WO2006038880A1

WO2006038880A1 - New composition and method

Info

Publication number: WO2006038880A1
Application number: PCT/SE2005/001493
Authority: WO
Inventors: Fariba Nayeri
Original assignee: Damavand'wound Ab
Priority date: 2004-10-08
Filing date: 2005-10-07
Publication date: 2006-04-13
Also published as: SE0402433D0

Abstract

The present invention relates to a pharmaceutical composition comprising HGF (Hepatocyte growth factor) and to the use of HGF for the manufacture of such pharmaceutical composition comprising HGF in a dose sufficient for the immunisation in a mammal suffering from cancer. The invention further relates to a method for the treatment cancer using the 5 pharmaceutical composition comprising HGF in a dose sufficient to cause an active immunisation in a mammal.

Description

New composition and method

Field of invention

The present invention relates to a pharmaceutical composition for the treatment or prevention of cancer comprising HGF (Hepatocyte growth factor) and to the use of HGF for the manufacture of such pharmaceutical composition comprising HGF in a dose sufficient for an active immunisation of a mammal. The invention further relates to a method for prevention or treatment of cancer using the pharmaceutical composition comprising HGF in a dose sufficient to cause an active immunisation in a mammal.

Background:

Hepatocyte growth factor (HGF) has been investigated widely over the past ten years. HGF is a heterodimeric protein of 96-110 kDa consisting of an α- and a β-chain. It is produced by mesenchymal cells as a single-chain precursor which is activated by the proteolytic action of an HGF-activator. The unique properties of this cytokine make it eligible to be involved in the recovery process after injuries. HGF exhibits several effects on epithelial cells: mitogenic (Matsumoto & Nakamura, 1991), motogenic (Nusrat et al., 1994), morphogenic (Montesano et al., 1991) and angiogenic (Bussolino et al, 1992). Through these properties HGF mediates regeneration and protection of various normal tissues, including the liver, lung, kidney, gastrointestinal tissues, nervous tissue, pancreas and heart (Matsumoto and Nakamura, 1997). Several researchers have attempted to treat induced hazardous injuries in animal models by HGF (Yanagita et al., 1993; Kondo et al., 1999). An increased level of HGF in serum during diseases, such as acute infectious diseases; gastroenteritis, sepsis, urinary tract infections and erysipelas, has been reported (Nayeri et al., 2002).

The effect of HGF on different malignant cells and the role of HGF in the development and progression of cancers as well as effect on invasiveness and metastasis of tumour cells is also an intense area of investigation (for recent review articles see Jiang et al., 1999; Comoglio et al., 2001; Birchmeier et al., 2003).

A comprehensive review on expression of HGF and c-Met in different cancer cells can be found at www.vai.org/HgfSf-Metandcancer. Expression of HGF by tumor cells is often correlated with poor prognosis and it has also been shown that high serum or blood leves of HGF in patients with different types of cancers are associated with poor survival. Good therapeutic response has been found to be associated with decreasing HGF-levels (Jakob C, et al., 2003; Dluzniewska J, et al., 2002; de Jong KP, et al., 2001; Beppu K₅ et al. 2000).

Some investigators have attempted to treat cancer by suppressing the interaction between HGF and the c-met receptor or by administration of antibodies against HGF (passive immunisation). However as far as we know there are no studies regarding active immunisation by HGF as a therapy model or as an explanation model for some diseases.

During our recent studies on HGF we observed that in mice, which were treated by repeated subcutaneous injections of HGF (0.35-0.70 λg/kg/day in 5 days), biopsies of testis showed reduced PCNA (proliferating cell nuclear antigen ) by imunohistochemical studies. Antibodies against human HGF (IgM and IgG) were detected in the serum of these mice. The mice that were observed in 9 months after the HGF injection, did not show signs of diseases or malignancy. Based on these data the following studies were carried out.

Summary of the invention

The object of the present invention is to find a treatment of cancer. This object is being achieved in providing the use of HGF (Hepatocyte growth factor) for the manufacture of a therapeutic composition comprising HGF in a dose sufficient for the active immunisation in a mammal suffering from cancer such as invasive prostate cancer and metastases as well as prevention of cancer in high risk subjects.

