WO2017036925A1

WO2017036925A1 - Mesenchymal stem cells for the treatment of metastatic liver disease

Info

Publication number: WO2017036925A1
Application number: PCT/EP2016/070112
Authority: WO
Inventors: Christine Günther; Sylvia Peter
Original assignee: Apceth Gmbh & Co. Kg
Priority date: 2015-08-28
Filing date: 2016-08-25
Publication date: 2017-03-09

Abstract

The invention relates to mesenchymal stem cells (MSC) and their use as a medicament in restoring liver function in subjects with metastatic cancer of the liver (metastatic liver disease).

Description

MESENCHYMAL STEM CELLS FOR THE TREATMENT OF METASTATIC LIVER DISEASE

DESCRIPTION

BACKGROUND OF THE INVENTION

Mesenchymal stem cells (MSCs) are cells of non-haematopoietic origin that reside in the bone marrow and other tissues. MSCs are commonly considered to be multipotent adult progenitor cells that have the ability to differentiate into a limited number of cell lineages, such as osteoblasts, chondrocytes, and adipocytes. Studies have been conducted on the use of MSCs as a therapeutic entity based on this capacity to differentiate directly into these end-stage phenotypes, including the use of MSCs to promote or augment bone repair and for the repair of cartilage defects. The isolation and cultivation of MSCs for a number of therapeutic indications has been described and represents a promising approach towards treating inflammation- associated disorders (for example WO 2010/1 19039).

MSCs are known to exhibit immune evasive properties after administration to a patient. MSCs have been shown to exhibit a beneficial immune modulatory effect in cases of transplantation of allogeneic donor material, thereby reducing a potentially pathogenic alloreactivity and rejection. Furthermore, MSCs are known to exhibit anti-tumorigenic effects, for example against Kaposi's sarcoma. MSCs treatment can also play a therapeutic role in wound healing. The therapeutic delivery of MSCs can be performed via systemic injection, followed by MSC homing to and engraftment within sites of injury.

Although it is clear that MSCs have a potentially therapeutic effect in some injured or inflamed tissues, their medical use in other clinical settings has not yet been fully explored.

Secondary hepatic malignancies (liver metastases) are more common than primary tumors in liver and account for approximately 95% of all hepatic malignancies. After the lymph nodes, the liver is the most common site for metastases from cancers arising in other sites in the body. For cancers that occur in the gastrointestinal tract the frequency of metastatic disease in the liver is high, assumedly due to the dual blood supply of the liver from the portal, and systemic circulation increases the likelihood of metastatic deposits in the liver.

Approximately 50% of patients with metastatic colorectal cancer will be diagnosed with liver metastases and involvement of the liver is typically associated with poor survival. Surgical resection is considered the only possibility to obtain long-term survival. It is generally admitted that 10% to 15% of patients with synchronous colorectal liver metastasis will benefit from hepatic resection. One additional factor in treating liver metastases is the added complication of reduced liver function, either before or after liver resection, due to the secondary tumors, thereby further contributing to reduced survival.

Some efforts have been made in administering MSCs in the treatment of medical conditions of the liver. Abdel Aziz et al (J Exp & Clin Can Res 201 1 , 30:49) disclose administering MSCs in a chemically induced hepatocellular carcinoma rat model, whereby the MSCs show tumor suppressive effects. Lukashyk et al (J Clin & Trans Hepatology 2014, 2:217) describe the use of MSCs in treating patients with HCV-related liver cirrhosis, demonstrating some functional improvement in the liver. Although positive effects of MSC treatment have been observed in some medical conditions of the liver, to the best knowledge of the inventors the use of MSCs in restoring liver function in patients with metastatic liver disease has not been previously described or suggested.

Genetically modified MSCs transfected with the sodium iodide symporter (NIS) under control of the RANTES promoter have been administered in the treatment of liver metastases of colon cancer in animal models (Knoop et al, J Nuclear Med, vol 56, No 4, 2015). The MSCs described therein achieve an anti-tumor therapeutic effect with respect to reduction of tumor load due to the tumor-specific uptake of ¹³¹1 via NIS expression under the RANTES promoter. However, Knoop et al do not disclose the restoration of liver function or the administration of MSCs to achieve this effect.

WO 2010/1 19039 also discloses the use of genetically modified MSCs transfected with a cytotoxic transgene under control of the RANTES promoter in the treatment of tumors. WO

2010/1 19039 does not disclose the restoration of liver function or the administration of MSCs to achieve this effect.

Genetically modified MSCs that express exogenous IL-12 have been described for the treatment of murine metastatic hepatoma (Jeong et al, Int J Cancer, 137, 721-730, 2015). Hepatocellular carcinoma (HCC) cells (hepatoma) were used to produce HCC mouse models, which were subsequently treated with IL-12-expressing MSCs, leading to a reduction in pulmonary metastases when treated in combination with irradiation. Jeong et al do not disclose the treatment of metastases in the liver or a restoration of liver function.

In light of the difficulties present in the treatment of metastatic liver disease, a significant need exists in the medical community for therapeutic options for both treating liver metastases and for restoring liver function in patients with liver metastases.

SUMMARY OF THE INVENTION

In light of the prior art the technical problem underlying the present invention is to provide alternative and/or improved means for restoring liver function in patients with liver metastases. This problem is solved by the features of the independent claims. Preferred embodiments of the present invention are provided by the dependent claims.

The invention therefore relates to a mesenchymal stem cell (MSC) for use as a medicament in restoring liver function in subjects with metastatic cancer of the liver (metastatic liver disease). The present invention therefore relates to the medical use of MSCs in restoring liver function in subjects with metastatic cancer of the liver, or treating metastatic liver disease, and to methods of treatment comprising administering MSCs to restore liver functions in subjects with metastatic liver disease.

It was entirely surprising that liver function in human subjects could be improved in patients with liver metastases via the administration of MSCs. Liver function is typically severely compromised in patients with metastatic liver disease and at present no effective means exist for restoring liver function.

In some embodiments, the present invention enables the treatment of liver metastasis, by attacking or inhibiting growth or migration of the tumor material in the liver, and/or by restoring liver function despite the presence of secondary liver tumors.

In particular, the present invention is defined by a surprising and beneficial medical (technical) effect, namely the reduction of disease-related elevated values in liver function tests (LFT), thereby indicating restoration of liver function, independent of an anti-tumor effect of the MSCs on tumor mass or growth.

As described in more detail in the examples herein, MSCs were administered in a phase I clinical trial in the treatment of patients with advanced metastatic disease of the liver. Tumor progression, in the form of stable or progressive disease, was evident after the treatment, although even at the low doses employed in the study a beneficial effect on the restoration of liver function could be observed. The present invention is therefore based on the finding that a mechanism of liver restoration is induced by the MSC treatment that is independent of a direct anti-tumor effect (limiting tumor progression), or occurs prior to a significant inhibition of tumor progression. This novel technical effect is therefore not to be equated with a beneficial side effect of tumor reduction in the liver of patients with metastatic liver disease, but rather represents a distinct effect independent of and/or prior to limiting tumor progression.

Despite the surprising independence of the restorative function of the MSCs from an anti-tumor effect, the observance of improved liver values despite continued tumor presence in the liver also represents a surprising and beneficial finding of the present invention. The novel technical effect described herein leads to a novel clinical situation. The potential of restoring liver function in patients with liver metastases without necessarily having to counteract tumor progression represents a novel medical use of MSCs that has not been previously suggested. The MSCs may therefore be applied in alleviating the symptoms of patients with liver metastasis caused by reduced liver function, thereby improving the quality of life of patients undergoing cancer treatment. It was entirely unexpected that even in patients with persisting liver metastases the function of the liver could be restored via MSC administration.

In a preferred embodiment of the invention the mesenchymal stem cell (MSC) for use in restoring liver function as described herein is characterized in that the subject exhibits, prior to treatment with said MSC, increased values in liver function tests (LFT) compared to a control, such as healthy subjects. A preferred technical effect of the present invention is the reduction of LFT values due to MSC administration. The present invention is based on observations from clinical trials in human subjects that showed increased liver values prior to treatment, and a reproducible reduction in liver values that correlates with the administration of MSCs. Some subjects exhibited at the beginning of the treatment regime significantly increased liver values compared to healthy subjects, as were measured via analytical tests, in particular liver function tests. These elevated levels are a known marker for reduced liver function and are commonly observed in patients with liver metastasis.

