQMC (SEQ ID NO: 47) GSTCRLREYYDQTAQMC (SEQ ID NO: 50) PGSTCRLREYYDQTAQMC (SEQ ID NO: 51) EPGSTCRLREYYDQTAQMC (SEQ ID NO: 52)

• · · · · · · · · ·

PEPGSTCRLREYYDQTAQMC (SEQ ID NO: 52) APEPGSTCRLREYYDQTAQMC (SEQ ID NO: 53) YAPEPGSTCRLREYYDQTAQMC (SEQ ID NO: 54) PYAPEPGSTCRLREYYDQTAQMC (SEQ ID NO: 55) TPYAPEPGSTCRLREYYDQTAQMC (SEQ ID NO: 5 6) FTPYAPEPGSTCRLREYYDQTAQMC (SEQ ID NO: 57) AFTPYAPEPGSTCRLREYYDQTAQMC (SEQ ID NO: 5 8) VAFTPYAPEPGSTCRLREYYDQTAQMC (SEQ ID NO: 59) QVAFTPYAPEPGSTCRLREYYDQTAQMC (SEQ ID NO: 60) AQVAFTPYAPEPGSTCRLREYYDQTAQMC (SEQ ID NO: 61) PAQVAFTPYAPEPGSTCRLREYYDQTAQMC (SEQ ID NO: 62) LPAQVAFTPYAPEPGSTCRLREYYDQTAQMC (SEQ ID NO: 63)

and wherein R 8 is a carboxy group of Cys ¹¹⁵ or a carboxy group of the carboxy terminus of amino acid residues selected from:

AND

AP

APL

APLR (SEQ NO NO: 64) APLRK (SEQ NO ID: 65) APLRKC (SEQ NO ID: 66) APLRKCR (SEQ NO NO: 67)

and variants thereof, but provided that when R ₄ represents a methionylated or unmethionylated amino group of the amino acid sequence TCRLREYYDQTAQMC or an N-terminal truncation of 1 to 15 residues, then R4- [Cys ³² -Cys ¹¹⁵ ] -R5 is not a variant of formula R4- [Cys ³² -Cys ^11:> ] -APLRKCRRg, wherein R 6 represents the carboxy group of the amino acid sequence Pro ¹²³ -Thr ¹⁷⁹ of Figure 8, or a truncation of its carboxy terminus.

In one aspect of the present invention, truncated sTNFRs can be prepared in glycosylated or non-glycosylated form. Truncated sTNFRs are prepared by recombinant genetic engineering techniques. In addition, truncated sTNFRs can be synthesized by chemical techniques or a combination of recombinant and chemical techniques.

• ·

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

Another aspect of the present invention encompasses various polynucleotides encoding truncated sTNFRs. Suitable nucleotide sequences include, for example, those specifically set forth in the figures, 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, where expression products or derivatives thereof are characterized by the ability to affect TNF activity.

Another aspect of the present invention are vectors comprising polynucleotides encoding truncated sTNFRs operatively linked to amplification and / or expression control sequences. Both prokaryotic and eukaryotic host cells can be stably transformed or transfected with such vectors to express truncated sTNFRs. The present invention further encompasses recombinant production of truncated sTNFRs. Host cells containing such polynucleotides are grown in a suitable nutrient medium, and cells expressed with truncated sTNFR are optionally isolated from host cells and / or nutrient medium.

Another aspect of the present invention includes pharmaceutical compositions comprising truncated sTNFR or derivatives thereof. Typically, truncated sTNFRs or derivatives thereof may be prepared in association with pharmaceutically acceptable carriers. A variety of materials can be used to facilitate production, storage, handling, transport and / or efficiency of truncated sTNFRs and variants thereof.

Another aspect of the present invention relates to methods of modulating TNF activity. In particular, TNF-mediated diseases (eg, TNF-α and / or TNF-β mediated diseases) can be treated by administering therapeutically effective amounts of truncated sTNFR or derivatives thereof.

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

• 9

The truncated sTNFRs of the present invention are particularly suitable for protein production in large quantities. For example, sTNFR-Ι has a deamidated site within amino acid sequence 111-126 (amino acids Asn ^ul -Gly ¹²⁶ ). The absence of this site is believed to increase the biochemical stability of the purified protein and reduce the possible degradation of the product, resulting in increased protein stability upon storage. Truncated sTNFRs have fewer disulfide bridges than other TNF inhibitor proteins previously discovered. For example, sTNFR-Ι has two disulfide bridges within amino acid sequence 111 to 126 and three disulfide bridges within amino acid sequence 127 to 161; sTNFR-II then has a disulfide bridge between Cys ¹²¹ and Cys ¹³⁹ , Cys ¹⁴² and Cys ¹⁵⁷ and Cys ¹⁶³ and Cys ¹⁷⁸ . A reduced number of disulfide bridges is important because a higher number of these bonds may complicate the protein refolding process. Surprisingly, truncated sTNFRs have fewer sites for antigenic epitopes than other TNF inhibitor proteins previously described (e.g., the truncated form of sTNFR-Ι with the first three domains has a new epitope created by revealing certain residues, see Example III), greatly reducing antigenicity and furthermore, there is a significant reduction in the rate of degradation upon repeated administration. The reduced immunogenicity of truncated sTNFRs is believed to be useful in the treatment of TNF-mediated diseases, particularly chronic inflammatory disease.

Other aspects and advantages of the invention will be apparent to those skilled in the art from the following description, which describes in detail the conventional methods of the present invention.

Detailed description of the invention

The present invention is based on the unexpected finding that both sTNFR-Ι and sTNFR-II can be reduced in size not only by removing the fourth domain, but also by part of the third domain and optionally part of the first domain while maintaining biological activity and reducing antigenicity. Production of these biologically active truncated sTNFRs or derivatives thereof is believed to be advantageous for at least the following reasons. First, these molecules may have one potentially destabilizing deamidated site less. Second, these molecules have fewer disulfide bridges and this will facilitate the eventual one

4. · · * 4 4 4 4 4 4 4 ' '4.. • '.4 .4. 4 4 4 • 4 4 4 4 4 4 4 4 4 4 4 4 • • • 4 4 • 4 4 4 · 4 4 · 4 4 · • 4 4 · 4 »·

purification and refolding. Third, these molecules have fewer sites for possible antigenic epitopes.

The term truncated sTNFR as used herein includes one or more biologically active, synthetic or recombinant molecules of the formula R ₁ - [Cys ¹⁹ -Cys ¹⁰³ ] -R 2 or R 4 - [Cys ³² -Cys ¹¹⁵ ] -R 5 and variants thereof ( including insertion, substitution, and deletion) as described below.

As used herein, the term biologically active means that the truncated sTNFR exhibits similar TNF inhibitory properties as sTNFR-Ι and / or sTNFR-II, although not necessarily all and not necessarily to the fullest extent. Generally truncated sTNFRs and derivatives thereof have the ability to inhibit TNF. Bioassays of truncated sTNFRs are described in Examples II below. The choice of particular TNF inhibitory properties depends on the desired use of the truncated sTNFR.

Truncated sTNFR proteins may advantageously be produced by recombinant techniques in bacterial, mammalian or insect cell systems and may be in either glycosylated or non-glycosylated form. Alternatively, they may be synthesized chemically. The presently preferred production methods are described in detail below.

All truncated sTNFRs can typically be isolated and purified to considerable purity and free of other proteinaceous materials (eg, non-truncated sTNFRs). Preferably, the truncated sTNFR is about 80% free of other proteins that may be present due to the production technique used to prepare truncated sTNFR. Preferably, the protein is even purer (greater than 90%, 95%, or greater than 98%). However, it is appreciated that the desired protein may be combined with other active components, chemical compounds and / or suitable pharmaceutical materials prior to administration, as described in more detail below.

Truncated sTNFR

The truncated sTNFRs of the present invention may be one or more proteins represented by the following formula:

R _{1 -} [Cys ¹⁹ -Cys ¹⁰³ ] -R ₂ wherein [Cys ¹⁹ -Cys ¹⁰³ ] represents residues 19-103 of sTNFR-Ι (in the designation of the amino acid residues used in Figure 1 (SEQ ID. 4 ', 4 4 · 4 4 · »··· · · 4 4 4 4 · ··· · ···

4 4 4 4 4 5

444 444 444 · 4 ·· «·

NO: 2)), wherein R _x represents methionylated or non-methionylated

amino group Cys ¹⁹ or amino acidic residue (residues) to amino end belonging to the following groups: C ZC SIC NSIC (SEQ ID NO: 15) NNSIC (SEQ ID NO: 16) QNNSIC (SEQ ID NO: 17) PQNNSIC (SEQ ID NO: 18) HPQNNSIC (SEQ ID NO: 19) IHPQNNSIC (SEQ ID NO: 20) YIHPQNNSIC (SEQ ID NO: 21) KYIHPQNNSIC (SEQ ID NO: 22) GKYIHPQNNSIC (SEQ ID NO: 23) QGKYIHPQNNSIC (SEQ ID NO: 24) PQGKYIHPQNNSIC (SEQ ID NO: 25) CPQGKYIHPQNNSIC (SEQ ID NO: 26) VCPQGKYIHPQNNSIC (SEQ ID NO: 27) SVCPQGKYIHPQNNSIC (SEQ ID NO: 28) DSVCPQGKYIHPQNNSIC (SEQ ID NO: 29) and wherein R ₂ represents a carboxy group, Cy _s 103 or amino acid the remainder of the carboxy terminus selected z follow ici groups:

F

FC

FCC

FCCS (SEQ ID NO: 30) FCCSL (SEQ ID NO: 31) FCCSLC (SEQ ID NO: 32) FCCSLCL (SEQ ID NO: 33)

and variants thereof, but provided that when R 1 represents a methionylated or unmethionylated amino group of the amino acid sequence VCPQGKYIHPQNNSIC or a truncation of the N-terminus of 1-15 residues, then

0 · 0 0 0

Ri ~ [Cys ¹⁹ -Cys ^103] -R 2 is not a variant of formula Rx- [Cys ¹⁹ -Cys ^103] -FCCSLCLR3 wherein R ₃ represents a carboxy group of the amino acid sequence Asn-Asn ^{lu 161} of Figure 1 or a carboxy terminal truncation.

In another case, the truncated sTNFR of the present invention may be one or more proteins represented by the following formula:

R ₄ - [Cys ³² -Cys ¹¹⁵ ] -R ₅ wherein [Cys ³² -Cys ¹¹⁵ ] represents the sTNFR-II residues Cys ³² to Cys ¹¹⁵ according to the amino acid residue numbering scheme in Figure 8 (SEQ ID NO: 35), wherein R ₄ represents a methionylated or unmethionylated Cys ³² amino group or an amino terminus (s) at the amino terminus belonging to the following group:

C

MC

QMC

AQMC (SEQ ID NO 36) TAQMC (SEQ ID NO 37) QTAQMC (SEQ ID NO 38) DQTAQMC (SEQ ID NO 39) YDQTAQMC (SEQ ID NO 40) YYDQTAQMC (SEQ ID NO 41) EYYDQTAQMC (SEQ ID NO 42) REYYDQTAQMC (SEQ ID NO 43) LREYYDQTAQMC (SEQ ID NO 44) RLREYYDQTAQMC (SEQ ID NO 45) CRLREYYDQTAQMC (SEQ ID NO 46) TCRLREYYDQTAQMC (SEQ ID NO 47) STCRLREYYDQTAQMC (SEQ ID NO 48) GSTCRLREYYDQTAQMC (SEQ ID NO 49) PGSTCRLREYYDQTAQMC (SEQ ID NO 50) EPGSTCRLREYYDQTAQMC (SEQ ID NO 51) PEPGSTCRLREYYDQTAQMC (SEQ ID NO 52) APEPGSTCRLREYYDQTAQMC (SEQ ID NO 53) YAPEPGSTCRLREYYDQTAQMC (SEQ ID NO 54) PYAPEPGSTCRLRFYYDQTAQMC (SEQ ID NO 55) TPYAPEPGSTCRLREYYDQTAQMC (SEQ ID NO 56) FTPYAPEPGSTCRLREYYDQTAQMC (SEQ ID NO 57)

AFTPYAPEPGSTCRLREYYDQTAQMC

VAFTPYAPEPGSTCRLREYYDQTAQMC

QVAFTPYAPEPGSTCRLREYYDQTAQMC

AQVAFTPYAPEPGSTCRLREYYDQTAQMC

PAQVAFTPYAPEPGSTCRLREYYDQTAQMC

LPAQVAFTPYAPEPGSTCRLREYYDQTAQMC

(SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID

NO: 58) NO: 5 9) NO: 60) NO: 61) NO: 62) NO: 63) and wherein R ₅ represents a Cys ¹¹⁵ carboxy group or a carboxy terminus residue selected from the following group:

amino acid

AND

AP APL APLR (SEQ ID NO: 64) APLRK (SEQ ID NO: 65) APLRKC (SEQ ID NO: 66) APLRKCR (SEQ ID NO: 67)

and variants thereof, but provided that when R ₄ represents a methionylated or unmethionylated amino group of the amino acid sequence TCRLREYYDQTAQMC or an N-terminal truncation of 1-15 residues, then R ₄ [Cys ³² -Cys ¹¹⁵ ] -R5 is not a variant of formula R4- [Cys ³² -Cys ¹¹⁵ ] -APLRKCR-R6, wherein R ₆ represents the carboxy group of the amino acid sequence Pro ¹²³ Thr ¹⁷⁹ of Figure 8, or a truncation thereof at the carboxy terminus.

Another aspect of the present invention includes one or more variants of R ₄ - [Cys ¹⁹ -Cys ¹⁰³ ] -R 2 and R ₄ - [Cys ³² -Cys ¹¹⁵ ] -R 5 either methionylated or unmethylated. Thus, the term truncated (truncated) sTNFR includes one or more naturally occurring allelic variants of R _x - [Cys ¹⁹ -Cys ¹⁰³ ] -R 2 and R 4 - [Cys ³² -Cys ¹¹⁵ ] -R 5, and one or more protein variants where the amino acids were residues in the amino acid sequences R ₄ - [Cys ¹⁹ -Cys ¹⁰³ J -R ₂ and R ₄ - [Cys ³² -Cys ¹¹⁵ ] -R 5 deleted (deletion variants), incorporated (insertion variants) or substituted (substitution variants).

Deletion of amino acid sequences typically range from about 20 amino acid residues; however, it is generally about 1-10 residues and most commonly 1-5 residues, so that the protein conformation is not disrupted. By deletion at the N-terminus, C-terminus, and internal delineation is meant. »- int ·· '·· * • · · Intrasquence. The number of total deletions and / or subsequent deletions will be selected to maintain the tertiary structure of the protein in the affected domain, eg, cysteine cross-linking.

Deletions within the amino acid sequence R ₁ - [Cys ¹⁹ -Cys ¹⁰³ J -R 2 and within the amino acid sequence R 4 - [Cys ³² -Cys ¹¹⁵ ] -R 5 can be performed in low homology with the sequences of other members of the NGF / TNF receptor family in the cell family surface membrane proteins. Deletions within the amino acid sequence R 1 - [Cys ¹⁹ -Cys ¹⁰³ ] -R 2 and within the amino acid sequence R 4 - [Cys ³² -Cys ¹¹⁵ ] -R 5 can also be performed in the region of significant homology with the sequences of other members of the NGF / TNF receptor family and then greater likelihood of significant modification of biological activity. In particular, the sequence similarity between members of the NGF / TNF receptor family is particularly high in the regions corresponding to the first two disulfide loops of domain 1, the entire domain 2, and the first disulfide loop of domain 3 (Banner et al. (1993), Cell, 73: 431-445) . Examples of two deletion variants Ri ~ [Cys ¹⁹ -Cys ^103] Rnjsou RI ^{[Cys19 (20} AThr) -Cys ^103] -R 2 and RJ [Cys ¹⁹ - (ACys ¹⁹ -Lys ²¹⁾ -Cys ^103] -R 2, wherein ri and R ₂ are as defined above. Examples of the three deletion variants of R ₄ - [Cys ³² -Cys ^115] -R 5 ~ R4 [Cys ³² - (ACys ¹¹⁵⁾ -Cys ^115] -R 5, R - [Cys ¹⁹ (ACys ¹¹⁵ -Lys ¹¹⁵⁾ Cys ^103] - R 2 and R 4 - [Cys ³² - UCys ¹¹⁵ -Arg ¹¹³ ) Cys ¹¹⁵ ] -R ₅ , wherein R ₄ and R ₂ are as defined above.

Addition of amino acid sequences may include fusions at the amino and / or carboxy terminus ranging in length from one residue to one hundred or more residues, as well as internal intra-sequence insertions of one or more amino acid residues. Internal additions generally range from 1 to 10 amino acid residues, more often from 1 to 5 amino acid residues, and most often from 1 to 3 amino acid residues.

Addition variants of the amino terminus include the addition of methionine (for example, as a result of direct protein expression in bacterial recombinant cell cultures) or additional amino acid residues or sequences. Another example of amino-terminal insertion involves fusing a signal sequence, sometimes with other pre-pro sequences, to facilitate protein secretion from recombinant host cells.

For prokaryotic host cells that do not recognize and process the original sTNFR-I and sTNFR-II signal sequences, the signal sequences may be replaced by prokaryotic signaling sequences.

9 9

9 99

sequences, for example from the group of alkaline phosphatases, penicilases, or the initial sequence of heat-resistant enterotoxin II. In yeast, the signal sequences may be selected, for example, from the group of yeast invertases, alpha factor, or the initial acid phosphatase sequence. In mammalian cells, expression of the original sTNFR-I and sTNFR-II signal sequences (EP 393 438 and EP 422 339) is sufficient, although other mammalian signal sequences may be appropriate (eg, sequences derived from other members of the NGF / TNF family).

Addition variants at the carboxy terminus do not include the addition of one or more amino acid residues that would affect the reconstitution of sTNFR-I or sTNFR-II. Desirably, the carboxy-terminal addition variants do not include the addition of one or more carboxylic acid residues that would affect the reconstitution of the third or fourth sTNFR-I or sTNFR-II domain. Examples of addition variants at the carboxy terminus may be chimeric proteins comprising the fusion of RJ [Cys ¹⁹ -Cys ^103] -R ₂ and R ₄ - [Cys Cys ^{3Z 115]} -R with all or part of the constant domain of the heavy or light chain of human immunoglobulin. Such chimeric proteins are preferred when their immunoglobulin portion comprises all domains except the first domain of the human immunoglobulin heavy chain constant region, such as IgG, IgA, IgM or IgE (particularly IgG, eg, IgG1 or IgG3). One of skill in the art will appreciate that (if the TNF binding portion still binds TNF and the immunoglobulin portion exhibits one or more of its characteristic properties) any amino acid of each immunoglobulin portion may be removed or replaced by one or more amino acids, or that one or more amino acids may be added.

Another group is amino acid substitution variants. All of these variants have at least one amino acid residue in R 1 -Cys ¹⁹ Cys ¹⁰³ ] -R 2 or R 4 - [Cys ³² -Cys ¹¹⁵ ] -R 5 removed and replaced by different residues at their sites. Substitution variants include allelic variants which are characterized by changes in the naturally occurring nucleotide sequences in the species population that may or may not result in amino acid changes. Those of skill in the art can use any known information about the binding or active site of a polypeptide to select possible sites for mutations.

One method for identifying amino acid residues or regions for protein mutagenesis is called "alanine scanning mutagenesis" (Cunningham and Wells (1989), Science, 244: 1081-1085, the disclosure of which is incorporated herein by reference). In this method, an amino acid residue or a group of target residues of a protein (e.g., charged residues such as Arg, Asp, His,

Lys and Glu) and replaced by a neutral or negatively charged amino acid (preferably alanine or polyalanine) to influence the interaction of the amino acids with the surrounding aqueous environment inside or outside the cell. Those residues that exhibit functional sensitivity to substitutions are further analyzed by incorporating additional or other residues at the site of substitution. Thus, the site for incorporation of the altered nucleic acid sequence is predetermined. Alanine screening or random mutagenesis can be performed to further optimize mutation properties at a given site. The resulting variant polypeptides are screened for the optimal combination of desired activity and degree of activity.

Interesting sites for substitutional mutagenesis include sites where the amino acids occurring in R _x - [Cys ¹⁹ -Cys ¹⁰³ ] -R 2 or R 4 - [Cys ³² Cys ¹¹⁵ ] -R 5 are significantly different (with respect to a substantial portion of the side chain, charge and (or hydrophobicity) from sTNFR-like proteins such as sTNFRs of various other species or other members of the NGF / TNF receptor family.

Other sites of interest include those in which certain residues are similar or identical to the corresponding sites for sTNFR-Ι-like proteins and sTNFR-II-like proteins. Such sites are generally important for the biological activity of the protein. For example, the skilled artisan understands that prior to the present invention, the effect of truncation of sTNFR-I and sTNFR-II on their respective three-dimensional structure was not predictable. However, on the basis of the results presented herein, one of ordinary skill in the art will appreciate that the first principles of developing a strategy for generating variant proteins could be based in part on previously explained data for full length sTNFR-I and sTNFR-II. Thus, the following information has been elucidated about sTNFR-Ι (Banner et al. (1993), supra and Fu et al. (1995), Protein Engineering, 8 (12): 1233-1241). The residues Tyr ⁹ , Thr ³⁹ , His ⁵⁵ domain 1, residues Phe ⁴⁹ , Ser ⁶³ , Asp ® ² domain 2 and residues Tyr ⁹² and Ser ¹⁰⁷ domain 3 have been identified as potentially important for stabilizing the corresponding structure of domains 1, 2 and 3. Pro ¹² and His ⁵⁵ were identified as potentially interacting Ser ^8s -Tyr ⁸⁷ on the TNFα subunit C. The residue of Glu ⁴⁵ -Phe ⁴⁹ was • 9 9 9

9 · · «999 9-9 ··

9 • 9 99 is identified as the nature of the loop that potentially interacts with the residue Leu ²⁹ -Arg ^{32 of} the TNFα subunit A. The Gly ⁴⁸ residue was identified as interacting with Asn ¹⁹ -Pro ²⁰ on the TNFα subunit A. His ⁵⁸ -Leu ⁶⁰ residue was identified as an important element in the conformation of the elongated strand and the side chain interaction with Arg ³¹ -Ala ³³ residue on the TNFα subunit A was potentially identified with His ⁵⁸ sTNFR-I residue that interacts specifically with Arg ³¹ . Lys ⁶⁴ -Arg ⁶⁶ residue was identified as an important element in elongated strand conformation and was found to exhibit side chain and backbone interactions with Ala ¹⁴⁵ -Glu ¹⁴⁶ and Glu ⁴⁵ residues on the TNFα subunit. A possible interaction with residue Tyr ¹¹⁵ on the TNFα subunit A has been identified in residue Met ⁶⁹ . His ⁹⁴ -Phe ¹⁰¹ residues were found to form a loop that interacted with the Thr ⁷² -Leu ⁷⁵ and Asn ¹³¹ residues of the TNFα subunit C and with the Trp ⁹⁶ sTNFR-I residue specifically interacting with the Ser ⁷¹ -Thr ⁷² residues on the subunit. C TNFα, Leu ¹⁰⁰ sTNFR-I (which is in close proximity to Asn ¹³⁷ residue on the C TNFα subunit) and Gin ¹⁰² sTNFR-I residue (which interacts specifically with residue Pro ¹¹³ on the TNFα subunit) (Residues His ⁹⁴ - Phe ¹⁰¹ has been identified as forming a loop which interacts with residues Thr ⁷² -Leu ⁷⁵ and Asn ¹³⁷ of subunit C of TNFα, with residue Trp ⁹ 6 of sTNFR-I specifically interacting with residues Ser ⁷¹ -Thr ⁷² on subunit C of TNFα , Leu ¹⁰⁰ of sTNFR-I is in close proximity with residue Asn ¹³⁷ on subunit C of TNFα and residue Gin ¹⁰² of sTNFR-I specifically interacting with residue Pro ¹¹³ on subunit A of TNFα). One skilled in the art will appreciate that these sites should first be modified by substitution in a relatively conservative manner.

Such conservative substitutions are listed in Table 1 under the heading "Preferred Substitutions". If such substitutions induce changes in biological activity, then more significant substitution changes (Sample substitutions, Exemplars substitutions) may be used and / or further additions / deletions and then analysis of the resulting products may be performed.

• * 1 I ··

Table 1: Amino Acid Substitution

Original Preferred Example substitution residue substitution Ala (A) Wall Wall; Leu; Ile Arg (R) Lys Lys; Gin; Asn Asn (N) Gin Gin; His; Lys; Arg Asp (D) Glu Glu Cys Ser Ser Gin (Q) Asn Asn Glu (E) Asp Asp Gly For For His (H) Arg Asn; Gin; Lys; Arg Ile (I) Leu Leu; Wall; Met; Ala; Phe; norleucine (norleucine) Leu (L) Ile norleucine (norleucine); Ile; Wall; Met; Ala; Phe Lys (K) Arg Arg; Gin; Asn Met (M) Leu Leu; Phe; Ile Phe (F) Leu Leu; Wall; Ile; Ala Pro (P) Gly Giy Ser (S) Thr Thr Thr (T) Ser Ser Trp (W) Tyr Tyr Tyr (Y) Phe Trp; .Phe; Thr; Ser Val (A) Leu Ile; Leu; Met; Phe; Ala; norleucine (norleucine)

The hydropathic index of amino acids may be considered in such changes of a similar nature. Each amino acid was assigned a hydropathic index based on its hydrophobicity and charge characteristics. The values are 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

-i44 <

¹

.. · 4 ... '

444 w · ··

4 · · 4 · · · í 4 4 4.

4

4 · ** (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9) and arginine (-4.5).

The importance of the hydropathic amino acid index in comparing interactive biological functions to proteins is generally clear to those skilled in the art (Kyte and Doolittle (1982), J. Mol. Biol., 157: 105131, the disclosure of which is incorporated herein by reference). It is known that certain amino acids may be substituted for others with a similar hydropathic index or condition and still retain a similar biological activity. When making changes based on the hydropathic idex, preference is given to amino acid substitutions whose hydropathic index is within ± 2, or more preferably within ± 1, or most preferably ± 0.5.

It is also known to those skilled in the art that the substitution of similar amino acids can be efficiently performed on the basis of hydrophilicity, especially when the biologically functional equivalent protein or peptide thus produced is used for immunological purposes, as in this case.

U.S. Patent 4,554,101 (the disclosure of which is incorporated herein by reference) states that the greatest local average hydrophilicity of a protein (as determined by the hydrophilicity of its adjacent amino acids) correlates with its immunogenicity and antigenicity, i.e., the biological properties of the protein.

As detailed in US Patent 4,554,101, the following hydrophilicity values have been assigned to individual 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) and tryptophan (-3.4).

When making changes based on similar values of hydrophilicity values, amino acid substitutions whose hydrophilicity values are within ± 2, or more preferably within ± 1, or most preferably ± 0.5, are preferred.

U.S. Pat. No. 4,554,101 also describes the identification and preparation of epitopes from a primary amino acid sequence based on hydrophilicity. The method disclosed in U.S. Pat. The patent 4,554,101 would be one of skill in the art to identify epitopes within the amino acid sequence such as the sTNFR sequences described herein. These regions are referred to as "epitopic core regions".

Many scientific publications have focused on secondary structure prediction and epitope identification by amino acid sequence analysis (Chou and Fasman (1974), Biochemistry, 13 (2): 222-245; Chou and Fasman, Biochemistry, 113 (2): 211-222; Chou and Fasman, (1978) Adv Enzymol Relat Areas Mol Biol, 47: 45-148;

Biochem., 47: 251-276 and Chou and Fasman (1979), Biophys. J., 26: 367-384 (the disclosures of which are incorporated herein by reference). In addition, computer programs are currently available to assist in predicting antigenic portions and epitope inner regions of proteins. Examples are programs based on Jameson-Wolf analysis (Jameson and Wolf (1998), Comput. Appl. Biosci., 4 (1): 181-186 and Wolf et al. (1988), Comput. Appl. Biosci., 4 (1): 1): 187-191, the disclosures of which are incorporated herein by reference), the PepPlot program (Brutlag et al. (1990), CABS, 6: 237-245 and Weinberger et al. (1985), Science, 228: 740-742, and other new programs for predicting tertiary structure of proteins (Fetrow and Bryant (1993), Biotechnology, 11: 479-483, the disclosure of which is incorporated herein by reference).

It is expected that conservative modifications of the amino acid sequences (and corresponding modifications of the nucleic acid coding sequence) of R 1 - [Cys ¹⁹ -Cys ¹⁰³ ] -R 2 or R 4 - [Cys ³² -Cys ¹¹⁵ ] -R 5 give proteins having similar functions and chemical characteristics to modified protein.

In contrast, a significant modification in the function and / or chemical characteristics of R 1 - [Cys ¹⁹ -Cys ¹⁰³ ] -R 2 and R 4 - [Cys ³² -Cys ¹¹⁵ ] R 5 may be achieved by selecting substitutions that differ significantly in their conservation effect: (a (b) the charge or hydrophobicity of the protein at the target site; (c) the major part of the side chain. Naturally occurring residues are divided into groups based on side chain properties:

1) hydrophobic: norleucine, Met, Ala, Val, Leu, Ile;

2) neutral hydrophilic: Cys, Ser, Thr;

3) acidic: Asp, Glu;

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

5) residues that affect chain orientation: Gly, Pro and

6) aromatic: Trp, Tyr, Phe.

Non-conservative substitutions may include exchanging a member of one of these groups for another. Such substitution may be introduced into the R 1 - [Cys ¹⁹ -Cys ¹⁰³ ] -R 2 and R 4 - [Cys ³ Z -Cys ¹¹⁵ ] -R 5 regions that are homologous or non-homologous to other members of the NGF / TNF receptor family.

Specific mutations of the R 1 - [Cys ¹⁹ -Cys ¹⁰³ ] -R 2 and R 4 - [Cys ³² -Cys ¹¹⁵ ] -R 5 sequences may include substitution of non-native amino acids at the N-terminus, C-terminus or at any point in the protein, which is modified by adding an N-linked or O-linked carbohydrate. Such modifications may be particularly useful, for example, when adding an amino acid (eg, cysteine), which is advantageous for attaching a water-soluble polymer to form derivatives as described below. An example is seen in Figure 5, which originally occurring Asn ¹⁰⁵ of sTNFR-Ι is changed to Cys to facilitate the attachment of polyethylene glycol molecule (Example I).

The sequence of R 1 - [Cys ¹⁹ -Cys ¹⁰³ ] -R 2 or R 4 - [Cys ³² -Cys ¹¹⁵ ] -R 5 may be further modified to add a glycosylation site or remove an N-linked or O-linked glycosylation site. The asparagin-linked glycosylation recognition site comprises a tripeptide sequence that is specifically recognized by a particular cellular glycosylation enzyme. These tripeptide sequences are Asn-Xaa-Thr or Asn-XaaSer, where Xaa can be any amino acid except Pro. Proven or predicted asparagine residues of sTNFR-I exist at positions 14, 105, and 111. Proven or predicted asparagine residues of sTNFR-II exist at positions 149 and 171. Replacement or addition of N-linked or O-linked glycosylation sites may be performed or amino acid removal; the result is a protein with altered glycosylation.

In a specific case, the variants are substantially homologous to the amino acid R ₁ - [Cys ¹⁹ -Cys ¹⁰³ ] -R 2 or R 4 - [Cys ³² -Cys ¹¹⁵ ] -R 5. The term "substantially homologous" as used herein means a degree of homology which is preferably over 70%, or more preferably over 80%, over 90%, and most preferably even over 95%. The percent homology described herein is calculated as the percentage of amino acid residues found in the smaller of the two sequences that match (which align) to identical amino acid residues in the sequence being compared, and four 100 amino acid gaps may be included to facilitate this alignment, as reported in Dayhoff (1972), in the Atlas of •

• ··

Protein Sequences and Structure, 5: 124, of the National Biochemical Research Foundation, Washington, D.C., the disclosure of which is incorporated herein by reference. Also included as substantially homologous are truncated sTNFRs that can be isolated by cross-reacting antibodies with the amino acid sequence of SEQ IF NO: 2 and SEQ ID NO: 35, or whose genes can be isolated by hybridization with the DNA of SEQ ID NO: 1a or SEQ. ID NO: 34 or segments thereof.

Examples of the sTNFR of the present invention include the following molecules: NH ₂ -MDSVCPQGKYIHPQNNSIC- [Cys ¹⁹ -Cys ¹⁰³ ] -FC-COOH (also referred to as sTNFR-Ι 2.6D / C105); NH2-MDSVCPQGKYIHPQNNSIC- [Cys ¹⁹ Cys ¹⁰³ ] -FNCSL-COOH (also referred to as sTNFR-Ι 2.6D / C106); NH2-MDSVCPQGKYIHPQNNSIC- [Cys ¹⁹ -Cys ¹⁰³ ] -FN-COOH (also referred to as sTNFR-Ι 2.6D / N105); NH2-MYIHPQNNSIC- [Cys ¹⁹ -Cys ¹⁰³ ] -FNCSL-COOH (also referred to as sTNFR-Ι 2.3D / d8); NH 2 -M- [Cys ¹⁹ -Cys ¹⁰³ ] -FNCSL-COOH (also referred to as sTNFR-Ι 2.3D / dl8); and NH ₂ -MSIS- [Cys ¹⁹ -Cys ¹⁰³ ] FNCSL-COOH (also referred to as sTNFR-Ι 2.3D / 15 15) either as methionylated or non-methionylated, and variants and derivatives thereof.

Production of truncated sTNFR variants is described in more detail below. Such variants can be prepared by incorporating the corresponding nucleotide changes into DNA encoding truncated sTNFR or by in vitro chemical synthesis of the desired truncated sTNFR. Those skilled in the art will appreciate that many combinations of deletions, insertions and substitutions can be made provided that the resulting sTNFRs are biologically active.

Mutagenesis techniques for exchanging, incorporating, or removing one or more selected amino acid residues are known to those skilled in the art (eg, U.S. Patent No. 4,518,584, the disclosure of which is incorporated herein by reference). In constructing each variant of the amino acid sequence, two basic variables are applied, namely the location of the mutation site and the nature of the mutation. In designing each variant, the location of each mutation and its nature will depend on the modified biochemical characteristics (or modified biochemical characteristics). Each mutation site can be modified individually or in series, eg (1) by first substituting the selected conservative amino acids and then with a more radical selection depending on the result achieved (2) • by removing the target amino acid residue (3) by incorporating amino acid residues adjacent to the selected site.

