Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Peptide containing doai
Reexamination Certificate
2000-09-08
2004-05-04
Kemmerer, Elizabeth (Department: 1647)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Peptide containing doai
C530S402000, C530S350000, C530S300000
Reexamination Certificate
active
06730657
ABSTRACT:
This application is the national phase under 35 U.S.C. §371 of PCT International Application No. PCT/EP99/00502 which has an International filing date of Jan. 28, 1999, which designated the United States of America.
FIELD OF THE INVENTION
The present invention relates to recombinant proteins obtained from the combination of structural domains derived from the &agr; subunits of hepatocyte growth factor (HGF) and macrophage stimulating protein (MSP).
In particular, the engineered factors of the invention are obtained by combining the hairpin loop and kringle domains of HGF &agr; chains and/or MSP, together with HGF or MSP &bgr; chains, to obtain a structure having two superdomains joined by an intervening linker sequence. Moreover, the invention relates to DNA sequences encoding the above mentioned recombinant proteins, to the expression vectors comprising said DNA sequences and to host cells containing said expression vectors. The recombinant proteins of the present invention are biologically active and protect epithelial cells and other cells from apoptosis induced by chemotherapic drugs. Therefore, these molecules can conveniently be used to prevent or treat the toxic side effects of the chemotherapeutical treatment of tumours.
STATE OF THE ART
Hepatocyte Growth Factor (HGF) and Macrophage Stimulating Protein (MSP) are highly related proteins both structurally and functionally (FIGS.
1
and
2
). Both these factors are secreted as an inactive precursor, which is processed by specific proteases which recognise a cleavage site inside the molecule, dividing the protein in two subunits. These subunits, named &agr; chain and &bgr; chain, are linked by a disulphide bond. Thus, the mature factor is an &agr;-&bgr; dimeric protein. Only the mature (dimeric) form of the factor is able to activate its receptor at the surface of the target cells (the Met tyrosine kinase in the case of HGF and the Ron tyrosine kinase in the case of MSP) and therefore to mediate biological responses (Naldini, L. et al., 1992, EMBO J. 11: 4825-4833; Wang, M. et al., 1994, J. Biol. Chem. 269; 3436-3440; Bottaro, D. et al., 1991, Science 25: 802-804; Naldini, L. et al., 1991, EMBO J. 10: 2867-2878; Wang, M. et al., 1994, Science 266: 117-119; Gaudino, G. et al., 1994, EMBO J. 13: 3524-3532).
The &agr; chain of both factors contains a hairpin loop (HL) structure and four domains with a tangle-like structure named kringles (K1-K4; Nakamura, T. et al., 1989, Nature 342: 440-443; Han, S. et al., 1991, Biochemistry 30: 9768-9780). The precursor also contains a signal sequence (LS) of 31 amino acids (in the case of HGF) or of 18 amino acids (in the case of MSP), removed in rough endoplasmic reticulum, which directs the neoformed peptide to the secretive pathway. The &bgr; chain contains a sequence box homologous to the typical catalytic domain of serine proteases, but it has no enzymatic activity (Nakamura, T. et al., 1989, Nature 342:440-443; Han, S. et al., 1991, Biochemistry 30:9768-9780). Both &agr; and &bgr; chains contribute to the binding of the growth factor to the respective receptor (Met for HGF and Ron for MSP).
HGF and MSP polypeptides are able to induce a variety of biological effects besides cell proliferation. The main biological activities of these molecules are: stimulation of cell division (mitogenesis); stimulation of motility (scattering); induction of polarisation and cell differentiation; induction of tubule formation (branched morphogenesis), increase of cell survival (protection from apoptosis). The tissues that respond to HGF and MSP stimulation are those containing cells that express the respective Met (HGF) and Ron (MSP) receptors. The most important target tissues of these factors are epithelia of different organs, such as liver, kidney, lung, breast, pancreas and stomach, and some cells of the hematopoietic and nervous systems. A detailed review of the biological effects of HGF and MSP in the various tissues can he found in: Tamagnone, L. & Comoglio, P., Cytokine & Growth Factor Reviews, 1997, 8: 129-142, Elsevier Science Ltd.; Zarnegar, R. & Michalopoulos, G., 1995, J. Cell Biol. 129: 1177-1180; Medico, E. et al., 1996, Mol. Biol. Cell, 7: 495-504; Banu, N. et al., 1996, J. Immunol. 156: S2933-2940.