Liver function tests (LFTs or LFs) are groups of tests, typically blood tests, which give information about the state of a patient's liver. These tests include prothrombin time (PT/INR), activated partial thromboplastin time (aPTT), albumin, bilirubin (direct and indirect), and others. Liver transaminases (such as gamma glutamyl transpeptidase (gammaGT or G-GT), aspartate transaminase (AST), serum glutamate-oxaloacetate transaminase (GOT), alanine transaminase (ALT) and/or serum glutamate-pyruvate transaminase (GPT)) are useful biomarkers of liver injury in a patient with some degree of intact liver function. The values obtained from LFTs are typically considered directly related to liver function.

In particular, the preferred tested values according to the present invention (and as demonstrated in the examples) are the transaminases gamma glutamyl transpeptidase (GGT), glutamate- pyruvate transaminase (GPT) and glutamate-oxaloacetate transaminase (GOT), and alkaline phosphatase and bilirubin. Increased levels of GGT and AP together with bilirubin typically represents a sign of cholestasis or closure of the bile duct (eg, inflammation, expansion, autoimmune processes). The transaminases are typically released upon cell damage and increase measurably in serum. Elevated transaminases can be found in all liver damage of toxic, infectious and/or inflammatory origin, but also in some patients with tumor infestation of the liver caused by increasing tissue destruction by metastatic tumors.

In one embodiment the mesenchymal stem cell (MSC) for use as a medicament as described herein, or the method of administering the MSC as described herein, is characterised in that the increased values in liver function tests (LFT) evident in a subject prior to treatment are selected from increased levels of gamma glutamyl transpeptidase (GGT), glutamate-pyruvate

transaminase (GPT) and/or glutamate-oxaloacetate transaminase (GOT). Increased levels refers preferably to an elevated level over a healthy person or population average.

In one embodiment the mesenchymal stem cell for use as a medicament as described herein, or the method of administering the MSCs as described herein, is characterised in that restoring liver function comprises reducing the values in liver function tests (LFT), wherein said LFT values that are reduced are gamma glutamyl transpeptidase (GGT), glutamate-pyruvate transaminase (GPT) and/or glutamate-oxaloacetate transaminase (GOT) levels.

In particular, a preferred effect of the invention is the reduction of GOT/GPT values due to the treatment of MSCs as described herein. Gamma GT provides useful information on liver function, but is typically more sensitive than GOT/GPT values, which are accordingly a more robust marker for liver function compared to gammaGT.

Typical values of gamma-GT in healthy individuals are below 50 U/L in females, and below 60 U/L in males. Typical values of GOT in healthy individuals are below 35 U/L in females, and below 50 U/L in males. Typical values of GPT in healthy individuals are below 35 U/L in females, and below 50 U/L in males. Any gamma-GT value above 50 U/L may in some embodiments be considered an elevated level of gamma-GT. Any GOT value above 35 U/L may in some embodiments be considered an elevated level of GOT. Any GPT value above 35 U/L may in some embodiments be considered an elevated level of GPT.

A reduction in the values of liver function tests as described herein may be determined by a skilled person without undue effort. For example, in one embodiment, the values for gamma-GT may be reduced during treatment by up to 200%, preferably 100%, more preferably 50%, of the initially elevated level in the diseased subject. In one embodiment this reduction may occur after 2 to 3 administrations of MSCs, preferably within a 1 to 4 week time window.

In some embodiments single MSCs administrations may lead to a reduction in gamma-GT levels between 0% and 40%, preferably between 10% and 30%, of the initially elevated level in the diseased subject. In some embodiments two or more MSCs administrations with about 1 -4 weeks between administrations may lead to a reduction in gamma-GT levels between 20% and 50%, preferably between 30% and 50%, of the initially elevated level in the diseased subject.

In some embodiments gamma-GT levels may be as high as 2000 U/L in diseased individuals, and reductions to about 1000 U/L may occur during treatment, for example after 2 to 3 administrations of MSCs, preferably within a 1 to 4 week time window. In other embodiments, gamma-GT levels may be increased to about 300 U/L in diseased individuals and reduced to about 150 U/L after treatment. In other embodiments, gamma-GT levels may be increased to about 400 U/L in diseased individuals and reduced to about 250 U/L after treatment. In other embodiments, gamma-GT levels may be increased to about 450 U/L in diseased individuals and reduced to about 200 U/L during treatment after treatment. In other embodiments gamma-GT levels may be reduced to or below typical values for healthy patients after treatment.

In one embodiment, the values for GOT and/or GPT may be reduced during treatment by up to 200%, preferably 100%, more preferably 50%, of initially elevated levels in the diseased subject. In one embodiment this reduction may occur after 2 to 3 administrations of MSCs, preferably within a 1 to 4 week time window. In some embodiments single MSCs administrations may lead to a reduction in GOT and/or GPT levels between 0% and 40%, preferably between 10% and 30%, of the initially elevated level in the diseased subject. In some embodiments two or more MSCs administrations with about 1 -4 weeks between administrations may lead to a reduction in gamma-GT levels between 20% and 60%, preferably between 30% and 50%, of the initially elevated level in the diseased subject.

In some embodiments GOT and/or GPT levels may be as high as 250 U/L in diseased individuals, and reductions to about 100 U/L may occur during treatment. Alternatively, in other embodiments, GOT and/or GPT levels may be increased to about 100 U/L in diseased individuals and reduced to about 50 U/L after treatment. In other embodiments, GOT and/or GPT levels may be increased to about 80 U/L in diseased individuals and reduced to about 20 U/L after treatment. In other embodiments, GOT and/or GPT levels may be increased to about 60 U/L in diseased individuals and reduced to about 30 U/L after treatment. In other embodiments GOT and/or GPT levels may be reduced to or below typical values for healthy patients. As used herein, the term restoring liver function may relate to improvement, stabilization or reducing deterioration of any one or more of the known functions of the liver. For example, the liver regulates the composition of blood, including the amounts of sugar (glucose), protein, and fat that enter the bloodstream, the liver removes bilirubin, ammonia, and other toxins from the blood, the liver processes nutrients absorbed by the intestines during digestion and converts those nutrients into forms that can be used by the body, and stores some nutrients, such as vitamin A, iron, and other minerals, the liver produces cholesterol and clotting factors, metabolizes alcohol and many drugs. Any one or more of these functions can be assayed by a skilled practitioner and may be used in assessing restoration of liver function. A preferred assessment of liver function relates to the use of LFTs, preferably those mentioned herein, in particular GOT/GPT.

It was entirely unexpected that subjects with liver malfunction due to metastatic cancer could be treated in a manner sufficient to show improved liver values over a short time frame. As is evidenced by the examples, the liver values showed clear improvement immediately after administration and continued improvement over two weeks of treatment comprising only three rounds of administration.

The effects observed indicate that either isolated or repeated administration may lead to beneficial effects, whereby prolonged or continued administration, for example a long term administration, is also preferred. An example of such long term application is that the MSCs are administrated to the patient in multiple events for at least two weeks, at least three weeks, at least four weeks, at least one month, at least two months, at least three months, at least four months, at least five months, at least six months, at least seven months, at least eight months, at least nine months, at least ten months, at least eleven months, at least one year, at least two years, or at least three years, or enduringly. Administration may, for example, be carried out as often as once or more per day, once per week, once every 7 to 14, or 7 to 21 days, or once per month, or once per two months, over a time period as mentioned above.

In a preferred embodiment of the invention the mesenchymal stem cell (MSC) for use in restoring liver function as described herein is characterized in that an inhibition in metastatic liver tumor growth and/or migration occurs due to treatment. Direct effects on the secondary tumor material with respect to inhibited or reduced growth, or inhibited cell migration and further metastasis, may be evident. An anti-tumor effect represents, without being limited by theory, one mode of action.

In a preferred embodiment of the invention the mesenchymal stem cell (MSC) for use in restoring liver function as described herein is characterized in that stimulation and/or support of liver regeneration is achieved by the treatment as described herein.

Liver regeneration occurs naturally in mammalian subjects, and can be enhanced in subjects suffering from metastatic liver disease where liver function and regeneration may be repressed, via the present invention. Liver regeneration may therefore be enhanced, or supported by the present invention, for example by stimulating hepatocytes to re-enter the cell cycle. Liver regeneration may be assessed via various techniques, such as those described in Assy et al (J Hepatol. 1997 Apr;26(4):945-52.).

