Liver damage occurs in a number of acute and chronic clinical conditions, including fulminant hepatic failure, hepatitis, partial hepatectomy, cirrhosis, hepatocyte transplant, transplant of artificial liver. In many instances, liver regeneration is vital to the survival of patients.
Liver cells have a good ability to regenerate. It is known that following partial hepatectomy, the liver size is usually restored to its exact original mass within about six days. Liver (hepatocyte) regeneration is believed to be controlled by various growth stimulatory and growth inhibitory cytokines of autocrine or paracrine origin, however, the exact role and action mechanism of these factors is far from entirely understood.
In vitro, DNA synthesis in isolated hepatocytes has been shown to be stimulated by growth factors such as insulin-like growth factor-I (IGF.sub.1), epidermal growth factor (EGF), type .alpha. transforming growth factor (TGF-.alpha.) and to be inhibited by members of the type .beta. transforming growth factor (TGF-.beta.) family, and transferrin (activin). Most recently, a further protein, named hepatocyte growth factor (HGF) has been shown to be a complete mitogen for primary hepatocytes. The observation that the level of HGF in the serum rapidly increases following experimental damage to the liver and in patients with fulminate hepatic failure suggests that it may be an important mediator of liver regeneration in vivo.
HGF was purified by Nakamura et al. from the serum of partially hepatectomized rats [Biochem. Biophys. Res. Comm. 122:1450-1459 (1984)]. Subsequently, HGF was purified from rat platelets, and its subunit structure was determined [Nakamura et al, Proc. Natl. Acad. Sci. USA. 83. 6489-6493 (1986); and Nakamura et al., FEBS Letters 224, 311-316 (1987)]. The purification of human HGF (hHGF) from human plasma was first described by Gohda et al., J. Clin. Invest. 81, 414-419 (1988). According to the results reported by Gohda et al. hHGF is more effective in the stimulation of cultured hepatocyte proliferation than human epidermal growth factor (hEGF) or insulin, and the effect of hHGF with the maximal effects of hEGF and insulin is "additive or synergistic". Similarly, Zarnegar et al., Cancer Research 49, 3314-3320 (1989) described the purification of a polypeptide growth factor, called human hepatopoietin A (HPTA) having very similar properties to hHGF as characterized in earlier publications. As the authors do not disclose the amino acid sequences of their purified proteins, the degree of the structural similarity between the two factors can not be determined.
Using partial amino acid sequence generated from hHGF purified from human plasma, the molecular cloning and expression of hHGF, including the nucleotide sequence of hHGF cDNA and the deduced amino acid sequence of the hHGF protein, have been reported by Miyazawa et al., Biochem. Byophys. Res. Comm. 163, 967-973 (1989) and Nakamura et al., Nature 342, 440-443 (1989). The reported sequences differ in several positions. Nakamura et al., Supra describe the effect of hHGF and hEGF as being additive.
The N-terminal amino acid sequence of rabbit HPTA was described by Zarnegar et al., Biochem. Biophys. Res. Comm. 163, 1370-1376 (1989).
The hHGF cDNA encodes a 728 amino acids polypeptide (pre-pro hHGF) having a molecular mass (M.sub.r) of about 82,000, and a heterodimeric structure, composed of a large .alpha.-subunit of 440 amino acids (M.sub.r 69,000) and a small .beta.-subunit of 234 amino acids (M.sub.r 34,000). The nucleotide sequence of the hEGF cDNA reveals that both the .alpha.- and the .beta.-chains are contained in a single open reading frame coding for a pre-pro precursor protein. In the predicted primary structure of mature hHGF, an interchain S-S bridge is formed between Cys 487 of the .alpha.-chain and Cys 604 in the .beta.-chain (see Nakamura et al., Nature, Supra). The N-terminus of the .alpha.-chain is preceded by 54 amino acids, starting with a methionine group. This segment includes a signal sequence and the prosequence. The .alpha.-chain starts at amino acid (aa) 55, and contains four Kringle domains. The Kringle 1 domain extends from about aa 128 to about aa 206, the Kringle 2 domain is between about aa 211 and about aa 288, the Kringle 3 domain is defined as extending from about aa 303 to about aa 383, and the Kringle 4 domain extends from about aa 391 to about aa 464 of the .alpha.-chain. It will be understood that the definition of the various Kringle domains is based on their homology with kringle-like domains of other proteins (prothrombin, plasminogen), therefore, the above limits are only approximate. The HGF .beta.-chain includes a serine-protease like domain. In a portion of cDNA isolated from human leukocytes in-frame deletion of 15 base pairs was observed. Transient expression of the cDNA sequence in COS-1 cells revealed that the encoded HGF molecule lacking 5 amino acids in the Kringle 1 domain was fully functional [Seki et al, Biochem. and Biophys. Res. Commun. 172, 321-327 (1990)]. This variant is referred to as "delta5 HGF". HGF contains four putative glycosylation sites, which are located at positions 294 and 402 of the .alpha.-chain and at positions 566 and 653 of the .beta.-chain.
Gamma interferon (IFN-.gamma.), which is also referred to as immune interferon, is a member of the interferon family, which exhibits the antiviral and anti-proliferative properties characteristic of interferons-.alpha., and -.beta. but, in contrast to those interferons, is pH 2 labile. IFN-.gamma. was originally produced upon mitogenic induction of lymphocytes. The recombinant production of IFN-.gamma. was first reported by Gray, Goeddel and co-workers [Gray et al., Nature 295, 503-508 (1982)], and is subject of U.S. Pat. Nos. 4,762,791, 4,929,544, 4,727,138 and 4,925,793. The recombinant IFN-.gamma. of Gray and Goeddel as produced in E. coli, consisted of 146 amino acids, the N-terminal portion of the molecule commencing with the sequence CysTyrCys. It has later been found that the native IFN-.gamma. (i.e., that arising from mitogen induction of human peripheral blood lymphocytes and subsequent purification) is a polypeptide which lacks the CysTyrCys N-terminus assigned by Gray et al., Supra.
It has previously been shown that IFN-.gamma. exhibits a synergistic effect with IFN-.alpha. or IFN-.beta. in assays for cells growth inhibition [EP 107,498, Czarnieski et al., J. Virology 49 (1984)], with lymphotoxin (EP 128,009), and with IL-2 (U.S. Pat. No. 5,082,658). Synergistic cytotoxic compositions comprising human interferon and TNF are disclosed in the U.S. Pat. No. 4,650,674.