WO2001005822A2

WO2001005822A2 - Molecules derived from interleukin-1 beta

Info

Publication number: WO2001005822A2
Application number: PCT/GB2000/002657
Authority: WO
Inventors: Sylvie Toulmond
Original assignee: The Victoria University Of Manchester
Priority date: 1999-07-16
Filing date: 2000-07-11
Publication date: 2001-01-25
Also published as: AU5997100A; WO2001005822A3; GB9916630D0

Abstract

The present invention relates to an isolated C-terminal fragment of pro-interleukin 1β comprising a protein sequence having a molecular weight of 20-22 kD or an N-terminal truncation thereof, incorporating an interleukin-1β converting enzyme (ICE) cleavage site, the fragment being cleavable by ICE to produce active IL-1β, and the cleavage product thereof.

Description

MOLECULES DERIVED FROM INTERLEUKINS

The present invention relates to molecules derived from interkeukins, and particularly to interleukin 1 beta (IL-l β)-derived proteins and peptides, nucleic acid molecules encoding such proteins and peptides and antibodies to such proteins and peptides and the use of the aforesaid in research, diagnosis and therapy.

IL-l β is a proinflammatory cytokine which plays a major role in peripheral inflammatory and immune responses. In the adult and in non-pathological conditions mature IL-lβ is not expressed or is expressed only in very low, basal, amounts. Bacterial or viral infections lead to induction of mature IL-lβ expression by immune cells, as part of a normal immune response. IL-lβ is a pyrogen and contributes to the rise in temperature involved in infection. Once the infection is eradicated IL-l β returns to basal levels. Several chronic (Alzheimer disease, Multiple sclerosis, AIDS and diabetes) and acute (stroke, head injury) pathologies are associated with a sustained increase in IL-l β expression, i.e. the expression rises sharply and does not return to basal levels, or does so very slowly. Understanding the mechanisms regulating the synthesis of active IL-l β is of primary interest for the development of therapeutic approaches.

The fully active mature form of the cytokine IL-l β (17 kD in rats and 17.5 kD in humans) is generated by cleavage of an inactive precursor, pIL-l β, by a cysteine protease called Caspase-1 or interleukin l β converting enzyme (ICE). The rat form of pIL-l β is 33 kD and the human form of pIL-lβ is 31 kD. The nucleic acid and protein sequences of both rat and human pILl-lβ are known. ICE recognises the consensus sequence XVXD and cleaves after aspartate (D) followed by a small amino acid (alanine (A) or valine (V)). The ICE consensus sequence XVXDA/V (X denotes any amino acid) is conserved between species of pIL-lβ from mouse, rat and human. ICE itself is generated from a precursor that requires processing to generate active ICE. Recent studies (Garcia-Calvo, M. et al., J. Biol. Chem. (1998) Dec 4; 273(49): 32608-13) suggests alternative consensus ICE cleavage sites, e.g. WEHD.

IL-1 β is an unusual protein in that neither pIL-1 β nor mature IL-1 β posses a secretion signal yet both can be released from IL-l β expressing cells. Virtually nothing is known regarding the intracellular trafficking and mechanisms of release of IL-lβ. As mature IL-lβ is thought to be involved in an array of conditions, further understanding of the activation and processing of IL-l β from pIL-lβ should lead to the identification of agents for the diagnosis and therapy of conditions thought to involve IL-l β, such as chronic and acute inflammation, arthritis, septicaemia, autoimmune diseases (e.g. inflammatory bowel disease, psoriasis), transplant rejection, graft versus host disease, infection, stroke, ischemia, acute respiratory distress syndrome, restenosis, brain injury, AIDS, bone diseases, cancer, atherosclerosis and Alzheimer^'s disease.

Studies into the activation of pIL-l β to active IL-lβ have shown the presence of an alternative active form of IL-l β in human psoriasis scales i.e. in the epidermis (Nylander, E.L. et al., Eur. J. Immunol. 1997 27:2165-71). Cells of the epidermis do not contain active ICE and thus the alternative active form is produced by cleavage of pIL-lβ by an enzyme other than ICE. According to the disclosure, the sites in the 31 kD precursor which are possibly cleaved by enzymes other than ICE to produce active IL-l β are at amino acid residues N-terminal of the ICE cleavage site at Asp 116, resulting in a slightly larger and more acidic protein than mature IL-l β (17.5 kD). The alternative active IL-l β is shown to be slightly larger (between 18.5 and 27.5 molecular weight markers on immunoblot) and more acidic than mature IL-lβ but the actual cleavage site is not disclosed. The alternative active form of IL-l β is hardly detectable when compared to the 31 kD and 17.5 kD forms produced in humans after endotoxin challenge. Perregaux, D.G and Gable, CA (Am. J. Physiol. 275 (Cell Physiol. 44): C1538- C1547, 1998) discloses another form of IL-l β, a 20 kD IL-l β polypeptide found in extracellular immunoprecipitate from human LPS activated monocytes. The identity of this 20 kD species is unknown as is its sequence. The 20 kD polypeptide may represent an N-, C- or central fragment of pIL-lβ.

Auron, P.E. et al., J. Immunol. 1987, vol. 138, 1447-56 discloses further cleavage products of pIL-lβ from human blood monocytes, the active IL-lβ products present in monocyte supernatant having molecular weights (m.w.) of 23 and 18 kD. The paper states that the mature N-terminus of a low m.w. form of IL-1 is alanine-proline, which corresponds to alanine-proline at position 1 17-1 18 in the human IL-lβ sequence. This site is located downstream from a series of basic residues as well as a non-basic sequence which have been conserved in all IL-1 cDNA molecules sequenced to date. It is suggested that processing at this site by a trypsin-like esterase activity may be responsible for generating the 23 kD form of IL-1. The alanine- proline sequence at the mature amino terminus of the low m.w. (18 kD) IL-1 peptide may be the result of a secondary cleavage, or a direct cleavage by a protease with a different specificity.

IL-l β actions are receptor mediated. Two receptors have been identified, type I and type II. Binding of an accessory protein to type I is required for signal transduction. The type II lacks an intracellular domain and is thought to act as a decoy. Signal transduction from type I receptors has been identified as acting through activation of the nuclear transcription factor NF-κB. NF-κB is itself synthesised as an inactive precursor that requires cleavage to become active. Processing of NF-κB is done in the proteosome, a multienzymatic complex, the primary function of which is the degradation of endogenous and exogenous proteins to generate short peptides presented as antigens by molecules of the major histocompatibility complex class I (MHC class I). Human monocytes are very efficient in releasing active (17.5 kD) IL-l β after endotoxin challenge. However, endotoxin-activated rat and mouse monocytes release very little 17 kD in comparison, although rodents are thought to be among the most resistant mammals to infection. This suggests different mechanisms of pIL-l β activation between humans and rodents.

It is an object of the present invention to identify further cleavage products of pIL-l β and provide for their use in treatment and diagnosis of conditions involving abnormal IL-lβ expression or activity.

According to the first aspect of the invention there is provided an isolated C-terminal fragment of pro-interleukin 1 β comprising a protein sequence having a molecular weight of 20 to 22 kD or an N-terminal truncation thereof, incorporating an interleukin- lβ converting enzyme (ICE) cleavage site, the fragment being cleavable by ICE to produce active IL-lβ.