Chemically modified derivatives or truncated sTNFRs can be prepared by one skilled in the art as described herein. Conjugates can be prepared using glycosylated, non-glycosylated or deglycosylated truncated sTNFRs. Typically, non-glycosylated truncated sTNFRs are used. Suitable moieties for the derivatization of truncated sTNFRs include water-soluble polymers.

Water-soluble polymers are suitable because the protein to which they are attached will not precipitate in an aqueous environment, such as a physiological environment. It is desirable that the polymer be pharmaceutically acceptable for the preparation of therapeutic products or compositions. One of ordinary skill in the art will be able to select the appropriate polymer depending upon the consideration of whether the polymer / protein conjugate will be used therapeutically and, if so, the desired dose, circulation time, and proteolysis resistance.

Suitable clinically acceptable water-soluble polymers include polyethylene glycol (PEG), polyethylene glycol propionaldehyde, ethylene glycol / propylene glycol copolymers, monomethoxy-polyethylene glycol, carboxymethyl cellulose, dextran, polyvinyl alcohol (PVA), polyvinyl pyrrolidone, poly-1,3 -dioxalan, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymer, poles (β-amino acid) (either homopolymer or random copolymers), polyesters (n-vinyl pyrrolidone) polyethylene glycol, propylene glycol homopolymers (PPG) and other polyalkylene oxides, polypropylene oxide / ethylene oxide copolymers, polyoxyethylated polyols (e.g., glycerol), polyoxyethylated sorbitol or polyoxyethylated glucose, colonic acids or other carbohydrate polymers, Ficoll or dextran, and mixtures thereof.

By polyethylene glycol is meant here any form that has been used to derivatize other proteins, such as mono- (C1-C10) alkoxy or aryloxy-polyethylene glycol. Polyethylene glycol propionaldehyde may be advantageous in manufacture due to its stability in water.

Each of the water-soluble polymers may be of any molecular weight and may be branched or unbranched. A typical average molecular weight of water-soluble polymers is about

• ·

I · ·· · between 2 kDa and 100 kDa (the term "probably indicates that in water-soluble protein preparations some molecules have a weight greater and some less than the stated molecular weight). The preferred average molecular weight of the water-soluble polymers is between about 5 kDa and 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 it is branched, the greater the polymer: protein ratio. Depending on the desired therapeutic profile (e.g., the duration of the desired release; the effect, if any, on biological activity; the ease of handling; the degree or absence of antigenicity and other known effects of water-soluble polymers on therapeutic proteins) use other sizes.

Each of the water-soluble polymers should be attached to the protein with respect to the effect on the functional or antigenic regions of the protein. In general, chemical modification can be performed under any suitable conditions used to react the protein with the activated polymer molecule. Activating groups that can be used to attach a water-soluble protein to one or more proteins include the following: sulfone, maleimide, sulfhydryl, thiol, triflate, tresylate, azidirine, oxirane, and 5-pyridyl.

All water-soluble proteins are generally attached to the protein at the α- or ε-amino group of the amino acid or to the reactive thiol group. The water-soluble group may be attached to any reactive group of the protein that is sufficiently reactive to attach to the water-soluble group under suitable reaction conditions. Amino acid residues with a free amino group may include lysine residues and amino acid residues at the N-terminus. Those with a free carboxy group may include aspartic acid residues, glutamic acid residues, and C-terminal amino acid residues. Those with a reactive thiol group include cysteine residues.

Methods for preparing proteins conjugated to water-soluble polymers will generally comprise the following steps: (a) reacting the protein with a water-soluble polymer under conditions where the protein is attached to one or more water-soluble polymers (b) to obtain a reaction product. The reaction conditions for each conjugation may be selected from any of the known ones or those that will be developed but so as to avoid or limit the action of reaction conditions (such as temperature, solvents or pH) that could inactivate the modified protein. In general, the optimum reaction conditions for the reactions are determined on a case-by-case basis depending on known parameters and the desired result. For example, the greater the ratio of water-soluble polymer: protein conjugate, the greater the percentage of conjugated product. The optimum ratio (in terms of reaction efficiency, so that there is no excess unreacted protein or polymer) can be given by factors such as the degree of derivatization (eg, mono-, di-, tri- etc.), the molecular weight of the selected polymer, if the polymer is branched or unbranched and the reaction conditions used. The ratio of water-soluble protein (eg, PEGu) to protein will generally be in the range of 1: 1 to 1: 100. One or more purified conjugates can be obtained from each mixture by standard isolation techniques (including, for example, dialysis, salting-out, ultrafiltration, ion chromatography, gel filtration, and electrophoresis).

There may be a requirement for a protein chemically modified at the N-terminus. The water-soluble polymer may be selected based on the molecular weight, branching, etc., the proportion of the water-soluble polymer to the protein (or peptide) molecules in the reaction mixture, the type of reaction to be performed, and the recovery method selected from the N-terminally modified protein. A method for obtaining an N-terminally chemically modified protein preparation (i.e., separating this active ingredient from other monoderivatized components, 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 accomplished by reductive alkylation which utilizes the different reactivity of different types of primary amino groups (lysine versus the N-terminus) available in a particular protein for derivatization. Under suitable reaction conditions, highly selective derivatization of the protein at the N-terminus with the carbonyl group-containing polymer is achieved. For example, it is possible to selectively attach a water-soluble polymer to the N-terminus of a protein by reacting at a pH that allows to take advantage of the different pK between the ε-amino group of lysine residues and the α-amino group of residues at the N-terminus.

• ·

Such selective derivatization controls the attachment of the water-soluble polymer to the protein: conjugation to the polymer occurs predominantly at the N-terminus of the protein and does not significantly modify other reaction groups such as amino groups on the lysine side chain. In reductive alkylation, the water-soluble polymer may be one of the types described above and should have one reaction aldehyde for coupling to the protein. Polyethylene glycol propionaldehyde containing one reactive aldehyde may be used.

In particular, the present invention relates to chemically modified proteins with mono- or poly- (eg 2-4) PEG components. PEGylation can be carried out by any alkylation reaction with PEG known to those skilled in the art. Methods for preparing PEGylated protein products generally include the steps of: (a) reacting the protein product with a polyethylene glycol (e.g., a reactive ester or a PEG aldehyde derivative) under conditions where the protein is attached to one or more PEG moieties; product (reaction products). In general, optimal reaction conditions for the reaction are determined on a case by case basis based on known parameters and the desired result.

A variety of attachment methods are known to those skilled in the art. An example is EP 0 401 384, the disclosure of which is incorporated herein 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, Frien Barnet Lane, London N20 OLD, UK); EP 0 154 316; EP 0 401 384; WO 92/16221; WO 95/34326; and other publications cited herein relating to PEGylations, the disclosure of which is incorporated herein by reference.

Specifically, PEGylation may be performed by an acylation reaction or an alkylation reaction with a reactive polyethylene glycol molecule. Thus, the protein products of the present invention also include PEGylated proteins wherein pegylated proteins are (are) PEG moiety (s) linked via an acyl or alkyl group. Such products may be mono- or poly-PEGylated (eg, containing 2-6 or preferably 2-5 PEG groups). PEG groups are generally attached to the protein at the α- or ε-amino groups of amino acids, but it should also be understood that PEG groups can be attached to any amino group attached to the protein that is sufficiently reactive to attach to the A PEG moiety under suitable reaction conditions.

• · ♦ · ··· ···

PEGylation generally involves reacting an active ester derivative of polyethylene glycol (PEG) with a protein. The polymer (s) selected for the acylation reaction should have one reactive ester group. Any known or subsequently discovered reactive PEG molecule can be used for PEGylation. A preferred activated PEG ester is PEG esterified to N-hydroxysuccinimide (NHS). "Acylation" refers to the following types of bonds (but not limited to) between a therapeutic protein and a water-soluble polymer (eg, PEG): amide, carbamate, urethane and the like (see Chamov (1994), Bioconjugate Chem., 5 (2)). ): 133-140, the disclosure of which is incorporated herein by reference). Reaction conditions may be selected from those known to those skilled in the art of PEGylation or possibly newly developed, but conditions that result in inactivation of the modified proteins (e.g., temperature, solvent, pH) should be avoided.

PEGylation by acylation generally results in proteins with multiple PEG molecules (poly-pegylated protein). Preferably, the linker is an amide. It is also preferred that the resulting product is for the most part (eg, greater than 95%) mono-, di- or tri- PEGylated. However, some products with a higher degree of PEGylation may also be formed in amounts depending on the specific reaction conditions used. If desired, purer PEGylated components (especially unreacted species) can be separated from the mixture by standard purification procedures including, for example, dialysis, salting-out, ultrafiltration, ion exchange chromatography, gel filtration, and electrophoresis.

PEGylation by alkylation generally involves reacting a terminal aldehyde derivative of PEGu with a protein in the presence of a reducing agent. For the reductive alkylation reaction, the selected protein (s) should have one reactive aldehyde group. An example of a reactive PEG aldehyde is polyethylene glycol propionaldehyde, which is water stable, or mono C 1 -C 10 alkoxy or aryloxy derivatives thereof (see U.S. Patent 5,252,714, the disclosure of which is incorporated herein by reference).

PEGylation by alkylation may also result in a protein with multiple PEG molecules (poly-pegylated protein). In addition, it is possible to influence the reaction conditions to substantially favor PEGylation of only the α-amino group at the N-terminus of the protein (i.e., a protein with a single PEG molecule). In both cases (monoPEGylation or polyPEGylation) are

Preferably, PEG groups are attached to the protein via a -CH ₂ -NH- group. As for the -CH ₂ - group, this type of linkage is referred to herein as an "alkyl bond."

Reductive alkylation to produce a highly homogeneous population of monopolymer / protein product will generally include the following steps: (a) reacting the protein with a reactive PEG molecule under conditions for reductive alkylation and at a pH allowing selective modification of the α-amino group at the amino terminus of the protein; of the reaction product (s). The preparation of derivatives by reductive alkylation to produce single PEG molecule products utilizes pKa differences between the lysine amino group and the α-amino group at the N-terminus (pKa is pH where 50% of the amino groups are protonated and 50% are not).

The reaction proceeds at a pH which makes it possible to exploit the pKa difference between the ε-amino groups of the lysine residues and the α-amino group of the residue at the N-terminus of the protein. In general, if the pH is lower, the greater the preponderance of the polymer to the protein will be desirable (i.e., the less reactive the N-terminal α-amino group is, the more polymer will be required to achieve optimal conditions). If the pH is higher, the polymer: protein ratio may not be as high (i.e., more reactive groups are available, so fewer polymer molecules are needed). For purposes of the present invention, the pH will generally be in the range of 3-9, or more preferably 3-6. For reductive alkylation, the reducing agent should be stable in aqueous solutions and also capable of reducing only the Schiff base formed at the beginning of the reductive alkylation process. Suitable reducing agents may be selected from sodium borohydride, sodium cyanoborohydride, dimethylamine borane, trimethylamine borane, piridine borane. A particularly suitable reducing agent is sodium cyanoborohydride. Other reaction parameters such as solvent, reaction time, temperature and method of purification of the product may be determined on a case by case basis and derived from published information relating to the preparation of protein derivatives with water-soluble polymers.

Such selective preparation of derivatives controls the attachment of a water-soluble polymer (which contains a reactive group such as an aldehyde) to the protein: conjugation with the polymer occurs predominantly at the N-terminus of the protein and does not undergo significant modification

other reactive groups such as amino groups of the lysine side chain. The composition will typically be comprised of greater than 90% monopolymer / protein conjugate (typically greater than 95% monopolymer / protein conjugate), with detectable unreacted molecules (i.e., protein without a polymer component) present.

A specific case of the present invention is an unbranched monomethoxy-polyethylene glycol aldehyde molecule with an average molecular weight of either about 20 kDa or about 33 kDa (eg, between 30 kDa and 35 kDa) or tertiary butyl polyethylene glycol aldehyde polyethylene (tertiarycolutene) ) with an average molecular weight of about 33 kDa (e.g., between 30 kDa and 35 kDa) conjugated by reductive alkylation to sTNFR-I 2.6D / N105.

PEGylation can also be performed specifically through water-soluble polymers with at least one reactive hydroxy group (eg, polyethylene glycol) that can react with a reagent having a reactive carbonyl, nitrile or sulfone group to convert the hydroxyl group to a reactive Michael acceptor. This creates an "activated linker" useful in modifying various proteins to provide better biologically active conjugates. "Reactive carbonyl, nitrile or sulfone" means a carbonyl, nitrile or sulfone group to which is attached a dicarbon group having a reactive site for a thiol specific bond on the other carbon from the carbonyl, nitrile or sulfone group (WO 92/16221).

The activated clutches can be monofunctional, bifunctional or multifunctional. Useful reagents with a reactive sulfone group that may be used in these methods are (but are not limited to) chlorosulfone, vinyl sulfone and divinylsulfone.

In a specific case, the water-soluble polymer can be activated by Michael acceptor. WO 95/13312 discloses (inter alia) water-soluble, sulfone-activated PEGs that are highly selective for binding to the thiol component instead of the amino components on the molecule and on the surface. These PEG derivatives are longer stable when hydrolyzed in an aqueous medium at pH 11 or lower and can form bonds with molecules to form conjugates which are also hydrolytically stable. The bond by which the PEGs and the biologically active molecule are conjugated include the sulfone moiety associated with the thiol moiety and have the structure PEG-SO ₂ -CH ₂ -CH ₂ -WW, wherein W represents the biologically active molecule and wherein the sulfone moiety is vinyl sulfone or active ethyl sulfone. Two particularly useful homobifunctional derivatives are PEG-bis-chlorosulfone and PEG-bis-vinyl sulfone.

PCT International Application US96 / 19459, the disclosure of which is incorporated herein by reference, discloses a method for preparing sulfone activated linkers by obtaining a compound having a reactive hydroxyl group and converting the hydroxyl group to a reactive Michael acceptor to form an activated linker. Tetrahydrofuran (THF) was used as the conversion solvent. PCT International Application US96 / 19459, the disclosure of which is incorporated herein by reference, discloses a procedure for purifying activated linkers that uses hydrophobic interaction chromatography based on the separation of linkers according to the size and functionality of the end group.

In particular, the present invention is directed to the following prokaryotic-expressed molecules chemically altered to contain poles- (e.g., 2-4) PEG components: sTNFR-I 2.6D / C105, sTNFR-I 2.6D / C106, sTNFR-I 2.6D / N105, sTNFR-I 2.3D / d8, sTNFR-I 2.3D / dl8 and sTNFR-I 2.3D / dl5, either methionylated or non-thionylated and variants thereof.

Polyvalent form (s)

Polyvalent form (s) can also be prepared, i.e. molecules containing more than one active moiety. In one instance, the molecule may have multiple tumor necrosis factor binding sites for a TNF ligand (eg, a combination of a truncated sTNFR product). The molecule further has at least one tumor necrosis factor binding site and (depending on the desired properties of the polyvalent form) at least one binding site for another molecule (eg, a combination of at least one truncated sTNFR product and at least one interleukin-1 receptor antagonist ("IL-1ra") ) as described below).

Polyvalent forms may also be formed, eg, by chemically linking at least one truncated sTNFR product to any

a clinically acceptable linker (eg, a water-soluble polymer as described below). As a rule, the linker should not impart new immunogenicity (due to new amino acid residues) or alter the hydrophobicity and charge of the construct so as to adversely affect its biodistribution and degradation.

Such polymers, when used as linkers, may be homopolymers, random or block copolymers and terpolymers based on the above monomers, unbranched or branched, substituted or unsubstituted. The polymer may be of any length or molecular weight, but these characteristics may affect biological properties. Polymers particularly suitable for reducing the degradation rate in pharmaceutical applications have an average molecular weight in the range of 2000 to 35000 daltons. In addition, the length of the polymer can be varied to optimize or generate the desired biological activities.

Activating groups that can be used to attach a water-soluble polymer to two or more proteins include the following: sulfone, maleimide, sulfhydryl, thiol, triflate, tresylate, azidirine, oxirane, and 5-pyridyl (sulfone, maleimide, sulfhydryl, thiol, triflate, tresylate, azidirine, oxirane and 5-pyridyl).

In a specific case, bifunctional or multifunctional activated couplings with at least one Michael acceptor may be prepared according to United States Patent Application No. 5,960,549. No. 08 / 473,809 and purified by United States Patent Application No. 08 / 473,809. 08/611 918.

The active ingredients may be coupled using conventional coupling techniques (see PCT Publication No. &lt; / RTI &gt;

WO 92/16221 and PCT Publication No. WO 95/34326, the disclosure of which is incorporated herein by reference). In addition, PCT Publication no. WO 92/16221 describes the preparation of various dimerized sTNFR-inhibitor inhibitor molecules, eg dimerized cl05 sTNFR-Ι. An example of a tumor binding tumor polyvalent factor of formula (sTNFR-I 2.6D / C106) _2- (20 kDa PEG) is given in Example I.

Alternatively, a bivalent molecule may consist of two tandem repeats of truncated sTNFR products separated by a polypeptide linker region. The design of polypeptide linkers is similar to the design of inserting a short loop sequence between domains of de novo proposed proteins (Mutter (1998), TIBS, 13: 260-265 and Regan and DeGrado (1988), Science, 241: 976-978, whose description is incorporated herein by reference). A linker suitable for single chain antibodies has been shown to be effective to generate a dimeric form of recombinant human sTNFR-II (Neve et al. (1996), Cytokine, 8 (5): 365-370, the disclosure of which is incorporated herein by reference). Several different linker constructs were prepared (and successfully used for antibodies); the most successful functional linkers are between 12 and 25 amino acids in size (amino acids with non-reactive side groups such as alanine, serine and glycine), which together form a hydrophilic sequence with several counter-charged residues to increase solubility and are adaptable (Whitlow and Filipula (1991), Methods: A Companion to Methods in Enzymology, 2: 97-105 and Bridgo et al (1993), J. Immunol., 150: 469-479, the disclosure of which is incorporated herein by reference).

In another aspect, truncated sTNFRs can be chemically attached to biotin, and biotin / truncated sTNFR conjugates can then bind to avidin to form a tetravalent avidin / biotin / truncated sTNFR molecule. Truncated sTNFRs may also be covalently attached to dinitrophenol (DNP) or tetranitrophenol (TNP) and the resulting conjugate precipitated by anti-DNP or anti-TNP IgM to form dimeric conjugates where the valence value for TNF binding sites is 10.

Also, recombinant sTNFR truncated fusion proteins can be prepared, wherein each recombinant chimeric molecule has an sTNFR sequence (as described above) substituted for the variable domains of either one or both parts of the immunoglobulin molecule (light or heavy chains) and complete or portions of constant domains ( but at least one constant domain) of the heavy or light chain of human immunoglobulins. For example, any such chimeric truncated sTNFR / IgG1 fusion protein may be prepared from two chimeric genes: the truncated sTNFR / human kappa light chain (truncated sTNFR / Ck) and the truncated sTNFR / human gamma-1 heavy chain (truncated sTNFR / Cg-1) ). After transcription and translation of the two chimeric genes as described below, the gene product can be folded into one chimeric molecule where the truncated sTNFR is bivalent. Further details regarding the construction of such chimeric molecules are given in United States Patent 5,116,964, PCT.

Publication no. PCT Publication No. WO 89/09622; WO 89/16437 and EP 315062, the disclosure of which is incorporated herein by reference.

In another aspect, recombinant sTNFR truncated fusion proteins can be prepared, wherein each recombinant chimeric molecule has the sTNFR sequence described above and at least a portion of the osteoprotegerin (OPG) region 186-401, as described in European Patent Application No. 5,768,149. 96309363.8.

Polynucleotides

The present invention also provides polynucleotides that encode truncated sTNFRs. Based on this interpretation using a codon table, one of ordinary skill in the art can readily determine all nucleotide sequences that encode the amino acid sequences of the truncated sTNFRs.

Presently preferred nucleic acid sequences include polynucleotide sequences encoding sTNFR-Ι 2.6D / C105, sTNFR-2.6 2.6D / C106, sTNFR-Ι 2.6D / N105, sTNFR-Ι 2.3D / d8, sTNFR-Ι 2.3D / dl8 and sTNFR-Ι 2.3D / dl5. Examples of various polynucleotides are shown in Figures 2, 3, 4, 5, 6 and 7.

For the preparation of these polynucleotides and for the expression of the encoded proteins, recombinant expression techniques performed in accordance with the descriptions explained below can be used. For example, by inserting a nucleic acid sequence that encodes truncated sTNFR into a suitable vector, one of ordinary skill in the art can readily generate a large number of nucleotide sequences of interest. These sequences can further be used to prepare detection probes or amplification primers. Poynucleotides encoding truncated sTNFRs may also be incorporated into expression vectors. By introducing expression vectors into a suitable host, the desired truncated sTNFR can be produced in large quantities.

As further described herein, a number of available host / vector systems exist for multiplying the desired nucleic acid sequence and / or producing truncated sTNFRs. These include, but are not limited to, plasmids, viral and insertion vectors, and prokaryotic and eukaryotic hosts. One of skill in the art may adapt a host / vector system capable of propagating or expressing heterologous DNA to produce or express the sequences of the present invention.

In addition, those skilled in the art will appreciate, with respect to the present disclosure, new nucleic acid sequences, including degenerate nucleic acid sequences encoding truncated sTNFRs with the sequences set forth in the Figures section, and those nucleic acid sequences that hybridize (preferably under stringent hybridization conditions) to these nucleic acid sequences ( Maniatis et al (1982), Molecular Cloning (A Laboratory Manual), Cold Spring Harbor Laboratory, pages 387-389). Exemplary stringent hybridization conditions are hybridization in 4x SSC at 62-67 ° C, followed by washing for about one hour in 0.1x SSC at 62-67 ° C. Other exemplary stringent hybridization conditions are hybridization in 45-55% formamide, 4x SSC at 40-45 ° C. Also included are DNA sequences that hybridize to the nucleic acid sequences depicted in Figures 1 and 9 under milder hybridization conditions and which encode truncated sTNFRs. Examples of such less stringent hybridization conditions are 4xSSC at 45-55 ° C or hybridization with 30-40% formamide at 40-45 ° C.

The present invention also provides recombinant DNA constructs comprising vector DNA with DNA sequences encoding truncated sTNFRs. In each such DNA construct, the nucleic acid sequence encoding a truncated sTNFR (with or without signal peptides) is operably linked (in operative association) to a suitable expression control or regulatory sequence capable of directing the replication and / or expression of the truncated sTNFR in the selected host.

Recombinant expression

Preparation of polynucleotides

Nucleic acid sequences encoding truncated sTNFRs can be readily obtained by a variety of methods including, but not limited to, chemical synthesis, cDNA or genomic library testing, expression library testing, and / or cDNA PCR amplification. These and other methods useful for isolating nucleic acid sequences are disclosed in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, (1989); in Ausubel et al., eds Current Protocols in Molecular Biology, Current Protocols Press, (1994); and in Berger and Kimmel, Methods in • · ·

Enzymology: A Guide to Molecular Cloning Techniques, Vol. 152, Academic Press, Inc., San Diego, CA, (1987), the disclosure of which is incorporated herein by reference.

Chemical synthesis of nucleic acid sequences that encode truncated sTNFRs can be accomplished, for example, using methods well known to those skilled in the art such as Engels et al., (1989) Angew. Chem. Intl. Ed., 28: 716-734 and Wells et al., (1985), Gene, 34: 315, the disclosure of which is incorporated herein by reference. These methods include, but are not limited to, phosphotriester, phosphoramidite and H-phosphonate (nucleic acid sequence synthesis) methods. Large nucleic acid sequences, for example longer than about 100 nucleotides, can be synthesized as several fragments. The fragments can then be ligated to create a nucleic acid sequence encoding a truncated sTNFR. A preferred method is polymer-supported synthesis using standard phosphoramidite chemistry.

Appropriate nucleic acid sequences can also be obtained by testing suitable cDNA libraries (i.e., libraries prepared from one or more tissues where protein expression is believed) or genomic libraries (library prepared from total genomic DNA). The source for a cDNA library is typically a tissue of any kind where expression of the desired protein is expected in reasonable amounts. The source for the genomic library may be any tissue or tissue from any mammal or other species where a gene encoding a truncated sTNFR is contemplated.

Hybridization media can be screened for the presence of DNA encoding truncated sTNFR using one or more nucleic acid probes (oligonucleotides, cDNAs or genomic DNA fragments having an acceptable degree of homology to a cDNA or cloned gene) that will hybridize selectively to the cDNA or gene (s). ) present in the library. Typical probes used for testing encode a small region of DNA sequence from the same or similar species as the species from which the library was prepared. The probes may also be degenerate as described below.

Hybridization is typically accomplished by annealing the oligonucleotide sequence or cDNA to the clones under such stringent conditions that eliminate non-specific binding but allow binding.

Those clones that have significant levels of probe or primer homology. Typical stringent hybridization and wash conditions depend in part on the size of the cDNA or oligonucleotide probe (i.e., the number of nucleotides) and whether the probe is degenerate. The likelihood of identifying a clone (i.e., whether a genomic or cDNA library is tested) is also taken into account when designing the hybridization medium.

When a DNA fragment (such as cDNA) is used as a probe, typical hybridization conditions include those described in Aubel et al. (1994), eds., Supra. After hybridization, the hybridization medium is washed off under suitable stringent conditions. These are selected depending on several factors, such as probe size, expected probe and clone homology, hybridization medium tested, number of clones tested, etc. Examples of stringent wash solutions (which typically have low ionic strength and are used at relatively high temperatures) are as follows. : 0.015 M NaCl, 0.05 M Na citrate and 0.1% SDS at 55-65 ° C or other - 1 mM Na ₂ EDTA, 40 mM NaHPO ₄ , pH 7.2 and 1% SDS at about 40-50 ° C or still another 0.2x SSC and 0.1% SDS at about 5065 C.

There are also exemplary protocols for stringent wash conditions where oligonucleotide probes are used to test hybridization media. For example, the first protocol uses 6x SSC with 0.05% sodium pyrophosphate at a temperature between about 35-63 ° C depending on the length of the probe. For example, a 14 base length probe is washed at 35-40 ° C, a 17 base length at 45-50 ° C, a 20 base length at 52-57 ° C, and a 23 base length at 57-63 ° C. The temperature can be increased by 2-3 ° C if the background due to non-specific binding of the probe is high. The second protocol uses tetramethyl ammonium chloride (TMAC) for washing. The stringent wash solution may thus have the following composition: 3M TMAC, 50mM Tris-HCl, pH 8 and 0.2% SDS.

Another suitable method for obtaining suitable nucleic acid sequences is polymerase chain reaction (PCR). Using this method, cDNA is prepared from poly (A) + RNA or total RNA using the reverse transcriptase enzyme. Two primers (oligonucleotides), typically complementary to two separate cDNA regions encoding a truncated sTNFR, are added to the cDNA together with a polymerase (such as

4

such as Taq polymerase) and that amplifies the cDNA region between two primers.

The oligonucleotide sequences selected as probes or primers should be of appropriate length and sufficiently unambiguous to minimize non-specific binding during assay or PCR amplification. The particular probe or primer sequence is typically based on conserved or highly homologous sequences or regions. Alternatively, the probes or primers may be wholly or partially degenerate, i.e. they may comprise a mixture of probes / primers, all of which encode the same amino acid sequence, but using different codons. An alternative to degenerate probes is the use of inosine at some or all of the codon positions, which vary by species. Oligonucleotide probes or primers can be prepared by chemical DNA synthesis methods as described above.

As described above, the alternative sequence is a natural (eg, allelic variant) or synthetic sequence that contains one or more nucleotide substitutions, deletions and / or insertions as compared to the sequences in Figures 2, 3, 4, 5, 6 and 7, which result in the expression of the variable amino acid sequences (as compared to the wild type amino acid sequence). The preparation of synthetic sequence variants is well known to those skilled in the art and is described, for example, in Sambrook et al. (1989), supra and Wells et al. (1985), Gene, 34: 315, the disclosure of which is incorporated herein by reference.

Vectors

DNA encoding truncated sTNFRs can be incorporated into vectors for further cloning (DNA amplification) or expression. Suitable vectors are commercially available or can be specially prepared. The choice or construction of a suitable vector will depend (1) on whether it will be used for DNA amplification or expression of DNA (2) on the size of the DNA to be incorporated into the vector (3) on the intended host cell to be transformed with the vector.

Each of the vectors comprises a nucleic acid sequence that encodes a desired protein operably linked to one or more expression control or regulatory sequences capable of directing or otherwise affecting the expression of the desired protein by the selected host cell. Each vector contains different components depending on its

function (DNA amplification or DNA expression) and its compatibility with the intended host cell. Typically, vector components include, but are not limited to: signal sequences, origin of replication, one or more signal (marker) genes, promoters, enhancer elements, transcription termination sequence, etc. These components may be obtained from natural sources or may be synthesized by known procedures.

Examples of suitable prokaryotic cloning vectors are bacteriophages (e.g., lambda-derived) or plasmids from E. coli. (eg, pBR322, colE1, pUC, F-factor, Bluescript® derivatives (Stratagene, LaJolla, CA)). A number of other suitable expression vectors known to those skilled in the art may be used for this purpose.

Signal sequence

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

The origin of replication

Both expression and cloning vectors generally include a nucleic acid sequence that allows the vector to replicate in one or more selected 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 an origin of replication or an autonomously replicating sequence. Such sequences are well known. The origin of replication from plasmid pBR322 is suitable for most Gram-negative bacteria. Various origins (eg, SV40, polyoma, adenovirus, VSV or BVP) are useful for cloning vectors in mammalian cells. Generally, an origin of replication is not necessary for mammalian expression vectors (eg, an SV40 origin is often used only because it contains an early promoter).

Selection gene

Both expression and cloning vectors typically contain a selection gene. This gene encodes a &quot; marker &quot; 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 a selection gene and therefore will not survive in the culture medium. Typical selection genes encode proteins that: (a) confer resistance to antibiotics or other toxins, eg, ampicillin, neomycin, methotrexate (metothrexate) or tetracycline, (b) supplement auxotrophic deficiency, or (c) provide essential nutrients that are not nutrient medium

Other selection genes may be used to amplify the genes to be expressed. Amplification is a process where genes that are more important for the production of proteins necessary for growth are repeated tandemly within the chromosomes of subsequent generations of recombinant cells. Examples of suitable selection markers for mammalian cells include, for example, dihydrofolate reductase (DHFR) and thimidine kinase. Cell transformants are subjected to selection pressure where only transformants are able to adapt and survive due to the markers present in the vector. Selection pressure is induced by growing the transformed cells under conditions where the concentration of the selection agent in the medium is gradually varied, thereby amplifying both the selection gene and the DNA that encodes truncated sTNFR. The result is an increased amount of truncated sTNFRs that are synthesized from the amplification DNA.

For example, cells transformed with the DHFR selection gene are first identified by growing all transformants in a nutrient medium containing methotrexate (a competitive DHFR analog). Using wild-type DHFR, a suitable host cell is a Chinese hamster ovary cell line lacking DHFR activity (Urlaub and Chasin (198), Proc. Natl. Acad. Sci., USA, 77: (7): 4216 -4220, the disclosure of which is incorporated herein by reference). The transformed cells are then exposed to elevated levels of methotrexate. This results in the synthesis of multiple copies of the DHFR gene and concomitantly multiple copies of other genes contained in the expression vector, such as DNA encoding a truncated sTNFR.

Promotors

Both expression and cloning vectors typically contain a promoter that is recognized by the host organism and is operably linked to the host.

a nucleic acid sequence encoding a truncated sTNFR. A promoter is an untranslated sequence located upstream of the (5 ') start codon of a structural gene (typically within 100 to 1000 bp) that directs the transcription and translation of a particular nucleic acid sequence that, for example, encodes truncated sTNFR. The promoter may suitably be classified into one of two classes - inducible promoters and constitutive promoters. Inducible promoters initiate increased transcription from DNA under their control in response to some nutritional or temperature changes. A variety of promoters are recognized by a variety of potential host cells. The promoter can be operably linked to DNA encoding a truncated sTNFR by removing restriction enzymes from the source DNA and inserting the desired promoter sequence. The original sTNFR-promot promoter sequence or the sTNFR-II promoter sequence can be used for direct amplification and / or expression of DNA encoding a truncated sTNFR. However, a heterologous promoter is preferred, as this results in greater transcription and a higher yield of the expressed protein compared to the original promoter and is compatible with the host cell system that has been selected for use. For example, any of the original promoter sequences of other members of the NGF / TNF family may be used to directly amplify and / or express DNA encoding a truncated sTNFR.

Promoters suitable for use in prokaryotic hosts include the beta-lactamase and lactose promoter systems; the alkaline phosphatase promoter; a tryptophan (trp) promoter system; a bacterial luminescent (luxR) gene system and hybrid promoters such as the tac promoter. Other known bacterial promoters are also suitable. Their nucleotide sequences have been published, thus allowing those skilled in the art to link them to the desired DNA sequence (s) using linkers and adapters when it is necessary to add the desired restriction site.

Suitable promoter sequences for use in mammalian host cells are also well known and include promoters derived from virus genomes such as polyoma virus, fowlpox virus, adenovirus (such as Adenovirus 2), bovine papiloma virus. papilloma virus), avian sarcoma virus, cytomegalovirus, retrovirus, jaundice B virus, and in particular simian virus 40 (SV40). Other suitable mammalian promoters include heterologous mammalian promoters, e.g. Heat-shock promoters and actin promoter.

Enhancer Element

Typically, both expression and cloning vectors will contain enhancer sequences to increase transcription of the DNA sequence encoding a truncated sTNFR in higher eukaryotes. Amplifiers are cis-acting components of DNA, generally 10-300 bp in length, which, by acting on a promoter, increase its transcription. The amplifiers are relatively orientation and position independent. They are found at both the 5 'and 3' ends of the transcriptional unit. The yeast enhancer is preferably coupled to yeast promoters. Several enhancer sequences available from mammalian genes (eg, globin, elastase, albumin, alpha fetoprotein, and insulin) are known. In addition, viral enhancers such as the SV40 enhancer, cytomegalovirus early promoter enhancer, polyoma enhancer, and adenovirus enhancers are examples of enhancer elements for activating eukaryotic promoters. Although the enhancer can be at both the 3 'and 5' ends of the DNA encoding the truncated sTNFR, it is typically at the 5 'position from the promoter.