In one embodiment of the invention the MSCs administered in restoration of liver function secrete Hepatocyte growth factor (HGF) and/or Keratinocyte growth factor (FGF-7). HGF is a paracrine cellular growth, motility and morphogenic factor, known to play a role in liver regeneration. FGF7 is a potent epithelial cell-specific growth factor, which is also known to play a role in liver regeneration. To the knowledge of the inventors, it was unknown at the time of the invention that the MSCs employed in the clinical study showed increased secretion of these growth factors and that these factors could play a therapeutic role in restoring liver function after systemic MSC administration. The expression and secretion of these factors, either alone or in combination, enables an improvement in liver function despite the persisting presence of metastatic tumors in the liver, thereby providing a surprising benefit to patients with metastatic liver disease.

Various cancers may lead to liver metastasis. As mentioned above, metastatic disease in the liver is frequent in cancer patients, assumedly due to the dual blood supply of the liver. The present invention therefore enables treatment of liver metastasis, in other words treatment of cancer of an origin outside the liver that has metastasized to the liver. The restoration of liver function, either dependency or independently of a direct anti-tumor effect, is enabled by the present invention for various cancer types.

In a preferred embodiment of the invention the mesenchymal stem cell (MSC) for use in restoring liver function as described herein is characterized in that the subject of treatment has metastatic gastrointestinal cancer. In a preferred embodiment the metastatic gastrointestinal cancer is colon, pancreatic or colorectal cancer, or Cholangiocarcinoma. It was entirely surprising that the use of MSCs in the context of treating subjects with gastrointestinal cancer, would not only target the primary gastrointestinal cancer, but also show a positive effect on liver function in cases where secondary liver cancer had developed. The present invention thereby provides means for potentially treating both primary cancers of various origins, and secondary cancers in the liver, via a single therapeutic agent.

In one embodiment of the invention, the MSCs described herein exhibit beneficial homing or migratory properties, thereby allowing the targeting of multiple tumor types by the MSCs

(administered preferably systemically, such as via intravenous administration) to any given site of the body. MSCs home, or migrate, preferably to areas of inflammation in the body of a subject, for example to tumors, and in some embodiments subsequently engraft into the tumor stroma, thereby providing a local and specific effect of the MSCs in the tumor environment. These beneficial properties lead in the present invention to synergistic beneficial effects, enabling the targeting of multiple tumors, or multiple sites of a metastasized tumor, in particular in the liver and the gastrointestinal tract.

In one embodiment of the invention the homing properties of the MSCs are not required for the beneficial therapeutic effect. MSCs administered systemically (such as via intravenous administration) circulate in the subject's blood stream and pass through the liver, where the MSCs pass through, remain and/or engraft into liver tissue. Local effects induced by the MSCs are then apparent.

For example, the MSCs may exhibit paracrine functions within the liver, for example after engraftment into liver tissue. The paracrine signaling represents a form of cell to cell

communication in which one cell, in this case the administered MSC, produces a signal to induce changes in nearby cells. The therapeutic MSCs described herein may provide a set of signals, such as in the form of cytokines or chemokines, to the physiological niche of the MSCs present in the liver, thereby providing signaling to nearby cells that lead to improved liver function and/or anti-tumor responses.

In a preferred embodiment of the invention the mesenchymal stem cell (MSC) for use in restoring liver function as described herein is characterized in that the subject of treatment has metastatic breast cancer. Breast cancer tends to metastasize primarily to the lungs, liver, brain, and regional lymph nodes, and the bone. According to some estimates, 20% to 30% of all women first diagnosed with localized breast cancer will usually develop it in other areas of the body.

Considering liver metastasis is a major product of metastatic breast cancer, novel means are for combatting this condition are required.

In a preferred embodiment of the invention the mesenchymal stem cell (MSC) for use in restoring liver function as described herein is characterized in that the subject of treatment has metastatic lung cancer. Lung cancer, for example small cell lung cancer, also shows a high rate of metastasis (in some studies up to 60% of subjects), whereby the liver is a common location for the existence of metastatic tumors.

In a preferred embodiment of the invention the mesenchymal stem cell (MSC) for use in restoring liver function as described herein is characterized in that the subject of treatment has metastatic pancreatic cancer. The liver is the most common location for pancreatic cancer to metastasize and represents a common cause of liver metastatic disease. The present invention surprisingly enables the treatment of liver metastatic cancer, and in particular restores liver function, independently of the original source of cancer.

The present invention relates to the use of MSCs as described herein, and comprises both the administration of MSCs without genetic modification and genetically modified MSCs, comprising modified MSCs either with or without a specific therapeutic transgene.

In a preferred embodiment of the invention the mesenchymal stem cell (MSC) for use in restoring liver function as described herein is characterized in that the MSC is not genetically modified.

In a further aspect of the invention the mesenchymal stem cell (MSC) for use in restoring liver function as described herein is characterized in that said MSCs comprise one or more exogenous nucleic acid molecule(s), wherein said exogenous nucleic acid molecule(s) comprise a region encoding a therapeutic transgene operably linked to a promoter or promoter/enhancer combination.

The therapeutic transgene may relate to any given gene that, when expressed, provides a protein or nucleic acid product capable of a positive therapeutic effect on patient in need thereof.

In one embodiment of the invention the therapeutic transgene is a cytotoxic transgene, preferably HSV-TK. In a further embodiment, or in combination with the HSV-TK-transgene, the promoter is induced in conditions of inflammation, and is preferably the RANTES promoter. In one embodiment of the invention the invention relates to the use of the cells described in WO

2010/1 19039. It was entirely surprising that the use of the HSV-TK- modified MSCs under control of the

RANTES promoter, in particular in the context of treating subjects with gastrointestinal cancer, would not only target the primary gastrointestinal cancer but also show a positive effect on liver function in cases where secondary liver cancer had developed. The present invention thereby provides means for potentially treating both primary cancers of various origins, and secondary cancers in the liver via a single therapeutic agent.

DETAILED DESCRIPTION OF THE INVENTION

The "mesenchymal stem cells" disclosed herein can give rise to connective tissue, bone, cartilage, and cells in the circulatory and lymphatic systems. Mesenchymal stem cells are found in the mesenchyme, the part of the embryonic mesoderm that consists of loosely packed, fusiform or stellate unspecialized cells. As used herein, mesenchymal stem cells include, without limitation, CD34-negative stem cells.

In one embodiment of the invention, the mesenchymal stem cells are plastic-adherent cells, also known as multipotent mesenchymal stromal cells, and include CD34-negative cells. For the avoidance of any doubt, the term mesenchymal stem cell includes a subpopulation of

mesenchymal cells, MSCs and their precursors, which subpopulation is made up of multipotent or pluripotent self-renewing cells capable of differentiation into multiple cell types in vivo.

As used herein, CD34-negative cell shall mean a cell lacking CD34, or expressing only negligible levels of CD34, on its surface. CD34-negative cells, and methods for isolating such cells, are described, for example, in Lange C. et al., "Accelerated and safe expansion of human

mesenchymal stromal cells in animal serum-free medium for transplantation and regenerative medicine". J. Cell Physiol. 2007, Apr. 25.

Mesenchymal stem cells can be distinguished from hematopoietic stem cells (HSCs) by a number of indicators. For example, HSCs are known to float in culture and to not adhere to plastic surfaces. In contrast, mesenchymal stem cells adhere to plastic surfaces.

In one embodiment the MSCs are genetically modified. MSC-based cellular therapy using genetically modified MSCs enables the delivery of therapeutic gene products to a specific region of interest in the body of a patient. For example, MSCs have been shown to migrate to areas of inflammation, such as tumors, and thereby locally exert therapeutic influence. MSCs typically have immune-modulatory effects that lead to immune suppression in the area of interest, thereby mediating or reducing inflammation to enhance recovery. The present invention makes use of MSCs, in particular for the restoration of liver function in subjects with metastatic liver disease.

The construction of genetically modified MSCs described herein may be carried out using techniques known to a person skilled in the art. Various methods are disclosed herein or in related applications, such as WO/2008/150368 and WO 2010/1 19039, which are incorporated in their entirety.

The MSCs of the present invention comprise in one embodiment one or more exogenous nucleic acid molecule(s), wherein said exogenous nucleic acid molecule(s) may comprise a region encoding a therapeutic transgene, in particular a cytotoxic protein, that is for example capable of exerting an anti-tumor effect. The therapeutic transgene is operably linked to a promoter or promoter/enhancer combination. Both inducible promoters and constitutive promoters are envisaged.