The term HGF as used herein refers to every naturally occuring form of HGF, biologically active or inactive, as well as to any recombinantly produced, chemically synthesized or modified peptide capable of raising specific anti-HGF antibodies in a mammal.

The present invention also provides a method for the prevention and/or treatment of cancer, comprising the steps of: injection of a dose of HGF (Hepatocyte growth factor) in a mammal, the dose of HGF being sufficient to cause an active immunisation in said mammal, and estimation of the formed antibodies against HGF (anti-HGF), measured as the increase of the presence of said anti-HGF in serum. According to another aspect of the invention, a pharmaceutical composition is provided, comprising a dose of HGF (Hepatocyte growth factor) for a mammal suffering from cancer, as well as prevention of cancer in high risk subjects, wherein the dose of HGF is sufficiently high to cause an active immunisation in said mammal.

Brief description of the drawings

The invention will now be described by means of the following drawings:

Fig 1 : Mouse with spontaneous tumour that received sc HGF injections (2 ng) repeatedly once a week. The size of tumour decreased as shown in the series of pictures. Dates are given in each picture.

Fig 2: Localization of c-Met receptor in CCL-53.1 cells by the immunofluorescence technique. As shown, there are no differences between HGF-treated (A) and control cells (B) in the presentation of receptor by immunofluorescence. The bare area has almost been covered in the HGF-treated (A) wells, but not in the non-treated control (B) wells. Fig 3: Different concentrations of HGF (1, 5, 10 ng/ml) and differences in restitution (Y-axis) after 24 hours.

Fig 4: Restitution in control and HGF-treated cells 24 and 48 hours after injury. Y-axis shows the measured injured area. Fig. 5: A comparison between the presence of serum HGF and anti-HGF in different patient groups. The figure shows serum HGF (logharitmic values) and anti-HGF in a healthy person vaccinated with HGF, one week and one month after vaccination, in a patient with renal insufficiency, in patients with cirrhosis and in a patient with prostate cancer with metastasis.

Detailed description of the invention

Humans produce autoantibodies against their own growth hormones, like hepatocyte growth factor (HGF) and this might be a defence against unwanted effects of this cytokine. In the situations that HGF rises in serum, antibodies against HGF raise after a short while. In patients with invasive cancer and metastasis, the serum HGF is high but serum anti-HGF is low and this might be one cause of cancer invasion.

Antibodies against hepatocyte growth factor have been suggested as a treatment against some cancer diseases but an active autoimmunisation by vaccination with recombinant HGF to produce autoantibodies against HGF in the body has not been reported. Vaccination with very low doses of HGF sc, causes rising anti-HGF antibodies, which can treat and even protect against some types of cancer.

The present invention provides the use of HGF for the manufacture of a therapeutic composition for the treatment of cancer or prevention of cancer in a high-risk subject. The composition comprises HGF in a dose sufficient for the active immunisation in a mammal suffering from cancer or a subject having a high risk of developing cancer. The cancer can be a cancer form wherein the patient exhibits high levels of serum HGF, a cancer form wherein the patient exhibits low serum levels of anti-HGF antibodies, or wherein the patient exhibits both high levels of serum HGF and low levels of anti-HGF antibodies. The cancer can be invasive cancer such as the invasive prostate cancer with metastases. The mammal is preferably a human but the mammal can also be a domestic animal such as a horse, a cow or a sheep or a pet such as a dog or a cat.

The present invention also provides a method for the treatment of cancer or prevention of cancer in a high-risk subject, comprising the steps of: injection of a dose of HGF in a mammal, the dose of HGF being sufficiently high to cause a immunisation in said mammal, and estimation of the formed antibodies against HGF (anti-HGF), measured as the increase of the presence of said anti-HGF in serum. The amount HGF given should be decided upon by the attending physician, but would usually be between 1 and 10 ng/day, preferably 2-4 ng/day. Preferably, also an estimation of the formed antibodies against HGF (anti-HGF) is made, measured as the increase of the presence of said anti-HGF in the patient's serum. The cancer can be a cancer form wherein the patient exhibits high levels of serum HGF, a cancer form wherein the patient exhibits low serum levels of anti-HGF antibodies, or wherein the patient exhibits both high levels of serum HGF and low levels of anti-HGF antibodies. The cancer can be invasive cancer such as invasive prostate cancer with metastases. The mammal is preferably a human but the mammal can also be a domestic animal such as a horse, a cow or a sheep or a pet such as a dog or a cat.

