EP1636264A2 - Tumour necrosis factor receptor molecules with reduced immunogenicity - Google Patents

Abstract

The invention relates to the modification of human soluble tumor necrosis factor receptor type 1 (sTNFR1) to result in sTNFR1 proteins, preferably fusion proteins comprising an immunoglobulin heavy chain constant region (Fc domain) and modified human sTNFR-I. These molecules are substantially non-immunogenic or less immunogenic than any non-modified counterpart when used in vivo by elimination or deletion of T-cell epitopes. The invention relates furthermore to T-cell epitope peptides derived from non-modified human sTNFR-I.

Description

TUMOUR NECROSIS FACTOR RECEPTOR MOLECULES WITH REDUCED IMMUNOGENICITY

FIELD The invention concerns human soluble tumour necrosis factor receptor type 1 (sTNFR-I) and in particular modified forms of sTNFR-I with improved properties. The improved proteins contain amino acid substitutions at specific positions. The invention provides modified sTNFR-I with improved biological activity concomitant with reduced immunogenic potential in the protein. The improved proteins are intended for therapeutic use in the treatment of diseases in humans.

BACKGROUND

Tumour necrosis factor alpha (TNF-alpha) is a proinflammatory cytokine and important mediator in the development of chronic and acute inflammatory diseases in man [Brenan, F.M. et al (1998) Seminars in Immunopathology 20: 133-147; Firestein, G.S. et al (1997) N Engl. J. Med. 337: 195-197]. TNF-alpha exerts its effects on cells by specific binding interaction with either the high affinity "p55" type I receptor (TNFR-I) or the lower affinity "p75" type II receptor (TNFR-II) on the cell surface. Inhibitors of TNF-alpha occur naturally and these TNF-binding proteins may consist of full length or truncated soluble forms of the TNFR-I protein. These proteins have been identified in active rheumatoid arthritic disease tissues, serum and synovial fluid. In some studies the presence of soluble TNFR-I has conelated with disease activity [Cope, AP et al (1995) J Rheumatol. 22: 382-384].

The gene for sTNFR-I has been cloned and the protein produced as a recombinant molecule [Gray, P.W. et al (1990) Proc. Nat. Acad. Sci. U.S.A. 87: 7380-7384; Loetschere, H. et al, (1990) Cell 61: 351-359; Schall, TJ. et al (1990) Cell 61: 361-370; Kohno, T. et al (1990) Proc. Nat. Acad. Sci. U.S.A 87: 8331-8335]. The protein comprises the extracellular domain of the intact receptor and exhibits an approximate molecular weight of 30KDa. Additional soluble TNF inhibitors and in particular a 40KDa form are also known [US 6,143,866].

Recombinant preparations of sTNFR-I are of significant potential therapeutic value for the treatment of diseases where an excess level of TNF-alpha is causing a pathogenic effect. Indications such as cachexia, sepsis and autoimmune disorders including multiple sclerosis and rheumatoid arthritis and others, may be targeted using sTNFR-I. Two versions of the molecule have previously been advanced into clinical trials. The first version was a dimerised form (termed TNF-bp) linked via a polyethyleneglycol (PEG) moiety at residue 105. The TNF-bp molecule was immunogenic in significant numbers of patients. Several deletion variants of the TNF-bp molecule have been tested in a baboon model for immunogenicity and functional activity [Solorazano et al (1998) J Appli. Physiol. 84: 1119-1130]. A second generation molecule has been produced which is monomeric and PEGylated at the N-terminus.

Clinical development of sTNFR-I has been hampered by the dual problems of short circulating half-life and immunogenicity. For individuals where an immune response to the protein has been mounted, this has occuned despite the fact that the protein may be normally present in the circulation. The pivotal feature leading to the induction of an immune response is the presence within the protein of peptides that can stimulate the activity of T-cells via presentation on MHC class II molecules. Such peptide sequences are "T-cell epitopes" and are commonly defined as any amino acid residue sequence with the ability to bind to MHC Class II molecules. Implicitly, a "T-cell epitope" means an epitope which when bound to MHC molecules can be recognised by a T-cell receptor (TCR), and which can, at least in principle, cause the activation of these T-cells by engaging a TCR to promote a T-cell response. Patients who develop antibodies to TPO possess T cells that are capable of recognising peptide fragments of TPO bound to MHC class II molecules in their T-cell repertoire.

From the foregoing there is clearly a continued need for sTNFR-I analogues with enhanced properties. There is a particular need for enhancement of the in vivo characteristics when administered to the human subject. In this regard, it is highly desired to provide sTNFR-I with reduced or absent potential to induce an immune response and enhanced biological potency in the human subject. It is a particular objective of the present invention to provide modified sTNFR-I proteins in which the immune characteristic is modified by means of reduced numbers of T-cell epitopes. Moreover, it is particularly desired to additionally provide modified sTNFR-I proteins with the capability of long circulating half-life coupled also with straightforward and efficient means for the recombinant production and purification of the molecules. Others have provided sTNFR-I molecules and analogues including chemically modified and truncated forms and fusion proteins.

US, 6,294,352; EP04223339B1 and EP0393438A3 describe TNF inhibitor proteins including TNFR-I and methods for obtaining the protein by recombinant DNA techniques. EP0417563B1 describes fusion proteins comprising fragments of TNFR-I with immunoglobulin constant region domains. Fusion molecules wherein the TNFR-I domain is oriented at the N-terminus are particularly contemplated.

EP04333900B1 describes the expression of whole TNFR-I molecule in CHO cells. US,6, 143,866 describes TNF inhibitors including pharmaceutical compositions based on modified TNFR-I proteins.

US,6,271,346 describes TNF receptor proteins with conservative amino acid substitutions.

US,6,221,675 also describes TNF receptor proteins for use in detection of TNF in biological samples.

US,6,417,158 provides methods for reducing the harmful effects of TNF by use of TNFR-I molecules.

US,6,306,820 also describes therapeutic methods for reducing the harmful effects of TNF exploiting molecules of the type R1-X-R2 where Rl or R2 could be TNFR molecules. None of these teachings recognise the importance of T cell epitopes to the immunogenic properties of the protein nor have been conceived to directly influence said properties in a specific and controlled way according to the scheme of the present invention.

WO 03/104263 does describe a single immunogenic epitope within the TNFR-I sequence comprising residues 106 - 120. The present invention is concerned with four epitope regions within the sTNFR-I molecule and provides modified sTNFR-I molecules in which the immunogenic properties of all epitopes are significantly reduced or eliminated.

Accordingly, the present invention is concerned with sTNFR-I molecules in which amino acid substitution and or combinations of substitution have been conducted. More especially, the molecules of the invention are fusion proteins comprising a human immunoglobulin constant region moiety(preferably a Fc region) linked with a sTNFR-I mutein. Linkage to the immunoglobulin constant region domain causes the protein to become dimeric and gain new properties. The new properties relate to the presence of the immunoglobulin domain and include high level expression in mammalian cells, straightforward purification of the protein and an expectation of a greatly enhanced in vivo half-life. This structure together with substitutions and combinations of substitutions in the sTNFR-I component confer the property of enhancing the biological activity of the molecule and also achieve a reduced immunogenic profile for the protein.

The general category of "human Fc fusion proteins" of which the present molecules are examples have been described previously [US, 5,541,087; US, 5,726,044 Lo et al (1998), Protein Engineering 11:495 - 500].

SUMMARY OF THE INVENTION

The invention provides sTNFR-I molecules containing amino acid substitutions. The amino acid substitutions confer improved properties to the protein. The improved properties concern the specific biological activity of the protein and also the immunogenic properties of the protein.

The molecules of the invention are fusion proteins comprising a human immunoglobulin heavy chain constant region moiety, preferably an Fc region optionally including a hinge region, linked with a sTNFR-I mutein, optionally via a linker molecule.

The sTNFR-I proteins of the invention are preferably expressed in mammalian cell-lines as a C-terminal fusion partner, linked to the Fc unit of human IgG₄. Thereby, the sTNFR-I sequence is fused preferably to the C-terminus of a binge modified/C_H2/C_H3 Fc region of human IgG₄, preferably via a 15 amino acid flexible linker between the C-terminus of the C_H3 and the N-terminus of sTNFR-I. The expressed fusion proteins are dimeric and have a stoichiometry of (hi ge-Cπ2-C_H3 -linker- sTNFR-T)₂. However, also the monomeric forms of these molecules are subject-matter of this invention.

The molecules of the invention have new properties. Such molecules may cause benefit for a patient with a TNF-alpha mediated inflammatory disease.

The molecules of the invention are characterised by the protein sequences defined herein as Ml to M58, F-Ml to F-M58, and F-L-Ml to F-L-M58, respectively, wherein Ml to M58 represent the protein sequences of differently modified human sTNFR-I, F-Ml to F-M58 represent the respective fusion proteins with the Fc portion of human IgG4 or optionally another human IgG form, and F-L-Ml to F-L-M58 represent the respective fusion proteins comprising a linker molecule between the Fc sequence and the sTNRF-I protein sequence. These molecules each show functional activity at least equal to that of a non-modified (wild- type) molecule and in some cases superior activity can be demonstrated. The most prefened molecules may be characterised further still by comprising sequences demonstrated to show reduced immunogenicity in human cells. In particular reduced immunogenicity as measured using a "T-cell assay" or a "time course assay" as defined herein.

The present invention provides for modified forms of sTNFR-I proteins that are expected to display enhanced properties in vivo. The present invention discloses the major regions of the sTNFR-I primary sequence that are immunogenic in man and provides modification to the sequences to eliminate or reduce the immunogenic effectiveness of these sites.

In one embodiment, synthetic peptides comprising the immunogenic regions can be provided in pharmaceutical composition for the purpose of promoting a tolerogenic response to the whole molecule.

In a further embodiment, the modified sTNFR-I molecules can be used in pharmaceutical compositions.