Transcription termination

All expression vectors used in eukaryotic host cells typically contain sequences necessary for termination of transcription and for stabilization of mRNA. Such sequences are commonly available from 5 'and sometimes 3' regions where eukaryotic DNA or cDNA is not translated. These regions contain nucleotide segments transcribed as polyadenylated fragments on the mRNA regions encoding truncated sTNFR where there is no translation.

Vector design

The construction of a suitable vector containing one or more of the aforementioned components (together with the truncated sTNFR coding sequence) is achieved by standard ligation techniques. Isolated plasmids or DNA fragments are cleaved, engineered, and reassembled in the desired order to generate the desired vector. To verify the correct sequence of the construct, the ligation mixture can be used to transform E.coli and successful transformants can be selected using the techniques described above. The vector is then prepared from the transformants in larger quantities and the vector is analyzed by restriction endonuclease digestion and / or sequencing to confirm the presence of the desired construct.

A vector that provides transient expression of DNA encoding a truncated sTNFR can also be used in mammalian cells. Generally, transient expression involves the use of an expression vector that is capable of replicating effectively in a host cell such that the host cell accumulates many copies of the expression vector and subsequently synthesizes high levels of the desired protein encoded by the expression vector. Any intermediate expression system consisting of an expression vector and a host cell is suitable for the positive identification of the proteins encoded by the cloned DNA, as well as for rapid testing of such proteins for the desired biological or physiological properties, i.e., the identification of biologically active truncated sTNFRs.

Host cells

The present invention also provides all recombinant host cells that contain nucleic acid sequences useful in expressing the protein of interest. Exemplary prokaryotic and eukaryotic host cells include bacterial, mammalian, insect, fungal and yeast cells, and plant cells.

Prokaryotic host cells include, but are not limited to, eubacteria such as Gram-positive or Gram negative organisms (e.g., E. coli (HB101, DH5α, DH10 and MC1061) bacilli such as B. subtilis; Pseudomans species such as P. aeruginosa Streptomyces subspecies Salmonella typhimurium or Serratia marcescans In a specific case, the protein of interest may be expressed in E. coli.

Moreover, in addition to prokaryotic host cells, eukaryotic microbes such as filamentous fungi or yeast may be suitable hosts for the expression of truncated sTNFRs. Among lower eukaryotic host organisms, Sacharomyces cerevisiae or common yeast is most commonly used, but many other classes, species and strains are well known and commonly available.

·· ·

Truncated sTNFRs may be expressed in glycosylated form by any of a number of suitable multicellular-derived host cells. Such host cells are capable of complex processing and glycosylation activities. In general, any eukaryotic cell culture can be used, whether from vertebrate or invertebrate cells, including plant and insect cells. In a particular case, the protein of interest may be expressed in baculovirus cells.

Vertebrate cells can be used because their proliferation in culture (tissue culture) is well known. Examples of suitable mammalian host cell lines include, for example, the monkey kidney CV1 line transformed by SV40 (COS-7), the embryonic human kidney line (293 cells or 293 cells subcloned for growth in suspension culture), hamster kidney cells and hamster ovary cells. . Other suitable mammalian cell lines include, for example, HeLa, mouse L-929 cells, 3T3 lines derived from Swiss, Balb-c or NIH mice, and hamster BHK or HaK cell lines. In a particular case, the protein of interest may be expressed in COS cells.

The host cell may be transfected or better transformed with the desired nucleic acid under appropriate conditions to express the nucleic acid. Selection of suitable host cells, methods for transforming, growing, amplifying, testing and producing the product and its purification are well known to those skilled in the art (Gething and Sambrook (1981), Nature, 293: 620-625 or Kaufman et al. (1985) Mol. Biol., 5 (7): 1750-1759, or US Pat.

419,446, the disclosures of which are incorporated herein by reference. For example, for a mammalian cell without a cell wall, a calcium phosphate precipital method may be used. Further, electroporation, micro-injection and other known techniques can be used.

It is also possible to produce truncated sTNFRs by homologous recombination or recombinant production methods using control elements introduced into cells that already contain DNA encoding truncated sTNFRs. Homologous recombination is a technique originally developed to target genes to induce or repair mutations in transcriptionally active genes (Kucherlapati (1989), Prog. In Nucl. Acid Res. And Mol. Biol., 36: 301, the disclosure of which is incorporated herein by reference). The basic technique has been 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 Doetschman et al. (1988), Proc. Natl. Acad. Sci., 85: 8583-8587, the disclosure of which is incorporated herein by reference), or to repair specific mutations within a defective gene (Doetschman et al. (1987), Nature, 330: 576-578, the disclosure of which is incorporated herein by reference). Exemplary techniques are described in U.S. Pat. Patent No. 5,272,071; WO92 / 01069; WO93 / 03183; WO 94/12650 and WO 94/31560, the disclosure of which is incorporated herein by reference.

By homologous recombination, the DNA sequence to be incorporated into the genome can be located in a particular region of the gene of interest by attaching it to the target DNA. The target DNA is DNA that is complementary (homologous) to the genomic DNA region. Small portions of the target DNA that are complementary to a specific region of the genome come into contact with the parent strand during the DNA replication process. The DNA that has been introduced into the cell generally hybridizes and therefore recombines with other regions of endogenous DNA by sharing homologous regions. If this complementary strand is attached to an oligonucleotide that contains a mutation or a different DNA sequence, it is also incorporated into the newly synthesized strand (as a result of recombination). Thanks to the proofreading function, it is possible that this new sequence serves as a template. In this way, the transferred DNA is incorporated into the genome.

If a particular gene sequence (e.g., a nucleic acid sequence truncated with sTNFR) is known, it may be synthesized or otherwise obtained (e.g., by appropriate restriction digestion of the original DNA at sites around the region of interest) an expression control sequence (a DNA segment that is complementary to the selected region) gene). This region serves as a target sequence for insertion into the cell and will hybridize to its homologous region within the genome. If this hybridization occurs during DNA replication, this DNA segment (and any other sequence so attached) will serve as an Okazaki fragment and will be inserted into the newly synthesized DNA daughter strand.

These regions of the target DNA are joined with DNA regions that can interact with the expression of truncated sTNFR. For example, a promoter / enhancer, suppressor, or exogenous transcriptional modulation element is inserted into the genome of a designated host cell in sufficient proximity and in the correct orientation to affect the transcription of DNA

· Β · • ··· encoding the desired truncated sTNFR. The control element does not encode a truncated sTNFR, but instead controls part of the DNA present in the host cell genome. Thus, expression of the truncated sTNFR can be accomplished not by transfecting the DNA that encodes truncated sTNFR but rather using a target DNA (containing homologous regions with the endogenous gene of interest) linked to a DNA regulatory region that provides an endogenous gene sequence with a recognizable signal for transcription of truncated sTNFR .

Growing of host cells

Methods for culturing all (one or more) recombinant host cells to produce the desired protein depend on many factors and considerations; the optimal manufacturing process for a given situation will be apparent to those skilled in the art after several experiments. Recombinant host cells are grown in a suitable medium and express truncated sTNFR, which is optionally isolated and purified from the nutrient medium (or from cells, if expressed intracellularly) by suitable procedures known to those skilled in the art.

In particular, each of the recombinant cells used to produce the desired truncated sTNFR may be grown in a medium suitable for inducing promoters, selecting suitable recombinant host cells, or amplifying the genes encoding the desired truncated sTNFR. The medium may be supplemented (if necessary) with hormones and / or other growth factors (such as insulin, transferrin or epidermal growth factor), salts (sodium chloride, calcium, magnesium and phosphate) buffers (such as HEPES), nucleotides ( such as adenosine and thymidine), antibiotics (such as gentamicin), trace elements (defined as inorganic compounds usually present in final concentrations in the order of micromoles) and glucose or other energy source. Other supplements may also be added at appropriate concentrations, in the opinion of experts. Suitable cultivation conditions such as temperature, pH, and the like are well known to those skilled in the art of selected hosts.

Pharmaceutical composition

Each pharmaceutical composition generally comprises a therapeutically effective amount of truncated sTNFRs and chemically modified derivatives

ti • · • 0

Truncated sTNFR (together, truncated sTNFR product (s)) with carrier additive. The carrier preferably comprises one or more pharmaceutically and physiologically acceptable material with the addition of a truncated sTNFR product (s) and a controlled release agent.

The primary solvent in the carrier may be either aqueous or non-aqueous in nature. The carrier may additionally contain other pharmaceutically acceptable substances for adjusting or maintaining the pH between 5-6.5, or more preferably between 5.5-6 (for example, buffers such as citrate, phosphate, and amino acids such as glycine); bulking agents (e.g., mannitol and glycine); osmomolarites (eg, mannitol or sodium chloride); surfactants (eg, polysorbate 20, polysorbate 80, triton and pluronics); viscosity; clarity; colors; sterility; stability (e.g., sucrose and sorbitol); antioxidants (eg, sodium sulfate and sodium hydrogen sulfite), preservatives (eg, benzoic acid and salicylic acid); fragrance of the preparation; flavorings and diluents; dissolution rates (eg, solvents and solubilizers such as alcohol, polyethylene glycol, and sodium chloride); release rates;

emulsifying agents; suspending agents; solvents; fillers; means of transport; diluents; inert substances and / or pharmaceutical enhancers. Other effective forms of administration such as parenteral slow-release formulation, inhalation mist, orally-active formulations or suppositories can also be envisaged. The composition may also contain individual preparations of polymeric compounds, such as bulk erosion polymers (e.g., lactic-co-glycolic (PLGA) copolymers, PEGA-blocking PLGA polymers, and lactic acid). and glycol, poles (cyanoacrylates)); surface erosion polymers (eg, poles (anhydrides) and poly (ortho esters)); hydrogel esters (eg, pluronic polyols, poly (vinyl alcohol), poles (vinylpyrrolidone), maleic copolymers) vinyl ether anhydride, cellulose, hyaluronic acid derivatives, alginate, collagen, gelatin, albumin, and starches and dextrans) and their composite forms, or liposomes or microspheres, such preparations may affect the physiological state, stability, the rate of in vivo release and the rate of degradation of those present

9 proteins and derivatives in vivo. The optimal pharmaceutical composition for the desired protein will be determined by one skilled in the art depending on the mode of administration and the desired dose. Examples of pharmaceutical compositions are provided in Remington's Pharmaceutical Sciences, 18th Ed. (1990), Mack Publishing Co., Easton, PA 18042, pages 1435-1712; Gombotz and Pettit (1995) Bioconjugate Chem., 6: 332-351; Leone-Bay, et al. (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 94121235, the disclosure of which is incorporated herein by reference.

Specific sustained release compositions are available from the following manufacturers: Depotech (Depofoam ™, multivesicular liposome): Alkermes (ProLease ™, PLGA microsphere). As used herein, hyaluronan (hyaluronan) refers to hyaluronan, hyaluronic acid (hyaluronic acid) and its salts (eg, sodium hyaluronate), esters, ethers, enzymatic derivatives and cross-linked hyaluronic acid gels (cross-linked) gels of hyaluronic acid), such as hylan (hylan). Examples of forms of hyaluronan are given in Peyron and Balazs (1974), Path. Biol., 22 (8): 731-736; Isdale et al. (1991), J. Drug Dev., 4 (2): 93-99; Larsen et al. (1993), Journal of Biomedical Materials Research, 27: 1129-1134; Namiki, et al. (1982),

International Journal of Clinical Pharmacology

Toxicology, 20 (11): 501 - 507; Meyer et al. (1,995), Journal of Controlled Release, 35: 67-72; Kikuchi et al. (1996), Osteoarthritis and Cartilage, 4: 99-110; Sakakibara et al. (1994), Clinical Orthopedics and Related Research, 299: 282-292; Meyers and Brandt (1995), 22 (9): 1732-1739; Laurent et al. (1995) Acta Orthop Scand

66 (266): 116-120; Cascone et al. (1995) Biomaterials, 16 (7): 569574; Yerashalmi et al. (1994), Archives of Biochemistry and Biophysics, 313 (2): 267-273; Bernatchez et al. (1993), Journal of Biomedical Materials Research, 27 (5): 677–681; Tan et al. (1990), Australian Journal of Biotechnology, 4 (1): 38-43; Gombotz and Pettit (1995), Bioconjugate Chem., 6: 332-351; U. S. 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, 5 356 8 83; European Patent Application Nos. 0 507 604 A2

• · · · · ·

And O 718 312 A2; and WO 96/05845, the disclosure of which is incorporated herein by reference. Certain hyaluronan mixtures are available from the following suppliers: BioMatrix, Inc. Ridgefield, NJ (Synvisc ™, 90:10 Hylan Fluid and Hylan Gel); Fidia SpA, Abano Terme, Italy (Hyalgan ™, disaggregated hyaluronic acid sodium (molecular weight about 500,000 to about 700,000)); Kaken Pharmaceutical Co., Ltd., Tokyo, Japan (Artz (TM), 1% disaggregated hyaluronic acid solution having a molecular weight of about 700,000); Pharmacia AB, Stockholm, Sweden (Healon ™, disaggregated hyaluronic acid having a molecular weight of about 4x10 ⁶ ); Genzyme Corporation, Cambridge, MA (Surgicoat (TM), recombinant hyaluronic acid); Pronova Biopolymer, Inc. Portsmouth, NH (Hyaluronic Acid FCH, high molecular weight, (e.g., about 1.5-2.2 x 10 °) hyaluronic acid prepared from a culture of Streptococcus zoaepidemicus; Sodium Hyaluronate MV (molecular weight about 1.0-1.6 x 10 ') and Sodium Hyaluronate LV (molecular weight about 1,5-2,2x10®) Calbiochem-Novabiochem AB, Lautelfingen, Switzerland (Hyaluronic Acid, sodium salt) (catalog number 1997 385908) prepared from the species Streptococcus); Intergen Company, Purchase, NY, split hyaluronic acid having a molecular weight of about 1 x 10 ⁶ ); Diosynth Inc, Chicago, IL; Amerchol Corp., Edison, NJ, and Kyowa Hakko Kogyo Co., Ltd., Tokyo, Japan.

Once given, the pharmaceutical composition can be stored in sterile ampoules as a solution, suspension, gel, solid or dehydrated emulsion or lyophilized powder. Such a composition may be stored either in a ready-to-use form or in a form requiring reconstitution (eg, lyophilized) before use.

In a specific case, the present invention is directed to kits for producing single dose dosage units. The kits may comprise a dried protein container and an aqueous formulation container. Kits within the scope of this invention include one or more pre-filled syringes; sample pre-filled syringes (eg liquid syringes), lyosyringes (such as Lyo-Ject,

Two-chamber pre-filled lyo-syringes) available from Vetter GmbH, Ravensburg, Germany.

Use

Truncated sTNFR products may be useful as research agents or as therapeutic and diagnostic agents. Truncated sTNFRs can be used in in vitro and / or in vivo diagnostic assays to quantify the amount of natural sTNFR-Ι or sTNFR-II in tissues or organ samples, or to identify and / or isolate cells that express TNF (Scallon et al. (1995) ), assays to quantify the amount of natural sTNFR-Ι or sTNFR-II in tissues or organ samples, or to determine and / or isolate cells that express TNF (Scallon et al. (1995), supra). In the tissue or organ assays, compared to the standard ¹²⁵ I-truncated sTNFR binding curve, less ¹²⁵ L of TNF-binding truncated sTNFR will be due to unlabeled natural TNF-binding sTNFR-Ι or sTNFR-II. Similarly, ¹²⁵ I-truncated sTNFRs can be used to detect the presence of TNF in various cell types.

The present invention also contemplates the use of truncated sTNFR products for the production of antibodies and the resulting antibody (particularly including those that will also bind to the original sTNFR-Ι or sTNFR-II). Antibodies can be prepared that bind to truncated sTNFR, for example, an epitope within the amino acid sequence R _T - [Cys ¹⁹ -Cys ¹⁰³ ] R 2 or within the amino acid sequence R 4 - [Cys ³² -Cys ¹¹⁵ ] -R 5. One of ordinary skill can use well known and published procedures to obtain monoclonal and polyclonal antibodies or recombinant antibodies that specifically recognize and bind to the various proteins encoded by the amino acid sequences of the present invention. Such antibodies can then be used to purify and characterize a full-length, 30 kDa TNF inhibitor and a full-length, 40 kDa TNF inhibitor.

The present invention also relates to methods of treating certain diseases and medical conditions (many of which can be characterized as inflammatory diseases) that are mediated by TNF (mediated by TNF). A disease or medical condition is considered to be a "TNF-mediated disease if the spontaneous or experimental disease is associated with elevated levels of TNF in body fluids or tissues." .

• ·· ··· · t ₍ "· · · · ·

99 · « _k , adjacent foci of the disease or occurs in the body. TNF-mediated diseases can also be distinguished by the following two conditions: (1) pathological findings associated with the disease can be induced experimentally in animals by administration of TNF, and (2) the pathology induced in experimental animal disease models can be inhibited or eliminated using agents that inhibit action TNF. Many TNF-mediated diseases fulfill two of these three conditions, while others fulfill all three. A non-exhaustive list of TNF-mediated diseases, together with corresponding downstream diseases and symptoms, all of which can be treated by the methods of the present invention are: adult respiratory distress syndrome; cachexia / anorexia (cachexia / anorexia); cancer (eg, leukemia); chronic fatigue syndrome; host graft rejection; hyperalgesia (hyperalgesia); bowel inflammatory disease; neuroinflammatory disease; ischemic / reperfusion injury including cerebral ischemia (brain damage as a result of trauma, epilepsy, bleeding or stroke, which in all cases may lead to neurodegenerations); diabetes (eg, onset of childhood diabetes mellitus type 1); multiple sclerosis; eye diseases; pain; pancreatitis; pulmonary fibrosis; rheumatic diseases (e.g. rheumatoid arthritis, osteoarthritis (osteoarthritis), pediatric (rheumatoid) arthritis, seronegative polyarthritis, ankylosing spondilitis (ankylosing spondylitis), Reiter's syndrome and reactive arthritis (lupus arthritis), lupus arthritis) ), polymyositis (polymyosithis), dermatomyositis (dermatomyositis), scleroderma (sclerodema), systemic sclerosis, vasculitis (vasculitis), cerebral vasculitis, Sjógren's syndrome, rheumatic fever and polychondritis (polychondritis) (polychondritis) giant cell arteritis), septic shock, side effects of radiation therapy; systemic lupus erytheumatous; temporary mandibular joint disease; thyroitis and tissue transplantation.

• · φφ ► ·· 'φφ •. φ · »φ-----------------..

Truncated sTNFR products may be administered to patients in therapeutically effective amounts for the treatment of TNF-mediated diseases (as defined above) including, for example, rheumatic diseases (eg Lyme disease, rheumatoid arthritis, osteoarthritis, psoriasis arthritis, rheumatoid arthritis and staphylococcal-induced staphylococci). septic) arthritis). The term "patient" means both animals (eg, cats, dogs, horses) and humans.

The truncated sTNFR product may be administered locally, enterally and parenterally including, but not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital (intraorbital), intracardial (intracardiac), intradermal, intraperitoneal, transtrachal, subcutaneous. (subcuticular), intra-articular, subcapsular, subarachnoid, intraspinal, intraventricular and intrasternal injection and infusion. The truncated sTNFR product may also be administered orally or through mucus membranes, i.e., intranasally, sublingually, buccally, or rectally; it is always a system application.

Preferably, truncated sTNFR products are administered by intra-articular, subcutaneous, intramuscular or intravenous injection. Truncated sTNFR products may also be administered by continuous infusion (for example, implanted or external infusion devices altering steady or intermittent flow) to provide a continuously desired level of truncated sTNFR in the blood over the course of administration. Preferably, this is achieved by means of a continuous infusion, eg a minipump, such as an osmotic minipump. In these methods, it is certain that the amount of drug is maintained at the desired level and that blood samples can be taken and the amount of drug in the blood stream monitored. Various pumps are commercially available from suppliers such as MiniMed Inc, Sylmar, CA (eg MT507) and AlzaCorp., Palo Alto, CA (eg Alzet osmotic pump, mdel 2MLI).

Other methods of continuous or near-continuous dosing are also contemplated. For example, chemical treatments can lead to sustained release forms of the protein, which also results in their constant presence in the bloodstream in a predicted amount based on the intended dosage regimen.

• ·

Methods of using truncated sTNFR products for treating TNF-mediated diseases (including inflammatory conditions of the joints (e.g., osteoarthritis, psoriasis arthritis and rheumatoid arthritis) are explained in European Patent Applic. 567566, the contents of which are incorporated herein by reference. In another specific case (in the treatment of rheumatoid arthritis, inflammatory bowel disease, cachexia / anorexia or multiple sclerosis), truncated sTNFR products may be administered subcutaneously or intramuscularly. In some cases, truncated sTNFRs may be administered intravenously to treat brain damage as a result of trauma, epilepsy, bleeding or stroke. intra-articular injections of truncated sTNFR products administered repeatedly as needed to prevent or treat outbreaking arthritis; and (2) repeated subcutaneous injections of truncated sTNFR products. Treatment initiation in septic shock should begin as soon as possible after blood poisoning (septicemia) or when the risk of blood poisoning is diagnosed. For example, treatment may begin immediately after surgery or accident or any other event associated with the risk of septic shock. Preferred treatments for adult respiratory distress syndrome include: (1) single or multiple intratracheal administration of truncated sTNFR products; and (2) bolus (bolus) or continuous intravenous infusion of truncated sTNFR products.

Alternatively, cell therapy is contemplated, eg, implantation of truncated sTNFR producing cells. This case of the present invention may involve the implantation of cells that are capable of synthesizing and secreting biologically active forms of truncated sTNFRs to a patient. Such truncated sTNFR producing cells may be cells that do not normally produce sTNFR but which have been modified to produce truncated sTNFR, or they may be cells whose ability to produce truncated sTNFR was enhanced by transformation with a polynucleotide suitable for expression and secretion of truncated sTNFR. To minimize the potential immunological response of patients by administration of truncated sTNFR of different species, preferably when the cells are of the same species as the patient (e.g., human), or when the cell may be encapsulated with material that provides a barrier to recognition by the immune system, or when the cells may be placed in an immunologically specific anatomical position such as testes, eye, or central nervous system.

Human or other cells may be implanted in patients in biocompatible, semipermeable polymeric enclosures or membranes that release truncated sTNFR but prevent cell destruction by the patient's immune system or other deleterious factors from surrounding tissues. Or, the patient's own cells transformed outside the body (ex vivo) to produce truncated sTNFR can be implanted directly into the patient without such envelopes. The methodology of enveloping living cells with a membrane is known to those skilled in the art, so that the preparation of the encapsulated cells and their implantation to patients can be performed.

Alternatively, in vivo gene therapy can be envisaged where the nucleic acid sequence encoding the truncated sTNFR is delivered directly to the patient. For example, a nucleic acid sequence encoding a truncated sTNFR is introduced into a target cell by local injection of a nucleic acid construct (with or without a suitable vector, for example an adeno-viral vector). Other vectors may include, but are not limited to, retroviruses, adenoviruses, herpes simplex virus vector, and papiloma virus vector. Physical transfer can be accomplished in vivo by local injection of the desired nucleic acid construct or other suitable delivery vector containing the desired nucleic acid sequence, liposomes mediated transfer, direct injection (naked DNA), receptor mediated transfer (ligand-DNA complex), or microparticle bombardment (gene gun) ).

Examples of cellular and gene therapy techniques are disclosed in U.S. Pat. Patent No. 4,892,538; in U.S. Pat. Patent No. 5,011,472; in U.S. Pat. Patent No. 5,106,627; in DE 4219626, WO 94/20517 and 96/22793, the disclosure of which is incorporated herein by reference.

Regardless of the route of administration, treatment of TNF-mediated diseases requires a dose or total dose regimen of truncated sTNFR effective to reduce or alleviate the symptoms of the disease. Other factors in determining the appropriate dosage may include the disease or condition to be treated or prevented, the severity of the disease, the route of administration, the age, sex and medical condition of the patient. Further refinement of the calculations necessary to determine a suitable dose for treatment is routinely done by those skilled in the art, particularly in light of the ad assays information contained herein. The dose may also be determined using known assays to determine the doses used in conjunction with appropriate dose response data. The dose is calculated based on 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. The truncated sTNFR may be administered once, or in the case of severe and prolonged complaints administered daily in less frequent doses or administered with an initial bolus followed by continuous or maintenance doses. For parenteral administration, each dose may be, for example, up to 10 mg, or more preferably up to 15 mg, or even more preferably up to 20 mg. When administered to the articular cavity, the pharmaceutical composition is preferably administered as a single injection (for example, a 3-10 ml syringe containing a dose between 5 mg / ml and 10 mg / ml) of truncated sTNFR dissolved in a solution of an isotonic phosphate buffered salt. The composition may be applied to the joint cavity, for example, once every 7 to 10 days. In this way, administration is carried out, for example, 4-5 times, and the dose can be adjusted (if necessary).

In some cases, truncated sTNFRs may be administered in addition to other therapies as well as other pharmaceutical preparations suitable for the indication being treated. The truncated sTNFR and any of the (or more) traditional or novel anti-inflammatory agents may be administered alone or in combination.

Truncated sTNFR products (eg, the proteins R 1 - [Cys ¹⁹ -Cys ¹⁰³ ] -R ₂ ) and any of the additional anti-inflammatory agents (or more) may be administered alone or in combination. Information on the following compounds can be found in The Merck Manual of Diagnosis and Therapy, Sixteen Editon, Merck, Sharp & Dohme Research Laboratories, Merck & Co., Rahway, NJ (1992) and in Pharmaprojects, PJB Publications Ltd.

Current treatments for TNF-mediated diseases (as defined above) including acute and chronic inflammations such as rheumatic

diseases (eg Lyme disease, childhood (rheumatoid) arthritis, osteoarthritis, psoriasis arthritis, rheumatoid arthritis and staphylococcal-induced (“septic) arthritis) include the first use of pain and inflammatory drugs classified as non-steroidal, anti-inflammatory drugs (NSAIDs) steroidal antiinflammatory). Secondary treatment includes corticosteroids, slow acting antirheumatic drugs (SAARDs) or disease modifying drugs (DM).

In a specific case, the present invention is directed to the use of a truncated sTNFR product (eg, the protein R 1 - [Cys ¹⁹ -Cys ^10j ] -R ₂ ) and any (one or more) NSAIDs for the treatment of TNF-mediated diseases as defined above, including acute and chronic inflammation such as rheumatic diseases (eg Lyme disease, childhood (rheumatic) arthritis, osteoarthritis, psoriasis arthritis, rheumatoid arthritis and staphylococcal-induced ("septic) arthritis); and graft-versus-host disease. NSAIDs owe their anti-inflammatory effects at least in part to the inhibition of prostagaldin synthesis (Goodman and Gilman in The Pharmaceutical Basis of Therapeutics, MacMillan, 7th Edition (1985)). NSAIDs can be divided into nine groups: (1) salicylic acid derivatives (2) propionic acid derivatives (3) acetic acid derivatives (4) fenamic acid derivatives (5) carboxylic acid derivatives (6) butyric acid derivatives (7) ) oxicams (8) pyrazoles (9) pyrazolones (pyrazolones)

In a specific case, the present invention is directed to the use of truncated sTNFR products (e.g., the protein R 1 - [Cys ¹⁹ Cys ¹⁰³ ] -R ₂ ) in combination (before, after, or concomitantly) with one or more salicylic acid derivatives, precursors thereof esters or pharmaceutically acceptable salts. The following salicylic acid derivatives, prodrug esters, or pharmaceutically acceptable salts thereof include (names not translated): acetaminosalol, aloxiprine, aspirin, benorylate, bromosaligenin, calcium acetylsalicylate, choline magnesium trisalicylate diflusinal, etersalate, fendosal, gentisic acid, glycol salicylate, imidazole , lysine acetylsalicylate, mesalamine, morpholine salicylate, 1-naphthyl salicylate, olsalazine, parsalmide, phenyl acetylsalicylate, phenyl salicylate, salacetamide, salicylamide O-acetic acid, salsalate and sulfasalazine. Structurally similar salicylic acid derivatives with similar analgesic and anti-inflammatory properties are also included in this group.

In a specific case, the present invention is directed to the use of truncated sTNFR products (e.g., R _{1 -} [Cys ¹⁹ Cys ¹⁰³ ] -R ₂ protein) in combination (before, after, or concurrently with) one or more propionic acid derivatives, precursors thereof esters or pharmaceutically acceptable salts. These propionic acid derivatives, prodrug esters or pharmaceutically acceptable salts thereof include (names not translated): alminoprofen, benoxaprofen, bucloxic acid, carprofen, dexindoprofen, fenoprofen, flunoxaprofen, fluprofen, flurbiprofen, furcloprofen, ibuprofen, ibuprofen aluminum, ibuprofen ind, ibuprofen aluminum, ibuprofen aluminum, ibuprofen ind, isoprofen, ketoprofen, loxoprofen, miroprofen, naproxen, oxaprozin, picetoprofen, pimeprofen, pirprofen, pranoprofen, antizinic acid, pyridoxiprofen, suprofen, thaprofenic acid and thioxaprofen. Structurally similar propionic acid derivatives with similar analgesic and anti-inflammatory properties are also included in this group.

In a specific case, the present invention is directed to the use of truncated sTNFR products (e.g., the protein R 1 - [Cys ¹⁹ Cys ¹⁰³ ] -R ₂ ) in combination (before, after, or concomitantly) with one or more acetic acid derivatives, precursors thereof esters or pharmaceutically acceptable salts. Acetic acid derivatives, prodrug esters thereof, or pharmaceutically acceptable salts thereof include, but are not limited to: acemetacin; alclofenac, amfenac, bufexamac, cinmetacin, clopirac, delmetacin, decclofenac sodium, etodolac, felbinac, fenclofenac, fenclorac, fenclozic acid, fentiazac, furofenac, glucametacin, ibufenac, indomethacin, oxofacolac, oxofacin, isoxepinacin, isoxepinacin, isoxepinacin , progiumethacin, sulindac, talmetacin, tiaramide, tivpinac, tolmetin, zidometacin and zomepirac. Structurally similar acetic acid derivatives with similar analgesic and anti-inflammatory properties are also included in this group.

• · ·

I

In a specific case, the present invention is directed to the use of truncated sTNFR products (e.g., the R 1 - [Cys ¹⁵ Cys 1 ^{O 3} ] -R ₂ protein) in combination (before, after, or concomitantly) with one or more fenamic acid derivatives ), prodrugs of their esters or pharmaceutically acceptable salts. The following fenamic acid derivatives, prodrug esters or pharmaceutically acceptable salts thereof include (not translated): enfenamic acid, etofenamate, flufenamic acid, isonixin, meclofenamic acid, meclofenamate sodium, medofenamic acid, mefanamic acid, niflumic acid, talniflumate, terofenamate, tolfenamic acid and ufenamate. Structurally similar derivatives of phenamic acid with similar analgesic and anti-inflammatory properties are also included in this group.

In a specific case, the present invention is directed to the use of truncated sTNFR products (e.g., the protein Ri-fCys ^{19 Cys103} ] -R ₂ ) in combination (before, after, or concurrently with) one or more carboxylic acid derivatives, precursors of their esters or pharmaceutically acceptable salts. Among these carboxylic acid derivatives, prodrug precursors or pharmaceutically acceptable salts thereof include (names not translated): clidanac, diflunisal, flufenisal, inoridine, ketorolac and tinoridine. Structurally similar carboxylic acid derivatives with similar analgesic and anti-inflammatory properties are also included in this group.

In a specific case, the present invention is directed to the use of truncated sTNFR products (e.g., the protein R 1 - [Cys ¹⁹ Cys ¹⁰³ ] -R ₂ ) in combination (before, after, or concomitantly) with one or more butyric acid derivatives, precursors thereof esters or pharmaceutically acceptable salts. Such butyric acid derivatives, prodrug esters, or pharmaceutically acceptable salts thereof include (names not translated): bumadisone, butibufen, fenbufen, and xenbucin. Structurally similar butyric acid derivatives with similar analgesic and anti-inflammatory properties are also included in this group.

In a specific case, the present invention is directed to the use of truncated sTNFR products (for example, the protein R _x - [Cys ¹⁹ Cys ¹⁰³ ] -R ₂ ) in combination (before, after, or concurrently with the treatment). With one or more oxicams, precursors of their esters or pharmaceutically acceptable salts. Oxicams, prodrug esters, or pharmaceutically acceptable salts thereof include (not translated) droxicam, enolicam, isoxicam, piroxicam, sudoxicam, tenoxicam and 4-hydroxyl-1,2-benzothiazine 1,1-dioxide 4 (N-phenyl) carboxamide . Structurally similar oxicams with similar analgesic and anti-inflammatory properties are also included in this group.

In a specific case, the present invention is directed to the use of truncated sTNFR products (e.g., the protein R _{1 -} [Cys ¹⁹ Cys ¹⁰³ ] -R ₂ ) in combination (before, after, or concomitantly) with one or more pyrazoles, precursors esters thereof or pharmaceutically acceptable salts thereof. Pyrazoles, prodrug esters or pharmaceutically acceptable salts thereof include (names not translated): diphenamizole and epirizole. Structurally similar pyrazoles with similar analgesic and anti-inflammatory properties are also included in this group.

In a specific case, the present invention is directed to the use of truncated sTNFR products (e.g., the protein R _{1 -} [Cys ¹⁹ Cys ¹⁰³ ] -R ₂ ) in combination (before, after, or concomitantly) with one or more pyrazolones (pyrazolones), precursors esters thereof or pharmaceutically acceptable salts thereof. Pyrazolones, prodrugs of its esters, or pharmaceutically acceptable salts include (names not translated): apazone, azapropazone, benzpiperylone, feprazone, mofebutazone, morazone, oxyphenbutazone, phenylbutazone, pipebuzone, propylphenazone, ramifenazone, suxibuzone and thuxibutone. Structurally similar pyrazolones with similar analgesic and anti-inflammatory properties are also included in this group.