A pharmaceutical composition is provided comprising a dose of HGF (Hepatocyte growth factor) for a mammal suffering from cancer, wherein the dose of HGF is sufficiently high to cause an immunisation in said mammal. The composition is preferably adapted for subcutaneous injection. The composition is further preferably adapted to deliver a dose of 1- 10 ng HGF per day to the patient, or 2-4 ng HGF per day. The cancer can be a cancer form wherein the patient exhibits high levels of serum HGF, a cancer form wherein the patient exhibits low serum levels of anti-HGF antibodies, or wherein the patient exhibits both high levels of serum HGF and low levels of anti-HGF antibodies. The cancer can be invasive cancer such as invasive prostate cancer and metastases. The mammal is preferably a human but the mammal can also be a domestic animal such as a horse, a cow or a sheep or a pet such as a dog or a cat.

Examples Mice with spontaneous tumours were treated successfully with HGF injections. The tumour size decreased and mouse continued a normal life in 4 months afterwards. See Example 1.

Serum from the mice that received sc HGF-injections repeatedly, stopped migration of mouse melanoma cells caused by HGF in vitro. See Example 2.

HGF was then injected subcutaneously to a healthy person. Increasing amounts of autoantibodies against human HGF was found in serum samples taken one week, two weeks and 4 weeks after administration. The serum HGF was low in the same sera (Example 3).

Thus, vaccination by subcutaneous injections of HGF can produce an active defence against tumours, cancer and metastasis.

Serum from patients with different infections was studied for presence of antibodies against HGF. High amounts of HGF as well as antibodies against HGF were found in these sera. High amounts of antibodies against HGF but low serum HGF were found in serum from patients with chronic hepatit C. The variation in antibodies against HGF was high in serum from normal blood donors but serum HGF was low in all these persons. In one patient with invasive prostate cancer and metastases the serum HGF was very high but antibodies against HGF were low.

The invention will now be described further by means of the following non-limiting examples. An ELISA method was provided to measure the antibodies against hepatocyte growth factor in sera of animals, patients and control subjects. Example 1

Mice with spontaneous tumours were treated successfully with HGF injections subcutaneously 2 ng/day in one week and injections weekly afterwards. The tumour size decreased (Fig 1) and mouse continued a normal life in 4 months afterwards. Pathological studies did not show signs of cancer invasion in the animals that were killed. Antibodies against human HGF was found in serum collected from treated animal.

The fertility decreased in mice treated by sc HGF injections in both sexes, and biopsies of testis showed reduced PCNA by immunohistochemical studies.

Example 2

A model of cell injury in mouse melanoma cell monolayer (CCL-53.1), which expresses HGF receptor: c-met, was developed in vitro. Recombinant human HGF (R&D systems) was added to the wells. HGF did not enhance cell proliferation, judged by [³H]-thymidine incorporation. The restitution of injured epithelial cells was significantly increased by HGF (Figure 2, 4).

This effect was dose-dependent (Figure 3), beginning at low concentrations (lng/ml) of HGF.

HGF enhanced migration of epithelial cells compared to untreated controls.

The actin structure was changed by HGF, with less F-actin in the dense peripheral structures compared to untreated cells. Corresponding morphological changes in cells were found showing spread morphology with smaller intracellular spaces after HGF addition (data not shown).

Mice (C57BL/6J) were injected subcutaneously by recombinant human HGF in 5 days. Serum from the immunised mice could inhibit completely the restitution of CCL-53.1 cells by HGF. Addition of serum from control mice did not affect the enhanced restitution of injured CCl-53.1 monolayer by HGF (data not shown).

We conclude that HGF might cause morphologic changes and enhanced motility of CCL-53.1 cells. Subcutaneous injection of recombinant human HGF contributes to production of substances in vivo, that might inhibit the effects of HGF completely. This active immunisation does not cause hazardous injuries in the mice.

