Recombinant proteins derived from HGF and MSP

Details Recombinant proteins derived from HGF and MSP Recombinant proteins derived from HGF and MSP

2000-09-15

2003-04-22

Kemmerer, Elizabeth (Department: 1647)

Drug, bio-affecting and body treating compositions

Designated organic active ingredient containing

Peptide containing doai

C530S300000, C530S350000, C530S388220, C530S399000, C530S402000, C536S023500, C536S023100, C435S069700

Reexamination Certificate

active

06551991

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to recombinant proteins obtained from the combination of structural domains derived from the a subunits of hepatocyte growth factor (HGF) and macrophage stimulating protein (MSP).
In particular, the engineered factors of the invention are obtained by combination of the hairpin loop and kringle domains of the &agr; chains of HGF and/or MSP, so as to obtain a structure having two superdomains with 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 their activity can be measured by determination of their ability to induce activation of the Met tyrosine kinase receptor, their “scattering” effect on epithelial cells, and their protective effect against cell death induced by chemotherapic drugs (vide infra). Therefore, these molecules can conveniently be used to prevent or treat the toxic side effects of the chemotherapeutical treatment of tumours, and to reduce iatrogenic cell damage induced by other types of drugs.
TECHNOLOGICAL BACKGROUND
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 box with a sequence homologous to that typical of serine proteases, but it has no catalytic 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 where cells express the respective Met (HGF) and Ron (MSP) receptors. The most important target tissues of these factors are epithelial cells 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 be found in Tamagnone, L. & Comoglio, P., 1997, Cytokine & Growth Factor Re-views, 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 NA et al., 1992, EMBO J., 11:2503-2510; 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 (NKI-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 regains mitogenic activity in the presence of heparin, a glucosaminoglycan that binds HGF through a domain contained in the first kringle and which 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. 27: 13110-13115). Anyway, a truncated factor is endowed with an activity markedly lower than the recombinant factors described in the invention, as shown in example 3.
In the case of MSP, the interaction sites with the Ron receptor are 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 would act stabilizing the complex (Wang MH 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. First of all, the pleiotropic character of these factors can causes poorly selective biological responses, which involve undesired side effects. For example, the use of HGF to prevent some side effects of the chemotherapeutic drug cisplatin has been proposed (Kawaida K et al., 1994, Proc. Natl. Acad. Sci. 91:4357-4361). Cancer patients treated with this drug can suffer kidney acute damage due to the cytotoxic action of cisplatin on proximal tubule epithelial cells. HGF is able to protect these cells against programmed death (apoptosis) induced by cisplatin, but at the same time it can induce an undesired proliferation of neoplastic cells. Other problems related to the pharmaceutical use of HGF and MSP are the necessity of their proteolytic activation and their stability, which causes technical problems. The NK1 and NK2 truncated forms of HGF do not require proteolytic activation, but they have a reduced biological activity.
SUMMARY OF THE INVENTION
The present invention provides recombinant molecules composed of a combination of structural domains derived from the &agr; chains of HGF and/or MSP, which overcome the problems of the prior art molecules described above. The molecules of this invention are composed of two superdomains connected by a linker. Each superdomain is composed of a combination of the HL and K1-K4 domains of the &agr; chain of HGF and/or MSP. These engineered factors induce selective biological responses, do not require proteolytic activation, are stable and are more active than the truncated forms of HGF described previously.
DETAILED DISCLOSURE OF THE INVENTION
The present invention relates to recombinant proteins (which will be hereinafter referred to indifferently as proteins, mo

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