The invention is based on the discovery of a new form of IL-lβ present in rats in vivo, having a molecular weight of approximately 21 kD as predicted by Western blotting. This fragment can be extracted from endotoxin-activated rat peripheral blood monocytes and bone marrow macrophages. Strong detergent and sodium dodecyl sulphate (SDS) are required for the extraction of the approximately 21 kD fragment of IL-l β in vivo, indicating that the fragment is membrane associated or undergoes strong interaction with a protein membrane complex. It is proposed that the approximately 21 kD fragment represents a storage form of IL-lβ in vivo. This approximately 21 kD fragment is not secreted and is expressed in much greater amounts than the 17 kD form in vivo. The approximately 21 kD fragment is also present in the injured brain (as evidenced in fluid percussion trauma).

The approximately 21 kD C-terminal fragment of rat pIL-lβ is generated by cleavage of the precursor at a site at the amino end of pIL-lβ not previously identified as a cleavage site. The fragment produced may be further cleaved by ICE to generate mature IL-l β and a peptide of approximately 25 to 40 amino acids in length that is highly acidic and negatively charged at neutral pH.

It is proposed that in rats pIL-l β is processed into the approximately 21 kD form of IL-1 by the proteosome, from where it is translocated by a transporter to the endoplasmic reticulum (ER), another subcellular compartment or the cell membrane. If the approximately 21 kD form of IL-l β is transported to the ER and active ICE is present, it cleaves the 21 kD IL-l β to generate active 17 kD IL-lβ which is released from the cell. Cleavage of the 21 kD form to the 17 kD form generates a short acidic peptide that may possess an unknown function (may be a positive or negative feedback signal for the regulation of IL-l β expression) or may be degraded. It is further proposed that if ICE is inactive or absent the 21 kD form of IL-lβ is either stored in the ER or recycled.

The approximately 21 kD form of IL-l β seen in rats is proposed to be a previously unrecognised intermediate formed in the conversion of pIL-lB to active IL-l β. This represents a target against which agents can be set to modulate the rate of active IL-lβ production (17 kD).

Preferably the isolated 20-22 kD fragment according to the present invention comprises a protein sequence having at least 80% identity over its entire length to the amino acid sequence of SEQ ID No. 1.

SEQ ID No. 1:

PWSFQDEDPS TFFSFIFEEE PVLCDSWDDD DLLVCD*VPIR QLHCRLRDEQ QKCLVLSDPC ELKALHLNGC NISQQVVFSM SFVQGETSND KIPVALGLKG LNLYLSCVMK DGTPTLQLES VDPKQYPKKK MEKRFVFNKI EVKTKVEFES AQFPNWYIST SQAEHRPVFL GNSNGRDIVD FTMEPVSS Amino acid residues shown in bold and underlined are predicted potential sites for phosphorylation by casein kinase II. * indicates the ICE cleavage site

The isolated 20-22 kD fragment according to the present invention may comprise a truncation of one or more amino acids from the N-terminus of the amino acid sequence of SEQ ID No. 1 or sequences having at least 80% identity over their entire length thereto. Such truncated sequences are shown as SEQ ID Nos. 2 — 11.

SEQ ID No. 2:

WSFQDEDPS TFFSFIFEEE PVLCDSWDDD DLLVCD*VPIR QLHCRLRDEQ QKCLVLSDPC ELKALHLNGC NISQQVVFSM SFVQGETSND KIPVALGLKG LNLYLSCVMK DGTPTLQLES VDPKQYPKKK MEKRFVFNKI EVKTKVEFES AQFPNWYIST SQAEHRPVFL GNSNGRDIVD FTMEPVSS

SEQ ID No. 3:

SFQDEDPS TFFSFIFEEE PVLCDSWDDD DLLVCD*VPIR QLHCRLRDEQ QKCLVLSDPC ELKALHLNGC NISQQVVFSM SFVQGETSND KIPVALGLKG LNLYLSCVMK DGTPTLQLES VDPKQYPKKK MEKRFVFNKI EVKTKVEFES AQFPNWYIST SQAEHRPVFL GNSNGRDIVD FTMEPVSS

SEQ ID No. 4:

FQDEDPS TFFSFIFEEE PVLCDSWDDD DLLVCD*VPIR QLHCRLRDEQ QKCLVLSDPC ELKALHLNGC NISQQVVFSM SFVQGETSND KIPVALGLKG LNLYLSCVMK DGTPTLQLES VDPKQYPKKK MEKRFVFNKI EVKTKVEFES AQFPNWYIST SQAEHRPVFL GNSNGRDIVD FTMEPVSS

SEQ ID No. 5:

QDEDPS TFFSFIFEEE PVLCDSWDDD DLLVCD*VPIR QLHCRLRDEQ QKCLVLSDPC ELKALHLNGC NISQQVVFSM SFVQGETSND KIPVALGLKG LNLYLSCVMK DGTPTLQLES VDPKQYPKKK MEKRFVFNKI EVKTKVEFES AQFPNWYIST SQAEHRPVFL GNSNGRDIVD FTMEPVSS

SEQ ID No. 6:

DEDPS TFFSFIFEEE PVLCDSWDDD DLLVCD*VPIR QLHCRLRDEQ QKCLVLSDPC ELKALHLNGC NISQQVVFSM SFVQGETSND KIPVALGLKG LNLYLSCVMK DGTPTLQLES VDPKQYPKKK MEKRFVFNKI EVKTKVEFES AQFPNWYIST SQAEHRPVFL GNSNGRDIVD FTMEPVSS

SEQ ID No. 7:

EDPS TFFSFIFEEE PVLCDSWDDD DLLVCD*VPIR QLHCRLRDEQ QKCLVLSDPC ELKALHLNGC NISQQVVFSM SFVQGETSND KIPVALGLKG LNLYLSCVMK DGTPTLQLES VDPKQYPKKK MEKRFVFNKI EVKTKVEFES AQFPNWYIST SQAEHRPVFL GNSNGRDIVD FTMEPVSS

SEQ ID No. 8:

DPS TFFSFIFEEE PVLCDSWDDD DLLVCD*VPIR QLHCRLRDEQ QKCLVLSDPC ELKALHLNGC NISQQVVFSM SFVQGETSND KIPVALGLKG LNLYLSCVMK DGTPTLQLES VDPKQYPKKK MEKRFVFNKI EVKTKVEFES AQFPNWYIST SQAEHRPVFL GNSNGRDIVD FTMEPVSS

SEQ ID No. 9:

PS TFFSFIFEEE PVLCDSWDDD DLLVCD*VPIR QLHCRLRDEQ QKCLVLSDPC ELKALHLNGC NISQQVVFSM SFVQGETSND KIPVALGLKG LNLYLSCVMK DGTPTLQLES VDPKQYPKKK MEKRFVFNKI EVKTKVEFES AQFPNWYIST SQAEHRPVFL GNSNGRDIVD FTMEPVSS

SEQ ID No. 10:

S TFFSFIFEEE PVLCDSWDDD DLLVCD*VPIR QLHCRLRDEQ QKCLVLSDPC ELKALHLNGC NISQQVVFSM SFVQGETSND KIPVALGLKG LNLYLSCVMK DGTPTLQLES VDPKQYPKKK MEKRFVFNKI EVKTKVEFES AQFPNWYIST SQAEHRPVFL GNSNGRDIVD FTMEPVSS

SEQ ID No. 11:

TFFSFIFEEE PVLCDSWDDD DLLVCD*VPIR QLHCRLRDEQ QKCLVLSDPC ELKALHLNGC NISQQVVFSM SFVQGETSND KIPVALGLKG LNLYLSCVMK DGTPTLQLES VDPKQYPKKK MEKRFVFNKI EVKTKVEFES AQFPNWYIST SQAEHRPVFL GNSNGRDIVD FTMEPVSS

Identity is a measure of the agreement between nucleotide or amino acid sequences and has a recognised meaning. A protein having an amino acid sequence having at least, for example, 80% identity to a referred amino acid sequence, for example SEQ ID No. 1 is intended to have an identical sequence to SEQ ID No. 1 except that it contains up to 20 amino acid alterations per 100 amino acids of SEQ ID No. 1. That is, to obtain a protein having an amino acid sequence at least 80% identical to a referred amino acid sequence, up to 20% of the amino acid residues in the reference sequence may be deleted or substituted with another amino acid, or number of amino acids up to 80% of the total amino acid residues of the reference sequence may be inserted.