In one embodiment, the genetically modified mesenchymal stem cell as described herein is characterized in that the exogenous nucleic acid comprises viral vector sequences, for example in the form of a viral expression construct.

In one embodiment, the genetically modified mesenchymal stem cell as described herein is characterized in that the exogenous nucleic acid is a non-viral expression construct.

As used herein, "nucleic acid" shall mean any nucleic acid molecule, including, without limitation, DNA, RNA and hybrids or modified variants thereof. An "exogenous nucleic acid" or "exogenous genetic element" relates to any nucleic acid introduced into the cell, which is not a component of the cells "original" or "natural" genome. Exogenous nucleic acids may be integrated or non- integrated in the genetic material of the target mesenchymal stem cell, or relate to stably transduced nucleic acids.

Any given gene delivery method is encompassed by the invention and preferably relates to viral or non-viral vectors, as well as biological or chemical methods of transfection, or combinations thereof. The methods can yield either stable or transient gene expression in the system used.

Genetically modified viruses have been widely applied for the delivery of genes into stem cells. Preferred viral vectors for genetic modification of the MSCs described herein relate to retroviral vectors, in particular to gamma retroviral vectors. The gamma retrovirus (sometimes referred to as mammalian type C retroviruses) is a sister genus to the lentivirus clade, and is a member of the Orthoretrovirinae subfamily of the retrovirus family. The Murine leukemia virus (MLV or MuLV), the Feline leukemia virus (FeLV), the Xenotropic murine leukemia virus-related virus (XMRV) and the Gibbon ape leukemia virus (GALV) are members of the gamma retrovirus genus. A skilled person is aware of the techniques required for utilization of gamma retroviruses in genetic modification of MSCs. For example, the vectors described Maetzig et al (Gammaretroviral vectors: biology, technology and application, 2001 , Viruses Jun;3(6):677-713) or similar vectors may be employed. For example, the Murine Leukemia Virus (MLV), a simple gammaretrovirus, can be converted into an efficient vehicle of genetic therapeutics in the context of creating gamma retrovirus-modified MSCs and expression of a therapeutic transgene from said MSCs after delivery to a subject.

Adenoviruses may be applied, or RNA viruses such as Lentiviruses, or other retroviruses.

Adenoviruses have been used to generate a series of vectors for gene transfer in the field of gene therapy and cellular engineering. The initial generation of adenovirus vectors were produced by deleting the El gene (required for viral replication) generating a vector with a 4kb cloning capacity. An additional deletion of E3 (responsible for host immune response) allowed an 8kb cloning capacity. Further generations have been produced encompassing E2 and/or E4 deletions. The use of any given adenovirus vector, for example those according to those described above, is encompassed by the present invention.

Lentiviruses are members of Retroviridae family of viruses (M. Scherr et al., Gene transfer into hematopoietic stem cells using lentiviral vectors. Curr Gene Ther. 2002 Feb; 2(1 ):45-55). Lentivirus vectors are generated by deletion of the entire viral sequence with the exception of the LTRs and cis acting packaging signals. The resultant vectors have a cloning capacity of about 8 kb. One distinguishing feature of these vectors from retroviral vectors is their ability to transduce dividing and non-dividing cells as well as terminally differentiated cells.

Non-viral methods may also be employed, such as alternative strategies that include conventional plasmid transfer and the application of targeted gene integration through the use of nuclease- based gene editing, integrase or transposase technologies. These represent approaches for vector transformation that have the advantage of being both efficient, and often site-specific in their integration. Physical methods to introduce vectors into cells are known to a skilled person. One example relates to electroporation, which relies on the use of brief, high voltage electric pulses which create transient pores in the membrane by overcoming its capacitance. One advantage of this method is that it can be utilized for both stable and transient gene expression in most cell types. Alternative methods relate to the use of liposomes or protein transduction domains. Appropriate methods are known to a skilled person and are not intended as limiting embodiments of the present invention.

The invention encompasses the use of more than one virus, or a virus and other gene editing event or genetic modification, including the use of mRNA, siRNA, miRNA, or other genetic modification in order to manipulate gene expression of any given relevant factor.

In one embodiment the genetically modified mesenchymal stem cell as described herein is characterized in that the promoter or promoter/enhancer combination yields constitutive expression of the exogenous nucleic acid.

In one embodiment the genetically modified mesenchymal stem cell as described herein is characterized in that the promoter is an EF1 alpha promoter, for example the EFI alphaS promoter, PGK promoter, or CMV or SV40 viral promoters.

In one embodiment, the genetically modified mesenchymal stem cell as described herein is characterized in that the transgene is expressed when the genetically modified mesenchymal stem cell comes into proximity with tumor tissue or tumor stromal tissue.

Given that mesenchymal stem cells can show a selective migration to different tissue

microenvironments in normal as well as diseased settings, the use of tissue-specific promoters, or other promoters linked to a particular disease microenvironment, or promoters induced by a differentiation pathway initiated in the recruited stem cell, is encompassed in the present invention and can be used to drive the selective expression of therapeutic genes specifically within a defined biological context.

In a preferred embodiment, the genetically modified mesenchymal stem cell as described herein is characterized in that the promoter or promoter/enhancer combination is induced upon differentiation of said cell post-administration. One example of differentiation post-administration is endothelial differentiation, wherein the MSC can engraft and subsequently differentiate into an endothelial or endothelial-like cell in or in proximity to the tumor tissue, thereby enabling expression of the therapeutic transgene in a local manner. In this embodiment, stem cells that are recruited to other tissue niches, but do not experience the disease region (the tumor environment), should not express the therapeutic gene. This approach allows a significant degree of potential control for the selective expression of the therapeutic gene within a defined microenvironment. Potential approaches to such gene modifications are disclosed in WO 2008/150368 and WO 2010/1 19039, which are hereby incorporated in their entirety.

In one embodiment, the genetically modified mesenchymal stem cell as described herein is characterized in that the promoter is the Tie2 promoter.

Promoters can be introduced that are selectively regulated in the context of inflammation or neovascularization. In this regard the Tie2-promoter, Flkl promoter and intronic enhancer, endothelin-1 promoter and the pre-proendothelin-1 promoter have been studied for endothelial specific expression (Huss, R, von Luttichau, I, Lechner, S, Notohamiprodjo, M, Seliger, C, Nelson, P (2004) [Chemokine directed homing of transplanted adult stem cells in wound healing and tissue regeneration]. Verh Dtsch Ges Pathol 88: 170-173).

Another embodiment of the invention provides mesenchymal stem cells that comprise a promoter or promoter/enhancer combination, which is inducible by inflammatory mediators and which controls the transcription of the therapeutic transgene. These inflammatory mediators can be released by the tumor's stromal tissue so that the expression of the cytotoxic protein in the mesenchymal stem cells is induced when the stem cells come into proximity with the tumor's stromal tissue. The inflammatory mediators can for example be cytokines, such as TNF alpha or I FN gamma. In particular the promoter can be the RANTES promoter, which can inter alia be induced by TNF alpha or IFN gamma (Nelson PJ, Kim HT, Manning WC, et al. Genomic organization and transcriptional regulation of the RANTES chemokine gene. J Immunol 1993; 151 (5) : 2601 -12; von Luettichau I, Nelson PJ, Pattison JM, et al. RANTES chemokine expression in diseased and normal human tissues. Cytokine 1996; 8(l):89-98; Nelson PJ,

Pattison JM, Krensky AM. Gene expression of RANTES. Methods Enzymol 1997; 287:148-62; Duell EJ, Casella DP, Burk RD, et al. Inflammation, genetic polymorphisms in proinflammatory genes TNF-A, RANTES, and CCR5, and risk of pancreatic adenocarcinoma. Cancer Epidemiol Biomarkers Prev 2006; 15 (4) :726-31 ; Marfaing-Koka, A., et al . , Regulation of the production of the RANTES chemokine by endothelial cells. Synergistic induction by IFN-gamma plus TNF- alpha and inhibition by IL-4 and IL-13. Journal of Immunology, 1995. 154(4): p. 1870-8).