In summary the invention is concerned with the following issues: • A modified sTNFR-I molecule (M) having essentially the same biological specificity and activity of human sTNFR-I when used in vivo containing one or more amino acid substitutions, wherein said modified sTNFR-I molecule is substantially non-immunogenic or less immunogenic than the parental non-modified human sTNFR-I and said amino acid substitutions cause a reduction or an elimination of one or more of T-cell epitopes within the sTNFR-I sequence which act in the parental non-modified molecule as MHC class II binding ligands and stimulate T-cells. • A modified sTNFR-I molecule as specified containing one or more of the amino acid substitutions I10Q, T20R, H23P, L56A, L108T, LI 10H and L149D within the sTNFR-I domain. • A modified sTNFR-I molecule as specified having the formula / structure

DSVCPQGKYX^PQNNSX^CX^CX^GTYLYNDCPGPGQDTDCRECESGSFTASENHX^HCX^C SKCRKE GQVEISSCTVDRDTVCGCRKNQYRHYWSENLX QCFNCSX⁸CX⁹NGTVHLSCQEKQNTVC TCHAGFFLRENECVSCSNCKKSX¹⁰ECTKLCLPQIEN wherein

X¹ is Q or S or N or E or A or G or K or P or R or I;

X² is Tor I;

X³isRorT; X⁴isPorH;

X⁵ is A or S or Q or L;

X⁶ is H or L;

X⁷isHorF;

X⁸isTorPorKorDorL; X⁹ is H or P or Q or L;

X¹⁰isDorEorTorL and whereby simultaneously X¹ = I, X² = I, X³ - T, X⁴ = H, X⁵ = L, X⁶ = L, X⁷ = F, X⁸ =

L, X⁹ = L and X¹⁰ = L are excluded, said meanings representing the native human sTNFR-I. • A modified sTNFR-I molecule (M) as specified having a protein sequence selected from the group consisting of Ml to M58, wherein Ml - M58 are specified in Table Al.

• A fusion protein of the structure

F-(L)n-M comprising a modified sTNFR-I molecule (M) as specified, fused directly (n = 0) or indirectly (n = 1) via a linker molecule (L) to a human immunoglobulin heavy constant region domain (F),

• A fusion protein as specified, wherein F is an Fc domain, optionally comprising a hinge region, wherein this hinge region may be modified.

• A fusion protein as specified wherein the C-terminus of the human immunoglobulin heavy constant region domain (Fc domain) is linked directly or indirectly to the N-terminus of the modified sTNFR-I.

• A dimeric fusion protein comprising two monomeric fusion protein chains as specified.

• A fusion protein as specified, wherein said sTNFR-I portion contains one or more of the amino acid substitutions I10Q, T20R, H23P, L56A, L108T, LI 10H and L149D within the sTNFR-I domain.

• A fusion protein as specified, wherein said sTNFR-I portion has the formula / structure:

DSVCPQGKYX^ΗPQNNSX^CX^CX^GTYLYNDCPGPGQDTDCRECESGSFTASENHX^HCX^C SKCRKEMGQVEISSCTVDRDTVCGCRKNQYRHY SENLX⁷QCFNCSX⁸CX⁹NGTVHLSCQEKQNTVC

TCHAGFFLRΞNECVSCSNCKKSX¹⁰ECTKLCLPQIEN wherein

X¹ is Q or S or N or E or A or G or K or P or R or I; X² is T or I;

X³ is R or T;

X⁴ is P or H;

X⁵ is A or S or Q or L;

X⁶ is H or L; X⁷ is H or F;

X⁸ is T or P or K or D or L;

X⁹ is H or P or Q or L;

X¹⁰ is D or E or T or L and whereby simultaneously X¹ = I, X² = I, X³ = T, X⁴ = H, X⁵ = L, X⁶ = L, X⁷ = F, X⁸ - L, X⁹ = L and X¹ ° = L are excluded.

• A fusion protein as specified in Table A5 or A6, wherein said sTNFR-I portion has a protein sequence selected from the group Ml to M58, wherein Ml - M58 are specified in Table Al.

• A fusion protein as specified in Table A5 or A6, wherein F has the sequence FI as specified in Table A3.

• A fusion protein as specified in Table A5 or A6, wherein L has the sequence LI as specified in Table A4.

• A fusion protein as specified selected from the group consisting of a member of Table A7.

• A peptide molecule selected from the group consisting of (A) GKYIHPQNNSICCTKCHKGTY,

(B) HLRHCLSCSKCRKEM,

(C) LFQCFNCSLCLNGTV,

(D) CK SLECTKLCLPQI or a sequence track consisting of at least 9 consecutive amino acid residues of any of said peptide molecules having a potential MHC class II binding activity and created from the primary sequence of non-modified human sTNFRl, whereby said peptide molecule or sequence track has a stimulation index of > 1.8 in a biological assay of cellular proliferation and said index is taken as the value of cellular proliferation scored following stimulation by a peptide and divided by the value of cellular proliferation scored in control cells not in receipt peptide and wherein cellular proliferation is measured by any suitable means.

• Use of said peptide molecule for the manufacture of a vaccine in order to reduce immunogenicity to sTNFR-I in a patient

• A modified peptide molecule deriving from any peptide molecule as specified having a reduced or absent potential MHC class II binding activity expressed by a stimulation index of less than 2, whereby said index is taken as the value of cellular proliferation scored following stimulation by a peptide and divided by the value of cellular proliferation scored in control cells not in receipt peptide and wherein cellular proliferation is measured by any suitable means.

• Use of said modified peptide molecule for the manufacture of a modified sTNFR-I molecule or a fusion protein comprising an Fc portion of an immunoglobulin and said modified sTNFR-I.

The mutant proteins of the present invention are readily made using recombinant DNA techniques well known in the art and the invention provides methods for the recombinant production of such molecules.

In as far as this invention relates to modified sTNFR-I, compositions containing such modified sTNFR-I proteins or fragments of modified sTNFR-I proteins and related compositions should be considered within the scope of the invention. In another aspect, the present invention relates to nucleic acids encoding modified sTNFR-I entities. In a further aspect the present invention relates to methods for therapeutic treatment of humans using the modified sTNFR-I proteins.

DETAILED DESCRIPTION OF THE INVENTION

The present invention concerns the human sTNFR-I molecule. The amino acid sequence of wilt-type human sTNFR-I (depicted as single-letter code) is as follows (Table A2: M59): DSVCPQGKYIHPQNNSICCTKCHKGTYLYNDCPGPGQDTDCRECESGSFTASENHLRHCLSCSKCRKEM GQVEISSCTVDRDTVCGCRKNQYRHY SENLFQCFNCSLCLNGTVHLSCQE QNTVCTCHAGFFLRENE CVSCSNCKKSLECTKLCLPQIEN

The term "sTNFR-I" is used herein to denote the human soluble tumour necrosis factor receptor type 1. In some instances the term is also used more broadly herein to include fusion proteins (see below) comprising a sTNFR-I moiety and or more especially a sTNFR-I mutein.

The term "mutein" is used herein to denote a sTNFR-I protein engineered to contain one or more amino acid substitutions differing from the above native sequence.

The term "peptide" as used herein, is a compound that includes two or more amino acids. The amino acids are linked together by a peptide bond.

A peptide bond is the sole covalent linkage between amino acids in the linear backbone structure of all peptides, polypeptides or proteins. The peptide bond is a covalent bond, planar in structure and chemically constitutes a substituted amide. An "amide" is any of a group of organic compounds containing the grouping -CONH-.

There are 20 different naturally occurring amino acids involved in the biological production of peptides, and any number of them may be linked in any order to form a peptide chain or ring. The naturally occurring amino acids employed in the biological production of peptides all have the L-configuration. Synthetic peptides can be prepared employing conventional synthetic methods, utilizing L-amino acids, D-amino acids, or various combinations of amino acids of the two different configurations. Some peptides contain only a few amino acid units. Short peptides, e.g., having less than ten amino acid units, are sometimes refened to as "oligopeptides". Other peptides contain a large number of amino acid residues, e.g. up to 100 or more, and are refened to as "polypeptides". By convention, a "polypeptide" maybe considered as any peptide chain containing three or more amino acids, whereas a "oligopeptide" is usually considered as a particular type of "short" polypeptide. Thus, as used herein, it is understood that any reference to a "polypeptide" also includes an oligopeptide. Further, any reference to a "peptide" includes polypeptides, oligopeptides, and proteins. Each different anangement of amino acids forms different polypeptides or proteins. The number of polypeptides, and hence the number of different proteins that can be formed, is practically unlimited.

Since the peptide bond is the sole linkage between amino acids, all peptides, polypeptides or proteins have defined termini conventionally refened to as the "N-terminus" or "N-terminal" residue and the "C-terminus" or "C-terminal residue". The N-terminal residue bears a free amino group, whereas the C-terminal residue bears a free carboxyl group.

All sequences of consecutive amino acids accordingly have an orientation N-terminal to C- terminal. Where fusion proteins are constituted or differing domains are connected within a protein species their relative orientation may be described as "N-terminal" or "C-terminal".

The term "fusion protein" is used herein to refer to a protein molecule comprising two or more functionally distinct protein domains within a single polypeptide chain. The protein moieties in the fusion protein may be directly coupled or may be joined via a linker peptide.

A "linker" or "linker peptide" refers herein to a peptide segment joining two moieties of fusion protein. An example of a linker peptide is provided by the amino acid sequence (G)₄S(G)₄S(G)₃SG. However also other linkers which are common in the art can used. The fusion proteins of the present invention contain such a linker but not all fusion proteins contain a linker.

Fusion proteins are commonly produced by means of recombinant DNA techniques and as such can be considered artificial proteins having no direct counterparts in nature (natural fusion proteins can arise, for example via chromosomal translocation, but are not considered here). An example of a fusion protein is a fusion in which an immunoglobulin Fc region is placed at the N-terminus of another protein such as sTNFR-I. Such a fusion is termed an "Fc- X" fusion, where X is a ligand (such as sTNFR-I) and Fc is the immunoglobulin constant region domain. Fc-X proteins have a number of distinctive, advantageous biological properties. In particular, whereas such fusion proteins can still bind the relevant Fc receptors on cell surfaces, when the ligand binds to its receptor, the orientation of the Fc region is altered such that antibody-dependent cell-mediated cytotoxicity (ADCC) and complement fixation are activated by the sequences present in the Fc domain should this be desired. Fc isotypes which do not significantly activate ADCC may be used where this is not desired. An example of a non-activating isotype is provided by Fc-gamma 4 (Fc-G4).

The term "immunoglobulin" is used herein to refer to a protein consisting of one or more polypeptides substantially encoded by immunoglobulin genes. The recognised immunoglobulin genes include the kappa, lambda, alpha, gamma (IgGl, IgG2, IgG3, IgG4), sigma, epsilon, and μ constant region genes and in nature multiple immunoglobulin variable region genes.

The term Fc is used herein to refer to an immunoglobulin heavy chain constant region domain.

The term "T-cell epitope" means according to the understanding of this invention an amino acid sequence which is able to bind MHC class II, able to stimulate T-cells and / or also to bind (without necessarily measurably activating) T-cells in complex with MHC class II.

Reference to "substantially non-immunogenic" or "reduced immunogenic potential" includes reduced immunogenicity compared to a parent protein or to a fusion protein containing the wild-type (WT) or native amino acid sequences of the test moiety.