A preferred protein according to the first aspect of the invention has at least 90% identity with one of the amino acid sequences shown as SED ID Nos. 1-11 over its entire length. More preferably the protein has a sequence of at least 95% identity with one of the amino acid sequences shown as SED ID Nos. 1-11 over its entire length. A protein with 97, 98, 99 or 100 % sequence identity is preferred.

The invention also encompasses variants of the sequences shown as SEQ ID Nos. 1- 1 1. Variant is a term defining a polypeptide or polynucleotide that differs from its reference sequence but retains essential properties. A variant and a reference sequence may differ in amino acid sequence by one or more substitutions, additions or deletions. A substituted or inserted residue may or may not be one encoded by the genetic code. Preferred variants are those that vary from the reference sequences by conservative amino acid substitutions, i.e. those that substitute a residue with another of like character. Typical conservative substitutions are among Ala, Val, Leu and He; among Ser and Thr; among the acidic residues Asp and Glu; among the basic residues Lys and Arg; among the aromatic residues Phe and Tyr and among Asn and Gin. Preferred are variants in which several, 5-10, 1-5 or 1-2 amino acids are substituted, deleted or added in any combination.

The IL-lβ fragment according to the present invention may be phosphorylated at one, two, three or four consensus casein kinase II phosphorylation sites. Preferred phosphorylation sites for SEQ ID Nos. 1-11 are indicated in bold and are underlined.

The IL-l β according to the present invention is preferably of mammalian origin, more preferably from a rodent or human source and most preferably isolated from rat

The protein according to the first aspect of the invention preferably has a molecular weight of about 20.5-21.5 kD and more preferably 20-21 kD.

The preferred protein according to the first aspect of the invention has a predicted molecular weight of 20. 893 Daltons as determined by Western blotting, for example as carried out according to Example Id that follows, allowing for error of +/- 100 daltons in interpreting the blot.

The protein according to the first aspect of the invention may be obtained by protein purification of cells or more preferably by recombinant nucleic acid techniques.

Accordingly the present invention in a second aspect provides an isolated nucleic acid molecule encoding the C-terminal fragment of pro-interleukin 1 β according to the first aspect of the invention. The invention also relates to vectors which comprise the nucleic acid according to the second aspect of the invention and host cells which are genetically engineered with said vectors and to the production of the protein according to the first aspect of the invention by recombinant techniques.

The 20-22kD IL-lβ protein according to the first aspect of the invention or cells expressing it can be used as immunogens to produce antibodies immunospecific for the 20-22 kD protein.

Accordingly, the third aspect of the invention provides antibodies raised against the C-terminal fragment of pro-interleukin l β according to the first aspect of the invention.

Antibodies generated against the 20-22 kD protein can be obtained by administering the protein or specific epitope bearing fragments, variants or cells to an animal using routine protocols. Preferred antibodies are monoclonal or humanised.

Knowledge of the cleavage site in pIL-l β which produces the 20-22kD protein is important for research. It is proposed that the cleavage site in rat pIL-lβ that produces the 20-22 kD protein according to the first aspect of the invention is located between P90 and T100, preferably between D95 and S99.

Specific antibodies and modified antibodies (e.g. biotin or fluorophore modified) may be directed against the cleavage site for immunocytochemistry and used to study cleavage of pIL-lβ and its effects in vitro and in vivo. Furthermore, inhibitors of cleavage and cleavage substrates (including recombinant cleavage sequence) may be used to identify the enzyme(s) responsible for cleavage and aid in characterisation of such enzyme(s) identified. The 20-22 kD protein according to the first aspect of the invention, the nucleic acid molecule encoding the protein, vectors containing the nucleic acid molecule, cells genetically modified with the vector and antibodies to the protein according to the first aspect of the invention have numerous uses in research, therapy and diagnosis.

Research.

The protein according to the first aspect of the invention, especially in recombinant form, may be used as a tool for indirect assessment of activation of the cleaving enzyme when identified and allow investigation into the activity and regulation of this enzyme.

Modified protein (with biotin, iodine, etc. labelling) may be used for localisation of binding sites, receptors, tracing studies etc. on the enzyme or the 20-22 kD protein.

Specific antibodies and modified antibodies (e.g. biotin or fluorophore labelled) may be directed against the 20-22 kD form for quantitation assay (e.g. ELISA, RIA), purification, immunocytochemistry etc.

Therapy

Conditions which are thought to involve either an excess or deficiency in IL-lβ expression include chronic and acute inflammation, arthritis, septicemia, autoimmuno diseases (e.g. inflammatory bowel disease, psoriasis), transplant rejection, graft versus host disease, infection, stroke, ischemia, acute respiratory disease syndrome, restenosis, brain injury, AIDS, bone diseases (e.g. osteoporosis), cancer (e.g. lymphoproliferative disorders), atherosclerosis, and Alzheimer's disease.

In some instances the activity of the 20-22 kD protein according to the first aspect of the invention may be positive or may be negative in relation to the expression of mature active IL-lβ. la. If the 20-22 kD form, or part of, is an intracellular feedback signal to suppress IL-lβ expression by the cell without inducing cell death, then it proposed that the 20- 22 kD protein may be of use in any pathology where IL-l β over expression is involved, as listed above.

lb. If the 20-22 kD form, or part of, is an intracellular feedback signal to increase IL-lβ expression without inducing cell death, then it proposed that the 20-22 kD protein may be of use in any pathology where deficiency in IL-lβ expression is involved, as listed above.

2a. If the 20-22 kD form, or part of, is an intracellular or extracellular signal (on its own and/or in association with HLA/MHC class I or class II molecules, receptor, transporter, etc.) preventing the death of the expressing cell by apoptosis (programmed cell death) or necrosis, then it is proposed that it may be of use in any pathology where apoptotic and/or necrotic cell death is observed (e.g. neuronal cell death in Parkinson's, Alzheimer's, AIDS, lupus, stroke, head injury, peripheral and central neuropathies, diabetes).

2b. If the 20-22 kD form, or part of, is an intracellular or extracellular signal (on its own and/or in association with HLA/MHC class I or class II molecules, receptor, transporter, etc.) inducing the death of the expressing cell by apoptosis (programmed cell death) or necrosis, then antibodies against 20-22 kD form or other inhibitors may be of use in any pathology where apoptotic and/or necrotic cell death is observed (e.g. neuronal cell death in Parkinson's, Alzheimer's, AIDS; lupus, stroke, head injury, peripheral and central neuropathies, diabetes).