In one embodiment, the genetically modified mesenchymal stem cell as described herein is characterized in that the promoter is the RANTES promoter. The "RANTES" promoter is also known in the art as the "CCL5" promoter. Further examples of promoters, which are inducible by pro-inflammation mediators are the NF-kB-responsive element and in general promoters, which can be induced by TNF. Additionally, promoters activated by anti-inflammatory mediators (e.g. TGF-beta) can be used to achieve a targeted expression of the cytotoxic protein in the mesenchymal stem cells. Examples are promoters which contain Smad-binding elements. Using promoters, which are inducible by inflammation mediators, enables a selective treatment of tumors, which have not yet undergone angiogenesis. Additionally, promoters activated in cancerous tissue, or activated by signals released by cancerous cells, can be used in the present invention to achieve selective expression of the encoded therapeutic transgene in the relevant location within the patient in order to avoid unwanted systemic effects.

As used herein, "tumor" shall include, without limitation, a prostate tumor, a pancreatic tumor, a squamous cell carcinoma, a breast tumor, a melanoma, a basal cell carcinoma, a hepatocellular carcinoma, a cholangiocellular carcinoma, testicular cancer, a neuroblastoma, a glioma or a malignant astrocytic tumor such as glioblastoma multiforme, a colorectal tumor, an endometrial carcinoma, a lung carcinoma, an ovarian tumor, a cervical tumor, an osteosarcoma, a

rhabdo/leiomyosarcoma, a synovial sarcoma, an angiosarcoma, an Ewing sarcoma/PNET and a malignant lymphoma. These include primary tumors as well as metastatic tumors (both vascularized and non-vascularized).

In the present invention tumors originating from esophageal cancer, gastric cancer, pancreatic cancer, intrahepatic cholangiocellular carcinoma (CCC) and colorectal cancer (plus rectal cancer), which have spread to the liver as metastases, are preferred cancer types to be treated according to the present invention.

The incidence of pancreatic cancer is 10 cases per 100.000 in Europe and 12 cases per 100.000 in the US (48.960 estimated cases in the US in 2015; 40.560 estimated deaths). This rate ends up in 3 % of all cancer cases. In 95% the exocrine pancreas is affected of whom 90 % are classified as adenocarcinomas. Pancreatic cancer is equally distributed with respect to gender. It metastasizes rather early and is growing invasively in the surrounding tissue. Usually, it is detected at a later stage because its growth does not cause any symptoms for a long time. A chance for a curative surgery (only 10-25 %) exists only in the UICC stages I and II (T1 -3 NO M0) and, possibly, in stage III, if the tumor responds to preoperative therapy. This clearly indicates the strong medical need for new efficacious treatments. Current "standard" chemotherapy consists of Gemcitabine, either alone, or in combination with Capecitabine or Erlotinib, a reversible tyrosine kinase inhibitor. Furthermore, Folfirinox and Folfox4 or Folfox6-regimen (contains platinum compounds) is applied. Nevertheless, the prognosis is infaust: Mean survival at stage T1 -3 Nx M0 is 4-6 months and with metastases it is below 3 months. It indicates why in this patient group "stable disease" (according to RECIST) can even be regarded as a therapeutic success.

The incidence of cholangiocellular carcinoma (CCC) is 1 -3 cases /100.000 in Europe and 1-2 cases/100.000 in the US. Women are affected three times more frequently than men. 90 % of all CCC are adenocarcinomas. 20-25% are located intrahepatically. The growth pattern is locally invasive. Primarily, CCC is being resected which is feasible in 30-40 % of all cases. Radiotherapy may be applied only in combination with chemotherapy. The latter consists of Gemcitabine alone, or in combination with cis-Platinum or Oxaliplatin. Chemotherapy can also be applied locally. The mean survival for unresectable tumors is in the range of 7-8 months. CCC is a tumor with a very high medical need for an improved treatment.

Colorectal cancer is the second most common cancer after lung cancer in men and ranks third in frequency in women in western countries (39610 estimated cases in the US in 2015; for estimated deaths no data available). Approximately 17% of patients have distant metastases at the time of presentation with the liver as the primary site for distant metastases. 90-95% are adenocarcinomas. 90 % of all CRC are resectable of which 50 % end up in a complete healing. A resection of metastases (liver or lung) is an option with a good survival rate as long as a R0 resection is possible and the remainder of the liver or lung remains sufficiently large. In this case the 5-year survival rate is 20-40 %. For this purpose a preoperative therapy may be useful. Another therapeutic option for liver metastases is the radiofrequency ablation.

For unresectable primary tumors or after surgery there are many therapeutic schemes on the basis of 5-FU, Oxaliplatin or Irinotecan. Over the last years targeted therapy was successfully introduced in the therapeutic armamentarium: the angiogenesis inhibitor bevacizumab (Avastin®) and the EGF-receptor inhibitors cetuximab (Erbitux®) and panitumumab (Vectibix®). Selective internal radiotherapy (SIRT) is another option for the treatment of liver metastases. Patients with advanced colorectal disease are usually present in good physical shape. In stage III the 5-years- survival rate is 10-30 % and in stage IV it is below 10 %. New treatment options are required.

The genetically modified cell(s) described herein may comprise different types of carriers depending on whether it is to be administered in solid, liquid or aerosol form, and whether it need to be sterile for such routes of administration as injection. The present invention can be administered intravenously, intradermal^, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intraprostaticaly, intrapleurally, intratracheal^, intranasally, intravitreally, intravaginally, intrarectally, topically, intratumorally, intramuscularly,

intraperitoneally, subcutaneously, subconjunctival, intravesicularlly, mucosally, intrapericardially, intraumbilically, intraocularally, orally, topically, locally, inhalation (e.g., aerosol inhalation), injection, infusion, continuous infusion, localized perfusion bathing target cells directly, via a catheter, via a lavage, in cremes, in lipid compositions (e.g., liposomes), or by other method or any combination of the forgoing as would be known to one of ordinary skill in the art (see, for example, Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, incorporated herein by reference). Local administration in the liver is contemplated.

The present invention encompasses treatment of a patient by introducing a therapeutically effective number of cells into a subject's bloodstream. As used herein, "introducing" cells "into the subject's bloodstream" shall include, without limitation, introducing such cells into one of the subject's veins or arteries via injection. Such administering can also be performed, for example, once, a plurality of times, and/or over one or more extended periods. A single injection is preferred, but repeated injections over time (e.g., quarterly, half-yearly or yearly) may be necessary in some instances. Such administering is also preferably performed using an admixture of CD34-negative cells and a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers are well known to those skilled in the art and include, but are not limited to, 0.01 -0.1 M and preferably 0.05 M phosphate buffer or 0.8% saline, as well as commonly used proprietary cryopreservation media.

Administration may also occur locally, for example by injection into an area of the subject's body in proximity to a tumor disease. MSCs have been shown to migrate towards cancerous tissue. Regardless, the local administration of the cells as described herein may lead to high levels of the cells at their site of action. Additionally, such pharmaceutically acceptable carriers can be aqueous or non-aqueous solutions, suspensions, and emulsions, most preferably aqueous solutions. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions and suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's and fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers such as Ringer's dextrose, those based on Ringer's dextrose, and the like. Fluids used commonly for i.v. administration are found, for example, in Remington: The Science and Practice of Pharmacy, 20th Ed., p. 808, Lippincott Williams S- Wilkins (2000). Preservatives and other additives may also be present, such as, for example, antimicrobials, antioxidants, chelating agents, inert gases, and the like.

As used herein, a "therapeutically effective number of cells" includes, without limitation, the following amounts and ranges of amounts: (i) from about 1 x 10² to about 1 x 10⁸ cells/kg body weight; (ii) from about 1 x 10³ to about 1 x 10⁷ cells/kg body weight; (iii) from about 1 x 10⁴ to about 1 x 10⁶ cells/kg body weight; (iv) from about 1 x 10⁴ to about 1 x 10⁵ cells/kg body weight; (v) from about 1 x 10⁵ to about 1 x 10⁶ cells/kg body weight; (vi) from about 5 x 10⁴ to about 0.5 x 10⁵ cells/kg body weight; (vii) about 1 x 10³ cells/kg body weight; (viii) about 1 x 10⁴ cells/kg body weight; (ix) about 5 x 10⁴ cells/kg body weight; (x) about 1 x 10⁵ cells/kg body weight; (xi) about 5 x 10⁵ cells/kg body weight; (xii) about 1 x 10⁶ cells/kg body weight; and (xiii) about 1 x 10⁷ cells/kg body weight. Human body weights envisioned include, without limitation, about 5 kg, 10 kg, 15 kg, 30 kg, 50 kg, about 60 kg; about 70 kg; about 80 kg, about 90 kg; about 100 kg, about 120 kg and about 150 kg. These numbers are based on pre-clinical animal experiments and human trials and standard protocols from the transplantation of CD34+ hematopoietic stem cells. Mononuclear cells (including CD34+ cells) usually contain between 1 :23000 to 1 :300000 CD34-negative cells.