Example 3 HGF (4 ng/day) was injected subcutaneously to a healthy person for 4 days. Increasing amounts of autoantibodies against human HGF was found in serum samples taken one week, two weeks and 4 weeks after administration. The serum HGF was low in the same sera. (See Fig 5). Increased ANA (anti-nuclear antibodies) and negative ANCA (antineutrophil cytoplasmic antibodides) tests was seen.

Example 4

Serum from patients with different disease conditions was studied for the presence of antibodies against HGF. High amounts of HGF as well as antibodies against HGF were found in these sera. High amounts of antibodies against HGF but low serum HGF were found in serum from patients with chronic hepatit C (cirrhosis) (n=10). In one patient with invasive prostate cancer and metastases the serum HGF was very high but antibodies against HGF were low. (See Fig. 5)

Example 5

Autoantibodies against HGF (IgG, IgM) were found in the sera of 50 healthy blood donors (25 women and 25 men between age range 18-51 years). The variation in antibodies against HGF was high in serum from normal blood donors but serum HGF was low in all the cases (Mean 0.67 ng/ml).

The invention is not limited to the above mentioned embodiments and examples, but shall be interpreted within the scope of the appending claims.

References:

1. Beppu K, Uchiyama A, Morisaki T, et al. Elevation of serum hepatocyte growth factor concentration in patients with gastric cancer is mediated by production from tumor tissue. Anticancer Res (2000) 20(2B):1263-7

2. Birchmeier C, Birchmeier W, Gherardi E, Vande Woude GF. Met, metastasis, motility and more. Nat Rev MoI Cell Biol (2003) 4:915-25 _o

8

3. Bussolino F, Di Renzo MF, Ziche M, Bochietto E, Oliviero M, Naldini L, Gaudino G, Tamagnone 1, Coffer A, Comoglio PM. Hepatocyte growth factor is a potent angiogenic factor which stimulates endothelial cell motility and growth. J Cell Biol 1992; 119:629-41

4. Comoglio PM, Boccaccio C. Scatter factors and invasive growth. Semin Cencer Biol (2001) 11:153-65

5. Dluzniewska J, Zolich D, Polanski J, Zajac L, Sitkiewicz D, Lukomska B. Hepatocyte growth factor levels in liver and blood, and post-operative liver cell proliferation in patients with benign and malignant liver tumors after partial hepatectomy. Med Sci Monit (2002) 8:CR690-6

6. Jakob C, Fleissner C, Zavrski I, Heider U, Possinger K, Sezer O. Circulating hepatocyte growth factor levels in multiple myeloma. Br J Haematol (2003) 121 :532;; de Jong KP, et al., Serum response of hepatocyte growth factor, insulin-like growth factor-I, interleukin-6, and acute phase proteins in patients with colorectal liver metastases treated with partial hepatectomy or cryosurgery. J Hepatol (2001) 34:422-

7;)

7. Jiang WG, Hiscox S, Matsumoto K, Nakamura T. Hepatocyte growth factor/scatter factor, its molecular, cellular and clinical implications in cancer. Crit Rev Oncol Hematol (1999) 29: 209-48

8. Kondo H, Tani T, Kodama M. Effects of deletion-type human hepatocyte growth factor on murine septic model. J Surg Res, 1999; 85:88-95

9. Matsumoto K, Nakamura T. Hepatocyte growth factor: molecular structure and implications for a central role in liver regeneration. J Gastroenterol Hepatol, 1991a; 6: 509-19

10. Matsumoto K, Nakamura T. Hepatocyte growth factor (HGF) as a tissue organizer for organogenesis and regeneration. Biochem Biophys Res Commun 1997; 239: 639-44. 11. Montesano R, Matsumoto K, Nakamura T, Orci L. Identification of a fibroblast- derived epithelial morphogen as hepatocyte growth factor. Cell, 1991; 67:901-8

12. Nayeri F, Nilsson I, Brudin L, Fryden A, Sδderstrδm C. High hepatocyte growth factor levels in the acute stage of community-acquired infectious diseases. Scand J