The term "immunogenicity" includes an ability to provoke, induce or otherwise facilitate a humoral and or T-cell mediated response in a host animal and in particular where the "host animal" is a human.

The terms "T-cell assay" and "immunogenicity assay" concern ex vivo measures of immune reactivity. As such these involve a test immunogen e.g. a protein or peptide being brought into contact with live human immune cells and their reactivity measured. A typical parameter of induced reactivity is proliferation. The presence of suitable control determinations are critical and implicit in the assay.

"Time course assay" refers to a biological assay such as a proliferation assay in which determinations of activity are made sequentially over a period of time. In the present context, a "time course T-cell assay", refers to the determination of T-cell proliferation in response to a test immunogen (peptide) at multiple times following exposure to the test immunogen. The terms "time course T-cell assay" and "time course immunogenicity assay" maybe used interchangeably herein.

One conventional way in which T-cell assays are expressed is by use of a "stimulation index" or "SI". The stimulation index (SI) is conventionally derived by division of the proliferation score (e.g. counts per minute of radioactivity if using for example ³H-thymidine incorporation) measured to a test immunogen such as a peptide by the score measured in cells not contacted with a test immunogen. Test immunogens (peptides) which evoke no response give SI = 1.0 although in practice SI values in the range 0.8 - 1.2 are unremarkable. The inventors have established that in the operation of such immunogenicity assays, a stimulation index equal to or greater than 2.0 is a useful measure of significant induced proliferation.

PBMC means peripheral blood mononuclear cells in particular as obtained from a sample of blood from a donor. PBMC are readily isolated from whole blood samples using a density gradient centrifugation technique well understood in the art and comprise predominantly lymphocytes (B and T cells) and monocytes. Other cell types are also represented.

"Relative activity" means according the present context activity measured for a test protein in any single assay expressed relative to the activity measured for a positive control protein in an identical assay and usually conducted in parallel. Thus if the test protein and the control protein have the same measured activity the relative activity is said to be 1.

A "TNF neutralisation assay" according to the present context means an in vitro assay able to provide a reading of the functional capability of the test protein. In the present instance this means the ability of a given sTNFR-I mutein or sTNFR-I fusion protein to evoke a specific measurable effect. A particularly suitable TNF neutralisation assay is exemplified herein using cells sensitive the lethal effect of TNF-alpha and wherein the molecules of the invention confer a protective effect on the indicator cells. Other cells and assay formats can be contemplated to also provide quantitative estimations of specific activity of the test molecules and permit EDs₀ determinations.

hi another aspect, the present invention relates to nucleic acids encoding modified sTNFR-I entities. Such nucleic acids are preferably comprised within an expression vector. The control sequences that are suitable for prokaryotes, for example, include a promoter, optionally an operator sequence, and a ribosome binding site. Eukaryotic cells are known to utilise promoters, enhancers and polyadenylation signals. Such nucleic acids in general comprise a selection means typically an additional gene encoding a protein able to provide for the survival of the host cell. An example of such a selection gene is the beta-lactamase gene suitable for some E.coli host cells and this and others are well known in the art ["Molecular Cloning: A Laboratory Manual", second edition (Sambrook et al, 1989); "Gene Transfer Vectors for Mammalian Cells" (J. M. Miller & M. P. Calos, eds., 1987); "Current Protocols in Molecular Biology" (F. M. Ausubel et al, eds., 1987)].

Nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally, "operably linked" means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in the same reading frame. However, enhancers do not have to be contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, the synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice.

In some embodiments the expression vector comprises a nucleic acid sequence encoding a sTNFR-I variant operably linked to an expression control sequence. In various embodiments the expression vector comprises a nucleic acid sequence encoding a protein selected from the group comprising inclusively Ml to M58, or F1-L1-M1 to F1-L1-M58. Such an expression vector will comprise at least the sTNFR-I encoding domain of one of the said proteins operably linked with suitable expression control and selection sequences. Such an expression vector would include degenerate versions of the nucleic acid wherein degeneracy in relation to polynucleotides refers to the fact well recognised that .in the genetic code many amino acids are specified by more than one codon. The degeneracy of the code accounts for 20 different amino acids encoded by 64 possible triplet sequences of the four different bases comprising DNA. Another aspect of the present invention is a cultured cell comprising at least one of the above- mentioned vectors.

A further aspect of the present invention is a method for preparing the modified sTNFR-I comprising culturing the above mentioned cell under conditions permitting expression of the sTNFR-I from the expression vector and purifying the sTNFR-I from the cell.

h a yet further aspect, the present invention relates to methods for therapeutic treatment of humans using the sTNFR-I compositions. For administration to an individual, any of the compositions would be produced to be preferably at least 80% pure and free of pyrogens and other contaminants. It is further understood that the therapeutic compositions of the sTNFR-I proteins may be used in conjunction with a pharmaceutically acceptable excipient. The pharmaceutical compositions according to the present invention are prepared conventionally, comprising substances that are customarily used in pharmaceuticals, e.g. Remington's Pharmaceutical Sciences, (Alfonso R. Gennaro ed. 18^th edition 1990), including excipients, carriers adjuvants and buffers. The compositions can be administered, e.g. parenterally, enterally, intramuscularly, subcutaneously, intravenously or other routes useful to achieve an effect. Conventional excipients include pharmaceutically acceptable organic or inorganic carrier substances suitable for parenteral, enteral and other routes of administration that do not deleteriously react with the agents. For parenteral application, particularly suitable are injectable sterile solutions, preferably oil or aqueous solutions, as well as suspensions, emulsions or implants, including suppositories. Ampulles are convenient unit dosages. The phannaceutical preparations can be sterilised and, if desired, mixed with stabilisers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers or other substances that do not react deleteriously with the active compounds.

The major embodiments of the present invention are encompassed by the protein sequences Ml - M58, Fl-Ml - F1-M58, and F1-L1-M1 - F1-L1-M58. The proteins are fusion proteins of the type "Fc-X" wherein X in this present instance comprise sTNFR-I muteins. The sTNFR-I proteins are expressed in mammalian cell-lines as a C-terminal fusion partner, linked to the Fc unit of human IgG . The sTNFR-I sequence is fused to the C-terminus of a hinge modified/C_H2/C_H3 Fc region of human IgG₄ via a 15 amino acid flexible linker between the C- terminus of the C_H3 and the N-terminus of sTNFR-I. The amino acid sequence of the linker was as follows: (G) S(G)₄S(G)₃SG. The expressed fusion protein had a stoichiometry of (hinge-C_H2-C_H3-linker-sTNFR-I)₂.

Human Fc-gamma 4 was used as the fusion partner in all prefened molecules, but it can be readily recognised that in principle other isotypes could equally be used. In the present instance, immune effector functions are not desirable for a therapeutic sTNFR-I molecule. In contrast to some other human Fc isotypes, the Fc-gamma 4 isotype does not support complement activation and antibody-dependent cell-mediated cytotoxicity (ADCC) and was therefore selected for as the most prefened fusion partner.

Where the "Fc-X" approach has been used in other molecules, such as for example Fc-ILIO and ILIO-Fc, the in vivo half-life in mice was extended from minutes to greater than 30 hours [Lo K-M, et al (1998) Protein Engineering; 11: 495-500; Gillies SD,et al (1999) Cancer Research; 59: 2159-2166; Zheng X. X. et al (1995) Journal of Immunology; 154: 5590-5600]. Similarly, where an "X-Fc" molecule has been used as a therapeutic in humans, the serum half-life is recorded at 3 days [Korth-Bradley JM, et al (2000) Annals of Pharmacotherapy; 34: 161-164].

The inventors have provided sTNFR-I fusion proteins that show increased activity compared to the fusion proteins containing the wild-type (WT) sTNFR-I moiety. The "WT" or "native" fusion proteins (sequences M59, F1-L1-M59) constructed herein has been designated clone IDs pcTNFR 4-3 / pcTNFR54 (these clones differ in respect to their non-coding regions only, pcTNFR54 lacks an intron).

It has been somewhat surprisingly found that some of the molecules of the invention demonstrate activity in a TNF-alpha neutralisation assay equivalent or better than the positive control preparation. In some cases the protective effect was in the order of 2 -3 fold better than WT (clone ID # pcTNFR45 / F1-L1-M36, ρcTNFR71 / F1-L1-M39). hi some cases the protective effect was greater than 10 fold better than WT. Clone ID pcTNFRlOl / Fl-Ll- M54; and pcTNFR108 / F1-L1-M55 each demonstrate significant enhanced activity over the WT counterpart molecule. The sTNFR-I muteins of the present were constructed to be less immunogenic than the parental molecule. The design of individual muteins was directed from immunological considerations as well as functional activity data. Four regions of immunological importance within the sTNFR-I molecule were defined using screening assays involving use of PBMC preparations from healthy donor subjects. This approach has proven to be a particularly effective method for the identification such biologically relevant immunogenic peptides and is disclosed herein as an embodiment of the invention. In the present study, the method has involved the testing of overlapping sTNFR-I-derived peptide sequences in a scheme so as to scan and test the sTNFR-I sequence. Such a scan required synthesis and use of 54 peptides each of 15 residues in length. The synthetic peptides were tested for their ability to evoke a proliferative response in human T-cells cultured in vitro. Where this type of approach is conducted using naϊve human T-cells taken from healthy donors, the inventors have established that a stimulation index equal to or greater than 2.0 is a useful measure of induced proliferation.

Four epitope regions were identified in these studies. Region 1 encompasses sTNFR-I residues 7 - 27 and comprises the sequence: GKYIHPQ NSICCTKCHKGTY.

Region 2 encompasses sTNFR-I residues 55 - 69 and comprises the sequence: HLRHCLSCSKCRKEM.

Region 3 encompasses sTNFR-I residues 100 - 114 and comprises the sequence:

LFQCFNCSLC NGTV.

Region 4 encompasses sTNFR-I residues 145 - 159 and comprises the sequence:

CKKSLECTKLCLPQI .

The Rl - R4 peptide sequences represent the critical information required for the construction of modified sTNFR-I molecules in which one or more of these epitopes is compromised. Equally, The Rl - R4 peptide sequences represent the critical information required for the production of tolerogemc peptides. Epitope regions Rl, R2, R3 and R4 are each embodiments of the invention.