Combined administration encompasses simultaneous treatment, co-treatment or joint treatment, and includes the administration of separate formulations of MSCs with anti-cancer therapies, whereby treatment may occur within minutes of each other, in the same hour, on the same day, in the same week or in the same month as one another. Sequential administration of any given combination of combined agents (for example MSCs and anti-tumor agents) is also encompassed by the term "combined administration". A combination medicament, comprising one or more of said MSCs with another medicament may also be used in order to co-administer the various components in a single administration or dosage.

As used herein, "treating" a subject afflicted with a disorder shall mean slowing, stopping or reversing the disorder's progression, or alleviating the symptoms of a disorder. A permanent removal or reduction of the disorder is not essential for a treatment. Treatment may therefore encompass a therapeutic effect for a limited period of time.

In the preferred embodiment, treating a subject afflicted with a disorder means reversing the disorder's progression, ideally to the point of eliminating the disorder itself. As used herein, ameliorating a disorder and treating a disorder are equivalent. The treatment of the present invention may also, or alternatively, relate to a prophylactic administration of said cells. Such a prophylactic administration may relate to the prevention of any given medical disorder, or the prevention of development of said disorder, whereby prevention or prophylaxis is not to be construed narrowly under all conditions as absolute prevention. Prevention or prophylaxis may also relate to a reduction of the risk of a subject developing any given medical condition, preferably in a subject at risk of said condition.

In this respect, the "restoration of liver function" relates preferably to an improvement, stabilization or reducing deterioration of any one or more of the known functions of the liver. For example, the liver regulates the composition of blood, including the amounts of sugar (glucose), protein, and fat that enter the bloodstream, the liver removes bilirubin, ammonia, and other toxins from the blood, the liver processes nutrients absorbed by the intestines during digestion and converts those nutrients into forms that can be used by the body, and stores some nutrients, such as vitamin A, iron, and other minerals, the liver produces cholesterol and clotting factors, metabolizes alcohol and many drugs. Any one or more of these functions can be assayed by a skilled practitioner and may be used in assessing restoration of liver function. Restoration of liver function can be determined by using liver function tests (LFTs).

Liver function tests (LFTs or LFs) are groups of molecular tests, such as blood tests, that give information about the state of a patient's liver. These tests include prothrombin (PT), aPTT, albumin, bilirubin and others. Liver transaminases (AST or GOT and ALT or GPT) are useful biomarkers of liver injury in a patient with some degree of intact liver function. This testing is preferably performed on a patient's blood sample. Some tests are associated with functionality (e.g., albumin), some with cellular integrity (e.g., transaminase), and some with conditions linked to the biliary tract (gamma-glutamyl transferase and alkaline phosphatase). Several biochemical tests are useful in the evaluation and management of patients with hepatic dysfunction. These tests can be used to detect the presence of liver disease, distinguish among different types of liver disorders, gauge the extent of known liver damage, and follow the response to treatment. A skilled person is aware of the necessary practical steps involved in conducting said liver tests, as is evident from Gowda et al (Pan Afr Med J. 2009; 3: 17) and Giannini et al (CMAJ. 2005 Feb 1 ; 172(3): 367-379). As available from Aigma Aldrich, kits may be obtained for assessing LFTs, for example MAK089 - γ-Glutamyltransferase (GGT) Activity Colorimetric Assay Kit, MAK055 - AST Activity Assay Kit, or MAK052 - ALT Activity Assay.

The term "stroma" as used herein refers to the supportive framework of a tissue or an organ (or gland, tissue or other structure), usually composed of extracellular matrix (ECM) and stromal cells. The stroma is distinct from the parenchyma, which consists of the key functional elements of that organ. Stromal cells (in the dermis layer) adjacent to the epidermis (the very top layer of the skin) release growth factors that promote cell division. Stroma is made up of the non- malignant host cells. Stroma provides an extracellular matrix on which tumors can grow or maintain existence or separate themselves from the immune environment.

As used herein, the term "tumor microenvironment" relates to the cellular environment in which any given tumor exists, including the tumor stroma, surrounding blood vessels, immune cells, fibroblasts, other cells, signalling molecules, and the ECM.

As used herein "cell migration" or "homing" is intended to mean movement of a cell towards a particular chemical or physical signal. Cells often migrate in response to specific external signals, including chemical signals and mechanical signals. The MSCs as described herein are capable of homing to tumor tissue or other inflammation signals. Chemotaxis is one example of cell migration regarding response to a chemical stimulus. In vitro chemotaxis assays such as Boyden chamber assays may be used to determine whether cell migration occurs in any given cell.

For example, the cells of interest may be purified and analysed. Chemotaxis assays (for example according to Falk et al., 1980 J. Immuno. Methods 33:239-247) can be performed using plates where a particular chemical signal is positioned with respect to the cells of interest and the transmigrated cells then collected and analysed. For example, Boyden chamber assays entail the use of chambers isolated by filters, used as tools for accurate determination of chemotactic behaviour. The pioneer type of these chambers was constructed by Boyden (Boyden (1962) "The chemotactic effect of mixtures of antibody and antigen on polymorphonuclear leucocytes". J Exp Med 1 15 (3): 453). The motile cells are placed into the upper chamber, while fluid containing the test substance is filled into the lower one. The size of the motile cells to be investigated determines the pore size of the filter; it is essential to choose a diameter which allows active transmigration. For modelling in vivo conditions, several protocols prefer coverage of filter with molecules of extracellular matrix (collagen, elastin etc.) Efficiency of the measurements can be increased by development of multiwell chambers (e.g. NeuroProbe), where 24, 96, 384 samples are evaluated in parallel. Advantage of this variant is that several parallels are assayed in identical conditions.

As used herein "engraftment" relates to the process of incorporation of grafted or transplanted tissue or cells into the body of the host. Engraftment may also relate to the integration of transplanted cells into host tissue and their survival and under some conditions differentiation into non-stem cell states.

Techniques for assessing engraftment, and thereby to some extent both migration and the bio- distribution of MSCs, can encompass either in vivo or ex vivo methods. Examples of in vivo methods include bioluminescence, whereby cells are transduced to express luciferase and can then be imaged through their metabolism of luciferin resulting in light emission; fluorescence, whereby cells are either loaded with a fluorescent dye or transduced to express a fluorescent reporter which can then be imaged; radionuclide labelling, where cells are loaded with radionuclides and localized with scintigraphy, positron emission tomography (PET) or single photon emission computed tomography (SPECT); and magnetic resonance imaging (MRI), wherein cells loaded with paramagnetic compounds (e.g., iron oxide nanoparticles) are traced with an MRI scanner. Ex vivo methods to assess biodistribution include quantitative PCR, flow cytometry, and histological methods. Histological methods include tracking fluorescently labelled cells; in situ hybridization, for example, for Y-chromosomes and for human-specific ALU sequences; and histochemical staining for species-specific or genetically introduced proteins such as bacterial β-galactosidase. These immunohistochemical methods are useful for discerning engraftment location but necessitate the excision of tissue. For further review of these methods and their application see Kean et al., MSCs: Delivery Routes and Engraftment, Cell-Targeting Strategies, and Immune Modulation, Stem Cells International, Volume 2013 (2013).

Progenitor or multipotent cells, such as the mesenchymal stem cells of the present invention, may be described as gene delivery vehicles, essentially enabling the localization and expression of therapeutic gene products in particular tissues or regions of the subject's body. Such therapeutic cells offer the potential to provide cellular therapies for diseases that are refractory to other treatments. For each type of therapeutic cell the ultimate goal is the same: the cell should express a specific repertoire of genes, preferably exogenous nucleic acids that code for therapeutic gene products, thereby modifying cell identity to express said gene product and provide a therapeutic effect, such as an immune stimulatory effect. The cells of the invention, when expanded in vitro, represent heterogeneous populations that include multiple generations of mesenchymal (stromal) cell progeny, which lack the expression of most differentiation markers like CD34. These populations may have retained a limited proliferation potential and

responsiveness for terminal differentiation and maturation along mesenchymal and non- mesenchymal lineages.