3a. If the 20-22 kD form, or part of, is an extracellular signal (on its own and/or in association with HLA/MHC class I or class II molecules, receptor, transporter, etc.) preventing the death of the target cell by apoptosis (programmed cell death) or necrosis, then the 20-22 kD form may be of use in any pathology were apoptotic and/or necrotic cell death is observed (e.g. neuronal cell death in Parkinson's, Alzheimer's. AIDS, lupus, stroke, head injury, peripheral and central neuropathies, diabetes).

3b. If the 20-22 kD form, or part of, is an extracellular signal (on its own and/or in association with HLA/MHC class I or class II molecules, receptor, transporter, etc.) inducing the death of the target cell by apoptosis (programmed cell death) or necrosis, then antibodies against 20-22 kD or other inhibitors may be of use in any pathology where apoptotic and/or necrotic cell death is observed (e.g. neuronal cell death in Parkinson's, Alzheimer^'s, AIDS, lupus, stroke, head injury, peripheral and central neuropathies, diabetes).

4. The 20-22 kD protein and antibodies thereto may be used to study inhibition of ICE activity, or inhibition of pro-ICE cleavage so as to prevent mature 17 kD IL-l β from being produced.

Diagnostic

The 20-22 kD protein, nucleotides encoding such protein and antibodies to the protein have particular use in diagnosis of conditions which are thought to involve either an excess or deficiency in IL-l β expression, as listed above.

Decreased or increased expression of IL-lβ can be measured at the RNA level using any of the methods known in the art for the quantitaion of polynucleotides, such as PCR, RT-PCR, RNase protection, Northern blotting and other hybridsation methods.

Assay techniques that can be used to determine the level of the 20-22 kD protein include radioimmunoassays, competitive binding assays, Western blot analysis and ELISA assays.

The components for carrying out the above assays may be incorporated into a kit. It is proposed that the 20-22 kD form of IL-lβ according to the first aspect of the present invention is membrane associated in vivo (as evidenced by the need to use detergents for its isolation). It is further proposed that the 20-22 kD protein is cleaved by ICE to generate active IL-l β and a minor fragment peptide with an acidic N- terminus.

According to the fourth aspect of the present invention there is provided an isolated peptide which is the minor fragment produced by cleavage of the 20-22 kD protein according to the first aspect of the invention with ICE.

Preferably the peptide according to the fourth aspect of the invention is 40 to 25 amino acid residues in length, more preferably 30 to 25 amino acid residues in length and most preferably 25 amino acid residues in length.

It is preferred that the peptide according to the fourth aspect of the invention comprises one of the sequences shown as SEQ ID Nos. 12 to 22 or a sequence with at least 80%) identity thereto. Identity is defined as described above.

SEQ ID No. 12

PWSFQDEDPS TFFSFIFEEE PVLCDSWDDD DLLVCD

SEQ ID No. 13

WSFQDEDPS TFFSFIFEEE PVLCDSWDDD DLLVCD

SEQ ID No. 14

SFQDEDPS TFFSFIFEEE PVLCDSWDDD DLLVCD

SEQ ID No. 15

FQDEDPS TFFSFIFEEE PVLCDSWDDD DLLVCD SEQ ID No. 16

QDEDPS TFFSFIFEEE PVLCDSWDDD DLLVCD

SEQ ID No. 17

DEDPS TFFSFIFEEE PVLCDSWDDD DLLVCD

SEQ ID No. 18

EDPS TFFSFIFEEE PVLCDSWDDD DLLVCD

SEQ ID No. 19

DPS TFFSFIFEEE PVLCDSWDDD DLLVCD

SEQ ID No. 20

PS TFFSFIFEEE PVLCDSWDDD DLLVCD

SEQ ID No. 21

S TFFSFIFEEE PVLCDSWDDD DLLVCD

SEQ ID No. 22

TFSFIFEEE PVLCDSWDDD DLLVCD

A preferred peptide according to the fourth aspect of the invention has at least 90% identity with one of the amino acid sequences shown as SED ID Nos. 12-22 over its entire length. More preferably the peptide has a sequence of at least 95% identity with one of the amino acid sequences shown as SED ID Nos. 12-22 over its entire length. A peptide with at least 97, 98, 99 or 100% sequence identity is most preferred.

The invention also encompasses variants of the sequences shown as SEQ ID Nos. 12- 22. A variant is defined as above. A preferred peptide according to the fourth aspect of the invention contains a WDDDD motif or any subset thereof, for example a DDD motif or a WD motif.

A preferred peptide according to the fourth aspect of the invention contains a XVXD motif, preferably a XVCD motif (where X denotes any amino acid), this motif defining a consensus for ICE cleavage. Preferably this motif is located at the C- terminus of the peptide.

The peptide according to the fourth aspect of the present invention may be phosphorylated at one or two consensus casein kinase II phosphorylation sites. Preferred phosphorylation sites for SEQ ID Nos. 12-22 are indicated in bold and are underlined.

The peptide according to the fourth aspect of the invention may be prepared synthetically, isolated from cells by protein purification and/or produced by recombinant nucleic acid techniques. Accordingly the fifth aspect of the invention provides an isolated nucleic acid molecule encoding peptide according to the fourth aspect of the invention.

The invention also relates to vectors which comprise the nucleic acid according to the fifth aspect of the invention and host cells which are genetically engineered with said vectors and to the production of the peptide according to the fourth aspect of the invention by recombinant techniques.

The peptide according to the fourth aspect of the invention or cells expressing it can be used as immunogens to produce antibodies immunospecific for the peptide.

Accordingly, the sixth aspect of the invention provides antibodies raised against the peptide according to the fourth aspect of the invention. Antibodies generated against the peptide can be obtained by administering the protein or specific epitope bearing fragments, variants or cells to an animal using routine protocols. Preferred antibodies are monoclonal or humanised.

There are several proposals with regard to the potential activity and uses of the peptide according to the fourth aspect of the present invention. All, some or none of these proposals may correctly define the activity of the peptide, alone or in combination.

1. The highly acidic peptide (as part of the 20-22 kD protein according to the first aspect of the invention) may interact with membrane phospholipids or other proteins in vivo and thus serve as a docking sequence for the 20-22 kD form of IL-l β. The cleavage of the 20-22 kD form into active (17 kD) IL-lβ may be either pre- or post- interaction. The interacting molecule may be a lipid, phospholipid or protein, possibly a transporter or a receptor, or an enzyme.

2. A region of the acidic peptide (as part of the 20-2 kD protein according to the first aspect of the invention) may interact with a putative transporter/receptor of the 20-22 kD form of IL-l β thereby preventing cleavage of the protein by ICE to produce active IL-lβ (17 kD). Prevention of cleavage could be direct (i.e. interaction with ICE) or indirect (interaction with enzymes that activate ICE).

In a preferred peptide, the region proposed to be involved in interaction is the DDDD sequence. This sequence is conserved between rats and mice but is present as ID in humans. It is known that rat and mouse monocytes release little 17 kD IL-lβ as compared to human monocytes. The sequence difference may be implicated in this phenomenon. 3. The acidic peptide may interact with the rest of the IL-l β sequence by, for example, interaction with cysteines or other amino acid interactions. This internal interaction within IL-lβ may result in altered activity as compared to IL-lβ (17 kD).

4. The most favourable proposal is that the acidic peptide is cleaved from the 20- 22 kD protein by ICE and then binds to an MHC molecule (preferably MHC class I). The MHC class I/petide complex may then be expressed at the cell surface where it may stimulate (or not if recognised as self) cytotoxic T lymphocytes. This may be involved in release of IL-l β (17 kD) from the cell.

