Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Patent
1993-05-10
1997-05-27
Ulm, John
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
435 697, 4352523, 435 371, 536 234, 536 235, C12N 510, C12N 1512
Patent
active
056331452
DESCRIPTION:
BRIEF SUMMARY
The present invention relates to recombinant proteins and their use.
Tumour necrosis factor-.alpha. (TNF.alpha.) is a potent cytokine which elicits a broad spectrum of biological responses. TNF.alpha. causes the cytolysis or cytostasis of many tumour cell lines in vitro, induces the haemorrhagic necrosis of transplanted tumours in mice, enhances the phagocytosis and cytotoxicity of polymorphonuclear neutrophils, and modulates the expression of many proteins, including lipoprotein lipase, class I antigens of the major histo-compatibility complex, and cytokines such as interleukin 1 and interleukin 6. TNF.alpha. appears to be necessary for a normal immune response, but large quantities produce dramatic pathogenic effects. TNF.alpha. has been termed "cachectin" since it is the predominant factor responsible for the wasting syndrome (cachexia) associated with neoplastic disease and parasitemia. TNF is also a major contributor to toxicity in gram-negative sepsis, since antibodies against TNF can protect infected animals.
The many activities of TNF.alpha. are mediated by binding to a cell surface receptor. Radioligand binding studies have confirmed the presence of TNF receptors on a wide variety of cell types. Although these receptors are expressed in limited numbers (1,000-10,000 receptors/cell), they bind TNF.alpha. with high affinity (Ka=10.sup.9 M.sup.-1 at 4.degree. C.). Lymphotoxin (LT, also termed TNF.beta.) has similar, if not identical, biological activities to TNF.alpha., presumably because both are recognized by the same receptor.
Recently, several laboratories have detected heterogeneity in TNF receptor preparations. Two distinct cell surface receptors which bind TNF.alpha. and TNF.beta. have recently been characterised at the molecular level. cDNA for one form of the receptor with a Mr of 55 kD was isolated utilising probes designed from the peptide sequence of a soluble form of the receptor (1,2). A second receptor of Mr 75 kD was cloned by a COS cell expression approach (3). Both receptors are members of a larger family of cytokine receptors which include the nerve growth factor receptor, the B cell antigen CD40, the rat T cell antigen MRC OX40. In addition these receptors are homologous to the predicted product of a transcriptionally active open reading frame from shope fibroma virus which appears to give rise to a secreted protein.
The most conserved feature amongst this group of cell surface receptors is the cysteine rich extracellular ligand binding domain, which can be divided into four repeating motifs of about forty amino acids. We have now generated four soluble receptor derivatives of the 55 kD TNF.alpha. receptor (TNFR). Each derivative is composed of the extracellular binding domain but without one of the cysteine rich subdomains. We have found that the derivative which lacks the membrane-proximal fourth subdomain retains the ability to bind TNF.alpha. with high affinity. This finding has general applicability.
Accordingly, the present invention provides a polypeptide which is capable of binding human TNF.alpha. and which consists essentially of: cysteine-rich subdomain, of the extracellular binding domain of the 55 kD or 75 kD receptor for human TNF.alpha.; or sequence (a).
The invention also provides: capable, when provided in a transformed host, of expressing the polypeptide of the invention encoded by the DNA sequence; and
In the accompanying drawings:
FIG. 1 shows the nucleotide sequence of the human TNF.alpha. cDNA (SEQ ID NO: 24) and encoded amino acid (SEQ ID NO: 25) sequence. The predicted signal sequence residues are numbered -40 to -1. The transmembrane domain is boxed and potential N-linked glycosylation sites are overlined. The sequence homologous with the designed oligonucleotide probe is found at nucleotide positions 477-533.
FIG. 2 is a Northern blot (lanes 1-3) of 10 .mu.g of oligo-dT selected RNA from human 293 cells (fibroblast cell line) (lane 1), placenta (lane 2) and spleen (lane 3) hybridised with the TNF receptor cDNA (Smal-EcoRI fragment). The Southern blot (lan
REFERENCES:
Yan et al, J. Biol. Chem. 266(18):12098-12104, Jun. 25, 1991.
Brennan et al., "Inhibitory Effect of TNF.alpha. Antibodies on Synovial Cell Interleukin-1 Production in Rheumatoid Arthritis," Lancet ii:244-247 (1989).
Haworth et al., "Applications of Cytokines in Human Immunotherapy" in Immunology and Molecular Biology of Cytokines (ed. A.W. Thompson) Academic Press, pp. 301-324 (1991).
Williams et al., "Anti-TNF Ameliorates Joint Disease in Murine Collagen-Induced Arthritis," Proc. Natl. Acad. Sci. USA 89:9784-9788 (1992).
Elliott et al., "Treatment of Rheumatoid Arthritis with Chimeric Monoclonal Antibodies to TNF.alpha.," Arth. Rheum. 36:1681-1690 (1993).
Elliott et al., "Randomised Double Blind Comparison of a Chimeric Monoclonal Antibody to Tumour Necrosis Factor .alpha.(cA2) versus Placebo in Rheumatoid Arthritis," Lancet 344:1105-1110 (1994).
Elliott et al., "Repeated Therapy with Monoclonal Antibody to Tumour Necrosis Factor .alpha.(cA2) in Patients with Rheumatoid Arthritis," Lancet 344:1125-1127 (1994).
Corcoran et al., "Characterization of Ligand Binding by the Human p55 Tumour-Necrosis-Factor Receptor, Involvement of Individual Cysteine-Rich Repeats," Eur. J. Biochem. 223:831-840 (1994).
Rankin et al., "The Therapeutic Effects of an Engineered Human Anti-Tumour Necrosis Factor Alpha Antibody (CDP571) in Rheumatoid Arthritis," Brit. J. of Rheum. 34:334-342 (1995).
Butler et al., "Modulation of Proinflammatory Cytokine Release in Rheumatoid Synovial Membrane Cell Cultures with an Anti TNF.alpha. Monoclonal: Comparison with Blockade of IL-1 using the Recombinant IL-1 Receptor Antagonist," European Cytokine Network (in press).
Schall et al., Cell (1990) 61:361-370.
Loetscher et al., Cell (1990) 61:351-359.
Gray et al., Proc. Natl. Acad. Sci. USA (1990) 87:7380-7384.
Brennan Fionula M.
Feldmann Marc
Gray Patrick W.
Turner Martin J. C.
The Mathilda and Terence Kennedy Institute of Rheumatology
Ulm John
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