Under the scheme of the present, the epitopes are compromised by mutation to result in sequences no longer able to function as T-cell epitopes. It is possible to use recombinant DNA methods to achieve directed mutagenesis of the target sequences and many such techniques are available and well known in the art. Broadly, the sTNFR-I muteins herein were constructed containing mutations within the four identified immunogenic regions. Individual residues were targeted based upon the known binding properties of HLA-DR molecules in that they have an almost exclusive preference for a hydrophobic amino acid in pocket 1 and that this is the most important determinant of peptide binding [Jardetzky, T.S. et al (1990), EMBO J. 9 1797-1803; Hill, CM. et al (1994) J Immunol. 152: 2890-2898]. Exhaustive mutational analysis identified those residues within these regions that could be altered without adversely affecting the activity of the fusion protein. Choice of alternate residue was guided comparison to other sTNFR-I proteins from other species. Buried residues were replaced with either alanine or similar sized non-hydrophobic residues whereas exposed residues were scanned with all possible non-hydrophobic alternatives.

The general method of the present invention leading to the modified sTNFR-I comprises the following steps:

(a) determining the amino acid sequence of the polypeptide or part thereof;

(b) identifying one or more potential T-cell epitopes within the amino acid sequence of the protein by any method including determination of the binding of the peptides to MHC molecules using in vitro or in silico techniques or biological assays; (c) designing new sequence variants with one or more amino acids within the identified potential T-cell epitopes modified in such a way to substantially reduce or eliminate the activity of the T-cell epitope as determined by the binding of the peptides to MHC molecules using in vitro or in silico techniques or biological assays. Such sequence variants are created in such a way to avoid creation of new potential T-cell epitopes by the sequence variations unless such new potential T-cell epitopes are, in turn, modified in such a way to substantially reduce or eliminate the activity of the T-cell epitope; and

(d) constructing such sequence variants by recombinant DNA techniques and testing said variants in order to identify one or more variants with desirable properties according to well known recombinant techniques.

Taken together, the inventors have been able to define improved sTNFR-I proteins which can be depicted by the following structure (M): DSVCPQGKYX¹HPQN SX²CCX³KCXKGTYLYNDCPGPGQDTDCRECESGSFTASE]SIHX⁵RHCX^SSCSKC R EMGQVEISSCTVDRDTVCGCRKNQYRHYWSENLX⁷QCFNCSX⁸CX⁹NGTλΛHLSCQΞKQNTVCTCHAGF FLRENECVSCSNCKKSX¹⁰ECTKLCLPQIEN wherein X¹ is Q or S or N or E or A or G or K or P or R or I;

X² is Tor I;

X³ is R or T;

X⁴isPorH;

X⁵ is A or S or Q or L; X⁶isHorL;

X⁷isHorF;

X⁸isTorPorKorDorL;

X⁹isHorPorQorL;

X¹⁰isDorEorTorL and whereby simultaneously X¹ = I, X² = I, X³ = T, X⁴ = H, X⁵ = L, X⁶ = L, X⁷ = F, X⁸ = L, X⁹ = L and X¹⁰ = L are excluded, said meanings representing the native human sTNFR-I;

or, alternatively, fusion proteins of the structure:

F-(L)n-M, wherein M has the meaning as specified above, F is an immunoglobuhn heavy chain constant region, preferably an Fc portion, and L is an optional linker molecule (n = 0, 1), preferably a peptide linker having 4-20 amino acid residues. Preferably the Fc region derives from human IgG4 an may be linked at its N-terminal to a hinge region, which may be modified in order to reduce immunogenicity or to improve other desired properties.

The following, figures, sequence listing and examples are provided to aid the understanding of the present invention. It is understood that modifications can be made in the procedures set fourth without departing from the spirit of the invention.

DESCRIPTION OF THE SEQUENCES

To aid the understanding of the invention, Table 1 below sets out a description of the fusion protein sTNFR-I muteins. The derivation and properties of these proteins are also more fully disclosed in the examples. Table 1

*The residue numbering for the sTNFR-I substitutions commences from residue 1 of the sTNFR-I reading frame and is independent of the Fc component. Table Al

Ml - M58: Modified human sTNFR-I

Ml

DSV CPQGKYQHPQ NNSICCRKCP KGTYLYNDCP GPGQDTDCRE CESGSFTASE NHARHCLSCS KCRKΞMGQVE ISSCTVDRDT VCGCRKNQYR HYWSENLFQC FNCSTCHNGT VHLSCQEKQN TVCTCHAGFF LRENECVSCS NCKKSDECTK LCLPQIEN

M2

DSV CPQGKYQHPQ NNSICCRKCP KGTYLYNDCP GPGQDTDCRE CESGSFTASE NHARHCLSCS KCRKEMGQVΞ ISSCTVDRDT VCGCRKNQYR HYWSENLFQC FNCSTCLNGT VHLSCQEKQN TVCTCHAGFF LRENECVSCS NCKKSDECTK LCLPQIEN

M3

DSV CPQGKYQHPQ NNSICCRKCP KGTYLYNDCP GPGQDTDCRE CESGSFTASE NHARHCLSCS KCRKEMGQVE ISSCTVDRDT VCGCRKNQYR HYWSENLFQC FNCSLCHNGT VHLSCQEKQN TVCTCHAGFF LRENECVSCS NCKKSDECTK LCLPQIEN

M4

DSV CPQGKYQHPQ NNSICCRKCH KGTYLYNDCP GPGQDTDCRE CESGSFTASE NHARHCLSCS KCRKEMGQVE ISSCTVDRDT VCGCRKNQYR HYWSENLFQC FNCSTCHNGT VHLSCQEKQN TVCTCHAGFF LRENECVSCS NCKKSDECTK LCLPQIEN

M5

DSV CPQGKYQHPQ NNSICCTKCP KGTYLYNDCP GPGQDTDCRE CESGSFTASE NHARHCLSCS KCRKEMGQVE ISSCTVDRDT VCGCRKNQYR HYWSENLFQC FNCSTCHNGT VHLSCQEKQN TVCTCHAGFF LRENECVSCS NCKKSDECTK LCLPQIEN

M6

DSV CPQGKYQHPQ NNSICCRKCP KGTYLYNDCP GPGQDTDCRE CESGSFTASE NHARHCLSCS KCRKEMGQVE ISSCTVDRDT VCGCRKNQYR HYWSENLFQC FNCSLCLNGT VHLSCQEKQN TVCTCHAGFF LRENECVSCS NCKKSDECTK LCLPQIEN

M7

DSV CPQGKYQHPQ NNSICCRKCH KGTYLYNDCP GPGQDTDCRE CESGSFTASE NHARHCLSCS KCRKEMGQVE ISSCTVDRDT VCGCRKNQYR HYWSENLFQC FNCSTCLNGT VHLSCQEKQN TVCTCHAGFF LRENECVSCS NCKKSDECTK LCLPQIEN

M8

DSV_CPQGKYQHPQ NNSICCRKCH KGTYLYNDCP GPGQDTDCRE CESGSFTASE NHARHCLSCS KCRKEMGQVE ISSCTVDRDT VCGCRKNQYR HYWSENLFQC FNCSLCHNGT VHLSCQEKQN TVCTCHAGFF LRENECVSCS NCKKSDECTK LCLPQIEN

M9

DSV CPQGKYQHPQ NNSICCTKCP KGTYLYNDCP GPGQDTDCRE CESGSFTASE NHARHCLSCS KCRKEMGQVE ISSCTVDRDT VCGCRKNQYR HYWSENLFQC FNCSTCLNGT VHLSCQEKQN TVCTCHAGFF LRENECVSCS NCKKSDECTK LCLPQIEN

M10 DSV CPQGKYQHPQ NNSICCTKCP KGTYLYNDCP GPGQDTDCRE CESGSFTASE NHARHCLSCS KCRKEMGQVE ISSCTVDRDT VCGCRKNQYR HYWSENLFQC FNCSLCHNGT VHLSCQEKQN TVCTCHAGFF LRENECVSCS NCKKSDECTK LCLPQIEN Mil

DSV CPQGKYQHPQ NNSICCTKCH KGTYLYNDCP GPGQDTDCRE CESGSFTASE NHARHCLSCS KCRKEMGQVE ISSCTVDRDT VCGCRKNQYR HYWSENLFQC FNCSTCHNGT VHLSCQEKQN TVCTCHAGFF LRENECVSCS NCKKSDECTK LCLPQIEN

M12

DSV CPQGKYQHPQ NNSICCRKCH KGTYLYNDCP GPGQDTDCRE CESGSFTASE NHARHCLSCS KCRKEMGQVE ISSCTVDRDT VCGCRKNQYR HYWSENLFQC FNCSLCLNGT VHLSCQEKQN TVCTCHAGFF LRENECVSCS NCKKSDECTK LCLPQIEN

M13

DSV CPQGKYQHPQ NNSICCTKCP KGTYLYNDCP GPGQDTDCRE CESGSFTASE NHARHCLSCS KCRKEMGQVE ISSCTVDRDT VCGCRKNQYR HYWSENLFQC FNCSLCLNGT VHLSCQEKQN TVCTCHAGFF LRENECVSCS NCKKSDECTK LCLPQIEN

M14

DSV CPQGKYQHPQ NNSICCTKCH KGTYLYNDCP GPGQDTDCRE CESGSFTASE NHARHCLSCS KCRKEMGQVE ISSCTVDRDT VCGCRKNQYR HYWSENLFQC FNCSTCLNGT VHLSCQEKQN TVCTCHAGFF LRENECVSCS NCKKSDECTK LCLPQIEN

M15

DSV CPQGKYQHPQ NNSICCTKCH KGTYLYNDCP GPGQDTDCRE CESGSFTASE NHARHCLSCS KCRKEMGQVE ISSCTVDRDT VCGCRKNQYR HYWSENLFQC FNCSLCHNGT VHLSCQEKQN TVCTCHAGFF LRENECVSCS NCKKSDECTK LCLPQIEN

M16

DSV CPQGKYQHPQ NNSICCTKCH KGTYLYNDCP GPGQDTDCRE CESGSFTASE NHARHCLSCS KCRKEMGQVE ISSCTVDRDT VCGCRKNQYR HYWSENLFQC FNCSLCLNGT VHLSCQEKQN TVCTCHAGFF LRENECVSCS NCKKSDECTK LCLPQIEN

M17

DSV CPQGKYQHPQ NNSICCTKCH KGTYLYNDCP GPGQDTDCRE CESGSFTASE NHLRHCLSCS KCRKEMGQVE ISSCTVDRDT VCGCRKNQYR HYWSENLFQC FNCSLCLNGT VHLSCQEKQN TVCTCHAGFF LRENECVSCS NCKKSLECTK LCLPQIEN

Ml 8

DSV CPQGKYIHPQ NNSICCRKCH KGTYLYNDCP GPGQDTDCRE CESGSFTASE NHLRHCLSCS KCRKEMGQVΞ ISSCTVDRDT VCGCRKNQYR HYWSENLFQC FNCSLCLNGT VHLSCQEKQN TVCTCHAGFF LRENECVSCS NCKKSLECTK LCLPQIEN