Preferably the acidic peptide interacts strongly with MHC class I allele HLA-A0205 (a computerised binding prediction).

The peptide according to the fourth aspect of the invention, the nucleic acid molecule encoding the peptide, vectors containing the nucleic acid molecule, cells genetically modified with the vector and antibodies to the peptide according to the fourth aspect of the invention have numerous uses in research, therapy and diagnosis.

Research.

The peptide, especially when produced recombinantly may be used for study of its activity in vitro and in vivo. The peptide may be modified (with biotin, iodine, etc. labelling) to investigate the localisation of binding sites and receptors and used to carry out tracing studies etc.

Specific antibodies and modified antibodies (e.g. biotin or fluorophore labelled) may be directed against the peptide for quantitation assay (e.g. ELISA, RIA), purification, immunocytochemistry etc. Antibodies to the peptide may also provide tool for indirect intracellular detection of active ICE, which is not currently possible as current commercial antibodies do not distinguish between pro-ICE and ICE.

Therapy. la. If the peptide, or part of, is an intracellular feedback signal to suppress IL-lβ expression by the cell without inducing cell death, then the peptide may be of use in any pathology where IL-l β overexpression is involved (see above).

In some instances the activity of the peptide according to the fourth aspect of the invention may be positive or may be negative in relation to the expression of mature active IL-l β.

lb. If the peptide, or part of, is an intracellular feedback signal to increase IL-l β expression without inducing cell death, then the peptide may be of use in any pathology where deficiency in IL-l β expression is involved (see above).

2a. If the peptide, or part of, is an intracellular or extracellular signal (on its own and/or in association with HLA/MHC class I or class II molecules, receptor, transporter, etc.) preventing the death of the expressing cell by apoptosis (programmed cell death) or necrosis, then the peptide may be of use in any pathology where apoptotic and/or necrotic cell death is observed (e.g. neuronal cell death in Parkinson's Alzheimer's, AIDS, lupus, stroke, head injury, peripheral and central neuropathies, diabetes).

2b. If the peptide, or part of, is an intracellular or extracellular signal (on its own and/or in association with HLA/MHC class I or class II molecules, receptor, transporter, etc.) inducing the death of the expressing cell by apoptosis (programmed cell death) or necrosis, then antibodies against the peptide or against the peptide in association with its corresponding MHC I receptor, transporter etc may be of use in any pathology where apoptotic and/or necrotic cell death is observed (e.g. neuronal cell death in Parkinson's, Alzheimer's, AIDS, lupus, stroke, head injury, peripheral and central neuropathies, diabetes).

3a. If the peptide, or part of, is an extracellular signal (on its own and/or in association with HLA/MHC class I or class II molecules, receptor, transporter, etc.) preventing the death of the target cell by apoptosis (programmed cell death) or necrosis, then the peptide may be of use in any pathology where apoptotic and/or necrotic cell death is observed (e.g. neuronal cell death in Parkinson's, Alzheimer's, AIDS, lupus, stroke, head injury, peripheral and central neuropathies, diabetes).

3b. If the peptide, or part of, is an extracellular signal (on its own and/or in association with HLA/MHC class I or class II molecules, receptor, transporter, etc.) inducing the death of the target cell by apoptosis (programmed cell death) or necrosis, then antibodies against the peptide or against the peptide in association with its corresponding MHC I, receptor, transporter, etc. may be of use in any pathology where apoptotic and/or necrotic cell death is observed (e.g. neuronal cell death in Parkinson's, Alzheimer's, AIDS, lupus, stroke, head injury, peripheral and central neuropathies, diabetes).

4. If the peptide, or part of, in association with HLA/MHC class I molecules is an extracellular signal inducing the selective killing of the expressing cell by cytotoxic T lymphocytes, then T lymphocytes could be primed in vitro by the peptide/MHC I complex and used to selectively destroy cells expressing the complex. This may have application in antitumor therapy, especially in the case of astrocytomas and gliomas, which have been shown to overexpress IL-l β. In this case labelled antibodies against the complex peptide/MHC I molecule could also be used as tracers for diagnosis. Drug Delivery. la. If peptide sequence or part of possess strong affinity for membrane, the sequence could be used to insert drugs into particles such as lipid vesicles

(liposomes).

lb. If peptide sequence or part of posses strong affinity for a specific membrane protein, the sequence could be used to insert drugs into particles such as liposomes with the specific membrane protein.

The peptide and antibodies thereto may be used to study inhibition of ICE activity, or inhibition of pro-ICE cleavage so as to prevent mature 17 kD IL-lβ from being produced.

Diagnostic

The peptide, nucleotides encoding such peptide and antibodies to the peptide have particular use in diagnosis of conditions which are thought to involve either an excess or deficiency in IL-l β expression, as listed above.

Decreased or increased expression of IL-l β can be measured at the RNA level using any of the methods known in the art for the quantitation of polynucleotides, such as PCR, RT-PCR, RNase protection, Northern blotting and other hybridisation methods.

Assay techniques that can be used to determine the level of the peptide include radioimmunoassays, competitive binding assays, Western blot analysis and ELISA assays.

The components for carrying out the above assays may be incorporated into a kit. The present invention will now be described, by way of example only, with reference to the accompanying drawing in which:

Figure 1 shows a photograph of a SDS PAGE gel, demonstrating the expression of pIL-lβ, mature IL-lβ and a 20-22 kD protein according to the first aspect of the invention by PBMC. PBMC were stimulated with LPS (lμg/ml) for 4 hours (Lanes B and C), and 24 hours (Lanes D and E). They were lysed in the presence of a cocktail of protease inhibitors, +/- z-VAD-fmk (Lanes C. E, G and B, D, F respectively). The expression of intracellular IL-l β was compared to that of normal, untreated cells (Lanes F and G) by SDS gel electrophoresis. rrlL-lβ was used as a molecular weight control, 25pg (Lane A).

Figure 2 shows a photograph of an SDS-PAGE gel demonstrating the presence of mature (17.5 kD) rat recombinant IL-l β with sheep anti-rat IL-lβ antibody at at least 12.5 pg. Detection of mature (17.5 kD), rat recombinant IL-l β (rrlL-l β) with the sheep anti rat IL-lβ antibody, S328/B4 (1/800). a biotinylated anti sheep secondary (1/10,000) and extravidin-peroxidase (1/16,000). Lane A, lOOpg; Lane B, 50pg; Lane C. 25pg and Lane D, 12.5pg of rrlL-l β. The detection limit is 12.5pg of rrlL-l β

Figure 3 demonstrates the specificity of protein detection on nitrocellulose membranes by S328. 33 kD and 17.5 kD products were detected in the concentrated cell culture medium of rat PBMC exposed to LPS for 24h (lane A). The 17.5 kD product co- migrated with recombinant rat IL-l β (25pg; lane B). Preadsorption with 5μM rat recombinant IL-l β led to the absence of protein detection in monocyte lysate (lane C), and the reduction of detection of recombinant rat IL-l β (25 pg; lane D). Specificity of the primary antibody (S328) was fully demonstrated by the absence of protein detection when the antibody was preadsorbed with lOμM rat recombinant IL-lβ (lane E: monocyte lysate; lane F: 25pg rat recombinant IL-lβ. Figure 4 a, b. c demonstrate the absence of cross reactivity of antibody S328 with proteins other than IL-1 β using the dot blot techniques.