As used herein "inducible expression" or "conditional expression" relates to a state, multiple states or system of gene expression, wherein the gene of interest, such as the therapeutic transgene, is preferably not expressed, or in some embodiments expressed at negligible or relatively low levels, unless there is the presence of one or more molecules (an inducer) or other set of conditions in the cell that allows for gene expression. Inducible promoters may relate to either naturally occurring promoters that are expressed at a relatively higher level under particular biological conditions, or to other synthetic promoters comprising any given inducible element. Inducible promoters may refer to those induced by particular tissue- or micro-environments or combinations of biological signals present in particular tissue- or micro-environments, or to promoters induced by external factors, for example by administration of a small drug molecule or other externally applied signal.

As used herein, in "proximity with" a tissue includes, for example, within 50 mm, 10 mm, 5 mm, within 1 mm of the tissue, within 0.5 mm of the tissue and within 0.25 mm of the tissue.

As used herein, "cytotoxic protein" shall mean a protein that, when present in, on and/or in proximity with a cell, causes that cell's death directly and/or indirectly. Cytotoxic proteins include, for example, suicide proteins (e.g. HSV-tk) and apoptosis inducers. Cytotoxic genes include null genes, siRNA or miRNA for gene knockdown (e.g. CCR5-/-). A number of suicide gene systems have been identified, including the herpes simplex virus thymidine kinase gene, the cytosine deaminase gene, the varicella-zoster virus thymidine kinase gene, the nitroreductase gene, the Escherichia coli gpt gene, and the E. coli Deo gene. Cytosine deaminase; Cytochrome P450;

Purine nucleoside phosphorylase; Carboxypeptidase G2; Nitroreductase. As detailed in: Yazawa K, Fisher WE, Brunicardi FC: Current progress in suicide gene therapy for cancer. World J Surg. 2002 Jul; 26(7):783-9. Cytotoxic factors include the following: (i) homing factors such as chemokines and mucin chemokine GPI fusions (chemokine derived agents can be used to facilitate the directed recruitment of engineered stem cells, see, e.g., PCT International

Application No. PCT/EP2006/01 1508, regarding mucin fusions anchored with GPI); (ii) viral antigens (measles, chicken pox) as cytotoxic proteins; and (iii) Her2/neu antigens which can be presented on the surfaces of engineered stem cells, followed by administration of her-2/neu antibody, and CamPath® (Alemtuzumab) directed against a CD52 epitope.

The invention therefore relates to the use of MSCs as a medicament for the treatment of diseases of the liver. Potential applications relate to the following conditions, without limitation, including treatment of liver tumors (primary tumors or metastases) with abnormal liver function tests and / or cholestasis, tumors of the bile duct or tract, such as Cholangiocellular carcinoma, Klatskin tumor or involvement in other tumors, inflammatory diseases of the liver in the context of liver inflammation or liver involvement in inflammatory diseases, toxic liver injury of various origins (toxic or medication-induced), autoimmune diseases of the liver and biliary tract: primary sclerosing cholangitis (PBC), cholangiopathy of unknown origin, or for the improvement of liver regeneration after surgical interventions. In particular the present invention relates to the treatment of disease of the liver in patients suffering from a tumor disease.

FIGURES

The following figures are presented in order to describe practical and in some cases preferred embodiments of the invention, by demonstrating a practical implementation of the invention, without being limiting to the scope of the invention or the concepts described herein.

Figure 1 . %-change in relation to baseline (screening visit day -7) in three LFT values are plotted over a 3 week treatment as per the examples of the present invention in four patients exhibiting similar patterns in GOT/GPT/gammaGT. The top left panel demonstrates gamma-GT levels in each of the 4 subjects, whereas the top right panel presents average levels of the four subjects for each of GOT/GPT/gammaGT.

Figure 2. Measurements of GOT/GPT and gammaGT in subject 101 -01 over a 56 day time window. Treatment with MSCs was conducted at the time points indicated by the arrows marked with a star. Treatment with a GCV infusion was conducted at the time points indicated by the remaining arrows. A reduction in both GOT/GPT and gammaGT is evident over the course of treatment.

Figure 3. Measurements of GOT/GPT and gammaGT in subject 101 -1 1 over a 56 day time window. Treatment with MSCs was conducted at the time points indicated by the arrows marked with a star. Treatment with a GCV infusion was conducted at the time points indicated by the remaining arrows. A reduction in both GOT/GPT and gammaGT is evident over the course of treatment.

Figure 4. Measurements of GOT/GPT and gammaGT in subject 101 -16 over a 56 day time window. Treatment with MSCs was conducted at the time points indicated by the arrows marked with a star. Treatment with a GCV infusion was conducted at the time points indicated by the remaining arrows. A reduction in both GOT/GPT and gammaGT is evident over the course of treatment.

Figure 5. Measurements of GOT/GPT and gammaGT in subject 101 -99 over a 56 day time window. Treatment with MSCs was conducted at the time points indicated by the arrows marked with a star. Treatment with a GCV infusion was conducted at the time points indicated by the remaining arrows. A reduction in both GOT/GPT and gammaGT is evident over the course of treatment.

Figure 6. Measurements of GOT/GPT and gammaGT in subject 101 -13 over a 56 day time window. Treatment with MSCs was conducted at the time points indicated by the arrows marked with a star. Treatment with a GCV infusion was conducted at the time points indicated by the remaining arrows. Subject 101-13 is provided as a control without liver metastases, in which GOT/GPT values are not elevated prior to treatment. No reduction in GOT/GPT is evident over the course of treatment.

Measurements of GOT/GPT and gammaGT in subject 101 -02 over a 56 day time window. Treatment with MSCs was conducted at the time points indicated by the arrows marked with a star. Treatment with a GCV infusion was conducted at the time points indicated by the remaining arrows. Subject 101-02 is provided as a control with liver metastases, in which GOT/GPT values are not elevated prior to treatment. No reduction in GOT/GPT is evident over the course of treatment.

Mean GOT measurements in four patients exhibiting reduced values over the course of treatment. Treatment with MSCs was conducted at the time points indicated by the arrows marked with a star. Treatment with a GCV infusion was conducted at the time points indicated by the remaining arrows. Significant changes are evident between GOT measurements conducted at early time points compared to GOT measured after the second MSC administration.

Mean GPT measurements in four patients exhibiting reduced values over the course of treatment. Treatment with MSCs was conducted at the time points indicated by the arrows marked with a star. Treatment with a GCV infusion was conducted at the time points indicated by the remaining arrows. Significant changes are evident between GPT measurements conducted at early time points compared to GPT measured after the second MSC administration.

Mean gammaGT measurements in four patients exhibiting reduced values over the course of treatment. Treatment with MSCs was conducted at the time points indicated by the arrows marked with a star. Treatment with a GCV infusion was conducted at the time points indicated by the remaining arrows. Significant changes are evident between gammaGT measurements conducted at early time points compared to gammaGT measured after the second MSC administration.

Overlay of GOT, gammaGT, GPT and lactate dehydrogenase (LDH) levels in subject 101-01 over the course of treatment. Treatment with MSCs was conducted at the time points indicated by the arrows marked with a star.

Treatment with a GCV infusion was conducted at the time points indicated by the remaining arrows. An increase in LDH is observed over the course of the treatment.

Overlay of measured values of leucocytes, erythrocytes, haemoglobin and thrombocytes in subject 101-01. Treatment with MSCs was conducted at the time points indicated by the arrows. An increase in leucocytes and thrombocytes is observed to correlate with MSC administration. EXAMPLES

The following examples are presented in order to describe potentially preferred embodiments of the invention, by demonstrating a practical implementation of the invention, without being limiting to the scope of the invention or the concepts described herein.

The present invention is exemplified by the following observations of clinical liver values obtained from a study involving treatment of patients with gastrointestinal cancer using genetically modified MSCs encoding HSV-TK under the RANTES promoter in combined administration with

Ganciclovir. Patients involved in these studies were undergoing an approved trial protocol for the treatment of advanced tumors of the gastrointestinal tract. Patients with adenocarcinomas, colorectal tumors, pancreatic tumors and tumor of the bile duct system were treated.