M19

DSV CPQGKYIHPQ NNSICCTKCP KGTYLYNDCP GPGQDTDCRE CESGSFTASE NHLRHCLSCS KCRKEMGQVE ISSCTVDRDT VCGCRKNQYR HYWSENLFQC FNCSLCLNGT VHLSCQEKQN TVCTCHAGFF LRENECVSCS NCKKSLECTK LCLPQIEN

M20

DSV CPQGKYIHPQ NNSICCTKCH KGTYLYNDCP GPGQDTDCRE CESGSFTASE NHARHCLSCS KCRKEMGQVE ISSCTVDRDT VCGCRKNQYR HYWSENLFQC FNCSLCLNGT VHLSCQEKQN TVCTCHAGFF LRENECVSCS NCKKSLECTK LCLPQIEN M21

DSV CPQGKYIHPQ NNSICCTKCH KGTYLYNDCP GPGQDTDCRE CESGSFTASE NHLRHCLSCS KCRKEMGQVE ISSCTVDRDT VCGCRKNQYR HYWSENLFQC FNCSTCLNGT VHLSCQEKQN TVCTCHAGFF LRENECVSCS NCKKSLECTK LCLPQIEN M22

DSV CPQGKYIHPQ NNSICCTKCH KGTYLYNDCP GPGQDTDCRE CESGSFTASE NHLRHCLSCS KCRKEMGQVE ISSCTVDRDT VCGCRKNQYR HYWSENLFQC FNCSLCHNGT VHLSCQEKQN TVCTCHAGFF LRENECVSCS NCKKSLECTK LCLPQIEN

M23

DSV CPQGKYIHPQ NNSICCTKCH KGTYLYNDCP GPGQDTDCRE CESGSFTASE NHLRHCLSCS KCRKΞMGQVE ISSCTVDRDT VCGCRKNQYR HYWSENLFQC FNCSLCLNGT VHLSCQEKQN TVCTCHAGFF LRENECVSCS NCKKSDECTK LCLPQIEN

M24

DSV CPQGKYAHPQ NNSICCTKCH KGTYLYNDCP GPGQDTDCRE CESGSFTASE NHLRHCLSCS KCRKEMGQVE ISSCTVDRDT VCGCRKNQYR HYWSENLFQC FNCSLCLNGT VHLSCQEKQN TVCTCHAGFF LRENECVSCS NCKKSLECTK LCLPQIEN

M25

DSV CPQGKYEHPQ NNSICCTKCH KGTYLYNDCP GPGQDTDCRE CESGSFTASE NHLRHCLSCS KCRKEMGQVE ISSCTVDRDT VCGCRKNQYR HYWSENLFQC FNCSLCLNGT VHLSCQEKQN TVCTCHAGFF LRENECVSCS NCKKSLECTK LCLPQIEN

M26

DSV CPQGKYGHPQ NNSICCTKCH KGTYLYNDCP GPGQDTDCRE CESGSFTASE NHLRHCLSCS KCRKEMGQVE ISSCTVDRDT VCGCRKNQYR HYWSENLFQC FNCSLCLNGT VHLSCQEKQN TVCTCHAGFF LRENECVSCS NCKKSLECTK LCLPQIEN

M27

DSV CPQGKYKHPQ NNSICCTKCH^' KGTYLYNDCP GPGQDTDCRE CESGSFTASE NHLRHCLSCS KCRKEMGQVE ISSCTVDRDT VCGCRKNQYR HYWSENLFQC FNCSLCLNGT VHLSCQEKQN TVCTCHAGFF LRENΞCVSCS NCKKSLECTK LCLPQIEN

M28

DSV CPQGKYNHPQ NNSICCTKCH KGTYLYNDCP GPGQDTDCRE CESGSFTASE NHLRHCLSCS KCRKEMGQVΞ ISSCTVDRDT VCGCRKNQYR H.YWSΞNLFQC FNCSLCLNGT VHLSCQΞKQN TVCTCHAGFF LRENECVSCS NCKKSLECTK LCLPQIEN

M29

DSV CPQGKYPHPQ NNSICCTKCH KGTYLYNDCP GPGQDTDCRE CESGSFTASE NHLRHCLSCS KCRKEMGQVE ISSCTVDRDT VCGCRKNQYR HYWSENLFQC FNCSLCLNGT VHLSCQEKQN TVCTCHAGFF LRENECVSCS NCKKSLECTK LCLPQIEN

M30

DSV CPQGKYRHPQ NNSICCTKCH KGTYLYNDCP GPGQDTDCRE CESGSFTASE NHLRHCLSCS KCRKEMGQVE ISSCTVDRDT VCGCRKNQYR HYWSENLFQC FNCSLCLNGT VHLSCQEKQN TVCTCHAGFF LRENECVSCS NCKKSLECTK LCLPQIΞN

M31

DSV CPQGKYSHPQ NNSICCTKCH KGTYLYNDCP GPGQDTDCRE CESGSFTASE NHLRHCLSCS KCRKEMGQVE ISSCTVDRDT VCGCRKNQYR HYWSENLFQC FNCSLCLNGT VHLSCQEKQN TVCTCHAGFF LRENECVSCS NCKKSLECTK LCLPQIEN M32

DSV CPQGKYIHPQ NNSTCCTKCH KGTYLYNDCP GPGQDTDCRΞ CESGSFTASE NHLRHCLSCS KCRKΞMGQVE ISSCTVDRDT VCGCRKNQYR HYWSENLFQC FNCSLCLNGT VHLSCQEKQN TVCTCHAGFF LRENECVSCS NCKKSLECTK LCLPQIEN M33

DSV CPQGKYIHPQ NNSICCTKCH KGTYLYNDCP GPGQDTDCRE CESGSFTASE NHHRHCLSCS KCRKEMGQVE ISSCTVDRDT VCGCRKNQYR HYWSENLFQC FNCSLCLNGT VHLSCQEKQN TVCTCHAGFF LRENECVSCS NCKKSLECTK LCLPQIEN

M34

DSV CPQGKYIHPQ NNSICCTKCH KGTYLYNDCP GPGQDTDCRE CESGSFTASΞ NHQRHCLSCS KCRKEMGQVE ISSCTVDRDT VCGCRKNQYR HYWSENLFQC FNCSLCLNGT VHLSCQEKQN TVCTCHAGFF LRENECVSCS NCKKSLECTK LCLPQIEN

M35

DSV CPQGKYIHPQ NNSICCTKCH KGTYLYNDCP GPGQDTDCRE CESGSFTASE NHSRHCLSCS KCRKEMGQVE ISSCTVDRDT VCGCRKNQYR HYWSENLFQC FNCSLCLNGT VHLSCQEKQN TVCTCHAGFF LRENECVSCS NCKKSLECTK LCLPQIEN

M36

DSV CPQGKYIHPQ NNSICCTKCH KGTYLYNDCP GPGQDTDCRE CESGSFTASE NHLRHCHSCS KCRKEMGQVE ISSCTVDRDT VCGCRKNQYR HYWSENLFQC FNCSLCLNGT VHLSCQEKQN TVCTCHAGFF LRENECVSCS NCKKSLECTK LCLPQIEN

M37

DSV CPQGKYIHPQ NNSICCTKCH KGTYLYNDCP GPGQDTDCRE CESGSFTASE NHLRHCQSCS KCRKEMGQVE ISSCTVDRDT VCGCRKNQYR HYWSENLFQC FNCSLCLNGT VHLSCQEKQN TVCTCHAGFF LRENECVSCS NCKKSLECTK LCLPQIEN

M38

DSV CPQGKYQHPQ NNSTCCTKCH KGTYLYNDCP GPGQDTDCRE CESGSFTASE NHLRHCLSCS KCRKEMGQVE ISSCTVDRDT VCGCRKNQYR HYWSENLFQC FNCSLCLNGT VHLSCQEKQN TVCTCHAGFF LRENECVSCS NCKKSLECTK LCLPQIEN

M39

DSV CPQGKYIHPQ NNSICCTKCH KGTYLYNDCP GPGQDTDCRE CESGSFTASE NHLRHCLSCS KCRKEMGQVE ISSCTVDRDT VCGCRKNQYR HYWSENLFQC HNCSLCLNGT VHLSCQEKQN TVCTCHAGFF LRENECVSCS NCKKSLECTK LCLPQIEN

M40

DSV CPQGKYQHPQ NNSICCTKCH KGTYLYNDCP GPGQDTDCRE CESGSFTASE NHARHCLSCS KCRKEMGQVE ISSCTVDRDT VCGCRKNQYR HYWSENLFQC FNCSLCLNGT VHLSCQEKQN TVCTCHAGFF LRENECVSCS NCKKSLECTK LCLPQIEN

M41

DSV CPQGKYQHPQ NNSICCTKCH KGTYLYNDCP GPGQDTDCRE CESGSFTASE NHLRHCHSCS KCRKEMGQVE ISSCTVDRDT VCGCRKNQYR HYWSENLFQC FNCSLCLNGT VHLSCQEKQN TVCTCHAGFF LRENECVSCS NCKKSLΞCTK LCLPQIEN

M42

DSV CPQGKYQHPQ NNSICCTKCP KGTYLYNDCP GPGQDTDCRE CESGSFTASE NHARHCLSCS KCRKEMGQVE ISSCTVDRDT VCGCRKNQYR HYWSENLFQC FNCSLCLNGT VHLSCQEKQN TVCTCHAGFF LRENECVSCS NCKKSLECTK LCLPQIEN M43

DSV CPQGKYIHPQ NNSICCTKCH KGTYLYNDCP GPGQDTDCRE CESGSFTASE NHLRHCLSCS KCRKEMGQVE ISSCTVDRDT VCGCRKNQYR HYWSENLFQC FNCSLCLNGT VHLSCQEKQN TVCTCHAGFF LRENECVSCS NCKKSEECTK LCLPQIEN M44

DSV CPQGKYIHPQ NNSICCTKCH KGTYLYNDCP GPGQDTDCRE CESGSFTASE NHLRHCLSCS KCRKEMGQVE ISSCTVDRDT VCGCRKNQYR HYWSENLFQC FNCSDCLNGT VHLSCQEKQN TVCTCHAGFF LRENΞCVSCS NCKKSLΞCTK LCLPQIΞN

M45

DSV CPQGKYIHPQ NNSICCTKCH KGTYLYNDCP GPGQDTDCRΞ CESGSFTASE NHLRHCLSCS KCRKEMGQVE ISSCTVDRDT VCGCRKNQYR HYWSENLFQC FNCSKCLNGT VHLSCQEKQN TVCTCHAGFF LRENECVSCS NCKKSLECTK LCLPQIEN