In the case of HGF, the hairpin loop and the first two kringles are known to contain the sites of direct interaction with the Met receptor (Lokker, N. et al., 1992, EMBO J. 11: 2503-251.0; Lokker, N. et al., 1994, Protein Engineering 7: 895-903). Two naturally-occurring truncated forms of HGF produced by some cells by alternative splicing have been described. The first one comprises the first kringle (NK1-HGF Cioce, V. et al., 1996, J. Biol. Chem. 271: 13110-13115) whereas the second one spans to the second kringle (NK2-HGF Miyazawa, K. et al., 1991, Eur. J. Biochem. 197: 15-22). NK2-HGF induces cell scattering, but it is not mitogenic as the complete growth factor is (Hartmann, G. et al., 1992, Proc. Natl. Acad. Sci. USA 89: 11574-11578). However, NK2-HGF exhibits mitocenic activity in the presence of heparin, a glycosaminoglycan that binds the first kringle of HGF and is likely to induce dimerization of NK2-HGF (Schwall, R. et al., 1996, J. Cell Biol. 133: 709-718). Moreover NK2-HGF, being a partial agonist of Met, behaves as a competitive inhibitor of HGF as far as the mitogenic activity is concerned (Chan, A. et al., 1991, Science 254: 1382-1385). NK1-HGF has also been described to exert partial stimulation of Met and competitive inhibition of HGF mitogenic activity (Cioce, V. et al., 1996, J. Biol. Chem. 271: 13110-13115).
In the case of MSP, the modality of interaction with the Ron receptor is less understood: some preliminary studies suggest a situation opposite of that of HGF, i.e. the &bgr; chain directly binds the receptor whereas the &agr; chain stabilises the complex (Wang, M. et al., 1997, J. Biol. Chem. 272: 16999-17004).
The therapeutical use of molecules such as HGF and MSP is potentially valuable in a wide range of pathologies (Abdulla, S., 1997, Mol. Med. Today 3: 233). Nevertheless, a number of technical as well as biological complications make the application of these molecules in clinics difficult.
For example, HGF was shown to protect kidney cells against programmed cell death (apoptosis) induced by cisplatinum, but at the same time it can induce an undesired proliferation of neoplastic cells. The natural truncated forms NK1 and NK2 of HGF show no problems of proteolytic activation, but they have a reduced biological activity.
SUMMARY OF THE INVENTION
The present invention provides recombinant molecules deriving from the combination of structural domains of HGF and MSP &agr; and &bgr; subunits, which overcome the problems of the prior art molecules described above.
The molecules of the invention are composed of two superdomains, one obtained combining HL and K1-K4 domains of HGF and MSP &agr; chains, the other corresponding to HGF or MSP &bgr; chain, connected by a linker which may contain a proteolytic cleavage site. This structure allows the recombinant proteins to interact with both Met and Ron receptors, in order to induce biological responses which are synergistic and selective compared with the natural factor and the truncated forms of the prior art.
DETAILED DISCLOSURE OF THE INVENTION
The present invention relates to recombinant proteins (which will be hereinafter referred to indifferently as proteins, molecules, engineered or recombinant factors) characterised by a structure that comprises two superdomains, one consisting of a combination of HL and K1-K4 domains derived from HGF or MSP &agr; chain, the other corresponding to HGF or MSP &bgr; chain, linked by a spacer sequence or a linker. In particular, the invention relates to proteins of general formula (I)
[A]-B-[C]-(D)
y
(I)
in which
[A] corresponds to the sequence (LS)
m
-HL-K1-(K2)
n
-(K3)
o
-(K4)
p
wherein (the numbering of the following amino acids being referred to the HGF and MSP sequences as reported in
FIG. 1 and 2
, respectively):
LS is an amino acid s
Caselli Gianfranco
Colessi Chiara
Comoglio Paolo
Medico Enzo
Michieli Paolo
Dompe' S.p.A.
Kemmerer Elizabeth
Wegert Sandra
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