In a specific case, the present invention is directed to the use of truncated sTNFR products (e.g., the protein R 1 - [Cys ¹⁹ -Cys ¹⁰³ ] -R ₂ ) in combination (before, after, or concomitantly) with one or more NSAIDs (names not translated) ): ε-acetamidocaproic acid, S-adenosylmethionine, 3-amino-4-hydroxybutyric acid, amixetrine, anitrazafen, anthraphenine, bendazac, bendazac lysine, benzydamine, beprozine, broperamole, bucolome, bufezolac, ciploquimate, ciproquimate , diphenpiramide, diphenpyramide, diphislamine, ditazole, emorphazone, fanetizole mesylate, fenflumizole, floctafenine, flumizole, flunixin, fluproquazone, fopirtoline, phosphosal, guaimesal, guaiazolene, lefetone, lefetamine, lefetamine, lefetamine , lysine clonixinate, meseclazone, nabumetone, nictindole, nimesulide, orgotein, orpanoxin, oxaceprolm, oxapadol, paranyline, perisoxal, perisoxal citrate, pifoxime, piproxen, pirazolac, pirfenidone, proquazone, proxazole, thielavine B, tiflamizole, timegadine, tolectin, tolpadol, tryptamide and substances labeled with company codes as 480156S, AA861, AD1590, AFP802, AFP860, AI77B, AP504, AU8001, BPPC , CN100, EB382, F1044, EK-506, GV3658, ITF182, KME4, LA2851, MR714, MR897, MY309, ONO3144, PR823, PV102, PV108, R830, RS2131, SCR152, SH440, SIR133, SIR133 , ST281,

SY6001, TA60, TAI-901 (4-benzoyl-1-indancarboxylic acid), TVX2706, U60257, UR2301, and WY41770. Structurally similar NSAIDs with similar analgesic and anti-inflammatory properties as the above-mentioned NSAIDs are also included in this group.

In a specific case, the present invention is directed to the use of truncated sTNFR products (e.g., R ₂ - [Cys ¹⁹ -Cys ¹⁰³ ] -R ₂ protein) in combination (before, after, or concomitantly) with one or more corticosteroids, precursors thereof esters or pharmaceutically acceptable salts for the treatment of TNF-mediated diseases (as defined above), including acute and chronic inflammation such as rheumatic diseases (eg Lyme disease, rheumatoid arthritis, osteoarthritis, psoriasis arthritis, rheumatoid arthritis and staphylococcal-induced ( "Septic) arthritis); and multiple sclerosis. Corticosteroids, prodrug esters or pharmaceutically acceptable salts thereof include hydrocortisone and hydrocortisone-derived compounds such as (names not translated): 21-acetoxypregnenolone, alclomerasone, algestone, amcinonide, beclomethasone, betamethasone, betamethasone valerate, budesnisolide, budesonide clobetasol propionate, clobetasone, clobetasone butyrate, clocortolone, cloprednol, corticosterone, cortisone, cortivazole, deflazacone, desonide, desoximerasone, dexamethasone, diflorasone, diflucortolone, difluprednate, fluoxoneone, fluoxone, fluoxone, fluoxone, fluoxone, fluoxone; ,. . ; ·.

.:. .:. ......

flumethasone, flumethasone pivalate, flunisolide, flucinolone acetonide, fluocinonide, fluorocinolone acetonide, fluocortin butyl, fluocortolone, fluorocortolone hexanoate, diflucortolone valerate, fluorometholone, fluperolone acetate, fluprednideneolide acetate, , hydrocortisone acetate, hydrocortisone butyrate, hydrocortisone phosphate, hydrocortisone 21-sodium succinate, hydrocortisone tebutate, mazipredone, medrysone, meprednisone, methylprednicolone, mometasone furoate, parametrhasone, prednicarbate, prednisolone, prednisolone acetate, prednisolone sodium sodium 21-msulfobenzoate, prednisolone sodium 21-stearoglycolate, prednisolone tebutate, prednisolone 21-trimethylacetate, prednisone, prednival, prednylidene, prednylidene 21-diethylaminoacetate, tixocortol, triamcinolone, triamcinolone aceto nide, triamcinolone benetonide and triamcinolone hexacetonide. Structurally similar corticosteroids with similar analgesic and anti-inflammatory properties are also included in this group.

In a specific case, the present invention is directed to the use of truncated sTNFR products (e.g., the protein R _x - [Cys ¹⁹ -Cys ^{10 :!} ] -R ₂ ) in combination (before, after, or concurrently with) one or more slow-acting anti-rheumatic drugs (SAARD) or disease-affecting anti-rheumatic drugs (DMARDS), their prodrug precursors or pharmaceutically acceptable salts for the treatment of TNF-mediated diseases (as defined above), including acute and chronic inflammation such as rheumatic diseases (eg Lyme disease, rheumatoid arthritis, osteoarthritis, psoriasis arthritis, rheumatoid arthritis and staphylococcal-induced ("septic"arthritis); and multiple sclerosis. SAARDs and DMARDS, their ester esters or pharmaceutically acceptable salts thereof include (names not translated): allocupreide sodium, auranofin, aurothioglucose, aurothioglycanide, azathioprine, brequinar sodium, bucillamine, calcium 3-aurothio-2-propanol-1-sulfonate, chlorambucil, chloroquine, clobuzarite, cuproxoline, cyclophosphamide, cyclosporine, dapsone, 15-deoxyspergualin, diacerein, glucosamine, gold salts * (eg cycloquine gold salt, gold sodium thiosulfate), hydroxychloroquine, hydroxyurea, kebuzone, levaritole, levamisole melittin, 6-mercaptopurine, methotrexate, mizoribine, mycophenolate mofetil, myoral, nitrogen mustard, D-penicillamine, pyridinol imidazoles such as SKNF86002 and SB203580, rapamycin, thiols, thymopoietin and vincristine. Structurally similar SAARDs or DMARDs with similar analgesic and anti-inflammatory properties are also included in this group.

In a specific case, the present invention is directed to the use of truncated sTNFR products (e.g., the protein R 1 - [Cys ¹⁹ -Cys ^10j ] -R ₂ ) in combination (before, after, or concomitantly) with one or more COX2 inhibitors, precursors thereof esters or pharmaceutically acceptable salts for the treatment of TNF-mediated diseases (as defined above), including acute and chronic inflammation. Examples of COX2 inhibitors, prodrug esters thereof, or pharmaceutically acceptable salts include celocoxib (name not translated). Structurally similar COX2 inhibitors with similar analgesic and anti-inflammatory properties are also included in this group.

In a specific case, the present invention is directed to the use of truncated sTNFR products (e.g., the protein R 1 - [Cys ¹⁹ -Cys ¹⁰³ ] -R ₂ ) in combination (before, after, or concomitantly) with one or more bacteriostats (antimicrobials), prodrugs of their esters or pharmaceutically acceptable salts for the treatment of TNF-mediated diseases (as defined above), including acute and chronic inflammation. Bacteriostatics include, for example, (names not translated): ampicillin, amoxycillin, aureomicin, hacitracin, ceftazidime, ceftriaxone, cefotaxime, cephachlor, cephalexin, cephradine, cprofloxacin, clavulanic acid, cloxacillin, cloxacillin, cloxacillin, cloxacillin, cloxacillin, cloxacillin, cloxacillin, oxacillan, penicillin and vancomycin. Structurally similar bacteriostats with similar analgesic and anti-inflammatory properties are also included in this group.

In a specific case, the present invention is directed to the use of truncated sTNFR products (e.g., the protein R _x - [Cys ¹⁹ -Cys ¹⁰³ ] -R ₂ ) in combination (before, after, or concomitantly) with one or more of the following compounds for treatment TNF-mediated diseases (as defined above), including acute and chronic inflammation (some names are not translated): granulocyte colony stimulating factor; thalidone; BN 50730; tenidap; E 5531; tiapafant PCA 4248; nimesulide; panavir; rolipram; RP 73401; peptide T; MDL 201 449A; (1R, 3S) -Cis-1- [9- (2,6-diaminopurinyl)] - 3-hydroxy-4-cyclopentene hydrochloride; (1R, 3R) trans-1- [9- (2,6-diamino) purine] -3-acetoxycyclopentane; (1R, 3R) trans-1- [9-adenyl] -3-azidocyclopentane hydrochloride and (1R, 3R) trans-1- (6-hydroxy-purin-9-yl) -3-azidocyclopentane.

In a specific case, the present invention is directed to the use of truncated sTNFR products (e.g., the protein R _x - [Cys ¹⁹ -Cys ¹⁰³ ] -R ₂ ) in combination (before, after, or concomitantly) with one or more other TNF inhibitors, for the treatment of TNF-mediated diseases (as defined above), including acute and chronic inflammation. TNF inhibitors include compounds and proteins that block in vivo synthesis or extracellular release of TNF, including the following compounds.

Other TNF inhibitors include anti-TNF antibodies (eg, MAK 195F Fab antibody (Holler et al. (1993), 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 disclosure of which is incorporated herein by reference); ΒΑΥ X 1351 murine anti-tumor necrosis factor monoclonal antibody (Kieft et al. (1995), CenTNF cA2 anti-TNF monoclonal antibody (Elliott et al. (1994), Lancet, 344: 1125-1127 and Elliott et al. (1994) 7th European Congress of Clinical Microbiology and Infectious Diseases, 9). , Lancet, 344: 1105-1110, the disclosure of which is incorporated herein by reference).

In a specific case, the present invention is directed to the use of truncated sTNFR products (for example, the protein R _{1 -} [Cys ¹⁹ -Cys ¹⁰³ ] -R ₂ ) in combination (before, after, or concurrently with) soluble recombinant human Fas antigen or its recombinant versions (WO 96/20206 and Mountz et al., J. Immunology, 155: 4829-4837 and EP 510 691), the disclosure of which is incorporated herein by reference.

WO 96/20206 discloses secreted human Fas Antigen (natural and recombinant, including Ig fusion protein) methods for gene isolation

responsible for encoding soluble recombinant human Fas antigen, methods for cloning a gene in a suitable vector and suitable cell types, and methods for producing inhibitors of gene expression.

EP 510 691 discloses DNA encoding human Fas antigen including soluble Fas antigen, vectors expressing the DNA and transformants transfected with the vector. For parenteral administration, the doses of Fas antigen fusion protein are between 1 µg / kg to 100 µg / kg.

In a specific case, the present invention is directed to the use of truncated sTNFR products (e.g., the R 1 - [Cys ¹⁹ Cys ¹⁰³ ] -R ₂ protein) in combination (before, after, or concomitantly) with one or more interleukin-1 inhibitors for treatment TNF-mediated diseases (as defined above) including acute and chronic inflammation such as rheumatic diseases (eg Lyme disease, childhood (rheumatic) arthritis, osteoarthritis, psoriasis arthritis, rheumatoid arthritis and staphylococcal-induced ("septic) arthritis); brain damage resulting from trauma, epilepsy, bleeding or stroke; and multiple sclerosis. Classes of interleukin-1 inhibitors include interleukin 1 receptor antagonists (any compound specifically preventing activation of cellular IL-1 receptors), such as IL-1ra, as described below; anti-IL-1 monoclonal antibody (e.g.

EP 623674, the disclosure of which is incorporated herein by reference); IL-1 binding proteins such as soluble IL-1 receptors (eg, U.S.P.

492,888, U.S.P. No. 5,488,032, U.S.P. No. 5,464,937, U.S.P. 5,319,071 and U.S.P. 5,180,812, the disclosure of which is incorporated herein by reference); anti-IL-1 monoclonal antibodies (eg, WO 9501997, WO 9402627, WO9006371, U.S.P. 4935343, EP 364778, EP 267611 and EP 220063, the disclosure of which is incorporated herein by reference); IL-1 receptor accessory proteins, e.g.

WO 96/23067 (the disclosure of which is incorporated herein by reference) and other compounds and proteins that block in vivo synthesis or extracellular release of IL-1.

The IL-1 receptor antagonist (IL-1ra) is a human protein that acts as a natural inhibitor of intereukin-1. Preferred receptor antagonists together with methods for their preparation and use are described in U.S. Pat. Patent No. 5,075,222 (referred to herein as the '222 Patent); WO 91/08285; WO 91/17184; AU 9173636; WO 92/16221;

· · · «« «« «« «« «« «« ««

WO93 / 21946; PCT International Application US97 / 02131 which discloses a pharmaceutical composition comprising (a) an effective amount of a controlled release polymer (eg, hyaluronic 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 disclosure of which is incorporated herein by reference. Proteins include glycosylated and non-glycosylated IL-1 receptor antagonists.

U.S. Pat. Patent No. No. 5,075,222, (Hannum et al. Entitled "Interleukin-1 Inhibitors), specifically, three preferred forms of IL-1ra (IL-1raa, IL-1rap, and IL-1rax), each derived from the same DNA encoding sequence. This U.S. Pat. The patent (referred to herein as the '222 patent) is specifically incorporated herein by reference. All three interleukin-1 inhibitors have similar functional and immunological activities. Also disclosed in the '222 patent are methods for producing IL-1 inhibitors, particularly IL-1ras. One method involves isolating inhibitors from human monocytes (where they are naturally produced). The latter method involves isolating the gene responsible for encoding IL-lras, cloning the gene in suitable types of vectors and cells, expressing the gene leading to the production of IL-lras, and obtaining IL-lras. A preferred method of the invention is the second method, which is a general example of recombinant DNA methods. In a specific case, IL-1ra contains a methionyl group at the N-terminus as a result of expression in E. coli. The present invention also encompasses modified IL-1ras. Modified IL-1ras include, for example, the muteins of such inhibitors, wherein the cysteine residue is replaced by an amino acid at one or more positions of the amino acid sequence of a naturally occurring inhibitor. Such muteins may then selectively react locally with functional units of polyethylene glycol (PEG) or other sulfhydryl-containing polyethers to form IL-1ra PEG species. PCT Publication No. WO 92/16221 discloses a number of modified IL-1ra species and methods for preparing these PEG-modified inhibitors.

Another class of interleukin-1 inhibitors include compounds capable of specifically preventing activation of cellular IL-1 rexeptors. Such compounds include IL-1 binding proteins, such as soluble receptors and monoclonal antibodies. Also included are monoclonal antibodies to receptors.

Another class of interleukin-1 inhibitors include compounds and proteins that block the in vivo synthesis and / or extracellular release of IL-1. These are substances that affect the transcription of the IL-1 gene or the modification of the unfinished IL-1 pre-protein.

The above examples do not preclude other treatments concurrently with these anti-inflammatory compounds known to those skilled in the art and which could be obtained by those skilled in the art using the guidelines set forth herein.

For ease of administration and uniformity of dosage, it is particularly advantageous to manufacture (to formulate) mixtures of additional anti-inflammatory compounds in the form of dosage units. Dosage unit form herein refers to a physically discrete unit suitable for unitary dosing of mammalian subjects to be treated, each unit containing a predetermined amount of complementary anti-inflammatory compounds intended to achieve (in association with the required pharmaceutical carrier) the desired therapeutic effect. "A pharmaceutically acceptable carrier includes all solvents, dispersion media, coatings, antimicrobial and antifungal agents, isotonic and absorption retarding agents, etc. that are compatible with the active ingredients, the mode of administration and the other ingredients of the composition and are not harmful to the recipient. The use of such agents and media is known to those skilled in the art (see, for example, Remington's Pharmaceutical Sciences, 18th Ed. (1990), Mack Publishing Co., Easton, PA 18042, pages 1435-1712, the disclosure of which is incorporated herein by reference). Additional active ingredients may also be incorporated into the mixture.

For oral therapeutic administration, additional anti-inflammatory compounds may be included with excipients (inert carriers) and used in the form of swallowable tablets, buccal (oral) tablets, lozenges, capsules, suspensions, syrups, wafers and the like, or may be incorporated directly into food. Tablets, lozenges, pills, capsules, and other forms may also contain: a binder, such as a 'gum tragacants', acacia, cereal starch or gelatin; inert excipients such as dicalcium phosphate; disintegrating agents such as cereal starch, alginic acid and the like; a lubricant such as magnesium stearate; a sweetening agent such as sucrose, lactose, or

00

0 saccharin; flavoring agents such as peppermint, wintergreen oil, cherry or orange flavor. If the dosage unit is a capsule, it may contain a liquid carrier in addition to the aforementioned substances. A variety of other materials can be used that modify the package or physical form of the dosage unit. For example, tablets, pills, or capsules may be coated with shellac, sugar, or both. Of course, all materials used to prepare any dosage unit must be pharmaceutically pure and non-toxic in the amounts used. In addition, additional anti-inflammatory compounds may be added to the sustained release formulations and compositions. The amount of these complementary anti-inflammatory compounds in a given therapeutically useful composition is selected to achieve a suitable dosage.

For parenteral therapeutic administration, all anti-inflammatory compounds may contain a sterile injectable solution. A sterile injectable solution may be prepared by combining the required amount of the additional anti-inflammatory agent in a suitable, pharmaceutically acceptable carrier with various other ingredients listed below (desired) and subsequent filter sterilization. All dispersions can be prepared by incorporating the anti-inflammatory compound into a sterile carrier that contains a basic dispersion medium and other desired ingredients from those enumerated above. All sterile injectable solutions can be prepared by combining the powder of the additional anti-inflammatory compound and (optionally) any other desired. ingredients from a previous preparation of their sterile solution. The powder is prepared by any suitable technique (e.g., vacuum drying and lyophilization).

The specific dose of the anti-inflammatory additive is calculated according to the approximate body weight or body surface area of the patient. Other factors may be involved in determining the appropriate dose - whether it is an acute or chronic inflammatory disease, or whether it is treatment or prevention, the severity of the disease, the route of administration, the age, sex and medical condition of the patient. Those skilled in the art will readily make more accurate calculations necessary for appropriate dosages in treatment, taking into account all of the above formulations. Dosage can also be determined using known dose determination assays used in conjunction with appropriate dose response data.

φ · ’··· ···

For example, it is an object of the present invention that doses of complementary anti-inflammatory compounds selected for the treatment of certain acute or chronic diseases such as rheumatic diseases (eg Lyme disease, childhood (rheumatoid) arthritis, osteoarthritis and staphylococcal-induced ("septic) arthritis) therapeutic effects diverse. When one of the anti-inflammatory additives has side effects, it can be administered to patients during the alternative treatment period of combination therapy. For example, long-term methotrexate treatment (chronic methotrexate treatment) is associated with gastrointestinal, hepatic, pulmonary and bone marrow toxicity (Sandoval et al., (1995) British Jouranal of Rheumatology, 34: 49-56, the disclosure of which is incorporated herein by reference.

Assays to monitor disease improvement may include specific assays aimed, for example, to determine systemic response to inflammation, including erythrocyte sedimentation rate (ESR), acute phase reactant (APR). Observe the swelling affected by part of the body. An improvement in stiffness and grip (if possible) and pain reduction may also be observed. If the patient's condition is stable, the same dose is given weekly and his condition is also evaluated weekly. If the patient's condition is stable, treatment may be continued. After six months of treatment, anatomical changes in the skeleton are detected by radiological imaging (e.g., X-ray).

At the end of each period, the patient is re-evaluated. Comparison of radiological evaluation before and after treatment, ESR and APR values indicate the efficacy of the treatment. Depending on the efficacy of the treatment and the patient's condition, the dosage may be increased or maintained at the same level during the treatment period.

The present invention is preferably directed to a method (optionally with one of the following combinations) for treating or preventing acute or chronic inflammatory diseases and conditions (as defined above), such as rheumatic diseases (eg, Lyme disease, childhood (rheumatoid) arthritis, osteoarthritis, psoriasis arthritis, rheumatoid arthritis and staphylococcal-induced ("septic" arthritis): a truncated sTNFR product (eg, protein R 1 - [Cys ¹⁹ -Cys ¹⁰³ ] -R ₂ ) and methotrexate (methotrexate); a truncated sTNFR product (e.g., a protein

4

R 1 - [Cys ¹⁹ -Cys ¹⁰³ ] -R 2) methotrexate and an inhibitor of IL-1, preferably IL-1ra; a truncated sTNFR product (eg, a protein R 1 - [Cys ¹⁹ -Cys ¹⁰³ ] -R 2) and one or more of the following - methotrexate, an immunosuppressant (eg, cyclosporine), ciproflaxin, Fas antigen and an inhibitor of IL-1, preferably IL-1ra; a truncated sTNFR product (eg, protein R ₁ - [Cys ¹⁹ -Cys ¹⁰³ ] -R 2) and methotrexate and an immunosuppressant (eg, cyclosporin); truncated sTNFR product (eg, protein Rx- [Cys ¹⁹ -Cys ¹⁰³ ] -R2) methotrexate and ciprofloxacin; a truncated sTNFR product (eg, protein R 1 - [Cys ¹⁹ Cys ¹⁰³ ] -R 2), methotrexate and an inhibitor of IL-1, preferably IL-1ra; a truncated sTNFR product (eg, R4- [Cys ¹⁹ -Cys ¹⁰³ ] -R2 protein) and one or more of the following - methotrexate, sulfasazine (sulphasazine), and hydroxychloroquinone (hydroxychloroquine); truncated sTNFR product (eg, protein R 1 - [Cys ¹⁹ -Cys ¹⁰³ ] -R 2) methotrexate and hydroxychloroquinone (hydroxychloroquine); a truncated sTNFR product (eg, protein R2 [Cys ¹⁹ -Cys ¹⁰³ ] -R2) methotrexate and sulfasazine.

In a specific preferred case, the method comprises administering (eg, intraarticular, subcutaneous or intramuscular) truncated sTNFR products (eg, protein R ₂ - [Cys ¹⁹ -Cys ¹⁰ · ¹ ] -R ₂ , optionally in a sustained release form ( (e.g., hyaluronan)) or in combination (before, after, or concurrently with) methotrexate and / or IL-1 inhibitor (eg, IL-1ra) and / or soluble human recombinant Fas antigen for the treatment of rheumatic diseases such as mentioned above (eg Lyme disease, childhood (rheumatoid) arthritis, osteoarthritis, psoriasis arthritis, rheumatoid arthritis and staphylococcal-induced (“septic) arthritis) and the symptoms associated with them.

In a specific preferred case, the method comprises administering (eg, intravenous or intraverticular) truncated sTNFR products (eg, protein R _x - [Cys ¹⁹ -Cys ¹⁰³ ] -R ₂ , optionally in a sustained release form (eg, hyaluronan)) and optionally in combination (pre-treatment, post-treatment or concomitant treatment) with a tissue plasminogen activator and / or IL-1 inhibitor (eg, IL-1ra) to treat brain damage resulting from trauma, epilepsy, bleeding or stroke, all leading to neurodegeneration .

. In a specific preferred case, the method comprises administering (eg, subcutaneous or intramuscular) truncated sTNFR products (eg, protein R 1 - [Cys ¹⁹ -Cys ¹⁰³ ] -R ₂ , optionally in sequential form.

9 9 9 9

9

9 99 9 99 9 99

9 «release (eg hyaluronan)) or in combination (before, after, or concomitantly) with (one or more substances) a corticosteroid, cyclosporin, FK-506, or interferon (eg alpha interferon, beta interferon, gamma interferon or consensus interferon) and / or IL-1ra for the treatment of multiple sclerosois.

In a specific preferred case, the method comprises administering (eg, subcutaneous or intramuscular) truncated sTNFR products (eg, protein R _x - [Cys ¹⁹ -Cys ¹⁰³ ] -R ₂ , optionally in sustained release form (eg, hyaluronan)) or in combination (pre-treatment, post-treatment or concomitant treatment) with G-CSF and / or IL-1ra for the treatment of inflammatory bowel disease.

In a specific preferred case, the method comprises administering (eg, subcutaneous or intramuscular) truncated sTNFR products (eg, protein R _x - [Cys ¹⁹ -Cys ¹⁰³ ] -R ₂ , optionally in sustained release form (eg, hyaluronan)) or in combination (pre-treatment, post-treatment or concomitant treatment) with leptin, Marinol (Marinol ™) or Megac (Megace ™) for the treatment of cachexia / anorexia.

In a specific preferred case, the method comprises administering (eg, subcutaneous, intraventricular or intrathecal) truncated sTNFR products (eg, protein R 1 - [Cys ¹⁹ -Cys ¹⁰³ ] -R ₂ , optionally in a sustained release form (eg, hyaluronan)) or in a combination (pre-treatment, post-treatment or concomitant treatment) with an NSAID (e.g.

indomethacin (indomethacin)) and / or an IL-1 inhibitor (eg IL-1ra) for the treatment of Alzheimer's disease.

In a specific preferred case, the method comprises administering (eg, subcutaneous, intraventricular, or intrathecal) truncated sTNFR products (eg, the protein R ₁ - [Cys ¹⁹ -Cys ¹⁰³ ] -R ₂ , optionally in a sustained release form (eg, hyaluronan)) or in a combining (before, after, or concurrently with) a soluble recombinant human Fas antigen for the treatment of cancer (eg, bowemias); baby dibetes mellitus type 1; host graft rejection; hepatitis; ischemic / reperfusion damage, including cerebral ischemia (brain damage as a result of trauma, epilepsy, bleeding or stroke, which can all lead to neurodegeneration); inflammatory neuro diseases; rheumatic diseases as defined above (eg Lyme disease, childhood (rheumatic) arthritis,

· <·. · * '·' '. *. * Osteoarthritis, psoriasis arthritis, rheumatoid arthritis and staphylococcal-induced ("septic") arthritis and tissue transplantation).

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

Overview of the drawings

Numerous aspects and advantages of the present invention will become apparent upon review of the drawings wherein:

Giant. 1 depicts a nucleic acid sequence (SEQ ID NO: 1) encoding Asp ¹ -Asn ¹⁶¹ -full length recombinant human sTNFR-.. Also shown is the amino acid sequence Asp ^ Asn ¹⁶¹ (SEQ ID NO: 2).

Giant. 2 depicts a nucleic acid sequence (SEQ ID NO: 3) encoding NH2-MDSVCPQGKYIHPQNNSIC- [Cys ¹⁹ -Cys ³⁰ ] -FC-COOH (also referred to as sTNFR-Ι 2.6D / C105). Also depicted is the amino acid sequence NH ₂ MDSVCPQGKYIHPQNNSIC- [Cys ¹⁹ -Cys ^30] -FC-COOH (SEQ ID NO: 4).

Giant. 3 shows the nucleic acid sequence (SEQ ID NO: 5) encoding NH2-MDSVCPQGKYIHPQNNSIC- [Cys ¹⁹ -Cys ¹⁰³ ] -FNCSL-COOH (also referred to as sTNFR-Ι 2.6D / C106). Also shown is the amino acid sequence of NH ₂ -MDSVCPQGKYIHPQNNSIC- [Cys ¹⁹ -Cys ¹⁰³ ] -FNCSL-COOH (SEQ ID NO: 6).

Giant. 4 shows the nucleic acid sequence (SEQ ID NO: 7) encoding _NH2 -MDSVCPQGKYIHPQNNSIC- [Cys ¹⁹ -Cys ^103] -FN-COOH (also referred to as sTNFR-Ι 2.6D / N105). Also depicted is the amino acid sequence NH ₂ MDSVCPQGKYIHPQNNSIC- [Cys ¹⁹ -Cys ^103] -FN-COOH (SEQ ID NO: 8).

Giant. 5 shows the nucleic acid sequence (SEQ ID NO: 11) encoding NH ₂ -MYIHPQNNSIC- [Cys ¹⁹ -Cys ^103] -FNCSL-COOH (also referred to as sTNFR-Ι 2.3D / d8). Also shown is the amino acid sequence of NH ₂ MYIHPQNNSIC- [Cys ¹⁹ -Cys ¹⁰³ ] -FNCSL-COOH (SEQ ID NO: 12).

Giant. 6 depicts a nucleic acid sequence (SEQ ID NO: 9) encoding NH ₂ -M- [Cys ¹⁹ -Cys ¹⁰³ ] -FNCSL-COOH (also referred to as sTNFR-I

2.3D / δ18). Also shown is the amino acid sequence of NH ₂ -M- [Cys ¹⁹ Cys ¹⁰³ ] -FNCSL-COOH (SEQ ID NO: 10).

Giant. 7 shows the nucleic acid sequence (SEQ ID NO: 13) encoding NH ₂ -MSIS- [Cys ¹⁹ -Cys ^103] -FNCSL-COOH (also referred to as sTNFR-1 2.3D / dl5). Also shown is the amino acid sequence of NH ₂ -MSIS- [Cys ¹⁹ Cys ¹⁰³ ] -FNCSL-COOH (SEQ ID NO: 14).

Giant. 8 depicts a nucleic acid sequence (SEQ ID NO: 34) encoding Leu ^x -Thr ^{179 of} ready-made recombinant human sTNFR-II. Also shown is the amino acid sequence of Leu ^ Thr ¹⁷⁹ (SEQ ID NO: 35).

Giant. 9 depicts the amount of swelling induced in a streptococcal cell wall-induced reactivation model as described in Examples II.

Giant. 10 depicts plasma profiles of sTNFR-D4D / C105db in healthy baboons after a two minute intravenous infusion of 0.2 mg / kg as described in Examples III.

Giant. 11 shows plasma profiles of sTNFR--3D / C105db in healthy baboons after a 2 minute intravenous infusion of 0.2 mg / kg as described in Examples III.

Giant. 12 depicts plasma profiles of sTNFR-Ι 2.6D / C105db in healthy baboons after a 2 minute intravenous infusion of 0.2 mg / kg as described in Examples III.

Giant. 13 shows the relationship between dose and degradation of various dimeric sTNFR-strukt constructs as described in Examples III.

DETAILED DESCRIPTION OF THE INVENTION

Standard methods forming the basis of the procedures described in the following examples, or other suitable procedures, are described in known molecular biological manuals, e.g.

Sambrook et al. (1989), supra, and Ausubel et al. (1990), supra. For clarity, the abbreviation "mL for milliliter" and "L for liter" is used.

Example I

The following example describes the production of various forms of truncated recombinant soluble TNFR-I: NH ₂ -MDSVCPQGKYIHPQNNSIC- [Cys ¹⁹ Cys ¹⁰³ ] - FC-COOH (sTNFR-Ι 2.6D / C105); NH2-MDSVCPQGKYIHPQNNSIC- [Cys ¹⁹ Cys ¹⁰³ ] - FNCSL-COOH (sTNFR-Ι 2.6D / C106); NH2-MDSVCPQGKYIHPQNNSIC [Cys ¹⁹ -Cys ¹⁰³ ] - FN-COOH (sTNFR-1 2.6D / N105); NH2-MYIHPQNNSIC- [Cys ¹⁹ Cys ¹⁰³ ] - FNCSL-COOH (sTNFR-1 2.3D / d8); NH2-M- [Cys ¹⁹ -Cys ¹⁰³ ] - FNCSLCOOH (sTNFR-Ι 2,3D / dl8) and NH ₂ -MSIS- [Cys ¹⁹ -Cys ¹⁰³ ] - FNCSL-COOH (sTNFR-Ι 2,3D / dl5) .

A. DNA Preparation:

1.sTNFR-I 2.6D / C106

The cloned DNA derived from the lambda clone - gt107ctnfbp (EP 422339) serves as a template in the PCR amplification reaction using the following PCR primers:

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

5 '- GGTTAGCCATATGGACAGCGTTTGCCCCCAA-3'

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

OLIGO # 1 and OLIGO # 2 contain NdeI and HindIII sites and hybridize to the 5 'end (OLIGO # 1) and the 3' end (OLIGO # 2) of the truncated gene. The PCR amplification reaction is run for 25 cycles; each cycle consists of 30 seconds at 94 ° C (denaturation), 15 seconds at 55 ° C (primer hybridization) and 1 minute at 72 ° C (polymerization) [type 2400 thermocycler (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 digested with NdeI and HindIII and then isolated from the gel using the QIAquick ™ Gel Extraction Kit (QIAGEN, Chatsworth, CA) according to the manufacturer's instructions. The PCR product isolated from the gel is

^{4 was} inserted into the vector pAMG11 (WO 95/26746) and introduced by transformation into E. coli FM 15 cells (ATCC 55765).

2. sTNFR-I 2.6D / C105

PCR amplification of sTNFR-Ι 2.6D / C105 is performed using sTNFR-Ι 2.6D / C106 plasmid DNA as a template and the following PCR primers:

OLIGO # 3: (SEQ ID NO: 70)

5 '-ACTCGA GGATCCGCGGATAAATAAGTAACGATCCGGTCCA- 3'

OLIGO # 4: (SEQ ID NO: 71)

5'-CAGGTCGGATCCTATCAGCAGAAGCACTGGAAAAGGTTTTC-3 '

OLIGO # 3 and OLIGO # 4 contain a BamHI site and a N (105) mutation followed by a stop codon. The primers are designed to fully amplify the matrix to provide a new BamHI site suitable for ligation. The PCR amplification reaction proceeds over 35 cycles; the first 10 cycles consisted of 10 seconds at 92 ° C (denaturation), 30 seconds at 55 ° C (primer hybridization) and 4 minutes at 68 ° C (polymerization); followed by 25 cycles of 10 seconds at 92 ° C (denaturation), 30 seconds at 55 ° C (primer hybridization) and 4 minutes + 20 seconds at 68 ° C (polymerization) [type 2400 thermocycler (Perkin-Elmer Cetus, Norwalk, CT)]. The PCR product is purified from the gel using the QIAquick ™ Gel Extraction Kit (QIAGEN, Chatsworth, CA) according to the manufacturer's instructions, digested with BamHI, extracted with phenol / chloroform and precipitated with ethanol. It is resuspended, inserted into the pAMG11 vector, and introduced by transformation into E. coli FM 15 cells.

3. sTNFR-1 2.6D / N105

PCR amplification of sTNFR-Ι 2.6D / N105 is performed using sTNFR-Ι 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)

5'-CGCGGATCCCTATTAATTGAAGCACTGGAAAAGG-3 '

OLIGO # 5 and OLIGO # 6 contain NdeI and BamHI sites and hybridize to the 5 'end (OLIGO # 5) and the 3' end (OLIGO # 6) of the truncated gene. The PCR amplification reaction proceeds over 30 cycles; each cycle consists of 45 seconds at 95 ° C (denaturation), one minute at 65 ° C (primer hybridization) and two minutes at 72 ° C (polymerization) [type 2400 thermocycler (Perkin-Elmer Cetus, Norwalk, CT)] .