M46

DSV CPQGKYIHPQ NNSICCTKCH KGTYLYNDCP GPGQDTDCRE CESGSFTASΞ NHLRHCLSCS KCRKΞMGQVE ISSCTVDRDT VCGCRKNQYR HYWSENLFQC FNCSPCLNGT VHLSCQEKQN TVCTCHAGFF LRENECVSCS NCKKSLECTK LCLPQIEN

M47

DSV CPQGKYIHPQ NNSICCTKCH KGTYLYNDCP GPGQDTDCRE CESGSFTASE NHLRHCLSCS KCRKEMGQVE ISSCTVDRDT VCGCRKNQYR HYWSENLFQC FNCSLCPNGT VHLSCQEKQN TVCTCHAGFF LRENECVSCS NCKKSLECTK LCLPQIEN

M48

DSV CPQGKYIHPQ NNSICCTKCH KGTYLYNDCP GPGQDTDCRE CESGSFTASE NHLRHCLSCS KCRKEMGQVΞ ISSCTVDRDT VCGCRKNQYR HYWSENLFQC FNCSLCQNGT VHLSCQEKQN TVCTCHAGFF LRENECVSCS NCKKSLECTK LCLPQIEN

M49

DSV CPQGKYQHPQ NNSICCTKCP KGTYLYNDCP GPGQDTDCRE CESGSFTASE NHLRHCHSCS KCRKEMGQVE ISSCTVDRDT VCGCRKNQYR HYWSENLFQC FNCSLCLNGT VHLSCQEKQN TVCTCHAGFF LRENECVSCS NCKKSLECTK LCLPQIEN

M50

DSV CPQGKYQHPQ NNSTCCTKCP KGTYLYNDCP GPGQDTDCRE CESGSFTASE NHARHCLSCS KCRKEMGQVE ISSCTVDRDT VCGCRKNQYR HYWSENLFQC FNCSLCLNGT VHLSCQEKQN TVCTCHAGFF LRENECVSCS NCKKSLECTK LCLPQIEN •

M51

DSV CPQGKYQHPQ NNSTCCTKCH KGTYLYNDCP GPGQDTDCRΞ CESGSFTASE NHLRHCLSCS KCRKEMGQVE ISSCTVDRDT VCGCRKNQYR HYWSENLFQC FNCSLCLNGT VHLSCQΞKQN TVCTCHAGFF LRENECVSCS NCKKSDECTK LCLPQIEN

M52

DSV CPQGKYQHPQ NNSICCTKCP KGTYLYNDCP GPGQDTDCRE CESGSFTASE NHARHCLSCS KCRKEMGQVΞ ISSCTVDRDT VCGCRKNQYR HYWSENLFQC HNCSLCLNGT VHLSCQEKQN TVCTCHAGFF LRENECVSCS NCKKSDECTK LCLPQIEN

M53

DSV CPQGKYQHPQ NNSICCTKCP KGTYLYNDCP GPGQDTDCRE CESGSFTASE NHARHCHSCS KCRKEMGQVΞ ISSCTVDRDT VCGCRKNQYR HYWSENLFQC FNCSTCHNGT VHLSCQEKQN TVCTCHAGFF LRENECVSCS NCKKSDECTK LCLPQIEN M54

DSV CPQGKYQHPQ NNSICCTKCP KGTYLYNDCP GPGQDTDCRE CESGSFTASE NHARHCHSCS KCRKΞMGQVΞ ISSCTVDRDT VCGCRKNQYR HYWSΞNLFQC HNCSTCHNGT VHLSCQΞKQN TVCTCHAGFF LRENECVSCS NCKKSDECTK LCLPQIEN M55

DSV CPQGKYQHPQ NNSICCTKCP KGTYLYNDCP GPGQDTDCRE CESGSFTASE NHARHCLSCS KCRKEMGQVE ISSCTVDRDT VCGCRKNQYR HYWSENLFQC HNCSTCHNGT VHLSCQΞKQN TVCTCHAGFF LRΞNECVSCS NCKKSDΞCTK LCLPQIΞN

M56

DSV CPQGKYQHPQ NNSICCRKCP KGTYLYNDCP GPGQDTDCRE CESGSFTASE NHARHCLSCS KCRKEMGQVE ISSCTVDRDT VCGCRKNQYR HYWSENLFQC HNCSTCHNGT VHLSCQΞKQN TVCTCHAGFF LRENECVSCS NCKKSDECTK LCLPQIEN

M57

DSV CPQGKYQHPQ NNSICCRKCH KGTYLYNDCP GPGQDTDCRE CESGSFTASE NHARHCLSCS KCRKEMGQVE ISSCTVDRDT VCGCRKNQYR HYWSENLFQC FNCSTCHNGT VHLSCQΞKQN TVCTCHAGFF LRΞNECVSCS NCKKSDΞCTK LCLPQIΞN

M58

DSV CPQGKYQHPQ NNSICCTKCH KGTYLYNDCP GPGQDTDCRE CESGSFTASE NHARHCLSCS KCRKEMGQVE ISSCTVDRDT VCGCRKNQYR HYWSENLFQC FNCSTCHNGT VHLSCQEKQN TVCTCHAGFF LRENECVSCS NCKKSDECTK LCLPQIEN

Table A2

M59 (wild-type human sTNFR-I)

DSV CPQGKYIHPQ NNSICCTKCH KGTYLYNDCP GPGQDTDCRE CESGSFTASE NHLRHCLSCS KCRKEMGQVE ISSCTVDRDT VCGCRKNQYR HYWSENLFQC FNCSLCLNGT VHLSCQΞKQN TVCTCHAGFF LRENECVSCS NCKKSLECTK LCLPQIEN

Table A3

FI (Fc domain human IgG4)

EPKSSDKTHT CPPCPAPΞFL GGPSVFLFPP KPKDTLMISR TPEVTCVWD VSQEDPEVQF NWYVDGVΞVH NAKTKPREEQ FNSTYRWSV LTVLHQDWLN GKEYKCKVSN KGLPSSIEKT

ISKAKGQPRE PQVYTLPPSQ ΞEMTKNQVSL TCLVKGFYPS DIAVEWESNG QPENNYKTTP

PVLDSDGSFF LYSKLTVDKS RWQQGNIFSC SV HEALHNH YTQKSLSLSP

Table A4 LI (linker peptide)

GAGGGGSGGG GSGGGSG

Table A5

Fusion proteins F - M (F is any immunoglobulin heavy chain constant region and M is a sequence of Table Al)

F-Ml, F-M2, F-M3,F-M4, F-M5, F-M6, F-M7, F-M8, F-M9, F-M10,F-M11, F-M12,F-M13,F-M14,F-M15,F-M16,F-M17,F-M18,F-M19,F-M20,F-M21, F-M22,F-M23,F-M24,F-M25,F-M26,F-M27,F-M28,F-M29,F-M30,F-M31, F-M32,F-M33,F-M34,F-M35,F-M36,F-M37,F-M38,F-M39,F-M40,F-M41, F-M42,F-M43,F-M44,F-M45,F-M46,F-M47,F-M48,F-M49,F-M50,F-M51, F - M52, F - M53, F - M54, F - M55, F - M56, F - M57, F - M58. Table A6

Fusion proteins F - L - M (F is any immunoglobulin heavy chain constant region, L is a any linker peptide, and M is a sequence of Table Al):

F-L-Ml, F-L-M2,F-L-M3,F-L-M4,F-L-M5,F-L-M6,F-L-M7,F-L-M8,

F-L-M9,F-L-M10,F-L-M11,F-L-M12,F-L-M13,F-L-M14,F-L-M15,

F-L-M16,F-L-M17,F-L-M18,F-L-M19,F-L-M20,F-L-M21,F-L-M22,

F-L-M23,F-L-M24,F-L-M25,F-L-M26,F-L-M27,F-L-M28,F-L-M29,

F-L-M29,F-L-M30,F-L-M31,F-L-M32,F-L-M33,F-L-M34,F-L-M35,

F-L-M36,F-L-M37,F-L-M38,F-L-M39,F-L-M40,F-L-M41,F-L-M42,

F-L-M43,F-L-M44,F-L-M45,F-L-M46,F-L-M47,F-L-M48,F-L-M49,

F-L-M50,F-L-M51,F-L-M52,F-L-M53,F-L-M54,F-L-M55,F-L-M56,

F-L-M57,F-L-M58.

Table A7 Fusion proteins Fl-Ll-M (FlisaFc portion from human IgG4 as indicated in Table A3, LI is the peptide linker of Table A4, and M is a sequence of Table Al)

F1-L1-M1,F1-L1-M2,F1-L1-M3,F1-L1-M4,F1-L1-M5,F1-L1-M6,

F1-L1-M7,F-L-M8,F1-L1-M9,F1-L1-M10,F1-L1-M11,F1-L1-M12,

FI -LI -M13, FI -LI -M14, FI -LI -M15, FI -LI -M16, FI -LI -M17, FI -LI -M18, FI -LI -M19, FI - LI -M20, FI -LI -M21, FI -LI -M22, FI -LI -M23, FI - LI -M24, FI -LI -M25, FI -LI -M26, FI -LI -M27, FI -LI -M28, FI -LI -M29, FI -LI -M29, FI -LI -M30, FI -LI -M31, FI -LI -M32, FI -LI -M33, FI -LI -M34, FI -LI -M35, F1-L1-M36,F1-L1-M37,F1-L1-M38,F1-L1-M39,F1-L1-M40,F1-L1-M41, F1-L1-M42,F1-L1-M43,F1-L1-M44,F1-L1-M45,F1-L1-M46,F1-L1-M47, F1-L1-M48,F1-L1-M49,F1-L1-M50,F1-L1-M51,F1-L1-M52,F1-L1-M53, FI -LI -M54, FI - LI -M55, FI -LI -M56, FI -LI -M57, FI -LI -M58.

Table A8

Fusion protein with wild-type human sTNFR-I (M59 of Table Al): F1-L1-M59

DESCRIPTION OF THE FIGURES Figure 1:

Identification of T-cell epitopes in sTNFR-I. Healthy donor samples were tested for reactivity with overlapping 15-mer synthetic peptides scanning the complete sTNFR-I sequence. The chart shows the frequency of response to each peptide. Peptides conesponding to epitope regions Rl, R2, R3 and R4 show response rates of 8-12% of donors tested.

Figure 2: Examples of individual donor responses in time course T-cell assays. Each panel shows the response to the WT peptide and modified peptides tested in parallel. Panel 2A shows the response of donor 15 to peptides from immunogenic epitope R2. Panel 2B shows the response of donor 19 to peptides from immunogenic epitope R2. Panel 2C shows the response of donor

11 to peptides from immunogenic epitope R4. Panel 2D shows the response of donor 8 to peptides from immunogenic epitope R4.