Figure 4 a shows Cross-reactivity test 1 : The sheep anti rat IL-lβ antibody (S328, dilution 1 :500) detected 250 ng of recombinant rat IL-l β applied on nitrocellulose membrane, but did not detect recombinant rat IL-lα (250ng), recombinant rat IL-lra (200ng), or recombinant rat CRF (250ng). After incubation with S328, the membrane was incubated sequentially with biotinylated Donkey anti-sheep IgGs (secondary antibody), avidin peroxidase.

Figure 4 b shows Cross-reactivity test 2: The sheep anti rat IL-lβ antibody (S328, dilution 1 :500) detected 20 ng of recombinant rat IL-lβ applied on nitrocellulose membrane, but did not detect recombinant rat IL-18 (200ng). After incubation with S328, the membrane was incubated sequentially with biotinylated Donkey anti-sheep IgGs (secondary antibody), avidin peroxidase. Lane A: no sample; Lane B: 20ng rrlL- lβ; Lane C: 200ng rrIL-18; Lane D: no sample; Lane E: 20 ng rrlL-lβ, no S328 applied; Lane F: 20 ng rrlL-l β, no secondary antibody applied (controls).

Figure 4 c shows Cross-reactivity test 3: The sheep anti rat IL-lβ antibody (S328, dilution 1 :500) detected 20 and 200ng of recombinant rat IL-l β applied on nitrocellulose membrane (Lane A), but did not detect recombinant rat IL-lra (Lane B), recombinant rat IL-6 (Lane C). recombinant rat TNF-α (Lane D) or recombinant rat CRF (Lane D). After incubation with S328, the membrane was incubated sequentially with biotinylated Donkey anti-sheep IgGs (secondary antibody) and avidin peroxidase. Lane A: no sample; Lane B: 20ng rrlL-lβ; Lane C: 200ng rrIL-18; Lane D: no sample; Lane E: 20 ng rrlL-l β, no S328 applied; Lane F: 20 ng rrlL-l β, no secondary antibody applied (controls). Figure 5 shows a photograph of an SDS-PAGE gel, demonstrating the expression of pIL-lβ, mature IL-l β and a 20-22 kD protein according to the first aspect of the invention by injured rat brain cortex. 10 micrograms of total protein from the supernatant obtained from homogenised cortices was analysed for expression of all forms of IL-l β (Lane A), using 25pg rrlL-lβ (Lane B) and 10 micrograms of total intracellular protein from PBMC (Lane C). The 20-22 kD form was present in the cortices, however in smaller amount than in the PBMCs.

Figure 6 shows a photograph of an SDS-PAGE gel, demonstrating the expression of pIL-l β, mature IL-l β and a 20-22 kD protein according to the first aspect of the invention by LPS stimulated blood monocyte macrophages. Blood monocyte macrophages (BMMφ) were stimulated with LPS (lμg/ml) for 4 hours (Lanes C, D) and lysed in the presence of a cocktail of protease inhibitors (Lanes A, C: + z-VAD- fmk; lanes B, D: - z-VAD-fmk). Lanes A, B: un-stimulated BMMφ.

Figure 7 shows a photograph of an SDS-PAGE gel demonstrating cleavage of pIL-l β and the 20-22 kD protein according to the first aspect of the invention by ICE. PBMC were stimulated with LPS (μg/ml) for 24 hours and lysed in the presence of a cocktail of protease inhibitors +/- z-VAD-fmk (Lanes C, D and A,B respectively). Ten micrograms of total protein was incubated with ICE (500 units) at 37°C for 1 hour and the cleavage products analysed by SDS gel electrophoresis (Lane B and D). Cleavage by ICE was inhibited by z-VAD-fmk (Lane D). Protein incubated with lOmM DTT alone (Lane E). Lane F, rrlL-lβ, 25pg.

EXAMPLES

1. Detection of 20-22 kD protein in LPS activated rat blood monocyte lysates by SDS page analysis followed by immunoblotting. a) Affinity purification of sheep anti rat IL-lβ antiserum

Sheep anti-rat IL-lβ antiserum (S328B4) was obtained by immunisation with recombinant rat IL-lβ as previously described (Bristow, A. et al, 1991, J. Mol. Endochrinol 7: 1-7). The serum obtained at the fourth bleeding (B4) of the sheep was affinity purified on a rat IL-lβ column, prepared using an AminoLink Immobilisation Kit (Pierce, UK), according to the manufacturer's guidelines.

Briefly, 2 ml of recombinant rat IL-l β (0.25 mg/ml PBS, NIBSC, UK) was mixed in a column with 2 ml of AminoLink gel and 200 μl of reducing agent, overnight at 4°C. The coupling solution was drained into a test tube, and the column was washed with 5 ml of PBS, pH 7.2. Remaining active sites were blocked by washing the column with 4 ml of quenching buffer and incubation with a mixture of 2 ml of the quenching buffer and 40μl of reducing agent, for 30 min at room temperature. The column was washed with 8 x 4 ml of 1 M NaCI, followed by 2 washes with 5 ml PBS 0.2% sodium azide. The rat IL-lβ column was stored at 4°C until use.

For affinity purification of sheep anti-rat IL-l β antiserum, the rat IL-lβ column was equilibrated with 5 ml 20 mM phosphate buffer, pH 8.0 (binding buffer). Sheep anti- rat IL-l β antiserum was decomplemented (45 min, 56°C), and centrifuged (20 min, 10,00 rpm). The supernatant (1.5 ml) was diluted 1 :1 with binding buffer and applied to the IL-1 β column for 3hours at room temperature. After extensive washes with the binding buffer, affinity purified sheep anti-rat IL-l β was eluted from the column with 1 mM Glycine/HCl solution, pH 2.5. One ml fractions were collected in tubes containing 100 μl binding buffer to partially restore the pH. Protein concentration was determined by ^A280 reading. Fractions with a concentration of 0.5 mg/ml were pooled and dialysed in a 3 ml Slide-A-Lyser dialysing cassette (Pierce) in 0.1 M PBS (1.5 h, room temperature). Final concentration of the affinity purified sheep anti-rat IL-l β after dialysis was 1.2 mg/ml, pH 7.5.

b) Isolation of rat peripheral blood monocytes (PBMC)

PBMC were obtained from the pooled blood of healthy Sprague Dawley rats. Blood was collected by cardiac puncture under sodium barbitone anaesthesia and mixed rapidly with heparin (15 U/ml). PBMC were isolated by poly sucrose/sodium diatrizoate density gradient centrifugation (Histopaque 1083; Sigma, UK) and washed three times in RPMI 1640 medium containing 4mM glutamine, lOOU/ml penicillin and lOOμg/ml streptomycin (wash medium). The cells were resuspended to a concentration of 9x10⁶ cells/ml in wash medium supplemented with 1% FCS (endotoxin free; Gibco) and 20 mM Hepes (culture medium). Cells were seeded at 1 ml/well in 8 well tissue culture plates (Nunc, UK). The monocytes were allowed to adhere for 1.5h at 37°C in humidified atmosphere (5%CO₂), washed three times with wash medium and stimulated for 4h to 24h with LPS (1 μg/ml Escherichia Coli, serotype 055. B5P, Sigma). Controls were unstimulated cells.