The PCR product is purified using the Wizard ™ Clean-Up System (Promega, Madison, WI) according to the manufacturer's instructions. The purified PCR product is digested with NdeI and BamHI, extracted with phenol / chloroform and precipitated with ethanol. It is then resuspended, inserted into the pAMG11 vector, and introduced by transformation into E. coli FM15 cells.

Those of ordinary skill in the art of molecular techniques are aware that various genetic materials and methods can readily be used or engineered for successful expression in a host cell (e.g., E. coli and other bacteria) based on the disclosure.

4.sTNFR 2,3D / dl8; sTNFR-Ι 2.3D / d8 and sTNFR-Ι 2.3D / dl5

PCR amplification of sTNFR-Ι 2.3D / dl8; sTNFR-Ι 2.3D / d8 and sTNFR-I 2.3D / dl5 are each performed using 2.6D / C1O6 plasmid DNA as a template using the following PCR primers:

sTNFR-Ι 2,3D / d8 PCR primers:

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

5 '-CCCCATATGTATATCCACCCTCAAAATAAT-3'

OLIGO # 8: (SEQ ID NO: 75)

5 '-CCCAAGCTTTTACAGAGAGCAATTGAAGCACTG-3' sTNFR-Ι 2,3D / dl5 PCR primers:

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

5 '-CCCCATATGTCGATTAGCTGTACCAAGTGCCACAAAGG-3'

OLIGO # 10: (SEQ ID NO: 77)

5 '-CCCAAGCTTTTACAGAGAGCAATTGAAGCACTG-3'

sTNFR-I 2,3D / dl8 PCR primers:

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

5'-CCCCATATGTGTACCAAGTGCCACAAAGGA-3 '

OLIGO # 12: (SEQ ID NO: 79)

5'-CCCAAGCTTTTACAGAGAGCAATTGAAGCACTG-3 '

Each of the primers OLIGO # 7, OLIGO # 9, and OLIGO # 11 contains an NdeI site and each of the primers OLIGO # a, OLIGO # 10 and OLIGO # 12 contains a HindIII site. The PCR amplification reaction is run for 25 cycles; each cycle consists of 45 seconds at 95 ° C (denaturation), one minute at 65 ° C (primer hybridization) and two minutes at 72 ° C (polymerization) [type 2400 thermocycler (Perkin-Elmer Cetus, Norwalk, CT)] . PCR products are purified using the Wizard ™ Clean-Up System (Promega, Madison, WI) according to the manufacturer's instructions. The purified PCR products are digested with NdeI and BamHI, extracted with phenol / chloroform and precipitated with ethanol. They are then resuspended, inserted into the pAMG11 vector, and introduced by transformation into E. coli FM15 cells.

B. Production in E. coli:

First, a fresh inoculum of a selected recombinant E.coli strain carrying the desired sTNFR-Ι 2.6D / N105 construct is propagated,

STNFR-I 2.6D / C105, sTNFR-Ι 2.6D / C106, sTNFR-I 2.3D / dl8, sTNFR-I 2.3D / d8 and sTNFR-Ι 2.3D / dl5. The entire contents of the frozen glycerol can in a stock ampoule (about 1.5 mL) is transferred to a 2 L container containing 500 mL of Luria (LB) soil. The bacterial culture is incubated at 37 ° C in a rotary shaker at 350 rpm. Culture density is determined by measuring absorbance at 660 nm (OD _E60 ). The inoculum is cultured to a density> 2.0 OD _6eo . At this point, 125 mL of culture is aseptically transferred to a 15 L production fermentor containing 10 L of sterile medium.

Production medium and fermentation conditions for self-production are described as complex medium and fermentation conditions by Sniff (1993) in a PhD thesis entitled “Chemically defined medium for overproduction of recombinant proteins in E. coli, Colorado State •

University. This work recommends the use of a complex medium containing casein hydrolyzate, salts, glycerol and antifoam. These medium components are sterilized in a fermenter. After cooling the fermenter to a temperature below 40 ° C, trace minerals and thiamine hydrochloride, sterilized by filtration, are added.

After stabilizing the temperature of the medium at 37 ° C, the medium is inoculated. Culture growth is monitored by measuring OD ₆₆₀ . The pH of the culture is maintained at 6.0 by the automatic addition of 5 M sodium hydroxide and 5 M hydrochloric acid. When the OD ₆₆₀ reaches 9.5 to 10.5, the culture is induced by aseptic addition of sterile isopropyl β-D thiogalactopyranoside (IPTG) to a final concentration of 0.50 mM. The culture is harvested after growth has stopped.

The culture medium and growth conditions are used as described by Sniff (1993), supra, with the following exceptions: ammonium sulfate (2.0 g / L) and L-cysteine hydrochloride monohydrate (1.0 g / L) are added to the medium. , tetracycline hydrochloride is omitted, pH is maintained at 6.0 with sodium hydroxide and hydrochloric acid instead of 7.0 maintained only with sodium hydroxide, cultivation temperature is increased to 37 ° C, inducer concentration increased from 0.15 mM to 0 , 50 mM IPTG, harvest time is determined at the time of growth arrest instead of the time elapsed since induction.

After fermentation, the bacterial cells are harvested by centrifugation in 500 mL cuvettes. They are centrifuged at 10,000 rpm for 30 minutes. The slurry bacterial pellet is diluted to a density of 15% solids in a lysis buffer composed of 50 mM Tris and 5 mM EDTA, pH 8.0. Resuspended cells are then lysed by passing the suspension three times through a homogenizer (APV Gaulin, Inc, Everett, MA) at a pressure of 57 MPa (8000 pounds per square inch). Inclusion bodies (IT) are obtained by centrifuging the homogenate at 10,000 rpm for 30 minutes. The inclusion bodies are washed in lysis buffer and centrifuged a third time at 10,000 rpm for 30 minutes. After resuspension of IT in deionized water (1: 1 ratio), they are last centrifuged at 10,000 rpm for 30 minutes. This step represents a second wash. The washed inclusion bodies of each protein are ready for dissolution, renaturation and purification. Each batch gives a yield of about 200-250 g IT.

Truncated sTNFR-I can be prepared by fermentation according to another method:

First, a fresh inoculum of a selected recombinant E.coli strain carrying the desired construct sTNFR-Ι 2.6D / N105 or sTNFR-Ι 2.6D / C106 is propagated. The entire contents of the frozen glycerol can in a stock ampoule (about 1.5 mL) is transferred to a 2 L flask containing 500 mL of BBL yeast extract (10 g / L), pH 7.0. The culture is incubated at 33 ° C in a rotary shaker at 300 rpm. Culture density is determined by measuring absorbance at 600 nm (OD _g0o ). The inoculum is cultured to a density> 2.0 OD 90 · At this point, 80 mL of culture is aseptically transferred to a 15 L production fermenter containing 7 L of sterile growth medium.

The production fermentation utilizes a process of continuous replenishment of nutrients into the medium. The production medium is a complex medium containing yeast extract, salts and antifoam. These medium components are sterilized in a fermenter. After cooling the fermentation vessel to a temperature below 40 ° C, trace minerals, glucose, magnesium sulfate and hexametaphosphate, sterilized by filtration, are added. Two nutrients are used, one is a carbon source (glucose / magnesium sulfate), the other is a yeast extract containing nitrogen.

After the temperature of the production medium has stabilized at 33 ° C, the medium is inoculated. Culture growth is monitored by measuring OD ₆₀₀ . The pH of the culture is maintained at 7.0 by the automatic addition of ammonium hydroxide and citric acid (48.7%). Upon reaching an OD 90/0 of 12.0, nutrient I is added at an exponential rate. When the OD ₆₀ o values are in the range of 30 to 40, the addition of nutrient II begins at a constant rate. When the OD _g values of 67-83 are reached, the culture is induced by aseptic addition of a sterile autoinductor (homoserine lactone) to a final concentration of 0.6 mg / L. At the time of induction, the nutrient addition rates I and II are changed to constant. The culture is harvested 16 hours (+ - 2) after induction.

After fermentation, the cells are harvested by centrifugation in 500 mL cuvettes at 10,000 rpm for 30 minutes. The slurry bacterial pellet is diluted to a density of 15% solids in a lysis buffer composed of 50 mM Tris and 5 mM EDTA, pH 8.0. Resuspended cells are then lysed by passing the suspension three times through the homogenizer (APV

Gaulin, Inc., Everett, MA) at a pressure of 57 MPa (8000 pounds per square inch). Inclusion bodies (IT) are obtained by centrifuging the homogenate at 10,000 rpm for 30 minutes. The inclusion bodies are washed in lysis buffer and centrifuged a third time at 10,000 rpm for 30 minutes. After resuspension of IT in deionized water (1: 1 ratio), they are last centrifuged at 10,000 rpm for 30 minutes. This step represents a second wash. The washed inclusion bodies of each protein are ready for dissolution, renaturation and purification.

C. Dissolution / Renaturation:

The washed ITs from each 10 L fermentation are dissolved in 800 mL of solubilization buffer (50 mM Tris, 8 M urea, 160 mM cysteine, pH 9.5). The pH of the solubilization mixture is adjusted to 9.5 with 10 N NaOH, the mixture is stirred at room temperature for 2-3 hours. Each batch yielded a yield of about 200-250 g IT.

Individual solubilization mixtures are diluted in ratio: 20 in cold renaturation buffer (50 mM Tris, 1.1 M urea). The final volume is about 16 L. Then the pH of the individual mixtures is adjusted to 9.7 with 6 N HCl. The mixtures are slowly stirred at 4 ° C for 2-3 days.

Then the pH of each mixture is adjusted to 5.0 with glacial acetic acid and 6 N HCl. The precipitate formed in each mixture is removed by centrifugation at 10,000 g on a Beckman type J2-HS centrifuge. The supernatant obtained is then filtered on a 5 µm and 0.22 µm filter.

D. Purification:

The refolded protein is ready for purification on an IX-1 SP Sepharose Big Bead ™ column (Pharmacia Biotech, Inc., Piscataway, NJ).

IX-1 SP Sepharose Big Bead ™ Column (4.4cm x 20cm)

Buffer A 25 mM acetate mM NaCl pH 5.0

Buffer B mM acetate

375 mM NaCl pH 5.0

Each column is equilibrated with 4-5 column volumes of Buffer A prior to refolding. The material from each batch is separately applied to the columns for purification. A maximum of 12 g protein per liter of polymer is loaded on each column. Then each column is washed with 3-4 column volumes of buffer A (unless the UV value returns to baseline). Proteins are eluted using a linearly increasing salt gradient in the range of 50- 375 mM NaCl with a total range of 8 column volumes. The whole protein fraction is collected together. The collection of the protein fraction on each column begins when the UV absorbance increases to 20% of the maximum. The eluate collection is stopped when 50% of the maximum UV absorbance is reached or when the absorbance stops falling.

Flow rate:

7.5 column volumes / hour - equilibration, column volume washing / hour - column volume / hour loading - elution

All column purification is performed at 4 ° C.

The pooled fractions from each IX-I column are ready for further purification on a Toyo Pearl ™ Butyl 650M HIC column (Toso Haas, Philadelphia, PA).

Column 300 mL - Toyo Pearl ™ Butyl 650M (4.4cm x 20cm)

Buffer A Dilution buffer Buffer B mM NaPO ₄ 40 mM NaPO ₄ Milli QH ₂ O

1.8 M NaCl, pH 6.0 4 M NaCl pH 6.0

Prior to loading the pooled fraction from the IX-1 column, the HIC column is equilibrated with 4-5 column volumes with buffer A. The pooled IX-1 fractions are diluted 1: 1 with dilution buffer and the pH is adjusted to 6.0. The diluted pool of IX-1 is loaded onto the column. A maximum of ten grams of protein per liter of polymer is loaded on each column. Each column is washed with 3 column volumes of buffer. Proteins are eluted from the column using a linear decreasing salt gradient in the range of 1.8 M NaCl to H ₂ O with a total range of 8 column volumes. Taking everyone

the protein fraction starts when the UV absorbance increases to 15-20% of the maximum. Harvesting is terminated when 50% of the maximum UV absorbance is reached or when the absorbance stops decreasing.

Flow rate:

column volumes / hour 3 column volumes / hour All purification at temperature.

equilibration, loading and column elution is performed at room temperature

Concentration / diafiltration (C / D)

A 1 square foot (30.5 cm x 30.5 cm) PLLC regenerated cellulose membrane with MVcutoff 5,000 (Milli-Pore, Bedford, MA) is used in the C / D step. Each pooled HIC column fraction is concentrated to a volume of about 200 mL and then diafiltered against 6-7 volumes of 20 mM NaPO ₄ pH 6.0 unless the conductivity is <4 mm / hr.

Concentration / diafiltration is performed at room temperature.

Each C / D sample is ready for purification on a IX-2 365 mL SP-Sepharose HP ™ column (Pharmacia Biotech, Inc., Piscataway, NJ).

IX-2 column - 365 mL SP-Sepharose HP ™ (5cm x 18.5cm) Equilibration Buffer A Buffer B buffer 20 mM Na NaPO ₄ 20 mM NaPO ₄ 20 mM NaPO ₄ pH 6.0 pH 6.3 50 mM NaCl pH 6.8

Prior to loading individual C / D samples, each column is equilibrated with 4 column volumes of equilibration buffer. Each C / D sample is applied separately to the column so that the amount applied does not exceed eight grams of protein per liter of polymer. Then each column is washed sequentially with 3 column volumes of equilibration buffer and 3 volumes of buffer A. Proteins are eluted using a linear pH gradient between 6.3 - 6.8 and a salt gradient between 0 - 50 mM NaCl (buffer B) with a total

range of 8 column volumes. Fraction collection starts at O.D. 1.0 at the start and end when 50% of the maximum is reached at the end.

The truncated sTNFR-Ι can be solubilized, renatured, and purified in another manner as described below.

C. l Dissolution / Renaturation:

The washed inclusion bodies (IT) are dissolved in a solution of 8 M urea, 60 mM Tris, 100 mM cysteine to a final concentration of 6.5 M urea, 50 mM Tris and 80 mM cysteine, pH 9.4 and 5. - 10 mg / mL truncated sTNFR-Ι. (The concentration of sTNFR-Ι is determined based on the amount of sTNFR-Ι in the washed IT in g / L.) The solution is stirred at room temperature for 90 minutes and then proteins are renatured by diluting 1:10 in cold (4 ° C - 8 ° C) buffer. of 0.85 M urea, 50 mM Tris, pH 9.8 (pH measured at 4 ° C 8 ° C).

The renatured solution is stirred for 24-72 hours at 4 ° C-8 ° C. At the end of this time, glacial acetic acid (~ 20 mM) is added and the pH is adjusted to 5.0. The precipitate formed is removed by centrifugation and the supernatant stored for loading on the first column.

D. l Purification

The clarified supernatant after acid precipitation is loaded onto an SP-Sepharose Big Bead ™ column (Pharmacia Biotech, Inc., Piscataway, NJ) which was equilibrated with 20 mM sodium acetate buffer, 75 mM NaCl, pH 5.0. The sample is applied to the column in an amount not exceeding 15 g of truncated sTNFR-Ι per L packing 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 a linear 9 column gradient ranging from 75 mM to 450 mM NaCl in 20 mM sodium acetate, pH 5.0. Purification on an SP-Sepharose Big Bead ™ column (SP-BB) is performed at 4 ° C - 8 ° C.

The pooled fractions from the SP-BB column are diluted 1: 1 in 2 M NaCl, 60 mM acetate, pH 4.5 and, if necessary, the pH is adjusted to 4.5. The pooled fraction from the SP-BB column is loaded onto a Toyopearl ™ Butyl 650M column (Toso Haas, Philadelphia, PA), equilibrated with 1M NaCl buffer, 30 mM acetate, pH 4.5. A truncated sTNFR-Ι is applied to the column in an amount of ~ 10-13 grams per liter of packed column. After

loading the sample, the column is washed with 3 column volumes of 1 M NaCl buffer, 30 mM acetate, pH 4.5 and eluted with a linear gradient (8 column volumes) between 1 M NaCl - 0 M NaCl in 30 mM acetate pH 4.5 .

The purified truncated sTNFR-akce fractions from the Butyl 650M column are pooled, diluted 1: 5 with water, and loaded onto a SP-Sepharose High Performance ™ (SP-HP) column (Pharmacia Biotech, Inc., Piscataway, NJ) equilibrated with 30 mM acetate, pH 4.5 (applied maximum ~ 15 g / L fill volume). The column is then washed with 3 volumes of 30 mM acetate, pH

4.5 and eluted with a linear gradient (12 column volumes) ranging from 100 mM to 400 mM NaCl in 30 mM acetate, pH 4.5. The purified truncated sTNFR-akce fractions are pooled and the pH adjusted to 5.0 with NaOH.

C. PEGylation:

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

To a cold (4 ° C) solution of sTNFR-2,6D / N105 (3.5 mg / mL) in 50 mM sodium acetate, pH 4, is added with triple molar excess of t-BuPEG (mono-t-butoxy-polyethylene glycol) , average mh = 33 kDa, Shearwater polymers, Inc.). NaCNBH ₃ is added to a final concentration of 20 mM and the reaction mixture is stirred for 18-24 hours at 7 ° C.

The extent of protein modification during the reaction is monitored by

SEC HPLC columns TSKG3000sw _XL (Toso Haas, Montgomeryville, PA) eluted 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 , BYE).

The pH of the reaction mixture is adjusted to about 3.5 with 1 M HCl and the reaction mixture is diluted with water to a final concentration of 1.5 mg / mL.

sTNFR-12,6D / N105-t-BuPEG (33 kDa) is separated from the remainder of t-BuPEG and other by-products by ion exchange chromatography on SP Sepharose HP 16/10 ™ (Pharmacia Biotech, Inc., Piscataway, NJ).

The reaction mixture is loaded onto the column and unreacted t-BuPEG is eluted with 3 column volumes of starter buffer A (20 mM sodium acetate, pH 4.0). sTNFR-I 2.6D / N105-t-BuPEG (33 kDa) is eluted with a linear gradient (20 column volumes) between 0-30% buffer B (1 M NaCl in 20 mM acetate, pH 4.0). The eluate is measured at 280 nm. Each fraction containing sTNFR-I 2.6D / N105-t-BuPEG (33 kDa) is analyzed

by SDS-PAGE on commercially prepared 420% gradient gels (Novex, San Diego, CA). Fractions are pooled according to SDS-PAGE results, concentrated and sterile filtered. All pooled fractions of purified sTNFR-I 2.6D / N105-t-BuPEG (33 kDa) are again analyzed by SDS-PAGE and SEC HPLC. The protein is transferred to a solution of 10 mM sodium phosphate, pH 6.5 and 20 mM NaCl.

2. Preparation of sTNFR-Ι 2.6D / N105-33 kDa (MePEG).

Acetic acid is added to the cooled (7 ° C) and stirred sTNFR-2,6D / N105 solution (4mg / ml) until the pH reaches 5.0. To this solution is added 15 mM NaCNBH ₃ and a 2-fold molar excess of t-butoxy PEG (t-butoxy-polyethylene glycol, average mh = 33 kDa, Shearwater polymers, Inc). The reaction mixture is stirred briefly at this temperature and then allowed to incubate for about 18 hours.

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

sTNFR-12,6D / N105-MePEG (33 kDa) is separated from the rest of MePEG and other by-products by ion chromatography on a SP Sepharose HP ™ column (Pharmacia Biotech, Inc., Piscataway, NJ).

The reaction mixture is loaded onto the column (maximum 8 mg / ml polymer) and unreacted MePEG is eluted with 3 column volumes of starter buffer A (20 mM sodium citrate, pH 3.0). sTNFR-I 2.6D / N105-MePEG (33 kDa) is eluted with a linear gradient (16 column volumes) ranging from 0.1-0.5 M NaCl in 20 mM citrate, pH 3.0. The eluate is measured at 280 nm. Each fraction containing sTNFR-I 2.6D / N105-MePEG (33 kDa) is analyzed by SDS-PAGE on commercially prepared 4-20% gradient gels (Novex, San Diego, CA). Fractions are pooled according to SDS-PAGE results, concentrated and sterile filtered. All pooled fractions of purified sTNFR-I 2.6D / N105-MePEG (33 kDa) are again analyzed by SDS-PAGE. Purified sTNFR-Ι 2.6D / N105MePEG (33 kDa) is concentrated to 5-20 mg / mL and converted into PBS, pH 6.5 (10 mM sodium phosphate, 35-100 mM NaCl), or 20 mM acetate , 100 mM NaCl, pH 5.0.

3. Preparation of sTNFR-I 2.6D / N105-MePEG (20 kDa)

The procedure of Step A of the 33 kDa (33 kDa) sTNFR-I 2.6D / N105-MePEG is essentially repeated except that MePEG (mono-methoxypolyethylene glycol, mean m = 33 kDa, Shearwater Polymers, Inc). is replaced by MePEG (mono-methoxy-polyethylene glycol, mean m.h = 20 kDa, Shearwater Polymers, Inc). This protein is transferred to a solution of 10 mM sodium phosphate, pH 6.5 and 20 mM NaCl.

4. Preparation of other conjugates.

Other conjugates with TNF-2,6D / N105 are prepared essentially the same as TNT-2,6D / N105-MePEG (33 kDa) using the following types of PEG aldehydes (Shearwater Polymers, Inc.)

Linear monofunction - m.h. 5 kDa, 6 kDa and 57 kDa

Branched monofunction - m.h. 10 kDa, 20 kDa and 40 kDa

Linear Multifunction - m.h. 8 kDa and 20 kDa

Branched trifunctional - m.h. 10 kDa

These proteins are converted into a solution of 10 mM sodium phosphate, pH 6.5 and 20 mM NaCl.

5. Other methods of pegylation.

Truncated sTNFR-molekuly molecules can also be pegylated and purified by other methods:

The eluate from the SP-HP column (3-5 mg / mL, pH adjusted to 5.0) is reacted with 2 moles of polyethylene glycol (eg MePEG or t-BuPEG) per mole of sTNFR-Ι 2.6D / N105 (~ 5 grams t -BuPEG per gram sTNFR-Ι 2.6D / N105). 10-20 mM cyanoborohydride is added after dissolution of the polyethylene glycol and the solution is incubated overnight at 7-15 ° C.

After pegylation (~ 18 hours), the reaction is terminated by the addition of 10 mM glycine.

The pegylation mixture is diluted with 4 volumes of 50 mM acetate, pH 4.0, and the final pH is adjusted to 4.0, if necessary. The mixture is loaded onto an SP-HP column equilibrated with 50 mM acetate, pH 4.0. A maximum of 8 grams of sTNFR-2,6D / N105 per liter of pack volume is loaded onto the column. The column is then washed with 3 column volumes of equilibration buffer and eluted with a linear gradient of 0-0.3 M NaCl in 50 mM acetate pH 4.0. The monopegylated fractions of sTNFR-2,6D / N105-30 kDa are collected, pH adjusted to 5.0, further concentrated and diafiltered into isotonic buffer. All purification steps are performed at room temperature. The protein is converted into either PBS, pH 6.5 (10 mM sodium phosphate, 35-100 mM NaCl), or 20 mM acetate, 100 mM NaCl, pH 5.0.

6. Preparation of sTNFR-Ι 2.6D / C105 and sTNFR-Ι 2.6D / C106

Sulfone-Activated Polyethylene Glycol (prepared and purified according to United States Patent Application No. 08/473, 809, filed June 7, 1995 and United States Patent Application No. 08 / 611,918, filed March 6, 1996) [PEG 20,000bis-vinylsulfone ] is used to dimerize proteins essentially according to the method described in PCT Publication No. WO 95/34326, except reduction and reaction conditions. Proteins are reduced by 4 moles of DTT per mole of protein at 5-6 ° C, pH 7.6 prior to polyethylene glycol attachment. All reactions are carried out in the presence of 30% glycerol. The dimerized protein is called sTNFR-Ι 2.6D / C105db and sTNFR-Ι 2.6D / C106db. Each protein is transferred to PBS, pH 6.5 (10 mM sodium phosphate, 35-100 mM NaCl), or to 20 mM acetate, 100 mM NaCl, pH 5.0.

7. Preparation of Comparative Molecules with TNFR-I (i). sTNFR-Ι4D / N105 is prepared as described in EP 422339. sTNFR-D4D / N105-t-BuPEG (33 kDa) is prepared by pegylation of sTNFR-I 4D / N105 essentially following the procedure above for pegylation of sTNFR-I 2, 6D / N105-t-BuPEG (33 kDa). sTNFR-D4D / N105-t-MePEG (33 kDa) is prepared by pegylating sTNFR-Ι4D / N105 essentially following the procedure described above for pegylating sTNFR-I 2.6D / N105-t-MePEG (33 kDa). The preparation of sTNFR-Ι 4D / C105 and sTNFR-Ι 4D / C105db is described in PCT Publication No. WO 95/34326. These proteins are dissolved in 10 mM sodium phosphate, pH 6.5 and 20 mM NaCl.

(ii). sTNFR-Ι 4D / C105-33 kDa (MePEG) is prepared by pegylating 4D / C105 essentially following the procedure above for pegylating sTNFR-I 2.6D / C105-33kDa (MePEG) except that the reaction proceeds at pH 7.5 with 1.3 moles of DTT per mole of sTNFR-Ι ~ 5-6 hours, followed by removal of DTT on an SP-Sepharose ™ FF column and PEGylation with 1.5-3 moles of PEG per mole of protein for at least 15 hours at

at room temperature. This protein is dissolved 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-Ι 3D / N105 (sTNFR-Ι 4D / N105 truncated by 34 amino acids at the C terminus) is prepared as follows. PCR amplification is performed with the sTNFR-Ι4D / N105 matrix and primers OLIGO # 13 (carrying the NdeI site, hybridizing to the 5 'end of the truncated gene) and OLIGO # 14 (carrying the HindIII site, hybridizing to the 3' end of the truncated gene). PCR amplification reactions run for 25 cycles; each cycle consists of 30 seconds at 94 ° C (denaturation), 15 seconds at 60 ° C (primer hybridization) and 1 minute at 72 ° C (polymerization) [type 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 is digested with NdeI and HindIII and then isolated from the gel using the QIAquick ™ Gel Extraction Kit (QIAGEN, Chatsworth, CA). The PCR product isolated from the gel is inserted into the pAMG11 vector and introduced by transformation into E. coli FM 15 cells.

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

5'-GGTTAGCCATATGGACAGCGTTTGCCCCCAA-3 '

3 'OLIGO # 14 (SEQ ID NO: 81)

5'-CCCAAGCTTTTAGGTGCACACGGTGTTCTGTTT-3 '

This protein is dissolved in 10 mM sodium phosphate, pH 6.5 and 20 mM NaCl.

(iv). sTNFR-Ι 3D / C105 (sTNFR-Ι 4D / C105 truncated by 34 amino acids at the C terminus) is prepared in the same way as sTNFR-3D 4D / C105. sTNFR-I 3D / C105 is dissolved 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.

(v). sTNFR-Ι 3D / C105db is prepared similarly to sTNFR-4 4D / C105db, but sTNFR-Ι 3D / C105 is used as a starting material instead of sTNFR-Ι 4D / C105. sTNFR-Ι 3D / C105db is dissolved 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

The ability to inhibit TNF activity is determined in various forms of truncated recombinant soluble TNFR-I.

A. WEHI Cytotoxic Assay

The WEHI assay is based on cell proliferation in vitro (Edwards et al. (1991), Endocrinology. 128: 989-996). Cell lines are sensitive to TNF-α (i.e., TNF-α is cytotoxic). In the presence of a TNFα inhibitor, the cells are protected from the cytotoxic effect and are therefore capable of proliferating.

Protocol:

TNF-sensitive WEHI 164 clone 13 cells (ATCC, Rockville, MD) are suspended at 20 x 10 ⁴ cells / mL in RPMI medium (Gibco, Grand Island, NY) supplemented with 5% Fetal Calf Serum (Hyclone, Ogden, UT) ) and penicillin (50 units / mL): streptomycin (50 mg / mL). One hundred microliters of this cell suspension is transferred to all wells of a 96-well flat bottom microtiter plate, and the cells are allowed to adhere for 4-6 hours at 37 ° C in 5% CO ₂ . 10 μΐ actinomycin D (0.0060 mg / mL, Sigma Chemical Co., St. Louis, MO) is added to each well. Ten microliters of recombinant human TNF-α at a concentration of 50 ng / ml (final concentration 5 ng / ml) is added to each well. A two-fold dilution series of different forms of sTNFR (sTNFR-Ι 2.6D / C106, sTNFR-Ι 4D / C105 and sTNFR-Ι 4D / C105db) is diluted in PBS and then added in duplicate to wells (10 μΕ / well) with adherent cell line WEHI 164, to which previously recombinant human TNF-α was added. WEHI-164 clone 13 cells are incubated for 18 hours at 37 ° C in 5% CO ₂ . At the end of the incubation, 10 mL of a 2 mg / mL solution of the organic dye MTT tetrazolium (3- [4,5-dimethylthiozol-2-yl] 2,5-diphenyl tetrazolium bromide; Sigma Chemical Co., St. Louis, MO) is added. ) and the cells are further incubated for 4-6 hours. Cells are solubilized by adding 50 μΐ of DMF / SDS solution (20% SDS and 50% N, N · N · N · N · N · N · N · N · N · N · N · N · N · N · N · N · N · N · N · N). The DMF / SDS solution is mixed by scooping and draining several times until all MTT crystals are dissolved and the cells are further incubated for 222 hours. Absorbance values are measured on a Vmax reader at 570. The specific cytotoxicity ratio is calculated from the optical density values according to the formula:% specific cytotoxicity = 100% x [abs (cells + medium) - abs (cells + sample)] / [abs (cells) + medium) - abs (cells + TX-100)]. The number of TNF units in each sample is determined by the proportion of mouse specific cytotoxicity as previously described.

The results of the WEHI test are summarized in Table 2 below:

TAB. 2: In vitro activity in WEHI assay

Substance IC50 (ng / mL) sTNFR-1 2.6D / C106 208 sTNFR-1 4D / C105 238 sTNFR-1 4D / C105db ON

Based on the WEHI test result, there are no significant differences in in vitro biological activity between sTNFR-Ι 2.6D / C106 and sTNFR-14D / C105.

B. L929 cytotoxic test:

The L929 cytotoxic assay is based on cell proliferation in vitro (Parmely et al. (1993), J. Immunol., 151: 389-396), also determines the cytotoxicity of TNF-α-sensitive killing. Cell lines are sensitive to TNF-α (i.e., TNF-α is cytotoxic). In the presence of a soluble TNF-α inhibitor, the cells are protected from the cytotoxic effect and are therefore capable of proliferating.

Protocol:

The L929 cell line is obtained from the American Type Culture Collection (CCL 1 Catalog Number, NCTC clone 929, strain L clone, connective tissue, mouse). RPMI Medium 1640 is used for cell multiplication • ·

supplemented with 10% FBS + 1% L-glutamine solution + 1% penicilinstreptomycin solution.

The assay is performed in 96-well microtiter plates (Corning) so that only 60 internal wells are used. The test is repeated three times on the same standard and test plate.

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

Samples are diluted in L92 9 growth medium, 10 ng / mL TNFα, 10 µg / mL actinomycin D (Sigma Chemical Co., St. Louis, MO) - Assay Diluent.

Plates are harvested with XTT / MEN solution (1.5 mg / mL XTT + 75 mM MEN)

On the first day, the cells are seeded in the assay plates. The cell suspension is prepared by trypsinizing and resuspending the cells at a concentration of 3.33 x 10 ⁴ cells / mL. 180 mL of this cell suspension is seeded into each of the inner 60 wells of the assay plate. 200 mL of growth medium is distributed in the outer 36 wells to avoid experimental evaporation errors. Plates, covered with foil and protected from drafts, are left at room temperature for about one hour. They are then placed in a high humidity incubator at 37 + 2 ° C, 5 + 1% CO ₂ . Plates are incubated for about 20-22 hours, then sTNFR-I is added at various dilutions.

The next day, the sTNFR-Ι 4D / N105 standard and test samples are prepared as follows: Dilute the sTNFR--4D / N105 standard and test samples to a concentration of about 2.0 mg / mL (or other appropriate concentration). Make serial dilutions of this concentration to form a curve dilution points of 10 values ranging from about 1.0 x 10® ng / mL to 1.0 x 10 ³ ng / mL, including a 0 ng / mL (Assay Diluent only). Other suitable concentrations may also be used. Add 1000 gL of each dilution in triplicate to each assay plate. Then incubate the plates in a high humidity incubator at 37 + 2 ° C, 5 + 1% CO ₂ for 20 + 1 hours.

On day 3, 50 μΐ per well of XTT / MEN solution is added to the 60 inner wells of the test plates. Plates are incubated in a high humidity incubator at 37 + 2 ° C, 5 + 1% CO ₂ (Falcon, New York, New York) for 24 + 0.5 hours.

• · '

On day 4, the optical density (O.D.) values of the assay plates at 450 nm minus 650 nm are determined on an ELISA reader (SpectraMAX, Beckman Instruments, Inc., Fullerton, CA). If, at these wavelengths, a value of 4000 OD is obtained for the wells on the plate, the plate must be read again at 490 nm minus 650 nm and used for calculation.

The standard dose-response logarithmic curve is determined using a four-parameter logistic approximation. Original concentrations of unknown samples are calculated from the standard curve, ED ₅₀ is calculated for the standard, the correlation coefficient of the standard curve is calculated.

Results:

The results of the L929 cytotoxic assay are summarized in Table 3.

TAB. 3: In vitro activity in the L929 cytotoxic assay.

Substance Concentration (mg / mL) ED ₅₀ (ng / mL)

sTNFR-Ι4D / C105db 7.8 1.0 + 0.1 sTNFR-Ι 2.6D / C105db 2.6 1.1 + 0.0 sTNFR-Ι 2.6D / C106db 2.2 1.0 + 0.1 sTNFR-Ι4D / N105-t-BuPEG (33 kDa) 2.0 229.2 + 8 sTNFR-Ι4D / C105-t-BuPEG (33 kDa) 1.1 325.5 + 147 sTNFR-I 2.6D / C105-t-BuPEG (33 kDa) 1.7 210.2 + 9

Internal standard: sTNFR-Ι 4D / C105

3.5

314.8 + 188.1

The data indicate that sTNFR-Ι4D / C105db, sTNFR-Ι2.6D / C105db and sTNFR-Ι2.6D / C106db are active and have a comparable dose response compared to the standard. Furthermore, these data suggest that sTNFR-Ι4D / N105-t-BuPEG (33kDa), sTNFR-Ι4D / C105-t-BuPEG (33kDa) and sTNFR-I 2.6D / C105-t-BuPEG (33kDa) they have almost two orders of magnitude lower activity, but are still active in this assay compared to TNFR-D4D / C105db.