Figure 3:

Exemplary plots showing functional activity data for a number of sTNFR-I muteins. In all panels A-C, the activity of the WT clone pcTNFR54 is shown. Activity is plotted as concentration of test protein (ug/ml) versus optical density (OD492nm). In Figure 3A, clones pcTNFRlOl, 106, 107 and 108 are plotted. Clones pcTNFRlOland 108 show greater activity than WT. Clone pcTNFR107 shows approximately equivalent activity to

WT whereas pcTNFR 106 (= substitution Y9E) shows no activity.

In Figure 3B, clones ρcTNFR95, 96, 97 and 98 show equivalent activity to WT.

In Figure 3C, clones ρcTNFR99 and pcTNFRlOO show activity better than or equal to the WT control. Clone pcTNFRl 02 comprising the substitution set II 0Q, H23P, L56A, LI 08T,

LllOH and L149D shows no activity.

EXPERIMENTAL EXAMPLES

EXAMPLE 1

Construction of Fc-sTNFR-I muteins

The modified sTNFR-I proteins of the present invention were made using conventional recombinant DNA techniques. The coding sequence for sTNFR-I was cloned from human genomic DNA using PCR. A listing of all synthetic oligonucleotides used for the construction of the wild-type Fc-sTNFR-I fusion protein (Table A8) are shown in Table 2.

The wild-type gene was used both as a control reagent and a template from which to derive modified sTNFR-I proteins by site directed mutagenesis. WT and modified genes were inserted into a modified version of the expression vector pdC-huFc [Lo K-M et al, (1998) Protein Eng Ll:495-500]. The sTNFR-I gene was excised with BamHl and cloned into a similarly cut preparation of the vector which had been modified such that the sTNFR-I sequence is fused to the C-terminus of a hinge modified/C_H2/C_H3 Fc region of human IgG₄ via a 15 amino acid flexible linker between the C-terminus of the C_H3 and the N-terminus of sTNFR-I. The amino acid sequence of the linker was as follows: (G) S(G) S(G)₃SG. The expressed fusion protein had a stoichiometry of (hinge-C_H2-C_H3-linker-sTNFR-I)₂. The final construct used in this study was designated pcTNFR4-3 ("M59", Table A8).

Table 2

Oligonucleotide sequences used in the construction of the WT sTNFR-I fusion protein.

With reference to Table 2, the sTNFR-I gene was amplified from human genomic DNA as two fragments, using OL-1402 + OL-1405 and OL-1404 + OL-1403 respectively. These fragments were joined together by overlap PCR using OL-1402 + OL-1403 as primers to give a 1180bp product. This gene encodes 161 amino acids of the sTNFR protein and includes 3 introns. The fragment was cloned into pCR4 TOPO (frivitrogen, Paisley, UK).

To aid subsequent step, an internal Xmal(Smal) site was removed by PCR mutagenesis using OL-1402 + OL-1407 and OL-1406 + OL-1403 in separate reactions. The resulting two fragments were joined by overlap PCR using OL-1402 + OL-1403 as primers. The modified gene was re-cloned into the pCR4 TOPO vector. The resulting construct was used as a template for generating a PCR fragment with an additional BamHI site at the 5' end using OL1524 + OL1403. BamHI digestion of this fragment allowed ligation into the BamHI digested vector pdCs-linker-Fc vector [Lo K-M et al, (1998) Protein Eng 11:495-500]. The resulting construct was designated pcTNFR4-3 (F1-L1-M59).

Construction of Fc-sTNFR Muteins

Variants of sTNFR-I linked to the Fc portion of human IgG4 were constructed containing mutations within the four immunogenic regions of the protein. Desired substitutions were introduced into the sTNFR-I sequence by overlap PCR using HiFi Expand polymerase. Cycles of mutational analysis involving construction and function testing identified those residues within these regions that could be altered without adversely affecting the activity of the Fc-linked protein. The assay as described herein (see Example 4) was the main screening tool in this aspect.

Muteins of Fc-sTNFR-I were made using pcTNFR4-3 as a template and the overlap PCR strategy according to standard methods [for example see Higuchi et al (1988) Nucleic Acids Res. 16: 7351]. A listing of all synthetic ohgonucleotide primers used for the construction of each mutein is provided in Table 3.

With reference to Table 3, individual substitutions were introduced using oligonucleotides OL-234 and OL-1689 as flanking primers in paired reactions using each in combination with a single specific mutational ohgonucleotide. The primary PCR fragments were joined by overlap PCR driven by OL-1689 and OL-234. These overlap fragments were digested with Xmal and Xhol and ligated into Xmal/ Xhol digested pdCs-linker-Fc vector. It became apparent that removal of the first intron would be beneficial in the construction of some mutants; and this was carried out using OL-234+OL-1858 and OL-1857+OL-1689 and the two fragments joined by overlap PCR driven using OL-234 and OL-1689. In the pdCs-lihker-Fc vector this construct was termed pcTNFR 54, and contained a WT sTNFR-I coding region interspersed with only two introns.

Muteins containing two or more desired substitutions were made using single mutant constructs as templates, generating primary PCR fragments and joining these by overlap PCR. DNA sequencing was conducted on all constructs. This was diligently performed to confirm introduction of desired substitutions and establish that no extraneous (undesired) substitutions had been introduced for example by PCR enor. Table 3

EXAMPLE 2

Transfection and purification of fusion proteins

Transient transfections were done using HEK293 (ATCC# CRL-1573) cells and Lipofectamine 2000 (Invitrogen, Paisley, UK) as described by the manufacturer. Stable transfectants were also made in HEK293 cells and selected in media containing increasing concentrations of methotrexate. All cell-lines were maintained in DMEM plus 10% FBS with antibiotics and antimycotics. Fusion proteins were purified via Prosep-A chromatography followed by size exclusion chromatography (SEC). Briefly, 1ml Prosep^®-A columns (Millipore, Watford, UK) were equilibrated in PBS pH 7.4 before being loaded with 0.2μM filtered cell-culture supernatants (up to 500ml) that had been pH adjusted with 1/20 volumes of IM Tris-HCl pH 7.4. The column was washed with 50ml PBS pH 7.4 and the fusion protein eluted with 0.1M citrate buffer pH 3.0 and 0.9ml fractions collected. The fractions were immediately neutralized with 0.1ml IM Tris-HCl pH 8.0. SEC was done with Superdex 200 (Amersham Pharmacia, Amersham, UK) in a 3.2/30 column equilibrated and run in PBS pH 7.4 containing 0.1% Tween 80. Fractions spanning the major peak were pooled and nision proteins quantified using molar extinction coefficients at 280nm calculated using Lasergene™ software (Dnastar, Madison, WI, USA). The concentrations were confirmed using a BCA protein assay (Pierce, Chester, UK).

EXAMPLE 3

Quantitation of fusion proteins in cell-culture supernatants Fusion proteins were quantified by detecting the amount of human IgG₄ Fc in an ELISA format as follows: ELISA plates (Dynex Immulon4) were coated with a mouse monoclonal anti-human IgG Fc specific antibody at a dilution of 1/1500 in PBS pH7.4, lOOμl well, for 2h at 37°C. The plate was washed x4 with lOOμl/well PBS/0.05% Tween 20. Human IgG standards (The Binding Site, Birmingham, UK) were diluted to 2μg/ml in PBS/2%BSA and duplicate two-fold dilutions made vertically down the plate. Test samples were diluted 1/100 and 1/500 in PBS/2% BSA and assayed in duplicate. The plate was incubated for lh at room temperature and washed as before. Detection was done using lOOμl/well goat anti-human IgG Fc-specific peroxidase conjugate (The Binding Site, Birmingham, UK) at a dilution of 1/1000 in PBS, the plate washed as before and colour developed using SigmaFast OPD, lOOμl/well (Sigma, Poole, UK). The colour reaction was stopped by the addition of 50μl 2M sulphuric acid and the absorbance measured at 492nm in an Anthos HTII plate reader.

EXAMPLE 4 Functional activity of Fc-sTNFR-I muteins; protection of TNF-alpha sensitive cells in vitro by TNF- alpha neutralisation.

The ability of the sTNFR-I muteins to neutralise the lethal effect of TNF-alpha on a cell line grown in vitro was tested using the scheme provided by Galloway [Galloway et al. 1991 J Immunol. Meth. 140:37-43]. The assay uses murine fibrosarcoma cell line WEHI164, a line which is very sensitive to the lethal effect of TNF-alpha.

For the assay, cells were grown overnight in the presence of a fixed, lethal concentration of TNF-alpha and a range of different test protein concentrations. The next day, the metabolic activity of cells was measured as an indication of survival. Muteins that neutralise TNF-alpha confer a protective effect to the cells and thereby a greater metabolic activity is measured in the assay.

WEHI164 were obtained from the European Collection of Animal Cell Cultures (ECACC #. 8702250) and grown in DMEM medium with Glutamax, (Gibco, Paisley, UK), 10% foetal calf serum (Perbio, Chester, UK) and containing antibiotic-mycotic (Gibco). On the day prior to assay, cells are sub-cultured to ensure active proliferation during the subsequent assay period. The assay was conducted in 96 well plates in duplicate for all treatments. Plates were prepared to contain dilutions of control protein, test protein and negative control with no added protein. Typically, doubling dilution series of the protein preparation were ananged across a plate. Proteins preparations were culture supernantants from transient transfections of HEK293 cells. The amount of sTNFR-I mutein present was determined using ELISA (Example 3).

A stock solution of TNF-alpha (PeproTech EC Ltd, London, UK) at 50μg/ml in medium containing 4μg/ml of actinomycin was prepared and added to the treatment wells. The TNF- alpha D solution was mixed by gently tapping the plate and the plate incubated for at least two hours at room temperature before the prepared solutions were transfened to the assay plate containing the cells.

The assay plate was prepared by seeding 2.5xl0⁴ cells in 50μl per well and incubating for at least lhour at 37°C, 5% CO . Following this, 50μl of the TNF-alpha/test protein mixture or control preparation was transfened from the plate used to dilute out the various treatments. The cell and treatment mixtures were mixed by gently tapping the plate and the plate incubated overnight at 37°C in a humidified atmosphere containing 5% CO . Next day, the metabolic activity of the cells in each well was assessed using a "CellTiter 96 Aqueous One Solution Cell Proliferation Assay" (Promega, Southampton, UK). Following addition of the assay solution, the plates were incubated for a further 90 minutes and the absorbance of the solutions in each well was read using a plate reader at 492nm. The absorbance figures are plotted versus antibody concentration. In all assays the positive control preparation was a sample of the WT fusion protein expressed from either pcTNFR 54 or pcTNFR4-3

A total of 58 different sTNFR-I variants demonstrated positive activity in the WEHI assay. The most prefened molecules of the invention demonstrate activity in a TNF-alpha neutralisation assay equivalent or better than the positive control preparation. In some cases the protective effect was in the order of 2 -3 fold better than WT (clone ID # pcTNFR45 / M36 (F1-L1-M36), pcTNFR71 / M39 (F1-L1-M39). hi some cases the protective effect was greater than 10 fold better than WT. Clone ID pcTNFRlOl / M54 (F1-L1-M54); and pcTNFR108 / M55 (F1-L1-M55) each demonstrate significant enhanced activity over the WT counterpart molecule.