After two washes in ice-cold PBS, the cell medium (supernatant) was removed and mixed with the following cocktail of protease inhibitors: leupeptin (lOμg/ml), benzamidine (ImM), pepstatin (lOμg/ml), aprotinin lμg/ml, AEBSF (02.mM), and the irreversible, non specific caspase inhibitor z-VAD-fmk (lOOμM) (Calbiochem, UK). The adherent cells were lysed in 25 mM TrisHCl/l%TritonX100/0.5% sodium deoxycholate/0.35M NaCl/10mM EDTA/0.1% SDS (lysis buffer, pH 7.4), containing the same cocktail of protease inhibitors. The lysates were concentrated (4 fold) in Microcon3 microconcentrators (Millipore, UK), then diluted in Laemmli sample buffer to give a final concentration of 5mg protein/ml.

c) SDS Page analysis

Each sample was boiled for 5 min, chilled on ice and analysed on acrylamide gels (stacking gel 4%, resolving gel 12%) in a Hoeffer Mighty Small II (Pharmacia, UK) gel electrophoresis unit. Ten microgram of sample or 10 μl of prestained molecular weight markers were added per well. Samples were separated for lh at 35mA, 250V. Protein transfer on 0.45 μm nitrocellulose membrane (Schleicher and Schwell, UK) was performed in an Hoeffer Mighty Small II Transfer Unit (Pharmacia), for lh at 400mA, 100V. The results are shown in Figure 1. d) Immunoblotting

After blocking of non-specific binding with 5% donkey serum/0.1%) BSA in TBS, membranes were sequentially incubated with sheep anti-rat IL-lβ (S328B4, NIBSC; 1 :800) overnight at 4°C, biotinylated donkey anti-sheep IgG (Chemicon, UK) diluted 1 :10000 in TBS/0. l%Tween RTM for 30 min at room temperature (RT), Extravidin (1 : 16000; Sigma) and ECL solution (ECL detection kit, Amersham, UK) for 1 min (RT) to induce chemoluminescence reaction. The membrane was washed extensively in TBS/0.1% Tween RTM between each step, except after blocking of non-specific binding. Detection was carried out using ECL hyperfilm (Amersham). Films were developed in a KodakX-omat M35 processor.

To estimate the detection limit of the SDS-Page/immunoblotting technique, a range of 17.5kD recombinant rat IL-l β concentrations (12.5pg to 5ng) were assayed. The detection limit was 12/5pg of rIL-1 β. The results are shown in Figure 2.

e) Specificity test

To assess the specificity of the protein detection on nitrocellulose membranes by S328B4, the antibody (diluted 1 :800) was preadsorbed with 5 or lOμM rrlL-l β before use. Preadsorption with 5μM rrlL-l β led to a decrease in the intensity of both proIL- lβ and IL-lβ bands, and preadsorption with lOμM rrlL-lβ completely abolished the signal. The results are shown in Figure 3.

f) Cross-reactivity test

The absence of cross reactivity of antibody S328B4 with proteins other than IL-lβ was verified using the dot-blot technique (Larrson, L.I. 1981, J. Histochem. Cytochem. 29: 408-410) on 0.45μm pore size nitrocellulose membranes (Schleicher and Schwell, UK) using a Hoeffer slot-blot mannifold (Pharmacia). One μl spots of the tested proteins (see list below) at concentrations of 50ng and 200ng were placed on a nitrocellulose membrane (0.45μm pore size, Schleicher and Schuell, Germany). The membrane was air-dried, fixed with 4% PFA for 15 min and washed thoroughly in TBS, and sequentially incubated in normal donkey serum (1 :5, 30 min), polyclonal sheep anti-rat IL-l β (lh 45 min), biotinylated rabbit anti-goat IgG (1 :3000, 30 min, Vector), avidin conjugated horseradish peroxidase (30 mins) and DAB. Washes were done in TBS containing 0.1% Tween 20. After the last wash in TBS, the nitrocellulose membrane was rinsed in distilled water and let to air dry.

Recombinant proteins tested for cross-reactivity of the antibody: rat IL-l β (NIBSC), rat IL-lra (NIBSC), rat IL-lα (NIBSC), rat IL-18 (Culhane et al, 1998), human IL-l β (Dupont-NEN), rat IL-6 (NIBSC), rat CRF (NIBSC). The results are shown in Figures 4 a, b and c.

2. Detection of 20-22 kD protein in lysates obtained from fluid percussion injured rat brain cortex homogenates. a) Fluid percussion trauma

Traumatic brain injury was induced in male Sprague Dawley rats (260-280g, Harlan- Olac, UK) under sodium pentobarbitone anaesthesia (60 mg/ml/kg; Rhδne-Merieux, France). Body temperature was maintained at 37°C during and until full recovery from anaesthesia, using a homeothermic blanket (Harvard Apparatus, UK). Cortical impact was induced by application of a pulse of pressurised sterile injectable water to the exposed dura of the right parietal cortex, at an applied pressure of 1.6-1.8 atm, as previously described (Toulmond, S. et al., 1993; Brain Res. 620: 24-31 ; Toulmond, S and Rothwell, N.J. 1995; Brain Res. 671 : 261-6). Sham operated rats were submitted to exposure of the dura only. Animals recovered consciousness within 1.5 hours and the percentage of recovery was 100%.

b) Obtention of lysates from cortex homogenates

Sham-operated and LFP injured rats were killed by decapitation at different times post-surgery (3 animals per group). Three non-operated rats (normal rats) were also included. Brains were removed and the right (site of injury) cortex was dissected out rapidly on ice, weight out, snap-frozen on dry ice and stored at -70°C. Each cortex was homogenised in 0.1M PBS (150 mg wet tissue/ml) containing a cocktail of protease inhibitors (see above). Homogenates were centrifuged for 15 min at 4°C, 13000 rpm. The supernatants were filtered through Anotop RTM 10 micro filters (Whatman, UK) and concentrated 2x in Microcon RTM 3 microconcentrators (Millipore, UK). The pellets were resupended in lysis buffer containing protease inhibitors (see above), left on ice for 30 min, centrifuged 15 min at 4°C, 13000 rpm, and the supernatant (lysate) concentrated 2x in Microcon RTM 3 microconcentrators. The protein concentration of supernatants and lysates obtained from brain tissue was assessed using a Biorad protein assay.

Samples were diluted in Laemmli buffer to give a concentration of 15μg protein/15μl, then processed and analysed as described in lb, c, d, e above. The results are shown in Figure 5.

3. Isolation of 20-22 kD IL-lβ from LPS-activated rat PBMC lysates.

An S328B4 affinity column was prepared using an AminoLink Immobilisation Kit (Pierce, UK), as described in la) for the preparation of the IL-lβ affinity column. Briefly, 2 ml of affinity purified S328B4 antibody (1 to 2 mg/ml PBS) was mixed in a column with 2 ml of AminoLink gel and 200 μl of reducing agent, overnight at 4°C. The coupling solution was drained into a test tube, and the column was washed with 5 ml of PBS, pH 7.2. Remaining active sites were blocked by washing the column with

4 ml of quenching buffer, and incubation with a mixture of 2 ml of the quenching buffer and 40μl of reducing agent, for 30 min at room temperature. The column was washed with 8 x 4 ml of 1 M NaCl, followed by 2 washes with 5 ml PBS 0.2% sodium azide and stored at 4°C until use.

For affinity purification of 20-22 kD IL-l β, the S328B4 column was equilibrated with

5 ml 20 mM phosphate buffer, pH 8.0 (binding buffer). PBMC lysates were diluted 1 : 1 with binding buffer, and applied to the S328B4 column for 3h, at room temperature. After extensive washes with the binding buffer, affinity purified IL-l β (all forms are extracted since the antibody is raised against the 17 kD sequence) was eluted from the column with a 1 mM Glycine/HCl solution, pH 2.5. Five hundred μl fractions were collected in tubes containing 100 μl binding buffer to partially restore the pH.