Attempt # 2 sTNFR-Ι 3D / C105db 0.2 2.27 + 0.3 sTNFR-Ι 3D / C105db 0.2 2,0 * sTNFR-Ι 3D / C105db 1.9 1,8 * sTNFR-Ι 3D / N105 2.4 413.3 * Internal standard: sTNFR-Ι4D / C105 3.5 115.9 + 42

• Value based on a single result

These data demonstrate that sTNFR-I 3D / C105db is active and ED ₅₀ values are within the values characterizing sTNFR-Ι4D / C105db (experiment # 1), sTNFR-Ι 2.6D / C105db (experiment # 1) and sTNFR- 2.6D / C106db (Experiment # 1). The data also show that sTNFR-Ι 3D / N105 is less active than the internal standard sTNFR-Ι 4D / C105.

C. Reactivation model experiment based on streptococcal cell wall induction:

A reactivation model trial of streptococcal cell wall induced arthritis in rats is performed according to known protocols (Esser et al. (1985), Arthritis And Rheumatism, 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 and 185 grams, are injected intraarticularly on the right ankle with a streptococcal cell wall preparative suspension containing peptoglycan-polysaccharide (SCW) (Lee Laboratory, Grayson, GA) at a dose of 1.5 mg / 10 mg per joint. Saline is injected into the contralateral joint as a control. Intra-articular injection of SCW causes acute arthritis of relatively short duration with swelling of the joint that culminates a day or two after injection. After twenty days, during which an acute inflammatory response subsides, the SCW at a dose of 200 mg / 200 mL per rat is again administered intravenously. The second dose is sufficient to reactivate inflammation in the ankle joint where SCW was injected before,

however, it has little effect on the ankle injected with saline. The extent of inflammation within 72 hours after intravenous injection of SCW is determined by measuring the posterior ankle joint using a caliper at 0, 24, 36, 48 and 72 hours after arthritis reactivation, after which the posterior contralateral cell is removed for histological examination (eg, inflammation , pannus formation, cartilage and bone damage.)

Results:

The effects of administration of sTNFR-I 2.6D / C106db on the development of joint swelling during arthritis reactivation are tested. The inhibitor and the blank are each administered in a single intravenous injection 24 hours prior to SCW reactivation.

sTNFR-I 2.6D / C106db shows statistically significant efficacy in reducing joint swelling, as evidenced by ANOVA, in the Fisher post-hoc test (Statview ^R ), at all four doses administered two or three days after reactivation and at all doses with except one (1.5 mg / kg) given on the first day. This swelling reduction is comparable to the positive control sTNFR-D4D / C105db administered daily at 0.5 mg / kg (i.e. 8.8 nM) over the time period from the first day before reactivation to the third day after reactivation. STNFR preparations also show significant efficacy when evaluating the extent of swelling as a whole over three days. The area delineated by the curve (AUC) is a dose-response pattern at all doses (see Figure 9, sTNFR-Ι 2.6D / C106db is designated sTNFR-2,6 2.6D and sTNFR-Ι 4D / C105db is designated sTNFR-Ι 4D ).

sTNFR-I 2.6D / N105-t-BuPEG (33 kDa) shows a significant reduction in joint width and histological indices compared to the disease control group in a model experiment.

D. Model experiment using D-galactosamine / lipopolysaccharide:

The D-galactosamine (D-GalNH ₂ ) / lipopolysaccharide (LPS) model (Parmely et al. (1993), supra) is an in vivo model experiment based on animal lethality dependent on TNF-α. In addition, MPS-Ipr / lpr autoimmune mice have been shown to be extremely susceptible to LPS or SEB-induced TNF-α (Mountz et al. (1995), J. Immunol., 155: 4829-4837).

Protocol:

ϋ 6-8 week old female mice (MRL-lpr / lpr) (Jakckson Laboratory, Bar Harbor, ME), after an overnight fast, an IP response is induced with the following pharmacological preparations: 25 mg DGalNH ₂ (Sigma Chemical Co., St. Louis, MO) suspended in Hanks Balanced Salt Solution (Gibco Laboratories, Inc., Grand Island, NY) (50 mg / mL); lipopolysaccharide (LPS) from E. coli serotype 0127: B8 (Sigma Chemical Co., St. Louis, MO) in sterile, endotoxin-free PBS (25 mg / mouse) medium; or SEB (Toxin Technologies, Sarasota, FL) in normal saline (50 mg / mouse). The various forms of sTNFR are administered in successive two-fold dilutions (doses in mg / kg) to obtain an ED ₅₀ curve using Macintosh statistical software (Statview ^R , Mountain View, CA). Lethality is monitored within 48 hours after inducing an IP response.

Results:

Table 4 shows that when sTNFR-Ι 2.6D / C106db is administered as described 1 hour prior to LPS / DGalNH ₂ , ED ₅₀ (ie the dose of sTNFR-Ι 2.6D / C106db required to rescue 50% mice) at 48 hours is ~ 50 gg / kg (N = 8 mice). Compared to TNFR->4D / C105db there is no significant difference (P> 0.05) in the ability of this form to prevent lethality (ED ₅₀ = ~ 50 gg / kg; N = 8 mice).

• · ·

TAB. 4: Comparison of sTNFR and optimized forms of sTNFR in the LPS / D-GalNH model experiment ₂ )

Preparation EDioo ed ₅₀ sTNFR-Ι4D / C105db ~ 100 μg / kg ~ 50 μg / kg sTNFR-I 2.6D / C106db ~ 100 μg / kg ~ 50 μg / kg sTNFR-I 2.6D / N105-t-BuPEG (33 kDa) ~ 2 mg / kg ~ 40 0 μg / kg sTNFR-I 2.6D / N105-MePEG (20kDa) ~ 800-1000 μg / kg ~ 1 mg / kg sTNFR-I 2.6D / N105-MePEG 2 mg / kg ~ 1-1.5 mg / kg (20 kDa branched) sTNFR-I 2.6D / N105-MePEG 1.5 mg / kg ~ 1 mg / kg (40 kDa branched)

These data show that sTNFR-Ι2.6D / C106db has the same activity as sTNFR-Ι4D / C105db, but sTNFR-I2.6D / N105-t-BuPEG (33 kDa) is less active in this trial (ED ₅₀ is about 400 gg / kg; n = 5 mice). Also, the activities of sTNFR-I 2.6D / N105-MePEG (20 kDa branched) and 2.6D / N105-MePEG (40 kDa branched) are lower in this experiment.

E. Experimental model of adjuvant-induced arthritis:

The adjuvant-induced rheumatoid arthritis induced in rats is in many ways similar to human rheumatoid arthritis. The purpose of this experiment is to demonstrate that systemic administration of truncated forms of sTNFR has a mitigating effect in the pathogenesis of adjuvant-induced arthritis in mice.

Protocol:

Male Lewis rats (5-7 per group) (Charles Laboratories, Inc., Wilmington, MA) weighing at least 200 g are cannulated with a SQ catheter and allowed to recover for several days. They are then placed in infusion cages where they get used to the week before the start of the infusion.

On day 0, all rats are injected with 100 μΐ of Freund's complete adjuvant (Sigma Chemical Co., St. Louis, MO) to which synthetic N, N-dioctyldecyldecyl N ', N-bis (2-hydroxy-ethyl) propanediamine, 50 mg / ml. On day 8, different groups of rats are given continuous SQ infusion with sTNFR-14D / C105 and sTNFR-Ι 2.6D / N105.

The results are summarized in Table 5.

TAB. 5: Adjuvant-induced arthritis.

Substance Dose (mg / kg / hour) AUC% Paw weight Inflammation HistO] % Inh. Res.Kosti pathology % Inh. % Inh. % Inh. Study # 1 sTNFR-Ι4D / C105 5 61 46 37 89 1 49 45 26 855 0.2 33 40 14 34 sTNFR-Ι 2.6D / N105 1 55 53 33 51 Study # 2 sTNFR-Ι 2.6D / N105 5 42 ND 19 Dec 67 1 38 ND 13 49 Study # 3 sTNFR-I 2.6D / N105 9 50 40 13 27 Mar: - MePEG (20kDa) 3 35 34 9 22nd 1 36 30 0 0 sTNFR-Ι 2.6D / N105 9 43 37 MePEG (33 kDa 3 38 33 1 24 20 May Forms sTNFR-I 2.6D / N105-t-BuPEG (33 kDa) and sTNFR-I 4D / Cl05db strangely enough if they adjuvant-induced arthritis in rats Lewis antiarthritic activity comparable to each other , though sTNFR-Ι4D / C105db is more efficient in in vitro cytotoxic tests WEHI - 164 and L929, as in the LPS / GalN experimental model. F.Artritida induced collagen: Arthritis induced collagen II in rats in many ways recalls rheumatoid arthritis u people. The purpose of this try them demonstrate that systemic administration abbreviated . forms of sTNFR has

a mitigating effect in the pathogenesis of type II collagen-induced arthritis in rats and mice.

• '' · · 4 '- 4 · 4 4' • · · 4 · 4 · 4 4 · φ 4 • 4 4 4 4 4 · 4 4 • 4 · 44 4 4 4 4 4 444

4 4 4

444 444 444

Protocol for rats:

Lewis female rats (Charles River Laboratories, Inc., Wilmington, MA) were implanted with SQ cannulas, and the rats were accustomed to attachment during continuous infusion. They are then immunized with cow type II collagen in Freund's complete adjuvant. On the 13th, 14th or 15th day after immunization, animals with developed arthritis are randomly assigned to groups of eight individuals. Infusion of different doses of sTNFR-I or blank is given to the test groups for seven days as described in Table 6. Inflammation in the paw is evaluated daily by measuring the ankle joints with a special scale (caliper). On day 7, the animals are gently killed and the paws are taken to determine the weight that serves as an index of inflammation. Ankle and knee joints are taken for histopathological assessment of arthritis parameters.

The results are shown in Tab. 6A.

• ·

TAB. 6A: Collagen-induced arthritis

Substance Dose AUC% % Inh. Paw weight % Inh. Inflammation Histoj % Inh. Res.Kosti > atologie % Inh. (mg / kg / hour) Study # 1 sTNFR-Ι4D / C105 5 65 81 ND ND 1 35 34 ND ND 0.2 19 Dec 22nd ND ND sTNFR-Ι 2.6D / N105 1 39 41 ND ND (mg / kg / hour) Study # 2 sTNFR-I 2.6D / N105 3 50 60 76 46 MePEG (33kDa) Study # 3 (mg / kg / hour) sTNFR-Ι 2.6D / N105 9 25 44 ND ND MePEG (33kDa) sTNFR-Ι 2.6D / N105 3 25 37 ND ND MePEG (33kDa) sTNFR-I 2.6D / N105 9 35 52 ND ND MePEG (20kDa) sTNFR-Ι 2.6D / N105 3 35 37 ND ND

MePEG (20kDa)

Interestingly, in the case of collagen-induced arthrosis, the efficacy was approximately the same for all treatment groups (e.g., curve shape, ratio of inhibition of the area defined by the curve (AUC) in the range of 30-59%, paw weight inhibition in the range of 40-64%). The untreated group differed significantly from all other groups in this arthritis model.

Mouse protocol:

Male DBA / 1 (Jackson Laboratories, Inc., Bar Harbor, ME) are immunized with cow type II collagen (Sigma Chemical Co., St. Louis, MO) in Freund's incomplete adjuvant. 24th and 26th day after immunization animals with developed arthritis are randomized ·· ·

9

9 divided into groups of eight animals. Subjects in the experimental groups are administered twice daily by the IP route either with saline or with sTNFR-I 2,6D / N105-MePEG (33 kDa) for three consecutive days (days +27, +28, +29). Inflammation in the paw is evaluated daily by measuring the ankle joints with a special scale (caliper). On day 34, the animals are gently killed and the paws are taken to determine the weight that serves as an index of inflammation. Ankle and knee joints are taken for histopathological assessment of arthritis parameters.

The results are summarized in Tab. 6B.

TAB. 6B: Collagen-induced arthritis

Substance

Dose (mg / kg 2D)

AUC% Total Histopathology (% Inh.) (% Inh.)

Study # 1 sTNFR-Ι4D / C105db 3 sTNFR-Ι4D / N105 3 -t-BuPEG (33 kDa)

Study # 2 sTNFR-Ι 2.6D / N105-9

MePEG (33 kDa) sTNFR-Ι 2.6D / N105-3

MePEG (33kDa)

ND

ND

G. LPS-induced TNF-α production affected by continuous infusion in rats:

sTNFR-Ι 2.6D / C105db and sTNFR-2,6 2.6D / C106db, sTNFR-Ι 2.6D / N105 and sTNFR-Ι 4D / N105 are administered IV to the jugular vein by Alzet ™ minipump (Alza Corp., Palo Alfo , CA) according to the manufacturer's instructions during a 48 hour infusion (1 mg / kg). Serum TNF-α levels as measured by ELISA (Genzyme, Cambridge, MA) are significantly reduced compared to controls +2 hours after LPS administration.

'- · ♦ ♦ · ·

• 4 · 4 · 444 · 44 · · · · · · · · · · · · · · · · · · ·

Example III: Immunogenic studies

The immunogenicity of various forms of truncated recombinant soluble TNFR-I is determined in several animal models.

A. Rodents:

sTNFR-I 2.6D / N105-t-BuPEG (33 kDa) and sTNFR-Ι4D / C105db are subcutaneously administered (4 mg / kg) on Sprague Dawley rats (Charles Rivers Labs, Wilmington, MA) on the first and fifth experimental days. (n = 6-8 per group). Blood samples are taken weekly until the 21st day after the start of the experiment. IgM and IgG antibody production is determined in the samples.

TAB. 7: Immunogenicity in rodents

Time [days] 0.01 7 14 21 GROUPS + ANIMALS IgM titer IgM titer IgM titer IgM titer sTNFR-I 2,6D / N105- 33 kDaPEG 1 NEG 0 0 0 2 NEG 0 0 0 3 NEG 0 0 0 4 NEG 0 0 0 6 NEG 0 0 0 sTNFR-I 2,6D / N105- 0 0.00 0.00 0.00 t-BuPEG (33 kDa) PULL 0 0.00 0.00 0.00 Control 7 0 0 50 0 8 0 0 50 0 3 0 0 100 ALIGN! 0 9 0 0 0 0 10 0 0 100 ALIGN! 50 11 0 0 100 ALIGN! 0 12 0 0 100 ALIGN! 0 13 0 0 0 0 Control 0 0 62.5 6.3 PULL 0 0 15.7 6.3

100 ALIGN!

Time [days]

0.01

GROUP + ANIMALS

IgM titer

IgM titer

IgM titer

IgM sTNFR-I 2,6D / Nl05t-BuPEG titer (33 kDa)

1 NEG NEG 0 0 2 NEG NEG 0 0 3 NEG NEG 0 0 4 NEG NEG 0 0 6 NEG NEG 0 0 sTNFR-Ι 2.6D / N105- 0, 00 0.00 0.00 0.00 t-BuPEG (33 kDa) PULL 0.00 0, 00 0, 00 0.00 Control 7 NEG NEG 0 200 8 NEG NEG 0 200 9 NEG NEG 0 0 10 NEG NEG 0 0 11 NEG NEG 200 400 12 NEG NEG 0 200 13 NEG NEG 200 800 14 NEG NEG 0 50 Control 0 0 50 231.3 PULL 0 0 32.7 94.0

Table 7 shows that sTNFR-D4D / C105db administered subcutaneously (SC) on days 1 and 5 provides higher titers of rat anti-sTNFR-I IgG antibodies by day 21 than sTNFR-I 2,6D / N105-t -BuPEG (33 kDa), which has almost zero antibody titers. Similar trends in immunogenicity are observed in rats producing anti-sTNFR-I IgM antibodies by day 21. sTNFR-I 2.6D / N105-t-BuPEG (33 kDa) did not induce the generation of rat anti-sTNFR-I IgM antibodies by day 21.

B.Papio anubis:

Phase A of Part 1 of this study is to determine the pharmacokinetics and immunogenicity of sTNFR-Ι4D / C105db (0.2 mg / kg body weight [BW]), sTNFR-I 3D / C105db (0.2 mg / kg BW) and sTNFR- Ι 2.6D / C105db (0.2 mg / kg BW) given twice IV to a healthy baboon at a 21 day interval.

Part 1 is divided into two phases. The aim of Phase A is to determine the pharmacokinetics and immunogenicity of various sTNFR-prepar preparations in a healthy person

101. · * · 0 · 9 99. 90 • ♦ ·· 909 9 9 99 9 • 9 999 9999

9 9 99 «9 900099

9 9 9 9 9

999 909 99 0 00 99 09 baboon in response to two applications. The twelve baboons are divided into three groups. Individuals of each group received anesthesia of 0.2 mg / kg BW with sTNFR-I 4D / C105db, sTNFR-Ι 3D / C105db and sTNFR-I 2.6D / C105db. Three baboons are studied in one experiment. The animals are followed for 21 days, then receive a second identical IV protein application and are studied for a further 21 days. The pharmacokinetics and immunogenicity are then determined at intervals.

The aim of phase B of this study is to determine the efficacy of these preparations in a well-investigated model of lethality caused by TNF-α (Espat et al., J. Surg. Res., 59: 153-158, 1995). In 16 animals divided into groups of 4, lethal E. coli bacteraemia is induced by administration of 5-10 x 10 ¹⁰ cfu / kg body weight. The placebo group is compared to baboons that have been pre-injected IV with sTNFR-Ι4D / C105db (0.2 mg / kg body weight [BW]), sTNFR-Ι 3D / C105db (0.2 mg / kg BW) and sTNFR-Ι 2.6D / C105db (1 mg / kg BW).

In both phases of Study 1, young adult Papio anubis babies, both male and female (6-11 kg), (Biomedical Research Foundation, San Antonio, TX) are fasted overnight. Animal anesthesia is performed with ketamine (10 mg / kg i.m.) and the head vein is cannulated percutaneously. Anesthesia is maintained by the initial administration of up to 35 mg / kg of sodium pentobarbital followed by repeated injections of 3-5 mg / kg / hour of sodium pentobarbital. The upper respiratory tract is insured by placing an endotracheal tube with a cuff, the animals breathe spontaneously. A catheter is placed percutaneously in the femoral artery to allow repeated collection of arterial blood samples as well as continuous monitoring of cardiac activity and mean arterial blood pressure using the Datascope 2000 Anesthesia and Cardiac Monitor (Datascope, San Antonio, TX). Blood samples from the artery are collected at intervals, and after addition of EDTA or heparin to prevent clotting, they are cooled on ice immediately after collection. The blood plasma fraction is collected by centrifugation at 4 ° C and stored at -70 ° C until analysis. The internal body temperature is monitored by a rectal probe. A non-swellable Foley catheter is placed in the urethra to monitor urine output and creatinine secretion. Hernodynamic parameters were monitored every 15 minutes by luminance. All animals receive 0.9% sodium chloride (4 ml / kg) to maintain i.v. of fluids. During phase B of the experiment, animals receive additional fluid (10 ml / kg every 15 minutes),

102 • 9 · 9 · 9 9 9 9 9 9 9 9 9 9 9

9 9 9 9 9

999 999 999 99 99 99 if two of the following physiological criteria are met:

(1) the mean arterial pressure decreases by more than 30%; 2) heart rate increases by more than 30% 3) urine output decreases to less than 1 ml / kg / hour. Protein infusion begins after blood samples representing baseline values are collected and at least one hour rest period to equilibrate.

During Phase A of this study, a cephalic vein is used to infuse recombinant proteins, the animals are followed for eight hours, after which all catheters are removed and the animals are returned to their cages for 21 days. After 24 and 48 hours, and on days 3, 5, 8, 11, 16, and 21, animals are lightly anesthetized with IM ketamine (10 mg / kg) and venous blood samples are taken. On day 21, a new anesthesia is performed, a second administration of protein is administered, and the whole procedure as described above from day zero is repeated for another 21 days. After this time, the animals are gently killed.

During Phase B of this study, four animals are randomly selected one hour prior to E. coli infusion, receiving either placebo or one of the above recombinant preparations. The animals are followed for eight hours, after which all catheters are removed, the animals are returned to their cages and any survival of lethal bacteraemia is observed. Animals that suffer excessively are gently killed. Excessive suffering is defined by IACUC as follows: 1) inability to stand or sit for 12 hours, 2) inability to receive food or water for 12 hours, 3) unmanageable bleeding from catheter sites, or 4) inability to respond to external stimuli. Venous blood samples are taken at -1, 0, 0.5, 1, 1.5, 2, 2.5, 3, 4, 5, 6, 7, 8, 24 hours, 48 hours and days 3, On days 5, 8, 11, 16, and 21, surviving animals are gently killed.

The presence of Papio antibodies against administered recorabinant proteins is determined by an ELISA sandwich assay. Briefly, ELISA plates are coated with sTNFR-Ι (lgg / ml), then dilute (1:50 to 1: 100,000) baboon plasma (100 μΐι) is added. After washing the samples, horseradish peroxidase-protein A conjugate (0.5 µg / ml) is added, and the reaction is visualized by TMB.

Results (Part I):

103 ·· '· * ·; »<. ·>» · 9 9 9 9 9 9 9

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The three recombinant preparations differed significantly in the plasma half-life. The disappearance curves are determined by a model independent method, with half-lives generally evaluated between 8 and 172 hours. In animals entering the experiment, the half-life in plasma is the longest in baboons receiving a 4-domain preparation (29 hours) and gradually decreases in baboons given sTNFR-Ι 3D / C105db (24.7 hours) and sTNFR-Ι 2, 6D / C105db (21.5 hr). The difference, although statistically significant, is only 26%. After the second administration of proteins to the respective baboons, plasma half-lives have an unexpected tendency to significantly shorten, suggesting faster elimination. This reduction in half-lives is most pronounced in sTNFR-Ι4D / C105db-influenced baboons, with half-lives shorter by 48% (p <0.01) [Fig.10]. The mean reduction in half-lives is in baboons affected by sTNFR-Ι 3D / C105db (31%) [Figure 11] and the lowest in animals treated with sTNFR-Ι 2.6D / C105db (14%) [Figure 12]. In baboons who received sTNFR-Ι 2.6D / C105db, the half-life reductions are not statistically different.

All preparations are immunogenic in baboons. However, the highest immunogenicity was found in sTNFR-4 4D / C105db-treated baboons, the median in sTNFR-Ι 3D / C105db-treated animals, and the lowest in sTNFR-Ι 2.6D / C105db-treated animals (Table 8).

104

TAB. 8: Peaks of antibody response ¹

First Median 21 days 26% -75% Of others Median 21 25 days % -75% sTNFR-Ι4D / C105db (n = 4) 3.20 3.20 3.20 3, 95 3, 50 4.40 sTNFR-Ι 3D / C105db (n = 4) 1.60 0. 00 3.65 3.50 1, 30 4.75 sTNFR-Ι 2.6D / C105db (n = 4) 0, 00 * 0,00 1,75 1.40 O, 00 3,50

¹ logarithmic scale (inverse of plasma dilution required to reach half the maximum absorbance in ELISA sandwich, see Experimental Methods) • p = 0.056, determined by Kruskal-Wallis two-parameter ANOVA (values expressed logarithmically were not applicable in normal distribution)

The antibody response generally develops around the eighth day after administration of the recombinant preparation and is present for 21 days. The antibody response becomes stronger after the second administration of the recombinant protein.

All four baboons who received sTNFR-Ι4D / C105db produced antibodies, two out of four animals given sTNFR-Ι 3D / C105db and one out of four baboons given sTNFR-Ι 2.6D / C105db produced antibodies. The size of the antibody response (expressed logarithmically) according to Kruskall-Wallis ANOVA varies significantly between the three groups as a function of time (p <0.05). Post-hoc analysis suggests that a significant difference in antibody response is predominantly between animals receiving sTNFR-Ι 4D / C105db and sTNFR-Ι 2.6D / C105db with moderate (and insignificant) responses in animals treated with sTNFR-I

3D / C105db.

A correlation is observed between antibody production and the change in secretion between the two 21-day studies (p <0.01). In animals with a strong antibody response to the first administration of recombinant

As expected, the protein is cleared more rapidly after the second administration. The change in elimination after the first and second administration is compared in animals that showed an antibody response (n = 7) and not (n = 5) [Fig.13].

Direct cytotoxicity on the ME-180 cell line and neutralization capacity in the L-929 assay was evaluated for antibodies found in baboon plasma in a selected number of animals. No cytotoxicity or neutralization was found in antibodies against the three recombinant proteins of interest.

As demonstrated during the A phase of baboon study, animals that develop the strongest antibody response show the fastest increase in secretion of the recombinant preparation after its second administration. Such findings suggest that the antibody response may shorten the half-life and hence the therapeutic efficacy of the recombinant preparation and therefore dose adjustment may be required. However, no adverse clinical response to the presence of antibodies after the second administration of the recombinant preparation appears to be demonstrated. Therapeutic efforts aimed at modifying recombinant preparations that would reduce immunogenicity and do not significantly affect the half-life of the efficacy are directed primarily at reducing the need to increase the dose, not reducing the risk of adverse clinical response.

Results of Part 1 of Phase B:

In conclusion, all three recombinant preparations are almost equally effective in untouched animals and prevent cytokine-induced damage during E. coli when administered at a dose of 1.0 mg / kg BW. Survivors survived one of the four placebo baboons, four of the four baboons treated with sTNFR-I 4D / C105db or sTNFR-Ι 3D / C105db, and three of the four baboons treated with sTNFR-I 2.6D / C105db survived. All three recombinant preparations prevent the biological activity of TNFα and have sufficient neutralizing capacity.

106

Part II:

The aim of the Part II study in baboons is to decide whether repeated exposure of animals (ie 3 separate applications) to different sTNFR-I proteins results in increased immunogenicity and reduced half-life. Furthermore, this study should compare the immunogenicity and pharmacokinetics of several sTNFR-I preparations including sTNFR-Ι4D / C105db and sTNFR-Ι 2.6D / C105db, as well as sTNFR-I 2.6D / N105-t-BuPEG (33 kDa) and sTNFR- Ι 4D / N105-t-BuPEG (33 kDa). Finally, this study is aimed at assessing the clinical significance of the antibody response and altered secretion to the subsequent response to TNFα (E. coli bacteraemia) damage.

On days 0, 21 and 42, various recombinant proteins (sTNFR-Ι4D / C105db, sTNFR-Ι2,6D / C105db, sTNFR-I 2,6D / N105-t- BuPEG (33 kDa) and sTNFR-D4D / N105-t-BuPEG (33 kDa) Baboons receive 2.0 mg / kg BW of the respective protein on day 63. On day 65 (i.e., 48 hours later) baboons are administered a lethal dose of E. coli as described above in Part I. The main findings of Part II are as follows.

Results (Part II):

In intact baboons, sTNFR-D4D / N105-t-BuPEG (33 kDa) and sTNFR-I 2.6D / N105-t-BuPEG (33 kDa) generally have longer half-lives than sTNFR-I 4D / Cl05db and sTNFR-Ι 2 , 6D / Cl05db regardless of the number of domains. The half-life is in the range of 30-35 hours for monopegylated forms of sTNFR-I as opposed to 10-20 hours for dimeric pegylated forms. In addition, sTNFR-Ι 4D / N105-t-BuPEG (33 kDa) and sTNFR-Ι 4D / C105db have longer half-lives than sTNFR-I 2, 6D / N105-t-BuPEG (33 kDa) and sTNFR-I

2.6D / C105db.

sTNFR-Ι 4D / C105db and sTNFR-2,6 2,6D / C105db are also immunogenic, with a slight trend towards lower immunogenicity in sTNFR-Ι 2.6D / C105db. However, only sTNFR-Ι4D / C105db is less excreted after repeated administration. sTNFR-Ι4D / N105-t-BuPEG (33 kDa) and sTNFR-1 2.6D / N105-t-BuPEG (33 kDa) are neither antigenic nor their secretion rate does not change significantly after repeated administration.

In vitro immunoreactivity (ELISA sandwich capture) to other recombinant proteins in serum, taken from each baboon (N = 3) on days 21, 42 and 61, is determined using a different recombinant

107 of the protein used as the capture antigen. E.g. baboon serum treated with sTNFR-2,6 2,6D / N105-t-BuPEG (33 kDa) Day 21 (Tab.9) does not react with tNFR-Ι 4D / C105db or with tNFR-Ι 4D / N105 used as capture (capture) antigens on an ELISA plate.

Antibody response with a titre> 1: 400 is considered positive. Data from day 42 and day 61 are summarized in Tab. 10 and 11. It is considered important that serum from 1 baboon given sTNFR-I 2.6D / N105-t-BuPEG (33 kDa) gave a positive in vitro response when tested with sTNFR-Ι4D / Cl05db capture antigen (tab.

11).

Antibody response with a titre> 1: 400 is considered positive. Data from day 42 and day 61 are summarized in Tab. It is considered important that serum from 1 baboon given sTNFR-I 2.6D / N105-t-BuPEG (33 kDa) gave a positive in vitro response when tested with sTNFR-Ι 4D / C105db capture antigen.

Depending on the efficacy of preventing TNFα-induced damage in baboons given three times a recombinant preparation, proteins can be sorted starting from the most effective as follows: (1) sTNFR-Ι4D / C105db, (2) sTNFR-Ι 2.6D / C105db, (3) sTNFR- Ι 4D / N105-t-BuPEG (33 kDa) and (4) sTNFR-I 2.6D / N105-t-BuPEG (33 kDa) (based on survival, systemic organ failure (MSOF), IL-6 and white blood cells (WBC) in serum). A defect in this study is that it did not take into account the differences in the ability of different recombinant proteins to neutralize TNF.

108

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ω 2 TI YEAH 1 H σ \ O R O <J1 Q. σ -> c> tn ω ϋ H) co \ 2 x - O 2 O H JO fl) o 1 - σ H 1 r + 1 CD C ti ra O - .. tm co σ i-3 co \ 2 Τ '2 τ Ο Η> yo »1 ο I - σι η 1 rt 1 03 / C ΤΙ W ο sTNFR-Ι 2.6D / C105db w a co · h3 ω σι Jg π σ τι α \ jo 0) 2 1 - Η Η Ο Cn (T 1 C0 C τ Μ Ο Number of animals: n = 3 3/3 neg sTNFR-I 2.6D / C105db 3/3 neg i _and sTNFR-I 2.6D / N105-t- BuPEG (32kDa) 3/3 neg STNFR-I 4D / N105-t- BuPEG (32kDa) sTNFR-I 4D / C105-t- BuPEG (32kDa) 1/3 reag. (400) 3/3 neg 3/3 neg 3/3 neg > Ě> w σ h \ 2 Ω TI JO »JO O 1 cn H and AT 3/3 neg 3/3 neg 3/3 neg 3/3 neg sTNFR-I 4D / N105

Baboon immune responses • · · · οττ

• · · · · · ·

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C. Chimpanzee

The aim of this study is to determine the immunogenicity of various forms of sTNFR-I that are repeatedly injected with I.V. by chimpanzees within 1 month. The following forms of sTNFR-Ι are tested: sTNFR-I 2.6D / C105db, sTNFR-Ι 4D / C105db, sTNFR-Ι 4D / ClO5-t-BuPEG (33 kDa), sTNFR-1 2, 6D / N105-t- BuPEG (33 kDa) and sTNFR-D4D / N105-t-BuPEG (33 kDa). There are three chimpanzees in one experimental group.

The dosing regimen and parameters of this study are as follows: Each chimpanzee receives a test sample as an intravenous injection (0.1 mg / kg) twice weekly on Mondays and Fridays for four weeks (a total of 8 doses). The dose volume varies depending on the concentration of the administered sample. 5 ml of serum is collected from each animal on day 0 prior to the start of the experiment. Additional serum samples are taken just prior to administration on day 7, 14, 21 and 28.

Basic data on immunogenicity in chimpanzees are shown in Tab. 12.

By day 28, all animals (N = 3) treated with sTNFR-I 4D / C105db or sTNFR-Ι 2.6D / C105db had a positive reaction (as determined by ELISA) with the highest titers measured 1:12 800 and 1: 3200 (Table 12). (Note: In this section of the experiment, all "immunization antigens are used as the corresponding capture antigens immobilized on an ELISA plate). One animal immunized with sTNFR-Ι4D / N105-t-BuPEG (33 kDa) had a positive antibody response on days 21 and 28 (Tab.12). Importantly, no animals immunized with sTNFR-D4D / C105-t-BuPEG (33 kDa) or sTNFR-I 2.6D / N105-t-BuPEG (33 kDa) during this experiment were observed to produce anti sTNFR-Ι antibodies. (tab.12).

Serum collected on day 28 for all chimpanzees (N = 3) immunized with various forms of sTNFR-Ι is tested for in vitro immunoreactivity against other recombinant sTNFR-protein proteins used as capture antigens in the ELISA as described above in baboon experiments. . A positive response is an antibody response with a titer greater than 1: 400.

Significantly, chimpanzee serum treated with sTNFR-I 2.6D / N105-t-BuPEG (33 kDa) "does not react with sTNFR-Ι 4D / C105db or sTNFR-Ι 4D / N105 when these proteins are used as capture antigens on the ELISA plate (Table 13). The same is observed in animals immunized with sTNFR-Ι4D / C105db, sTNFR-Ι4D / C105-t-BuPEG (33 kDa) and administered sTNFR-Ι4D / N105-t-BuPEG (33 kDa) (tab.13)

112

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Chimpanzee Immune Results • ·

Example IV

EAE is an acute or chronic relapsing inflammatory demyelinating CNS disease that results from the action of neuroantigens such as myelin basic protein (MBP) on genetically sensitive animals. EAE is a recognized and frequently used model of acute human MS.

Lewis rats (Jackson Laboratories, Bar Harbor, ME) are immunized on left hind foot pedal (day 0) with 0.1 ml of an emulsion containing myelin basic protein (MBP) in complete Freund's adjuvant dissolved in PBS with the same volume of complete Freund's adjuvant (CFA) containing 5 mg / ml Mycobacterium tuberculosis H37Rα (Difco Lab MI). Control rats are given 0.1 ml of PBS / CFA emulsion without MBP into the left hind paw pedal.