Infect Dis, 2002;34: 127-30

13. Nusrat A, Parkos CA, Bacarra AE, Godowski PJ, Delp-Archer C, Rosen EM, Madara JL. Hepatocyte growth factor effects on epithelia. Regulation of intracellular junctions in transformed and nontransformed cell lines, basolateral polarization of c-met receptor in transformed and natural intestinal epithelia, and induction of rapid wound repair in a transformed model epithelium. J Clin Invest, 1994; 93:2056-65

14. Yanagita K, Matsumoto K, Sekiguchi K, Ishibashi H, Nihol Y, Nakamura T. Hepatocyte growth factor may act as a pulmotrophic factor on lung regeneration after acute ling injury. J Biol Chem, 1993;268:21212-7

Claims

1. Use of HGF (Hepatocyte growth factor) for the manufacture of a pharmaceutical composition for the treatment or prevention of cancer, which composition comprises HGF in a dose sufficient to cause an active immunisation in a mammal.

2. Use according to claim 1, wherein the mammal suffering from cancer exhibits high levels of serum HGF.

3. Use according to claim 1 or 2, wherein the mammal suffering from cancer exhibits low serum levels of anti-HGF antibodies.

4. Use according to any of the preceding claims, wherein the cancer is invasive prostate cancer and metastases.

5. Use according to any of the preceding claims, wherein is the composition is adapted to be administered by subcutaneous injection.

6. Use according to any of the preceding claims, wherein the composition is adapted to deliver a dose of HGF of about 1-10 ng/day, preferably 2-4 ng/day.

7. Use according to any of the preceding claims, wherein the mammal is a human.

8. Use according to any of the preceding claims, wherein the mammal is a domestic animal such as a horse, a cow or a sheep.

9. Use according to any of the preceding claims, wherein the mammal is a pet such as a dog or a cat.

10. Method for prevention of cancer in high risk mammalian subjects, comprising injection of a dose of HGF (Hepatocyte growth factor) in a mammal, the dose of HGF being sufficiently high to cause a immunisation in said mammal.

11. Method for treatment of cancer, comprising injection of a dose of HGF (Hepatocyte growth factor) in a mammal, the dose of HGF being sufficiently high to cause an immunisation in said mammal.

12. Method according to claim 10 or 11, wherein the mammal suffering from cancer exhibits high levels of serum HGF.

13. Method according to any of claims 10-12, wherein the mammal suffering from cancer exhibits low serum levels of anti-HGF.

14. Method according to any of the claims claim 10-13, wherein the cancer is invasive prostate cancer and metastases.

15. Method according to any of the claims 10-14, wherein HGF is administered be subcutaneous injection.

16. Method according to any of the claims 10-15, wherein the dose of HGF is about 1-10 ng/day, preferably 2-4 ng/day.

17. Method according to any of the claims 10-16, wherein the mammal is a human.

18. Method according to any of the claims 10-16, wherein the mammal is a domestic animal such as a horse, a cow or a sheep.

19. Method according to any of the claims 10-16, wherein the mammal is a pet such as a dog or a cat.

20. A pharmaceutical composition comprising a dose of HGF (Hepatocyte growth factor) for a mammal suffering from cancer, wherein the dose of HGF is sufficiently high to cause an immunisation in said mammal.

21. Pharmaceutical composition according to claim 20, wherein the mammal suffering from cancer exhibits high levels of serum HGF.

22. Pharmaceutical composition according to claim 20 or 21, wherein the mammal suffering from cancer exhibits low serum levels of anti-HGF.

23. Pharmaceutical composition according to any of the claims 20-22, wherein the cancer is invasive prostate cancer and metastases.

24. Pharmaceutical composition according to any of the claims 20-23, wherein the composition is adapted to be administered by subcutaneous injection.

25. Pharmaceutical composition according to any of the claims 20-24, wherein the composition is adapted to deliver a dose of HGF of about 1-10 ng/day, preferably 2-4 ng/day.

26. Pharmaceutical composition according to any of the claims 20-25, wherein the mammal is a human.

27. Pharmaceutical composition according to any of the claims 20-25, wherein the mammal is a domestic animal such as a horse, a cow or a sheep.

28. Pharmaceutical composition according to any of the claims 20-25, wherein the mammal is a pet such as a dog or a cat.