A list of active muteins is provided in Table 4. Table 4 Active muteins

FI is the sequence of Table A3, LI is the sequence of Table A4 and Ml - M5δ are the sequences of Table Al.

* Activity = "+" if the relative activity is between 1 and 4. Activity = "++" if activity is > WT

Exemplary activity plots showing activity of several Fc-sTNFR muteins are provided in Figure 3.

EXAMPLE 5

Identification of T- cell epitopes in human sTNFR-ID

All blood samples used in this study were obtained with approval of the Addenbrooke's Hospital Local Research Ethics Committee. T-cell epitope mapping was performed using human PBMCs isolated from blood obtained from the National Blood Transfusion Service (Addenbrooke's Hospital, Cambridge, UK). PBMCs from 20 healthy donors were isolated by Ficoll density centrifugation and stored under liquid nitrogen. Each donor was tissue-typed using an Allset™ PCR based tissue-typing kit (Dynal) and T cell assays were performed by selecting donors according to individual MHC haplotypes. 15mer peptides staggered by three amino acids and spanning the human sTNFR-I sequence were purchased from Pepscan Systems BV (NL). Using this scheme, total of 54 peptides were required to scan the sTNFR-I residues of interest. The sequence and peptide number of these peptides are provided in Table 5.

Table 5

Peptides used to map immunogenic epitopes within sTNFR-I

For each donor sample, PBMCs were thawed and resuspended in AIM-V (frivitrogen) containing 100 units/ml penicillin, lOOμg/ml streptomycin and ImM glutamine. Triplicate cultures of 2x10⁵ PBMC/well of flat-bottomed 96 well plate were incubated with peptides at a final concentration of 1 μM and lOμM. Cells were incubated for 7 days before pulsing with lμCi/well tritiated thymidine for 18 hours. Cultures were harvested onto glass fibre filter mats using a Tomtec Mach HI plate harvester and cpm values determined by scintillation counting using a Wallac Microbeta TriLux plate reader. Regions of immunogenicity were determined by identifying peptides that induced donors to respond with stimulation indexes >2 and by determination of the donor response rate for each peptide (Figure 1). Peptides located within four separate regions were able to induce T cell proliferation. Region 1 (Rl) encompasses sTNFR-I residues 7 - 27 and comprises the sequence:

GKYIHPQNNS I CCTKCHKGTY.

Region 2 (R2) encompasses sTNFR-I residues 55 - 69 and comprises the sequence:

HLRHCLSCSKCRKEM.

Region 3 (R3) encompasses sTNFR-I residues 100 - 114 and comprises the sequence:

LFQCFNCSLCLNGTV.

Region 4 (R4) encompasses sTNFR-I residues 145 - 159 and comprises the sequence:

CKKSLECTKLCLPQI .

hi all assays, each donor was also tested for their ability to respond to two positive control peptides influenza haemagglutinin A amino acids 307-319 [Krieger JI, et al (1991) Journal of Immunology; 146: 2331-2340] and chlamydia HSP60 amino acids 125-140 [Cenone MC, et al (1991) Infection and Immunity; 59: 79-90]. Keyhole limpet haemocyanin, a well documented potent T cell antigen was also used as a control.

EXAMPLE 6

Analysis of immunogenic regions by time-course T-cell assays

Bulk cultures of 2-4x10⁶ PBMC/well were established from 20 healthy donor samples in 24 well plates. Cells were incubated for 6 to 9 days with WT and variant peptides spanning the immunogenic regions (see Table 6). T cell proliferation was assessed by tritiated thymidine incorporation on days 6, 7, 8 and 9. Proliferation was assessed at each time point, by gently resuspending the bulk cultures and removing samples of PBMC, that were then incubated in triplicate wells of U-bottomed 96 well plate with lμCi/well tritiated thymidine for 18 hours as described above.

The time course assay was used to test variant peptides containing substitutions over WT. Substitutions were made at key locations where there was expectation that the substitution would prevent binding to MHC class II and therefore, subsequent T cell proliferation in the assay. Particular substitutions were made based on information from various models of MHC class II binding motifs. The favoured mutations were large basic residues such as arginine or lysine but where structural models predicted severe affects on the protein structure we used alanine instead. All substitutions tested as synthetic peptides in the time-course assay were mutations known to be compatible with functional activity. The peptide sequences tested are listed in Table 6.

Table 6.

Sequences of peptides used in time-course assays

significant induced proliferation in any to the donor samples tested. By contrast WT peptides and peptides with some substitutions do induce proliferation with an SI > 2 in one or more donors.

Exemplary time course assay results depicted as SI plotted versus time are shown in Figure 2. The results are consistent with the assertion that substitutions within T-cell epitopes are able to remove the immunogenic properties of the sequences in which they are contained. As these substitutions are also compatible with functional activity in whole molecules of sTNFR, these substitutions where present in a sTNFR mutein will confer to the protein a significantly reduced immunogenic potential. It is contemplated that by use of the methods set out herein that the immunogenic potential of the modified protein may be tested using time-course assays and whole purified protein as test immunogen.

Claims

Patent Claims:

1. A fusion protein of the structure

F - (L)n- M, having essentially the same biological specificity and activity of human sTNFR-I, comprising an immunoglobulin heavy chain constant region (F) and a human sTNFR-I molecule (M) modified by one or more amino acid substitutions, wherein said fusion protein is substantially non-immunogenic or less immunogenic than the parental fusion protein comprising the non-modified human sTNFR-I, and said amino acid substitutions have been carried out in one or more of the sequence tracks

^' (i) GKYIHPQNNSICCTKCHKGTY,

(ii) HLRHCLSCSKCRKEM,

(iii) LFQCFNCSLCLNGTV,

(iv) CKKSLECTKLCLPQI within the wild-type sTNFR-I molecule and cause a reduction or an elimination of one or more of T-cell epitopes, which act in the parental non-modified fusion molecule as MHC class II binding ligands and stimulate T-cells, said immunoglobulin heavy chain constant region is fused directly (n = 0) or indirectly (n = 1) via a linker molecule (L) to said modified human sTNFR-I molecule (M).

2. A fusion protein according to claim 1, wherein F is an Fc domain.

3. A fusion protein of claim 1 or 2, wherein F comprises a hinge region.

4. A fusion protein according to any of the claims 1 - 3, wherein the C-terminus of the human immunoglobulin heavy constant region domain is linked directly or indirectly to the N- terminus of the modified sTNFR-I molecule.

5. A fusion protein according to any of the claims 1 -4, wherein said modified sTNFR-I molecule contains one or more of the amino acid substitutions

I10Q, T20R, H23P , L56A, L108T, LllOH and L149D within the sequence tracks (i) - (iv).

6. A fusion protein according to any of the claims 1-4, wherein said sTNFR-I molecule in said fusion protein has the formula / structure:

DSVCPQGKYX^XHPQNNSX²CCX³KCX⁴KGTYLYNDCPGPGQDTDCRECESGSFTASENHX⁵RHCX^SS

CSKCRKEMGQVΞISSCTVDRDTVCGCRKNQYRHYWSΞNLX⁷QCFNCSX^aCX⁹NGTVHLSCQEKQN TVCTCHAGFFLRFNΞCVSCSNCKKSX¹⁰ECTKLCLPQIEN wherein

X¹ is Q or S or N or E or A or G or K or P or R or I;

X² is Tor I;

X³ is R or T; X⁴isPorH;

X⁵ is A or S or Q or L;

X⁶isHorL;

X⁷isHorF;

X⁸isTorPorKorDorL; X⁹isHorPorQorL;

X¹⁰ is D or E or T or L and whereby simultaneously X¹ = I, X² = I, X³ = T, X⁴ = H, X⁵ = L, X⁶ - L, X⁷ = F,

X⁸ = L, X⁹ = L and X¹⁰ = L are excluded.

7. A fusion protein according to any of the claims 1-6, wherein F is in Fc domain of human IgG4.

8. A fusion protein according claim 7, wherein L is a peptide linker having 4-20 amino acid residues.

9. A fusion protein according to any of the claims 1-8, wherein said sTNFR-I molecule has a protein sequence selected from the group consisting of Ml to M58 of Table Al.

10. A fusion protein according to any of the claims 1-8 selected from the group consisting of F1-L1-M1 to F1-L1-M58 of Table A7, wherein FI is a Fc domain of human IgG4 comprising a modified hinge region, LI is a peptide linker having the sequence GAGGGGSGGG GSGGGSG, and Ml to M58 have the sequences as specified in Table Al.

11. A dimeric fusion protein comprising two identical monomeric fusion protein chains according to any of the claims 1 - 10.

12. A peptide molecule selected from the group consisting of (i) GKYIHPQNNSICCTKCHKGTY,

(ϋ) HLRHCLSCSKCRKEM, (iϋ) LFQCFNCSLCLNGTV, (iv) CKKSLECTKLCLPQI or a sequence track consisting of at least 9 consecutive amino acid residues of any of said peptide molecules having a potential MHC class II binding activity and created from the primary sequence of non-modified human sTNFRl, whereby said peptide molecule or sequence track has a stimulation index of > 1.8 in a biological assay of cellular proliferation and said index is taken as the value of cellular proliferation scored following stimulation by a peptide and divided by the value of cellular proliferation scored in control cells not in receipt peptide.

13. Use of a peptide molecule according to claim 12 for the manufacture of a vaccine in order to reduce immunogenicity to sTNFR-I in a patient.

14. A peptide molecule modified by one or more amino acid substitutions deriving from any peptide molecule according to claim 11 and having a reduced or absent potential MHC class II binding activity expressed by a stimulation index of less than 2, whereby said index is taken as the value of cellular proliferation scored following stimulation by a peptide and divided by the value of cellular proliferation scored in control cells not in receipt peptide.

15. Use of a modified peptide molecule according to claim 14 for the manufacture of a modified sTNFR-I molecule Ml to M58 of Table Al, or a fusion protein comprising an Fc portion of an immunoglobulin and said modified sTNFR-I molecule.