4. Isolation of 20-22 kD from LPS activated bone marrow monocytes.

This follows an adaptation of the procedure of Boltz-Nitulescu et al., 1987; J. Leukoc. Biol. 41 :83-91. Male Sprague Dawley rats were terminally anaesthetised by fluothane inhalation. The femurs were removed aseptically and the marrow plugs flushed out with wash medium (RPMI supplemented with penicillin (lOOU/ml), streptomycin (100 μg/ml) and L-glutamine (2mM). A single cell suspension was obtained by gentle aspiration through a syringe. The cells were resuspended in lysis buffer (Sigma), to remove contaminating erythrocytes. The cells were washed in washing buffer and resuspended at 10⁶ cells/ml in RPMI supplemented with 15% fetal calf serum, 5% horse serum, 30% crude L929 (murine fibroblast cell line) supernatent, penicillin, streptomycin and L-glutamine. The cells were seeded into petri dishes at 15ml/petri dish or 24 well plates at 1 ml/well. Plated cells were maintained at 37°C, 5% CO₂ in a humidified atmosphere, with half of the medium replaced by fresh medium every 3-4 days.

For exposure to LPS, the cells were washed twice to remove non adherent cells and stimulated with 1 μg/ml LPS (E. Coli serotype 026:B6) for 0 to 24h. The cell medium was collected and the cells lysed as described in 1 above. Samples were diluted in Laemmli buffer to give a concentration of 15μg protein/15μl, then processed and analysed as described in lb, c, d, e above. Results are shown in Figure 6.

6. Characterisation of new cleavage site to generate 20-22 kD IL-lβ.

The cleavage site can be characterised using the MALDI-TOF technique (Jan, J.X. et al, Electrophoresis 1999 Apr-May; 20(4-5): 749-54 and Houston, C.T. & Reilley, J.P.

Rapid. Commun. Mass Spectrom. 1997; 11(13): 1435-9) if sufficient amount of the protein is available. 7. Demonstration that ICE cleaves 20-22 kD to generate IL-lβ.

Cell lysates were obtained as described in 1 above and divided in two batches prior addition of the protease inhibitor cocktail. One batch was mixed with the cocktail including the caspase-1 inhibitor Z-VAD -fmk, the other batch was mixed with the cocktail excluding Z-VAD -fmk.

Five hundred units of recombinant human caspase-1 (a gift from N. Thornberry, Merck, USA) was added to each lysate batch in the sample activation buffer required for enzyme activation (buffer formulation provided by N. Thornberry). The samples were incubated at 25°C for 30 min (Culhane et al., 1998) and then analysed on acrylamide gels as described in 1 above. The results are shown in Figure 7.

Claims

CLAIMS:

1. An isolated C-terminal fragment of pro-interleukin 1 β comprising a protein sequence having a molecular weight of 20-22 kD or an N-terminal truncation thereof, incorporating an interleukin- l β converting enzyme (ICE) cleavage site, the fragment being cleavable by ICE to produce active IL-lβ.

2. An isolated fragment according to claim 1 from a mammalian source.

3. An isolated fragment according to claim 2 from a rodent or human source.

4. An isolated fragment according to claim 2 from rat.

5. An isolated fragment according to claim 1 comprising a protein sequence having at least 80% identity over its entire length to the amino acid sequence of SEQ ID No. 1.

6. An isolated fragment according to claim 5 comprising a protein sequence having at least 80% identity over their entire length to any one of SEQ ID Nos. 2 - 1 1

7. An isolated fragment according to claim 1 comprising a protein sequence having at least 90% identity with one of the amino acid sequences shown as SED ID Nos. 1-1 1 over its entire length.

8. An isolated fragment according to claim 7 comprising a protein sequence having at least 95% identity with one of the amino acid sequences shown as SED ID Nos. 1-11 over its entire length. JJ

9. An isolated fragment according to claim 8 comprising a protein sequence having at least 97, 98, 99 or 100% sequence identity with one of the amino acid sequences shown as SEQ ID Nos. 1-11.

10. An isolated fragment according to any preceding claim comprising a protein sequence variant of one of the amino acid sequences shown as SEQ ID Nos. 1- 1 1 as herein before defined.

1 1. An isolated fragment according to any preceding claim which is phosphorylated at one, two, three or four consensus casein kinase II phosphorylation sites.

12. An isolated nucleic acid molecule encoding the isolated fragment of any one of claims 1 to 11.

13. A vector comprising the nucleic acid molecule of claim 12.

14. A host cell transformed with the nucleic acid molecule of claim 12 or the vector of claim 13.

15. Production of the isolated fragment according to any one of claims 1 to 11 by culturing the cells according to claim 14.

16. Antibodies raised against the isolated fragment according to any one of claims 1 to 11.

17. Use of the isolated fragment according to any one of claims 1 to 11, the nucleic acid molecule of claim 12, vectors according to claim 13, host cells according to claim 14 or antibodies according to claim 16 for research, therapy or diagnosis.

18. Use of the isolated fragment according to any one of claims 1 to 11, the nucleic acid molecule of claim 12, vectors according to claim 13, host cells according to claim 14 or antibodies according to claim 16 in diagnosis and therapy of conditions involving either an excess or a deficiency in IL-l β.

19. An isolated peptide which is the minor fragment produced by cleavage of the 20-22 kD protein according to any one of claims 1 to 1 1 with ICE.

20. An isolated peptide according to claim 19 of 40 to 25 amino acid residues in length.

21. An isolated peptide according to claim 20 of 30 to 25 amino acid residues in length.

22. An isolated peptide according to claim 19 having at least 80 % identity to any one of the sequences shown as SEQ ID Nos. 12 to 22 over its entire length.

23. An isolated peptide according to claim 22 having at least 90% identity to any one of the amino acid sequences shown as SED ID Nos. 12-22 over its entire length.

24. An isolated peptide according to claim 23 having at least 95% identity with one of the amino acid sequences shown as SED ID Nos. 12-22 over its entire length.

25. An isolated peptide according to claim 24 having at least 97, 98, 99 or 100% sequence identity with one of the amino acid sequences shown as SEQ ID Nos. 12-22 over its entire length.

26. An isolated peptide according to claim 19 comprising a peptide sequence variant of one of the amino acid sequences shown as SEQ ID Nos. 12-22 as herein before defined.

27. An isolated peptide according to any one of claims 19 to 26 which is phosphorylated at one or two consensus casein kinase II phosphorylation sites.

28. An isolated nucleic acid molecule encoding the isolated peptide of any one of claims 19 to 27.

29. A vector comprising the nucleic acid molecule of claim 28.

30. A host cell transformed with the nucleic acid molecule of claim 28 or the vector of claim 29.

31. Production of the isolated peptide according to any one of claims 19 to 27 by culturing the cells according to claim 30.

32. Antibodies raised against the isolated peptide according to any one of claims 19 to 27.

33. Use of the isolated peptide according to any one of claims 19 to 27, the nucleic acid molecule of claim 28, the vector according to claim 29, host cells according to claim 30 or antibodies according to claim 32 for research, therapy or diagnosis.

34. Use of the isolated peptide according to any one of claims 19 to 27, the nucleic acid molecule of claim 28, the vector according to claim 29, host cells according to claim 30 or antibodies according to claim 32 for diagnosis and therapy of conditions involving either an excess or a deficiency in IL-lβ.