Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Patent
1995-09-15
1999-04-06
Draper, Garnette D.
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
4352525, 4353201, 536 235, 530351, 424 851, C12N 1519, C07K 1452
Patent
active
058916792
DESCRIPTION:
BRIEF SUMMARY
The present invention relates to TNF-.alpha.-muteins, a process for preparing them and their use as active substances in pharmaceutical compositions.
BACKGROUND OF THE INVENTION
Tumor necrosis factor-.alpha. (TNF-.alpha.), also known as cachectin, is a multifunctional cytokine produced mainly by activated macrophages. In vitro, it has diverse biological effects (reviewed by Manogue et Cerami, 1988) including killing of transformed cells (Carswell et al., 1975), stimulation of granulocytes and fibroblasts (Old, 1985; Vilchek et al., 1986; Beutler and Cerami, 1987), damage to endothelial cells (Sato et al, 1986), and anti-parasitic effects (Taverne et al., 1984). In vivo, it plays a key role as an endogenous mediator of inflammatory, immune and host defence functions and it is involved in a number of pathological conditions in man and mouse such as septic shock, cachexia, capillary leak syndrome, hemorhagic necrosis of multiple organs, etc. It is capable of acting independently and in conjunction with other factors affecting a whole plethora of different body functions. These effects can either be beneficial or life-threatening to the host. Some of these effects are direct, others may be mediated via the induction of other secreted factors. The biological effects of TNF-.alpha. are mediated via binding to specific cell surface receptors.
The primary structure of human and mouse TNF-.alpha. (Pennica et al., 1984; Fransen et al., 1985) and of two different TNF-receptors (p55-TNF-R and p75-TNF-R) has been deduced from the nucleotide sequence of the cloned cDNA (Dembic et al., 1990; Loetscher et al., 1990). Both receptors bind not only TNF-.alpha. but also TNF-.beta. or lymphotoxin with high affinity (Schoenfeld et al., 1991). TNF-.beta. is a related lymphocyte product (TNF-.alpha. and TNF-.beta. share 32% homology on the amino acid sequence level) that exhibits pleiotropic activities very similar to those of TNF-.alpha.. X-ray crystallographic analysis revealed that the tertiary structure of both molecules is virtually identical except that the TNF-.beta. trimer creates a molecule that is less elongated than the TNF-.alpha. trimer and the latter has a top-region that flares open (Eck et al, 1992).
Besides the cell receptor interaction, TNF-.alpha. has been shown to have a lectin-like property for the oligosaccharide ligands chitobiose and Man(.alpha.1,3)-(Man(.alpha.1,6)-Man (Hession et al., 1987; Sherblom et al., 1988). They further demonstrated that the TNF-.alpha. protein has at least two different binding sites, one lectin-like and the other directed at cell surface receptors. Several TNF-.alpha. mutants were described for which the binding to the cellular TNF-receptor p55 and/or p75 was hampered. All these mutations are located in the lower half of the pyramidal structure of the biologically active TNF-.alpha. trimer (Van Ostade et al., 1991; Van Ostade et al., 1992; EP-A-0 486 908).
The availability of recombinant TNF has enhanced its use in tumor therapy. However, the in vivo tumoricidal effects of TNF-.alpha. have always been accompanied by toxic side effects. Different approaches were followed to overcome these noxious effects, including the use of monoclonal antibodies as well as fragments thereof, which neutralize the in vitro and in vivo toxic properties of TNF-.alpha. (e.g. EP-A-0 350 690).
In addition to its immunomodulating activity, TNF-.alpha. has also been shown to be involved in the control of growth and differentiation of various parasites. Upon infection of the host, parasites are capable of inducing the secretion of different cytokines such as TNF which may affect the course of the disease. For instance, in the case of malaria, TNF-.alpha. can be protective in certain circumstances, such as inhibiting parasite survival in rodent malaria (Clark et al., 1987; Taverne et al., 1987). By contrast, its overproduction can be detrimental to the host and can contribute to the pathology of the disease (Clark, 1987; Grau et al., 1989).
In schistosomiasis, the parasite uses the host-derived immunor
REFERENCES:
Goh et al., Protein Engineering 4(2) 1991, pp. 785-791.
Kircheis et al, Inmunology 1992, 76, pp. 433-438.
Protein Engineering, vol. 3, 1990, pp. 713-719, J. Yamagishi et al Mutational analysis of structure-activity relationships in human tumor necrosis factor-alpha *abstract; Table II(a) amd (b).
Protein Engineering, vol. 3, 1990, pp. 721-724, T. Arakawa et al; `Alteration in folding efficiency and conformation of recombinant human tumor necrosis factor-alpha by replacing cysteines 69 and 101 with aspartic acid 69 and arginine 101` *abstract.
Science, vol. 263, 11 Feb. 1994, pp. 814-817, R. Lucas et al; `Mapping the lectin-like activity of tumor necrosis factor` *whole document.
Biochimica et Biophysica Acta, 1096 (1991) pp. 245-252, Ito et al; `Novel muteins of human tumor necrosis factor .beta.`.
Tibtech, vol. 8, Jun. 1990, Undercurrents, Complementarity of peptides specified by `sense` and `antisense` strands of DNA.
De Baetselier Patrick
Fransen Lucia
Lucas Rudolph
Sablon Erwin
Draper Garnette D.
N.V. Innogenetics S.A.
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