Clinical evaluation of the disease is based on the usual 0-5 scoring system. The following states are assigned to each value: 0, normal; 0.5, partial tail tone loss; 1, complete tail tone loss; 2, dragging one hind limb; 3, paralysis of both hind limbs; 4, dying; 5, death. Injections of recombinant sTNFR-Ι preparations or blank are administered by S.C. at a dose of 1 mg / kg every other day starting on day 9 after immunization. The experiment is completed on day 21 of all animals. The results are expressed in two forms, both as a clinical severity score over time and the integrated score of each rat throughout the course of the disease is calculated as the area defined by the daily clinical score versus time curve. The values of the integrated clinical scores of the treated groups are compared to those of the control group by means of the Mann-Whitney test.

The animals that were given the blank showed the onset of the disease around day 10, the disease peaked on day 16 and then subsided. sTNFR-Ι4D / C106db alleviates clinical signs by about 73% compared to blank treated animals. sTNFR-1 4D / C105t-BuPEG (33 kDa) also alleviates clinical symptoms by about 85%. sTNFR-Ι4D / C105-t-BuPEG (33 kDa) and sTNFR-I 2.6D / N105-t-BuPEG (33 kDa) are equally effective in relieving clinical symptoms (64 and 57%).

It can be concluded that truncated forms of sTNFR appear to be clinically effective in this animal model of MS.

115 ·· ·

The present invention has been described above generally and in the form of recommended specific procedures and experiments. However, it will be apparent to those skilled in the art that other variations and modifications can be made based on this disclosure.

116 φ · · · · · · · · · · · ♦ · · · <· φ · <· <·

LIST OF SEQUENCES (1) GENERAL INFORMATION:

(i) APPLICANT: AMGEN INC.

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117 · φ ((((((2 2 2 2 2 2 2 2 2 2 2 2 2

(i) Sequence characteristics (A) Length: 483 base pairs (B) Type: nucleic acid (C) Number of strands: unknown (D) Topology: unknown (ii) Molecule type: cDNA (ix) Properties:

(A) Name / Key: CDS (B) Location: 1..483 (xi) Description of sequence: SEQ ID NO: 1:

GAT Asp 1 AGT Ser GTG Wall TGT Cys ccc For 5 CAA GGA AAA MELT Tyr ATC CAC CCT For CAA Gin AAT Asn AAT Asn 15 Dec TCG Ser 48 Gin Gly Lys Ile 10 His ATT TGC TGT ACC AAG TGC CAC AAA GGA ACC TRAY TTG TRAY AAT GAC TGT 96 Ile Cys Cys Thr 20 May Lys Cys His Lys Gly 25 Thr Tyr Leu Tyr Asn 30 Asp Cys CCA GGC CCG GGG CAG GAT ACG GAC TGC AGG GAG TGT GAG AGC GGC TCC 144 For Gly For 35 Gly Gin Asp .Thr Asp 40 Cys Arg Glu Cys Glu 45 Ser Gly Ser TTC ACC GCT TCA GAA AAC CAC CTC AGA CAC TGC CTC AGC TGC TCC AAA 192 Phe Thr 50 Ala Ser Glu Asn His 55 Leu Arg His Cys Leu 60 Ser Cys Ser Lys TGC CGA AAG GAA ATG GGT CAG GTG GAG ATC TCT TCT TGC ACA GTG GAC 240 Cys 65 Arg Lys Glu Met Gly 70 Gin Wall Glu Ile Ser 75 Ser Cys Thr Wall Asp 80 CGG GAC ACC GTG TGT GGC TGC AGG AAG AAC CAG TRAY CGG CAT MELT TGG 288 Arg Asp Thr Wall Cys 85 Gly Cys Arg Lys Asn 90 Gin Tyr Arg His Tyr 95 Trp AGT GAA AAC CTT TTC CAG TGC TTC AAT TGC AGC CTC TGC CTC AAT GGG 336 Ser Glu Asn Leu 100 ALIGN! Phe Gin Cys Phe Asn 105 Cys Ser Leu Cys Leu 110 Asn Gly ACC GTG CAC CTC TCC TGC CAG GAG AAA CAG AAC ACC GTG TGC ACC TGC 384 Thr Wall His 115 Leu Ser Cys Gin Glu 120 Lys Gin Asn Thr Wall 125 Cys Thr Cys CAT GCA GGT TTC TTT CTA AGA GAA AAC GAG TGT GTC TCC TGT AGT AAC 432 His Ala 130 Gly Phe Phe Leu Arg 135 Glu Asn Glu Cys Wall 140 Ser Cys Ser Asn TGT AAG AAA AGC CTG GAG TGC ACG AAG TTG TGC CTA CCC CAG ATT GAG 480 Cys 145 Lys Lys Ser Leu Glu 150 Cys Thr Lys Leu Cys 155 Leu For Gin Ile Glu 160

AAT 483

Asn

118 (2) Information about SEQ ID NO: 2:

(i) Sequence characteristics (A) Length: 161 amino acids (B) Type: amino acid (D) Topology: linear (ii) Molecule type: protein (xi) Sequence description: SEQ ID NO: 2:

Asp 1 Ser Val Cys For 5 Gin Gly Lys Tyr Ile 10 His For Gin Asn Asn 15 Dec Ser Ile Cys Cys Thr Lys Cys His Lys Gly Thr Tyr Leu Tyr Asn Asp Cys 20 May 25 30 For Gly For Gly Gin Asp Thr Asp Cys Arg Glu Cys Glu Ser Gly Ser 35 40 45 Phe Thr Ala Ser Glu Asn His Leu Arg His Cys Leu Ser Cys Ser Lys 50 55 60 Cys Arg Lys Glu Met Gly Gin Wall Glu Ile Ser Ser Cys Thr Wall Asp 65 70 75 80 Arg Asp Thr Wall Cys Gly Cys Arg Lys Asn Gin Tyr Arg His Tyr Trp 85 90 95 Ser Glu Asn Leu Phe Gin Cys Phe Asn Cys Ser Leu Cys Leu Asn Gly 100 ALIGN! 105 110 Thr Wall His Leu Ser Cys Gin Glu Lys Gin Asn Thr Wall Cys Thr Cys 115 120 125 His Ala Gly Phe Phe Leu Arg Glu Asn Glu Cys Wall Ser Cys Ser Asn 130 135 140 Cys Lys Lys Ser Leu Glu Cys Thr Lys Leu Cys Leu For Gin Ile Glu 145 150 155 160

Asn (2) Information about SEQ ID NO: 3:

(i) Sequence characteristics (A) Length: 332 base pairs (B) Type: nucleic acid (C) Number of strands: unknown (D) Topology: unknown (ii) Molecule type: cDNA (ix) Properties:

(A) Name / Key: CDS (B) Location: 4..324

119

.. · · -. ·. 00 »0 • 0 '00, •. • * • 0 '· 0 '' • '· • • 0 • 9 • • · • · • 0 • «· 99 9 • • · · • 0 0 · «· • · · 0 0 • • • · • 0 «

(xi) Description sequence SEQ ID NO :: 3: CAT ATG GAC AGC GTT TGC CCC CAA GGA Met 1 Asp Ser Wall Cys 5 For Gin Gly AAT TCG ATT TGC TGT ACC AAG TGC CAC Asn Ser lle Cys Cys 20 May Thr Lys Cys His GAC TGT CCA GGC CCG GGG CAG GAT ACG Asp Cys For Gly 35 For Gly Gin Asp Thr 40 GGC TCC TTC ACC GCT TCA GAA AAC CAC Gly Ser Phe 50 Thr Ala Ser Glu Asn 55 His TCC AAA TGC CGA AAG GAA ATG GGT CAG Ser Lys 65 Cys Arg Lys Glu Met 70 Gly Gin GTG GAC CGG GAC ACC GTG TGT GGC TGC Wall 80 Asp Arg Asp Thr Wall 85 Cys Gly Cys MELT TGG AGT GAA AAC CTT TTC CAG TGC Tyr Trp Ser Information about Glu SEQ Asn Leu ID NO: 3: Phe Gin Cys

(i) Sequence characteristics

AAA TAC ATC CAC CCT CAA AAT

Lys Tyr lle His Gin Asn

15 Dec

AAA GGA ACC TAC

Lys Gly Thr Tyr Leu Tyr Asn

30

GAC TGC AGG GAG

Asp Cys Glu Cys Glu Ser

CTC AGA CAC TGC

Leu Arg His Cys

GTG GAG ATC TCT TCT TGC ACA 240

Val Glu Ser Ser Cys Thr

AGG AAG AAC CAG TAC CGG CAT 288

Arg Lys Asn Gin Tyr

95

TTC TGC TGA TAGGATCC 332

Phe Cys *

105 (A) Length: 107 amino acids (B) Type: amino acid (D) Topology: lineami (ii) Molecule type: protein (xi) Sequence description: SEQ ID NO: 4:

Met Asp Ser Val Gys Gly Lys Tyr lle His Gn Asn Asn 15 10 15

Ser Lle Cys Thys Lys Cys His Lys Gly Thr Tyr Leu Tyr Asp 20 25 30

Cys Pro Gly Pro Gly Gin Asp Thr Asp Cys Arg Glu Cys Glu Ser Gly 35 40 45

Ser Phe Thr Ala Ser Glu As His His Leu Arg His Cys Leu Ser Cys Ser 50 55 60

Lys Cys Arg Lys Glu Met Gly Gin Val Glu L Ser Ser Cys Thr Val 65 70 75 80

120. ·: · # ··. .. * · ··· ..

• · · · · <<*..............

· · · · · · · · · · · · · · · · · · · · · · · ·

Asp Arg Asp Thr Wall Cys Gly Cys Arg Lys Asn Gin Tyr 65 90 95 Trp Ser Glu Asn Leu Phe Gin Cys Phe Cys * 100 ALIGN! 105 Information on SEQ ID NO: 6:

(i) Sequence characteristics (A) Length: 339 base pairs (B) Type: nucleic acid (C) Number of strands: unknown (D) Topology: unknown (ii) Molecule type: cDNA (ix) Properties:

(A) Name / Key: CDS (B) Location: 4..333

(xí: ) Description of sequence: SEQ id: NO: 5: CAT ATG GAC AGC GTT TGC ccc CAA GGA AAA MELT ATC CAC CCT CAA AAT 48 Met Asp Ser Wall Cys For Gin Gly Lys Tyr Ile His For Gin Asn 1 5 10 15 Dec AAT TCG ATT TGC TGT ACC AAG TGC CAC AAA GGA ACC TRAY TTG TRAY AAT 96 Asn Ser Ile Cys Cys Thr Lys Cys His Lys Gly Thr Tyr Leu Tyr Asn 20 May 25 30 GAC TGT CCA GGC CCG GGG CAG GAT ACG GAC TGC AGG GAG TGT GAG AGC 144 Asp Cys For Gly For Gly Gin Asp Thr Asp Cys Arg Glu Cys Glu Ser 35 40 45 GGC TCC TTC ACC GCT TCA GAA AAC CAC CTC AGA CAC TGC CTC AGC TGC 192 Gly Ser Phe Thr Ala Ser Glu Asn His Leu Arg His Cys Leu Ser Cys 50 55 60 TCC AAA TGC CGA AAG GAA ATG GGT CAG GTG GAG ATC TCT TCT TGC ACA 240 Ser Lys Cys Arg Lys Glu Met Gly Gin Wall Glu Ile Ser Ser Cys Thr 65 70 75 GTG GAC CGG GAC ACC GTG TGT GGC TGC AGG AAG AAC CAG TRAY CGG CAT 288 Wall Asp Arg Asp Thr Wall Cys Gly Cys Arg Lys Asn Gin Tyr Arg His 80 85 90 95 MELT TGG AGT GAA AAC CTT TTC CAG TGC TTC AAT TGC TCT CTG TAA 333 Tyr Trp Ser Glu Asn Leu Phe Gin Cys Phe Asn Cys Ser Leu * 100 ALIGN! 105 110

AAGCTT 339

121 · · · · · · · · · · · · · · · · φ · φ · · · φ · φ · · · 2 · 2 · 2 · 2 · 2 · 6:

(i) Sequence characteristics (A) Length: 110 amino acids (B) Type: amino acid (D) Topology: linear (ii) Molecule type: protein

(xi) Description of the sequence Ince: SEQ ID NO: 6 Met Asp Ser Wall Cys Pro Gin Gly Lys Tyr Ile His For Gin Asn Asn 1 5 10 15 Dec Ser Ile Cys Cys Thr Lys Cys His Lys Gly Thr Tyr Leu Tyr Asn Asp 20 May 25 30 Cys For Gly For Gly Gin Asp Thr Asp Cys Arg Glu Cys Glu Ser Gly 35 40 45 Ser Phe Thr Ala Ser Glu Asn His Leu Arg His Cys Leu Ser Cys Ser 50 55 60 Lys Cys Arg Lys Glu Met Gly Gin Wall Glu Ile Ser Ser Cys Thr Wall 65 70 75 80 Asp Arg Asp Thr Val Cys Gly Cys Arg Lys Asn Gin Tyr Arg His Tyr 85 90 95 Trp Ser Glu Asn Leu Phe Gin Cys Phe Asn Cys Ser Leu * 100 ALIGN! 105 110

(2) Information about SEQ ID NO: 7:

(i) Sequence characteristics (A) Length: 333 base pairs (B) Type: nucleic acid (C) Number of strands: unknown (D) Topology: unknown (ii) Molecule type: cDNA (ix) Properties:

(A) Name / Key: CDS (B) Location: 4..324 (xi) Sequence Description: SEQ ID NO: 7:

CAT ATG Met 1 GAC Asp AGC Ser GTT TGC CCC CAA GGA AAA TAT ATC CAC CAA Gin AAT Asn 15 Dec 48 Wall Cys 5 For Gin Gly Lys Tyr 10 Ile His For AAT TCG ATT TGC TGT ACC AAG TGC CAC AAA GGA ACC TRAY TTG TRAY AAT 96 Asn Ser Ile Cys Cys Thr Lys Cys His Lys Gly Thr Tyr Leu Tyr Asn 20 May 25 30

122 • t.:.:. ··

• · • · • · • · · · · · • • • · ♦ · • · • · · • • « • • • · ... »· ' »· • · · • • · GAC TGT CCA GGC CCG GGG CAG GAT ACG GAC TGC AGG GAG TGT GAG AGC Asp Cys For Gly For Gly Gin Asp Thr Asp Cys Arg Glu Cys Glu Ser 35 40 45 GGC TCC TTC ACC GCT TCA GAA AAC CAC CTC AGA CAC TGC CTC AGC TGC Gly Ser Phe Thr Ala Ser Glu Asn His Leu Arg His Cys Leu Ser Cys 50 55 60 TCC AAA TGC CGA AAG GAA ATG GGT CAG GTG GAG ATC TCT TCT TGC ACA Ser Lys Cys Arg Lys Glu Met Gly Gin Wall Glu Ile Ser Ser Cys Thr 85 70 75 GTG GAC CGG GAC ACC GTG TGT GGT TGC AGG AAG AAC CAG TRAY CGG CAT Wall A3p Arg Asp Thr Wall Cys Gly Cys Arg Lys Asn Gin Tyr Arg His 80 85 90 95 MELT TGG AGT GAA AAC CTT TTC CAG TGC TTC AAT TAA TAGGGATCC Tyr Trp Ser Glu Asn Leu Phe Gin Cys Phe Asn * 100 ALIGN! 105

144

192

240

288

333 (2) Information about SEQ ID NO: 9:

(i) Sequence characteristics (A) Length: 107 amino acids (B) Type: amino acid (D) Topology: linear (ii) Molecule type: protein (xi) Sequence description: SEQ ID NO: 8:

Met Asp Ser Wall Cys For Gin Gly Lys Tyr Ile His For Gin Asn Asn 1 5 10 15 Dec Ser Ile Cys Cys Thr Lys Cys His Lys Gly Thr Tyr Leu Tyr Asn Asp 20 May 25 30 Cys For Gly For Gly Gin Asp Thr Asp Cys Arg Glu Cys Glu Ser Gly 35 40 45 Ser Phe Thr Ala Ser Glu Asn His Leu Arg His Cys Leu Ser Cys Ser 50 • 55 60 Lys Cys Arg Lys Glu Met Gly Gin Wall Glu Ile Ser Ser Cys Thr Wall 65 70 75 80 Asp Arg Asp Thr Wall Cys Gly Cys Arg Lys Asn Gin Tyr Arg His Tyr 85 90 95 Trp Ser Glu Asn Leu Phe Gin Cys Phe Asn *

100 105

123 • · · 4

4 444 444 444 · 4 4 · (2) Information about SEQ ID NO: 9:

(i) Sequence characteristics (A) Length: 285 base pairs (B) Type: nucleic acid (C) Number of strands: unknown (D) Topology: unknown (ii) Molecule type: cDNA (ix) Properties:

(A) Name / Key: CDS (B) Location: 4..279 (xi) Sequence Description: SEQ ID NO: 9:

CAT ATG TGT ACC Thr AAG Lys TGC Cys 5 CAC His AAA Lys GGA Gly ACC Thr TRAY Tyr 10 TTG Leu TRAY Tyr AAT Asn GAC Asp TGT Cys 15 Dec 48 Met 1 Cys CCA GGC CCG GGG CAG GAT ACG GAC TGC AGG GAG TGT GAG AGC GGC TCC 96 For Gly For Gly Gin Asp Thr Asp Cys Arg Glu Cys Glu Ser Gly Ser 20 May 25 30 TTC ACC GCT TCA GAA AAC CAC CTC AGA CAC TGC CTC AGC TGC TCC AAA 144 Phe Thr Ala Ser Glu Asn His Leu Arg His Cys Leu Ser Cys Ser Lys 35 40 45 TGC CGA AAG GAA ATG GGT CAG GTG GAG ATC TCT TCT TGC ACA GTG GAC 192 Cys Arg Ly3 Glu Met Gly Gin Wall Glu Ile Ser Ser Cys Thr Wall Asp 50 55 60 CGG GAC ACC GTG TGT GGC TGC AGG AAG AAC CAG TRAY CGG CAT MELT TGG 240 Arg Asp Thr Wall Cys Gly Cys Arg Lys Asn Gin Tyr Arg His Tyr Trp 65 70 75 AGT GAA AAC CTT TTC CAG TGC TTC AAT TGC TCT CTG TAA AAGCTT 285 Ser Glu Asn Leu Phe Gin Cys Phe Asn Cys Ser Leu 80 85 90 (2) Information on SEQ ID NO: 0:

(i) Sequence characteristics (A) Length: 92 amino acids (B) Type: amino acid (D) Topology: linear (ii) Molecule type: protein (xi) Sequence description: SEQ ID NO: 10:

Met Cvs Thr Lys Cys His Lys Gly Thr

5 10 15

Gly Pro Gly Gin Ser Gly Ser Phe

25 30

124 ·· .. ·· ·,. · 3 3. «3 · 9 ·· '·' · ..... '·· 1» - · ·· - ·' * · «·· * · · · · 3 ··· · 3 · 3 3 · 3 333 3 3 3 · · 33 3 ·

Thr Ala Ser Glu Asn His Leu Arg His Cys Leu Ser Cys Ser Lys Cys 35 40 45 Arg Lys Glu Met Gly Gin Wall Glu Ile Ser Ser Cys Thr Wall Asp Arg 50 55 60 Asp Thr Wall Cys Gly Cys Arg Lys Asn Gin Tyr Arg His Tyr Trp Ser 65 70 75 80 Glu Asn Leu Phe Gin Cys Phe Asn Cys Ser Leu * 85 90

(2) Information ο SEQ ID Ν0: 11:

(i) Sequence characteristics (A) Length: 315 base pairs (B) Type: nucleic acid (C) Number of strands: unknown (D) Topology: unknown (ii) Molecule type: cDNA (ix) Properties:

(A) Name / Key: CDS (B) Location: 4..309 (xi) Sequence Description: SEQ ID NO: 11:

CAT ATG TAT ATC CAC CCT CAA Pro Gin 5 AAT AAT TCG Ser ATT Ile 10 TGC Cys TGT Cys ACC AAG TGC 48 Met 1 Tyr Ile His Asn Asn Thr Lys Cys 15 Dec CAC AAA GGA ACC TRAY TTG TRAY AAT ' GAC TGT CCA GGC CCG GGG CAG GAT 96 His Lys Gly Thr Tyr Leu Tyr Asn Asp Cys For Gly For Gly Gin Asp 20 May 25 30 ACG GAC TGC AGG GAG TGT GAG AGC GGC TCC TTC ACC GCT TCA GAA AAC 144 Thr Asp Cys Arg Glu Cys Glu Ser Gly Ser Phe Thr Ala Ser Glu Asn 35 40 45 CAC CTC AGA CAC TGC CTC AGC TGC TCC AAA TGC CGA AAG GAA ATG GGT 192 His Leu Arg His Cys Leu Ser Cys Ser Lys Cys Arg Lys Glu Met Gly 50 55 60 CAG GTG GAG ATC TCT TCT TGC ACA GTG GAC CGG GAC ACC GTG TGT GGC 240 Gin Wall Glu Ile Ser Ser Cys Thr Wall Asp Arg Asp Thr Wall Cys Gly 65 70 75 TGC AGG AAG AAC CAG TRAY CGG CAT MELT TGG AGT GAA AAC CTT TTC CAG 288 Cys Arg Lys Asn Gin Tyr Arg His Tyr Trp Ser Glu Asn Leu Phe Gin 80 85 90 95 TGC TTC AAT TGC TCT CTG TAA AAGCTT 315 Cys Phe Asn Cys Ser Leu »»

100 ALIGN!

125 • · 4 · · ·

4 · 44 * 44

4 «4 (2) Information about SEQ ID NO: 12:

(i) Sequence characteristics (A) Length: 102 amino acids (B) Type: amino acid (D) Topology: linear (ii) Molecule type: protein (xi) Sequence description: SEQ ID NO: 12:

Met 1 Tyr Ile His For 5 Gin Asn Asn Ser Ile 10 Cys Cys Thr Lys Cys 15 Dec His Lys Gly Thr Tyr 20 May Leu Tyr Asn Asp Cys For Gly For Gly Gin 30 Asp Thr Asp Cys Arg 35 Glu Cys Glu Ser Gly Ser Phe Thr Ala Ser 45 Glu Asn His Leu Arg His Cys Leu Ser Cys Ser Lys Cys Arg Lys C. C \ Glu Met Gly Gin 50 55 oU Wall 65 Glu Ile Ser Ser Cys Thr Val Asp Arg Asp 75 Thr Wall Cys Gly Cys 80 Arg Lys Asn Gin Tyr 85 Arg His Tyr Trp Ser 90 Glu Asn Leu Phe Gin 95 Cys

Phe Asn Cys Ser Leu

100 (2) Information about SEQ ID NO: 13:

(i) Sequence characteristics (A) Length: 294 base pairs (B) Type: nucleic acid (C) Number of strands: unknown (D) Topology: unknown (ii) Molecule type: cDNA (ix) Properties:

(A) Name / Key: CDS (B) Location: 4..288 (xi) Sequence Description: SEQ ID NO: 13:

CAT ATG Met 1 TCG Ser ATT Ile AGC Ser TGT ACC AAG TGC CAC AAA GGA ACC TAC 48 Cys 5 Thr Lys Cys His Lys 10 Gly Thr Tyr Leu Tyr 15 Dec AAT GAC TGT CCA GGC CCG GGG CAG GAT ACG GAC TGC AGG GAG TGT GAG 96 Α3Π Asp Cys For Gly For Gly Gin Asp Thr Asp Cys Arg Glu Cys Glu 20 May 25 30

126 ·

»E e e e e e e e e« ««> · * * * * * * * * * * * * * * ·· «·

AGC Ser GGC Gly TCC Ser TTC Phe 35 ACC Thr GCT Ala TCA Ser GAA AAC CAC CTC Leu AGA Arg CAC His TGC Cys 45 CTC Leu AGC Ser - 144 Glu Asn 40 His TGC TCC AAA TGC CGA AAG GAA ATG GGT CAG GTG GAG ATC TCT TCT TGC 192 Cys Ser Ly3 Cys Arg Lys Glu Met Gly Gin Wall Glu Ile Ser Ser Cys 50 55 60 ACA GTG GAC CGG GAC ACC GTG TGT GGC TGC AGG AAG AAC CAG TRAY CGG 240 Thr Wall Asp Arg Asp Thr Wall Cys Gly Cys Arg Lys Asn Gin Tyr Arg 65 70 75 CAT MELT TGG AGT GAA AAC CTT TTC CAG TGC TTC AAT TGC TCT CTG TAA 288 His Tyr Trp Ser Glu Asn Leu Phe Gin Cys Phe Asn Cys Ser Leu * 80 85 90 95

AAGCTT (2) Information about SEQ ID NO: 14:

(i) Sequence characteristics (A) Length: 95 amino acids (B) Type: amino acid (D) Topology: linear (ii) Molecule type: protein (xi) Sequence description: SEQ ID NO: 14:

Met 1 Ser Ile Ser Cys 5 Thr Lys Cys His Lys 10 Gly Thr Tyr Leu Tyr 15 Dec Asn Asp Cys For Gly 20 May For Gly Gin Asp Thr 25 Asp Cys Arg Glu Cys 30 Glu Ser Gly Ser Phe 35 Thr Ala Ser Glu Asn 40 His Leu Arg His Cys 45 Leu Ser Cys Ser Lys 50 Cys Arg Lys Glu Met 55 Gly Gin Wall Glu Ile 60 Ser Ser Cys Thr Wall 65 Asp Arg Asp Thr Wall 70 Cys Gly Cys Arg Lys 75 Asn Gin Tyr Arg His 80 Tyr Trp Ser Glu Asn 85 Leu Phe Gin Cys Phe 90 Asn Cys Ser Leu AND 95

(2) Information about SEQ ID NO: 15:

(i) Sequence characteristics (A) Length: 4 amino acids (B) Type: amino acid (C) Number of chains: unknown (D) Topology: unknown

127 * · (ii) Molecule type: protein (xi) Sequence description: SEQ ID NO: 15:

Asn Ser Ile Cys 1 (2) Information about SEQ ID NO: 16:

(i) Sequence characteristics (A) Length: 5 amino acids (B) Type: amino acid (C) Number of chains: unknown (D) Topology: unknown (ii) Molecule type: protein (xi) Sequence description: SEQ ID NO: 16:

Asn Asn Ser Ile Cys 1 5 (2) Information about SEQ ID NO: 17:

(i) Sequence characteristics (A) Length: 6 amino acids (B) Type: amino acid (C) Number of chains: unknown (D) Topology: unknown (ii) Molecule type: protein (xi) Sequence description: SEQ ID NO: 17:

Gin Asn Asn Ser Ile Cys 1 5 (2) Information about SEQ ID NO: 18:

(i) Sequence characteristics (A) Length: 7 amino acids (B) Type: amino acid (C) Number of chains: unknown (D) Topology: unknown (ii) Molecule type: protein (xi) Sequence description: SEQ ID NO: 18:

Pro Gin Asn Asn Ser Ile Cys 1

128 * · t · t 99 · ·

• 9 ♦ · (2) Information about SEQ ID NO: 19:

(i) Sequence characteristics (A) Length: 8 amino acids (B) Type: amino acid (C) Number of chains: unknown (D) Topology: unknown (ii) Molecule type: protein (xi) Description of sequence: SEQ ID NO: 19:

His Pro Gin Asn 2

Asn Ser Ile Cys 5 (2) Information about SEQ ID NO: 20:

(i) Sequence characteristics (A) Length: 9 amino acids (B) Type: amino acid (C) Number of chains: unknown (D) Topology: unknown (ii) Molecule type: protein (xi) Sequence description: SEQ ID NO: 20:

Ile His Pro Gin Asn Asn Ser Ile Cys 1 5 (2) Information about SEQ ID NO: 21:

(i) Sequence characteristics (A) Length: 10 amino acids (B) Type: amino acid (C) Number of chains: unknown (D) Topology: unknown (ii) Molecule type: protein (xi) Sequence description: SEQ ID NO: 21:

Tyr Ile His Gin Asn Asn Ser Ile Cys 15 10 (2) Information about SEQ ID NO: 22:

(i) Sequence characteristics (A) Length: 11 amino acids (B) Type: amino acid (C) Number of chains: unknown (D) Topology: unknown (ii) Molecule type: protein

129 (xi) Sequence description: SEQ ID NO: 22:

Lys Tyr Ile His Gin Asn Asn Ser Ile Cys 1 5 10.

(2) Information about SEQ ID NO: 23:

(i) Sequence characteristics (A) Length: 12 amino acids (B) Type: amino acid (C) Number of chains: unknown (D) Topology: unknown (ii) Molecule type: protein (xi) Description of sequence: SEQ ID NO: 23:

Gly Lys Tyr Xle His Pro Gin Asn Asn Ser Ile Cys 15 10 (2) Information about SEQ ID NO: 24:

(i) Sequence characteristics (A) Length: 13 amino acids (B) Type: amino acid (C) Number of chains: unknown (D) Topology: unknown (ii) Molecule type: protein (xi) Sequence description: SEQ ID NO: 24:

Gin Gly Lys Tyr Ile His For Gin Asn Asn Ser Ile Cys 15 1 ° (2) Information about SEQ ID NO: 25:

(i) Sequence characteristics (A) Length: 14 amino acids (B) Type: amino acid (C) Number of chains: unknown (D) Topology: unknown (ii) Molecule type: protein (xi) Sequence description: SEQ ID NO: 25:

Pro Gin Gly Lys Tyr Ile His

(2) Information about SEQ ID NO: 26:

(i) Sequence characteristics (A) Length: 15 amino acids (B) Type: amino acid (C) Number of chains: unknown (D) Topology: unknown (ii) Molecule type: protein (xi) Sequence description: SEQ ID NO: 26:

Cys Pro Gin Gly Lys Tyr Ile His Pro Gin Asn Asn Ser Ile Cys 1 5 10 15 (2) SEQ ID NO: 27:

(i) Sequence characteristics (A) Length: 16 amino acids (B) Type: amino acid (C) Number of chains: unknown (D) Topology: unknown (ii) Molecule type: protein (xi) Sequence description: SEQ ID NO: 27:

Val Cys Gin Gly Lys Tyr Ile His Gin Asn Asn Ser Ile Cys 1 5 10 15 (2) SEQ ID NO: 28:

(i) Sequence characteristics (A) Length: 17 amino acids (B) Type: amino acid (C) Number of chains: unknown (D) Topology: unknown (ii) Molecule type: protein (xi) Sequence description: SEQ ID NO: 28:

Ser Val Cys Pro Gin Gly Lys Tyr Ile His Gin Asn Asn Ser Ile 15 10 15

Cys

131 φ · '- · · · · · φ φ φ φ • •

2 Φφ φ »(2) Information ο SEQ ID ΝΟ: 29:

(i) Sequence characteristics (A) Length: 18 amino acids (B) Type: amino acid (C) Number of chains: unknown (D) Topology: unknown (ii) Molecule type: protein (xi) Description of sequence: SEO ID NO: 29:

Asp Ser Val Cys Pro Gin Gly Lys Tyr Ile His Pro Gin Asn Asn Ser $ 1

Xle Cys (i) Characteristics of sequence (A) Length: 4 amino acids \ i ~ j / (C) Number of strings: unknown (D) Topology: unknown (n) Molecule type: protein (xi) Description of sequence: SEO ID NO: 30 :

Phe Cys Cys Ser 1 (2) Information about SEQ ID NO: 31:

(i) Sequence characteristics (A) Length: 5 amino acids (B) Type: affiino acids (C) Number of chains: unknown (D) Topology: unknown (ii) Molecule type: protein (xi) Sequence description: SEO ID NO: 31:

Phe Cys Cys Ser Leu

132 φ φ · • · · · 'φ φ φ φ • φ φ • • • φ φ φ φ φ φ φ φ φ φ φ φ Informace Informace Informace Informace

(i) Sequence characteristics (A) Length: 6 amino acids (B) Type: amino acid (C) Number of chains: unknown (D) Topology: unknown (ii) Molecule type: protein (xi) Sequence description: SEQ ID NO: 32:

Phe Cys Cys Ser Leu Cys 1 5 (2) Information about SEQ ID NO: 33:

(i) Sequence characteristics (A) Length: 7 amino acids (B) Type: amino acid (C) Number of chains: unknown (D) Topology: unknown (ii) Molecule type: protein (xi) Sequence description: SEQ ID NO: 33:

Phe Cys Cys Ser Leu Cys Leu 1 5 (2) Information about SEQ ID NO: 34:

(i) Sequence characteristics (A) Length: 705 base pairs (B) Type: nucleic acid (C) Number of strands: unknown (D) Topology: unknown (ii) Molecule type: cDNA (ix) Properties:

(A) Name / Key: CDS (B) Location: 1..705 (xi) Sequence Description: SEQ ID NO: 34:

TTG Leu 1 CCC For GCC Ala CAG Gin GTG Wall 5 GCA Ala TTT Phe ACA Thr CCC For TRAY Tyr 10 GCC Ala CCG For GAG Glu CCC For GGG Gly 15 Dec AGC Ser ACA Thr TGC Cys CGG Arg CTC Leu 20 May AGA Arg GAA Glu TRAY Tyr MELT Tyr GAC Asp 25 CAG Gin ACA Thr GCT Ala CAG Gin ATG Met 30 TGC Cys TGC Cys

133 • 9 * 99 · »9 99 '' 9 · - 9 9 ..... · '9' 9 '' '9 • 9 9 9 · 9 9

9,999 9 · 9,999

9

999 98 9 «99

AGC Ser AAG TGC TCG CCG GG CAA CAT GCA AAA GTC TTC TGT ACC AAG ACC 144 Lys Cys 35 Ser For Gly Gin His 40 Ala Lys Wall Phe Cys 45 Thr Lys Thr TCG GAC ACC GTG TGT GAC TCC TGT GAG GAC AGC ACA TRAY ACC CAG CTC 192 Ser Asp Thr Wall Cys Asp Ser Cys Glu Asp Ser Thr Tyr Thr Gin Leu 50 55 60 TGG AAC TGG GTT CCC GAG TGC TTG AGC TGT GGC TCC CGC TGT AGC TCT 240 Trp Asn Trp Wall For Glu Cys Leu Ser Cys Gly Ser Arg Cys Ser Ser 65 70 75 80 GAC CAG GTG GAA ACT CAA GCC TGC ACT CGG GAA CAG AAC CGC ATC TGC 288 Asp Gin Wall Glu Thr Gin Ala Cys Thr Arg Glu Gin Asn Arg Ile Cys 85 90 95 ACC TGC AGG CCC GGC TGG TRAY TGC GCG CTG AGC AAG CAG GAG GGG TGC 336 Thr Cys Arg For Gly Trp Tyr Cys Ala Leu Ser Lys Gin Glu Gly cys 100 ALIGN! 105 110 CGG CTG TGC GCG CCG CTG CGC AAG TGC CGC CCG GGC TTC GGC GTG GCC 384 Arg Leu Cys Ala For Leu Arg Lys Cys Arg For Gly Phe Gly Wall Ala 115 120 125 AGA CCA GGA ACT GAA ACA TCA GAC GTG GTG TGC AAG CCC TGT GCC CCG 432 Arg For Gly Thr Glu Thr Ser Asp Wall Wall Cys Lys For Cys Ala For 130 135 140 GGG ACG TTC TCC AAC ACG ACT TCA TCC ACG GAT ATT TGC AGG CCC CAC 480 Gly Thr Phe Ser Asn Thr Thr Ser Ser Thr Asp Ile Cys Arg For His 145 150 155 160 CAG ATC TGT AAC GTG GTG GCC ATC CCT GGG AAT GCA AGC AGG GAT GCA 528 Gin Xle Cys Asn Wall Wall Ala Xle For Gly Asn Ala Ser Arg Asp Ala 165 170 175 GTC TGC ACG TCC ACG TCC CCC ACC CGG AGT ATG GCC CCA GGG GCA GTA 576 Wall Cys Thr Ser Thr Ser For Thr Arg Ser Met Ala For Gly Ala Wall 180 185 190 CAC TTA CCC CAG CCA GTG TCC ACA CGA TCC CAA CAC ACG CAG CCA ACT 624 His Leu For Gin For Wall Ser Thr Arg Ser Gin His Thr Gin For Thr 195 200 205 CCA GAA CCC AGC ACT GCT CCA AGC ACC TCC TTC CTG CTC CCA ATG GGC 672 For Glu For Ser Thr Ala For Ser Thr Ser Phe Leu Leu For Met Gly 210 215 220 CCC AGC CCC CCA GCT GAA GGG AGC ACT GGC GAC 705 For Ser For For Ala Glu Gly Ser Thr Gly Asp 225 230 235

134

(2) Information about SEQ ID NO: 35:

(i) Sequence characteristics (A) Length: 235 amino acids (B) Type: amino acid (D) Topology: linear (ii) Molecule type: protein (xi) Sequence description: SEQ ID NO: 35:

Leu Pro 2 Ala Gin Wall 5 Ala Phe Thr For Tyr 10 Ala Pro Glu For Gly 15 Dec Ser Thr Cys Arg Leu Arg Glu Tyr Tyr Asp Gin Thr Ala Gin Met Cys Cys 20 May 25 30 Ser Lys Cys Ser For Gly Gin His Ala Lys Wall Phe Cys Thr Lys Thr 35 40 45 Ser Asp Thr Wall Cys Asp Ser Cys Glu Asp Ser Thr Tyr Thr Gin Leu 50 55 60 Trp Asn Trp Wall For Glu Cys Leu Ser Cys Gly Ser Arg Cys Ser Ser 65 70 75 80 Asp Gin Wall Glu Thr Gin Ala Cys Thr Arg Glu Gin Asn Arg lle Cys 85 90 95 Thr Cys Arg For Gly Trp Tyr Cys Ala Leu Ser Lys Gin Glu Gly Cys 100 ALIGN! 105 110 Arg Leu Cys Ala For Leu Arg Lys Cys Arg For Gly Phe Gly Wall Ala 115 120 125 Arg For Gly Thr Glu Thr Ser Asp Wall Wall Cys Lys For Cys Ala For 130 135 140 Gly Thr Phe Ser Asn Thr Thr Ser Ser Thr Asp lle Cys Arg For His 145 150 155 160 Gin lle Cys Asn Wall Wall Ala lle For Gly Asn Ala Ser Arg Asp Ala 165 170 175 Wall Cys Thr Ser Thr Ser For Thr Arg Ser Met Ala For Gly Ala Wall 180 185 190 His Leu For Gin For Wall Ser Thr Arg Ser Gin His Thr Gin For Thr 195 200 205 For Glu For Ser Thr Ala For Ser Thr Ser Phe Leu Leu For Met Gly 210 215 220 For Ser For For Ala Glu Gly Ser Thr Gly Asp

+420 225 230 235

135 (2) Information of SEQ ID NO: 36:

(i) Sequence characteristics (A) Length: 4 amino acids (B) Type: amino acid (C) Number of chains: unknown (D) Topology: unknown (ii) Molecule type: protein (xi) Sequence description: SEQ ID NO: 36:

Ala Gin Met Cys 1 (2) Information about SEQ ID NO: 37:

(i) Sequence characteristics (A) Length: 5 amino acids (B) Type: amino acid (C) Number of chains: unknown (D) Topology: unknown (ii) Molecule type: protein (xi) Description of sequence: SEQ ID NO: 37:

Thr Ala Gin Met Cys 15 (2) Information about SEQ ID NO: 38:

(i) Sequence characteristics (A) Length: 6 amino acids (B) Type: amino acid (C) Number of chains: unknown (D) Topology: unknown (ii) Molecule type: protein (xi) Sequence description: SEQ ID NO: 38:

Gin Thr Ala Gin Met Cys 15 (2) Information about SEQ ID NO: 39:

(i) Sequence characteristics (A) Length: 7 amino acids (B) Type: amino acid (C) Number of chains: unknown (D) Topology: unknown

136 (ii) Type of molecule: protein (xi) Description of sequence: SEQ ID NO: 39:

Asp Gin Thr Ala Gin Met Cys 1 5 (2) Information about SEQ ID NO: 40:

(i) Sequence characteristics (A) Length: 8 amino acids (B) Type: amino acid (C) Number of chains: unknown (D) Topology: unknown (ii) Molecule type: protein (xi) Sequence description: SEQ ID NO: 40:

Tyr Asp Gin Thr & Ala Gin Met Cys. 15 (2) Information about SEQ ID NO: 41:

(i) Sequence characteristics (A) Length: 9 amino acids (B) Type: amino acid (C) Number of chains: unknown (D) Topology: unknown (ii) Molecule type: protein (xi) Description of sequence: SEQ ID NO: 41:

Tyr Tyr Asp Gin Thr Ala Gin Met Cys 1 5 (2) Information about SEQ ID NO: 42:

(i) Sequence characteristics (A) Length: 10 amino acids (B) Type: amino acid (C) Number of chains: unknown (D) Topology: unknown (ii) Molecule type: protein (xi) Sequence description: SEQ ID NO: 42

Glu Tyr Tyr Gin Thr Ala Gin Met Cys 15 ¹⁰

137 • · ·

(2) SEQ ID NO: 43 information:

(i) Sequence characteristics (A) Length: 11 amino acids (B) Type: amino acid (C) Number of chains: unknown (D) Topology: unknown (ii) Molecule type: protein (xi) Sequence description: SEQ ID NO: 43:

Arg Glu Tyr Tyr Asp Gin Thr Ala Gin Met Cys 15 10 (2) Information about SEQ ID NO: 44:

(i) Sequence characteristics (A) Length: 12 amino acids (B) Type: amino acid (C) Number of chains: unknown (D) Topology: unknown (ii) Molecule type: protein (xi) Sequence description: SEQ ID NO: 44:

Leu Arg Glu Tyr Asp Gin Thr Ala Gin Met Cys 15 10 (2) Information about SEQ ID NO: 45:

(i) Sequence characteristics (A) Length: 13 amino acids (B) Type: amino acid (C) Number of chains: unknown (D) Topology: unknown (ii) Molecule type: protein (xi) Description of sequence: SEQ ID NO: 45:

Arg Leu Arg Glu Tyr Asp Gin Thr Ala Gin Met Cys 1 5 10 (2) Information about SEQ ID NO: 46:

(i) Sequence characteristics (A) Length: 14 amino acids (B) Type: amino acid (C) Number of chains: unknown (D) Topology: unknown

138 ·

• 99 (ii) Type of molecule: protein (xi) Description of sequence: SEQ ID NO: 46:

Cys Arg Leu Arg Glu Tyr Asp Gin Thr Ala Gin Met Cys (2) Information of SEQ ID NO: 47:

(i) Sequence characteristics (A) Length: 15 amino acids (B) Type: amino acid (C) Number of chains: unknown (D) Topology: unknown (ii) Molecule type: protein (xi) Sequence description: SEQ ID NO: 47:

Thr Cys Arg

Asp Gin Thr Ala Gin Met Cys 10 15 (2) Information about SEQ ID NO: 48:

(i) Sequence characteristics (A) Length: 16 amino acids (B) Type: amino acid (C) Number of chains: unknown (D) Topology: unknown (ii) Molecule type: protein (xi) Sequence description: SEQ ID NO: 48:

Ser Thr Cys Arg Leu Arg Glu Tyr Asp Gin Thr Ala Gin Met Cys 15 10 15 (2) SEQ ID NO: 49 info:

(i) Sequence characteristics (A) Length: 17 amino acids (B) Type: amino acid (C) Number of chains: unknown (D) Topology: unknown (ii) Molecule type: protein

139

4 · · • 4 * (xi) Description of sequence: SEQ ID NO: 49:

Gly Ser Thr Cys Arg Leu Arg Glu Tyr

Cys (2) Information about SEQ ID NO: 50:

(i) Sequence characteristics (A) Length: 18 amino acids (B) Type: amino acid (C) Number of chains: unknown (D) Topology: unknown (ii) Molecule type: protein (xi) Description of sequence: SEQ ID NO: 50:

For Gly Ser Thr Cys Arg Leu Arg Glu Tyr Tyr Asp Gin Thr Ala Gin 15 10 ¹⁵ Met Cys (2) SEQ ID NO: 51 info:

(i) Sequence characteristics (A) Length: 19 amino acids (B) Type: amino acid (C) Number of chains: unknown (D) Topology: unknown (ii) Molecule type: protein (xi) Sequence description: SEQ ID NO: 51:

Glu Pro Gly Ser Thr Cys

Gin Met Cys (2) Information about SEQ ID NO: 52:

(i) Sequence characteristics (A) Length: 20 amino acids (B) Type: amino acid (C) Number of chains: unknown (D) Topology: unknown (ii) Molecule type: protein

140 (xi) Description of sequence: SEQ ID NO: 52:

For Glu For Gly Ser Thr Cys Arg Leu Arg Glu Tyr Tyr Asp Gin Thr! 5 10 15

Ala Gin Met Cys 20 (2) Information about SEQ ID NO: 53:

(i) Sequence characteristics (A) Length: 21 amino acids (B) Type: amino acid (C) Number of chains: unknown (D) Topology: unknown (ii) Molecule type: protein (xi) Sequence description: SEQ ID NO: 53:

Ala Glu Pro Gly Ser Thr Cys Arg Leu Arg Glu Tyr Asp Gin 1 5 10 15

Thr Ala Gin Met Cys 20 (2) Information about SEQ ID NO: 54:

(i) Sequence characteristics (A) Length: 22 amino acids (B) Type: amino acid (C) Number of chains: unknown (D) Topology: unknown (ii) Molecule type: protein (xi) Sequence description: SEQ ID NO: 54:

Tyr Ala Glu Pro Gly Pro Thr Cys Arg Leu Arg Glu Tyr Asp 15 10 15

Gin Thr Ala Gin Met Cys

141 (2) Information about SEQ ID NO: 55:

(i) Sequence characteristics (A) Length: 23 amino acids (B) Type: amino acid (C) Number of chains: unknown (D) Topology: unknown (ii) Molecule type: protein (xi) Sequence description: SEQ ID NO: 55:

Pro Tyr Ala Pro Glu Pro Gly Ser Thr Cys Arg Leu Glu Tyr Tyr 1 5 10 15

Asp Gin Thr Ala Gin Met Cys 20 (2) Information about SEQ ID NO: 56:

(i) Sequence characteristics (A) Length: 24 amino acids (B) Type: amino acid (C) Number of chains: unknown (D) Topology: unknown (ii) Molecule type: protein (xi) Sequence description: SEQ ID NO: 56:

Thr Pro Tyr Ala Pro Glu Pro Gly Ser 1

Tyr Asp Gin Thr Ala Gin Met Cys 20 (2) Information about SEQ ID NO: 57:

(i) Sequence characteristics (A) Length: 25 amino acids (B) Type: amino acid (C) Number of chains: unknown (D) Topology: unknown (ii) Molecule type: protein (xi) Sequence description: SEQ ID NO: 57:

Phe Thr Pro Tyr Glu Pro Gly Pro ^{5 5}

Tyr Tyr As Gin Thr Ala Gin Met Cys 20 ²⁵

Thr Cys Arg Leu Arg Glu 10 ¹⁵

Tyr

Ser Thr Cys Arg

142 V V V 99 99 99 99 (((((((((((((((((((((99 (2) :

(i) Sequence characteristics (A) Length: 26 amino acids (B) Type: amino acid (C) Number of chains: unknown (D) Topology: unknown (ii) Molecule type: protein (xi) Sequence description: SEQ ID NO: 58:

Ala Phe Thr For Tyr Ala For Glu For Gly Ser Thr Cys Arg Leu Arg 1 5 10 15 Dec Glu Tyr Tyr Asp Gin Thr Ala Gin Met Cys 20 May 25

(2) Information about SEQ ID NO: 59:

(i) Sequence characteristics (A) Length: 27 amino acids (B) Type: amino acid (C) Number of chains: unknown (D) Topology: unknown (ii) Molecule type: protein (xi) Description of sequence: SEQ ID NO: 59:

Val Ala Phe Thr Pro Tyr Ala Pro Glu Pro Gly Ser Thr Cys Arg Leu 15 10 ¹⁵

Arg Glu Tyr Asp Gin Thr Ala Gin Met Cys 20 25 (2) Information about SEQ ID NO: 60:

(i) Sequence characteristics (A) Length: 28 amino acids (B) Type: amino acid (C) Number of chains: unknown (D) Topology: unknown (ii) Molecule type: protein (xi) Sequence description: SEQ ID NO: 60:

Gin Val Ala Phe Thr Cys Arg 15 ¹⁰

Leu Arg Glu Tyr Tyr Asp Gln Thr Ala Gln Met Cys 20 ²⁵

143 4 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 SEQ ID NO: 61:

(i) Sequence characteristics (A) Length: 29 amino acids (B) Type: amino acid (C) Number of chains: unknown (D) Topology: unknown (ii) Molecule type: protein (xi) Description of sequence: SEQ ID NO: 61:

Ala 1 Gin Wall Ala Phe 5 Thr For Tyr Ala For 10 Glu For Gly Ser Thr Cys Arg Leu Arg Glu 20 May Tyr Tyr Asp Gin Thr 25 Ala Gin Met Cys

(2) Information about SEQ ID NO: 62:

(i) Sequence characteristics (A) Length: 30 amino acids (B) Type: amino acid (C) Number of chains: unknown (D) Topology: unknown (ii) Molecule type: protein

(xi) Sequence description: SEQ ID NO; ; 62: For Ala Gin Val Ala Phe Thr For Tyr Ala For Glu For Gly Ser Thr 1 ⁵ 10 15 Cys Arg Leu Arg Glu Tyr Tyr Asp Gin Thr & Ala Gin Met Cys 20 May 25 30

(2) Information about SEQ ID NO: 63:

(i) Sequence characteristics (A) Length: 31 amino acids (B) Type: amino acid (C) Number of chains: unknown (D) Topology: unknown (ii) Molecule type: protein

144 • 4 44 44 444 · 4 4 · · «

444 444 444 · 4 44 ·· (xi) Sequence description: SEQ ID NO: 63:

Leu For Ala Gin Wall Ala Phe Thr For Tyr Ala For Glu For Gly Ser 1 5 10 15 Dec Thr Cys Arg Leu Arg Glu Tyr Tyr Asp Gin Thr Ala Gin Met Cys 20 May 25 30

(2) SEQ ID NO: 64 information:

(i) Sequence characteristics (A) Length: 4 amino acids (B) Type: amino acid (C) Number of chains: unknown (D) Topology: unknown (ii) Molecule type: protein (xi) Sequence description: SEQ ID NO: 64:

Ala Pro Leu Arg 1 (2) Information about SEQ ID NO: 65:

(i) Sequence characteristics (A) Length: 5 amino acids (B) Type: amino acid (C) Number of chains: unknown (D) Topology: unknown (ii) Molecule type: protein (xi) Sequence description: SEQ ID NO: 65:

Ala Pro Leu Arg Lys 15 (2) Information about SEQ ID NO: 66:

(i) Sequence characteristics (A) Length: 6 amino acids (B) Type: amino acid (C) Number of chains: unknown (D) Topology: unknown (ii) Molecule type: protein (xi) Sequence description: SEQ ID NO: 66:

Ala Pro Leu Arg Lys Cys

145

• · · »

(2) Information about SEQ ID NO: 67:

(i) Sequence characteristics (A) Length: 7 amino acids (B) Type: amino acid (C) Number of chains: unknown (D) Topology: unknown (ii) Molecule type: protein (xi) Sequence description: SEQ ID NO: 67:

Ala Pro Leu Arg Lys Cys Arg 15 (2) Information about SEQ ID NO: 68:

(i) Sequence characteristics (A) Length: 31 base pairs (B) Type: nucleic acid (C) Number of strands: unknown (D) Topology: unknown (ii) Molecule type: cDNA (xi) Sequence description: SEQ ID NO: 68:

GGTTAGCCAT ATGGACAGCG TTTGCCCCCA A (2) Information about SEQ ID NO: 69:

(i) Sequence characteristics (A) Length: 33 base pairs (B) Type: nucleic acid (C) Number of strands: unknown (D) Topology: unknown (ii) Molecule type: cDNA (xi) Sequence description: SEQ ID NO: 69:

CCCAAGCTTT TACAGAGAGC AATTGAAGCA CTG

146

(2) Information about SEQ ID NO: 70:

(i) Sequence characteristics (A) Length: 40 base pairs (B) Type: nucleic acid (C) Number of strands: unknown (D) Topology: unknown (ii) Molecule type: cDNA (xi) Sequence description: SEQ ID NO: 70:

ACTCGAGGAT CCGCGGATAA ATAAGTAACG ATCCGGTCCA 40 (2) Information about SEQ ID NO: 71:

(i) Sequence characteristics (A) Length: 41 base pairs (B) Type: nucleic acid (C) Number of strands: unknown (D) Topology: unknown (ii) Molecule type: cDNA (xi) Sequence description: SEQ ID NO: 71:

CAGGTCGGAT CCTATCAGCA GAAGCACTGG AAAAGGTTTT C (2) SEQ ID NO: 72 info:

(i) Sequence characteristics (A) Length: 31 base pairs (B) Type: nucleic acid (C) Number of strands: unknown (D) Topology: unknown (ii) Molecule type: cDNA (xi) Sequence description: SEQ ID NO: 72:

GGTTAGCCAT ATGGACAGCG TTTGCCCCCA A -31 (2) Information about SEQ ID NO: 73:

(i) Sequence characteristics (A) Length: 34 base pairs (B) Type: nucleic acid (C) Number of strands: unknown (D) Topology: unknown (ii) Molecule type: cDNA

147 4 · 4 4 4 4 4 4

4-. . .4.4. .4 4 ..4 4. 4 4 *. ·

4,444,444

4 4 44 4 444444

4 4 4 4 4 5

444 444 444 44 44 44 (2) Information ο SEQ ID NO: 77:

(i) Sequence characteristics (A) Length: 33 base pairs (B) Type: nucleic acid (C) Number of strands: unknown (D) Topology: unknown (ii) Molecule type: cDNA (xi) Sequence description: SEQ ID NO: 77:

CCCAAGCTTT TACAGAGAGC AATTGAAGCA CTG (2) Information about SEQ ID NO: 78:

(i) Sequence characteristics (A) Length: 30 base pairs (B) Type: nucleic acid (C) Number of strands: unknown (D) Topology: unknown (ii) Molecule type: cDNA (xi) Sequence description: SEQ ID NO: 78

CCCCATATGT GTACCAAGTG CCACAAAGGA 30 (2) Information about SEQ ID NO: 79:

(i) Sequence characteristics (A) Length: 33 base pairs (B) Type: nucleic acid (C) Number of strands: unknown (D) Topology: unknown (ii) Molecule type: cDNA (xi) Sequence description: SEQ ID NO: 79:

• 33

CCCAAGCTTT TACAGAGAGC AATTGAAGCA CTG (2) Information about SEQ ID NO: 80:

(i) Sequence characteristics (A) Length: 31 base pairs (B) Type: nucleic acid (C) Number of strands: unknown (D) Topology: unknown (ii) Molecule type: cDNA

148

• • · »» · '· '·' • • · • * · 4 · • 4 • · • • • • · • • • · · • · · • · · · • • ·

(xi) Sequence description: SEQ ID NO: 73:

CGCGGATCCC TATTAATTGA AGCACTGGAA AAGG 34 (2) Information about SEQ ID NO: 74:

(i) Sequence characteristics (A) Length: 30 base pairs (B) Type: nucleic acid (C) Number of strands: unknown (D) Topology: unknown (ii) Molecule type: cDNA (xi) Sequence description: SEQ ID NO: 74:

CCCCATATGT ATATCCACCC TCAAAATAAT 30 (2) Information about SEQ ID NO: 75:

(i) Sequence characteristics (A) Length: 33 base pairs (B) Type: nucleic acid (C) Number of strands: unknown (D) Topology: unknown (ii) Molecule type: cDNA (xi) Sequence description: SEQ ID NO: 75:

CCCAAGCTTT TACAGAGAGC AATTGAAGCA CTG (2) Information about SEQ ID NO: 76:

(i) Sequence characteristics (A) Length: 38 base pairs (B) Type: nucleic acid (C) Number of strands: unknown (D) Topology: unknown (ii) Molecule type: cDNA (xi) Sequence description: SEQ ID NO: 76:

CCCCATATGT CGATTAGCTG TACCAAGTGC CACAAAGG

149

·

4

(xi) Sequence description: SEQ ID NO: 80:

GGTTAGCCAT ATGGACAGCG TTTGCCCCCA A (2) Information about SEQ ID NO: 81:

(i) Sequence characteristics (A) Length: 33 base pairs (B) Type: nucleic acid (C) Number of strands: unknown (D) Topology: unknown (ii) Molecule type: cDNA (xi) Sequence description: SEQ ID NO: 81:

CCCAAGCTTT TAGGTGCACA CGGTGTTCTG TTT

150

• · ·

Claims (31)

A truncated sTNFR having the following formula: R 1 - [Cys ¹⁹ -Cys ¹⁰³ ] -R ₂ , wherein [Cys ¹⁹ -Cys ¹⁰³ ] represents residues 19-103 of sTNRF-I (the amino acid residue numbering scheme is shown in Fig. 1 (SEQ. ID NO: 2) to facilitate comparison), and wherein further R 1 represents the methionylated or non-methionylated amino group of Cys ¹⁹ or the amino acid residue (s) of the amino acid terminus selected from: C IC SIC NSIC (SEQ ID NO 15) NNSIC (SEQ ID NO 16) QNNSIC (SEQ ID NO 17) PQNNSIC (SEQ ID NO 18) HPQMMSIC (SEQ ID NO 19) IHPQNNSIC (SEQ ID NO 20) YIHPQNNSIC (SEQ ID NO 21) KYIHPQNNSIC (SEQ ID NO 22) GKYIHPQNNSIC (SEQ ID NO 23) QGKYIHPQNNSIC (SEQ ID NO 24) PQGKYIHPQNNSIC (SEQ ID NO 25) CPQGKYIHPQNNSIC (SEQ ID NO 26) VCPQGKYIHPQNNSIC (SEQ ID NO 27) SVCPQGKYIHPQNNSIC (SEQ ID NO 28) DSVCPQGKYIHPQNNSIC (SEQ ID NO 29) and wherein R ₂ is a carboxy group of Cys ¹⁰³ or a carboxy group of a carboxy-terminal amino acid residue selected from the group of: F FC FCC FCCS (SEQ ID NO: 30) FCCSL (SEQ ID NO: 31) FCCSLC (SEQ ID NO: 32) • · FCCSLCL (SEQ ID NO: 33) and the derivatives or derivatives thereof derived therefrom, but provided that when R 1 represents a methionylated or unmethionylated amino group of the amino acid sequence VCPQGKYIHPQNNSIC or a N terminal truncation of 1 to 15 residues derived therefrom, then R ₃ [Cys ¹⁹ -Cys ^103] -R 2 is added variant having the formula Rx-tCys ¹⁹ -Cys ^103] -FCCSLCL-R 3 wherein R ₃ represents a carboxy group of the amino acid residues Asn ¹⁶¹ -Asn FIGs ^m or a truncation at the carboxyl end of Asn Asn ^{161 of} FIG ⁿⁱ Tumor necrosis binding protein according to claim 1, selected from the group consisting of sTNFR-Ι 2.6D / C105, sTNFR-Ι 2.6D / 106, sTNFR-Ι 2.6D / N105, sTNFR-Ι 2.3D / d8, sTNFR- Ι 2.3D / dl8, sTNFR-I 2.3D / dl5 or derivatives or derivatives derived therefrom. A truncated sTNFR having the formula R ₄ - [Cys ³² -Cys ¹¹⁵ ] -R ₅ , wherein [Cys ³² Cys ¹¹⁵ ] represents Cys ³² to Cys ¹¹⁵ residues of a total 40 kDa full-length TNF inhibitor, the amino acid residue numbering scheme is shown in Fig. 8 (SEQ ID NO: 35) to facilitate comparison. Wherein R 4 is a methionylated or unmethionylated amino group of Cys ³² or an amino acid residue (or residues) of an amino acid terminus selected from: C MC QMC AQMC (SEQ ID NO: 3 6) TAQMC (SEQ ID NO: 37) QTAQMC (SEQ ID NO: 38) DQTAQMC (SEQ ID NO: 3 9) YDQTAQMC (SEQ ID NO: 0 0) YYDQTAQMC (SEQ ID NO: 41) EYYDQTAQMC (SEQ ID NO: 4 2) REYYDQTAQMC (SEQ ID NO: 43) LREYYDQTAQMC (SEQ ID NO: 4 4) RLREYYDQTAQMC (SEQ ID NO: 4 5) • · CRLREYYDQTAQMC (SEQ ID NO: 46) TCRLREYYDQTAQMC (SEQ ID NO: 47) STCRLREYYDQTAQMC (SEQ ID NO: 4 8) GSTCRLREYYDQTAQMC (SEQ ID NO: 4 9) PGSTCRLREYYDQTAQMC (SEQ ID NO: 0 0) EPGSTCRLREYYDQTAQMC (SEQ ID NO: 51) PEPGSTCRLREYYDQTAQMC (SEQ ID NO: 52) APEPGSTCRLREYYDQTAQMC (SEQ ID NO: 53) YAPEPGSTCRLREYYDQTAQMC (SEQ ID NO: 5 4) PYAPEPGSTCRLREYYDQTAQMC (SEQ ID NO: 55) TPYAPEPGSTCRLREYYDQTAQMC (SEQ ID NO: 5 6) FTPYAPEPGSTCRLREYYDQTAQMC (SEQ ID NO: 57) AFTPYAPEPGSTCRLREYYDQTAQMC (SEQ ID NO: 5 8) VAFTPYAPEPGSTCRLREYYDQTAQMC (SEQ ID NO: 59) QVAFTPYAPEPGSTCRLREYYDQTAQMC (SEQ ID NO: 60) AQVAFTPYAPEPGSTCRLREYYDQTAQMC (SEQ ID NO: 61) PAQVAFTPYAPEPGSTCRLREYYDQTAQMC (SEQ ID NO: 62) LPAQVAFTPYAPEPGSTCRLREYYDQTAQMC (SEQ ID NO: 63) and wherein R ₅ represents a carboxy group of Cys ¹¹⁵ or a carboxy group of a carboxy-terminal amino acid residue selected from: AND AP APL APLR (SEQ ID NO: 65) APLRK (SEQ ID NO: 66) APLRKC (SEQ ID NO: 65) APLRKCR (SEQ ID NO: 66) and variations derived therefrom, but provided that when R ₄ represents the methionylated or unmethionylated amino group of the amino acid sequence TCRLREYYDQTAQMC or the N-terminal truncation derived therefrom from 1 to 15 residues, then R ₄ - [Cys ³² -Cys ¹¹⁵ ] -R5 is not an added variant having formula R 4 - [Cys ³² -Cys ¹¹⁵ ] -APLRKCR-RS, wherein R ₆ represents the carboxy group of the Pro ¹²³ Thr residue ¹⁷⁹ Figs. 8 or truncation at the carboxyl end of Pro ¹²³ -Thr ¹⁷⁹ Fig. 8. Tumor necrosis binding protein according to any one of claims 1 to 3, wherein said amino acid sequences are non-glycosylated. Tumor necrosis binding protein according to any one of claims 1 to 3, wherein said amino acid sequences are glycosylated. Tumor necrosis binding protein according to any claim 1-5 wherein the protein is conjugated to a water-soluble polymer. A tumor necrosis polyvalent binding protein comprising at least one tumor necrosis binding protein according to any one of claims 1 to 6. 8. Tumor necrosis polyvalent binding protein having the formula Rx-XR? wherein: X is comprised of a linker and said linker is a water-soluble polymer and wherein R 1 AR ₂ are biologically active molecules covalently bound to said water-soluble polymer and wherein at least one of Ry and R ₂ is a tumor necrosis tumor binding protein according to any one of claims 1 to 15 6. Tumor necrosis polyvalent binding protein according to claim 8 wherein the water-soluble polymer is polyethylene glycol. The tumor necrosis polyvalent binding protein of claim 9, wherein the protein is selected from the group consisting of sTNFR-I 2.6D / C105db and sTNFR-I 2.6D / C106db. • · Tumor necrosis binding protein according to any one of claims 1 to 10 for use in the treatment of a TNF-mediated disease. Tumor necrosis binding protein according to any one of claims 1 to 10 used for the treatment of arthritis. A polynucleotide encoding a tumor necrosis binding protein according to any one of claims 1 to 3. 14. A nucleic acid sequence comprising a tumor necrosis factor binding protein encoded by a nucleotide sequence selected from the following: and) sequence cDNA how Yippee shown on Giant. 2 (b) sequence cDNA how Yippee shown on Giant. 3 C) sequence cDNA how Yippee shown on Giant. 4 (d) sequence cDNA how Yippee shown on Giant. 5 E) sequence cDNA how Yippee shown on Giant. 6 F) sequence cDNA how Yippee shown on Giant. 7 g) a sequence that is degenerate in the coding regions or parts thereof of sequences a), b), c), d), e) and f) (h) sequences that hybridize to sequences (a), (b), (c), (d), (e) and (f); and i) sequences that are complementary to the sequences a), b), c), d), e) and f), but assuming that the nucleic acid does not encode a protein having a formula Rj.- [Cys Cys ^{103 i9]} -FCCSLCL-R 3 wherein [Cys ¹⁹ -Cys ¹⁰³ ] represents residues 19-103 of sTNRF-I (the amino acid residue numbering scheme is shown in Figure 1 (SEQ ID NO: 2) for ease of comparison), wherein Rx is a methionylated or unmethionylated amino group of the NNSIC-containing amino acid sequence and R3 is carboxy amino acid Asn Asn ^lu FIGs ^LCL or a truncation at the carboxyl end of Asn Asn ^{161 of} FIG ^lu 15. A polynucleotide having the sequence as set forth in 2,3,4,5,6,7, or having a portion thereof. A vector comprising the polynucleotide of any one of claims 13 to 15, effectively linked to an expression control sequence. A prokaryotic or eukaryotic host cell comprising the polynucleotide of any one of claims 13 to 15. A method comprising growing host cells according to claim 17 in a suitable nutrient medium and, optionally, isolating said truncated sTNFR from said cells or said medium The method of producing a tumor necrosis binding protein according to claim 18, wherein said host cells are E. coli cells. The method of producing a tumor necrosis factor binding protein of claim 18, wherein said host cells are Chinese hamster ovary cells. A method comprising the following steps: (a) growing prokaryotic or eukaryotic host cells according to claim 17 (b) maintaining said host cells under conditions permitting expression of the truncated sTNFR by said host cells; and c) optionally isolating a truncated sTNFR expressed by said host cells. 22nd 23. 24. 25. 26. 27 Mar: 28. 29. · * * * * * * * * * * * * * * Tumor necrosis binding protein, which is the product of recombinant expression of prokaryotic or eukaryotic host cells comprising the polynucleotide of any one of claims 13 to 15. A pharmaceutical composition comprising the tumor necrosis factor binding protein of any one of claims 1 to 10 in association with a pharmaceutically acceptable carrier. A pharmaceutical composition comprising a tumor necrosis factor binding protein produced according to the method of claim 18 in association with a pharmaceutically acceptable carrier. A pharmaceutical composition comprising a tumor necrosis binding protein produced according to the method of claim 21 in association with a pharmaceutically acceptable carrier. A method of treating diseases caused by TNF comprising administering the pharmaceutical compositions of claims 23 to 25 to patients. The method of claim 26, wherein the TNF-mediated disease is arthritis. A method of preparing a pharmaceutical composition wherein the therapeutically effective amount of the tumor necrosis factor binding protein of any one of claims 1 to 10 is admixed with one or more pharmaceutically acceptable carrier. Use of a tumor necrosis factor binding protein according to any one of claims 1 to 10 for the treatment of diseases caused by TNF. 4 »* 4 • 5 4 4 4 4 4 44 4 » 4 4 4 4 4 4 4 4 444 444 4 4 44 44 Use of a factor binding protein according to claim 29 for the treatment of tumor necrosis arthritis A kit for preparing a protein solution of a composition comprising the tumor necrosis factor binding protein of any one of claims 1 to 10 and a second container containing a physiologically acceptable solvent.