Some subjects exhibited at the beginning of the treatment regime significantly increased liver values compared to healthy subjects, as were measured via analytical liver function tests. In particular, the tested values included the transaminases G-GT, GPT and GOT, and alkaline phosphatase and bilirubin. LDH was also tested. GGT, AP and bilirubin are markers for cholestasis or closure of the bile duct (eg, inflammation, expansion, autoimmune processes). The transaminases are released upon cell damage and increase measurably in serum. Elevated transaminases can be found in all liver damage of toxic, infectious and/or inflammatory origin, but also in tumor infestation of the liver caused by increasing destruction of liver tissue due to tumor growth.

As described in more detail in the data provided herein, a therapeutic effect is observed on the measured LFT values. After combined i.v. administration of MSC on day 1 , 8, 15, followed by Ganciclovir on days 3-5, 10-12 and 17-19, the previously elevated liver values drop significantly in a continuous and reproducible manner. The reduction in these values directly correlates with the temporal administration of the MSC, and in some cases in combination with GCV. After completion of the therapy, the liver values increased again, indicating that MSC administration is preferably conducted in greater numbers of cells, for greater numbers of repetition, or for longer periods of time, in order to maintain reduced liver values.

The effect of reducing disease-indicative liver values occurred in individuals with elevated liver values (Figures 2-5).

Subjects with normal liver values showed no significant changes (Figures 6-7).

The effect observed may be due to the cytoprotective effect of the MSCs, an immunomodulatory effect, liver regeneration and/or a cytotoxic effect, potentially in combination with Ganciclovir. The reduction in the liver values assessed reveals a restoration of liver function in patients with liver metastatic disease.

Elevated LDH levels indicate increased cell death, potentially due to tumor destruction due to the MSCs and/or Ganciclovir treatment, and/or cell turnover, and may be indicative of a regenerative effect.

To the present date, according to the knowledge of the inventor, the effects described herein have not been previously described in either animal or human subjects. Example 1: Changes in liver values after treatment with MSC aoceth 101 in the Phasa I trial:

The study used as an example for the present invention involves administration of a suspension of autologous bone-marrow derived mesenchymal stromal/stem cells (MSC) which are expanded and modified in vitro by introducing a gene for the expression of HSV-Thymidine Kinase (HSV- TK). These MSCs (MSC_apceth_101 ) home to and/or accumulate in tumor tissue and/or in metastases after intravenous administration. Subsequently tlhe prodrug Ganciclovir (GCV) is given and is converted by HSV-TK into the active cytotoxic compound GCV -triphosphate, a process which is restricted to the tumor tissue due to the preferential migration or location of MSCs to and in injured and inflamed (tumor) tissue. The bone marrow aspiration is performed at least 10 weeks before the intended administration of the IMP under a separate approved protocol.

In the study, patients suffering from advanced, recurrent or metastatic gastrointestinal adenocarcinoma were enrolled. The study consists of a phase I and a phase II.

In phase I (= run-in phase) the safety of MSC_apceth_101 was investigated in 6 patients mainly with liver metastases due to colorectal carcinoma ("advanced patients" or patient group 1 ). The administration was shown to be safe. In phase II 16 patients with advanced gastrointestinal adenocarcinoma are enrolled.

The objective of the study was to demonstrate safety of the combination of genetically modified MSC and GCV. Furthermore, first hints on efficacy were to be shown keeping in imind :hat the number of patients in this first clinical study with MSC/GCV had to be small. Efficacy parameters were the RECIST (Response Evaluation Criteria In Solid Turmors) criteria and tumor markers (patient group 1 ). In addition, in resected tumor material from patients of group 2 MSCshould be detected for the proof of the assumed mode of action. Furthermore, various additional clinical parameters were assessed, including liver values capable of indicating liver function.

The initial 6 patients were treated with MSC_apceth_101 in a 3 weeks dose regimen. These patients received between 1.5 x10e6 to 3x10e6 cells/kg in total (range: 1 -2x10e6 cells/kg) divided in three doses (week 1 , 2, 3) intravenously according to the regular dose regimen for

MSC_apceth_101.

Table 1 : The regular treatment of patients was performed with MSC_apceth_101 (M) and Ganciclovir (G) as follows:

^*The MSC_apceth_101 infusions and the first Ganciclovir infusion of one respective treatment cycle is preferably at least 48 and not more than 72 hours apart from each other.

In all MSC_apceth_101 treated patients the Ganciclovir dose was kept unchanged at 2x5 mg/kg/day over 3 days (time interval: 12±3 hours) and given as a slow intravenous infusion. Surprisingly, the analysis of the study data revealed a measurable and reproduced reduction in liver values that correlates with MSC and GCV treatment (refer to figures 1 to 1 1 ), thereby indicating a restoration in liver function.

Figure 1 demonstrates an overlay of gamma-GT and GOT/GPT levels in four patients with elevated liver values at the beginning of the study. Significant reduction of the measured values of the gamma-GT and GOT/GPT levels is evident across the course of the study.

Figures 2-5 demonstrate raw data obtained from the four patients of interest (101-01 , 101 -1 1 , 101-16, 101-99). Figures 6-7 represent patients who did not exhibit increased liver values prior to MSC treatment initiation (101-13, 101-02).

Figures 8-10 show the relevance of the observed effects in the patients interrogated for each of the three LFT used.

Figures 1 1 and 12 indicate increase LDH levels, thereby indicating increased turnover in cells (unspecific cell death), in addition to increased leucocyte and thrombocyte levels and decrease in erythorycytes and hemoglobin (possible indicator for hemolysis and/or unspecific cell death in conjuction with raised LDH) in one patient that correlate with MSC administration.

Example 2: Evaluation of tumor progression after treatment with MSC apceth 101:

The observation of improved liver values in patients with initially elevated liver values represents a surprising and unexpected technical effect. Of note is the additional observation that tumor staging according to the RECIST criteria and measurement of the change in target lesions showed no significant effect of the MSCs administered at low doses and in advanced patients on tumor progression. Patients of both groups (four patients of interest with initially elevated liver values (101 -01 , 101-1 1 , 101 -16, 101-99) and patients who did not exhibit increased liver values prior to MSC treatment initiation (101-13, 101-02)) showed either stable disease or disease progression.

A significant anti-tumor effect was most likely not evident due to the low dose of MSCs employed in the present Phase I trial, which was designed primarily to assess the safety of the combination of genetically modified MSC and GCV. A further factor that may lead to the absence of a significant reduction in tumor size is likely the advanced stage of patients enrolled in the study. The clinical efficacy of the MSCs combined with GCV against tumors is therefore still predicted to occur, although in particular in patients treated with higher doses and with relatively less advanced cancer stage.

Importantly, the beneficial effect on the liver values and liver function of the patients with metastatic liver disease occurred independently of an anti-tumor effect. Tumor progression was evident after the treatment described above, although even at the low doses employed in this study a beneficial effect on the restoration of liver function could be observed. These data therefore indicate a mechanism of liver restoration that is independent of limiting tumor progression and/or occurs prior to a significant inhibition of tumor progression. This novel technical effect is therefore not to be equated to a beneficial side effect of tumor reduction in the liver of patients with metastatic liver disease, but rather to a beneficial effect that occurs via a mechanism distinct from limiting tumor progression. Despite this apparent independence of the restorative function of tie MSCs from an anti-tumor effect, the observance of improved liver values despite continued .umor presence in the liver also represents a surprising finding of the present invention.

Table 2: Data obtained for tumor staging accord ng to the RIECIST criteria in addition > the measurement in the change of target and non-target lesions. As can be observed in both patient groups, either tumor progression occurs or the disease is classified as stable (PD: progressive disease = 20% increase in the sum of the longest diameter of target lesions ; SD: stable disease = small changes that do not meet above criteria). Treatment of patients with MSC_apceth_101 in the first-in-human safety trial does not lead to tumor shrinkage at the administered dose.

Nevertheless beneficial effects on liver parameters are seen at these doses .

Example 3: Protein secretion profiling in MSC apceth 101:

The data presented above indicate beneficial properties of the MSCs independent of their anti - tumor effect. In order to further assess the properties of the IMSCs employed, protein secretion by the MSC_apceth_101 was assessed to determine if molecular factors could be identified that indicate an underlying basis for the ability of the VISCs to restore I ver function.

The average increase/decrease of various proteins secreted into the culture media by 3 different batches of MSC employed was compared to the protein levels determined in the culture media.

Protein secretion was assessed via a Human Angiogenesis Array Kit from R&D Systems (Cat.- No. ARY007). Pooled unconditioned and conditioned media (i.e. media after MSC were cultivated) were collected and assessed for the presence or absence of 55 different pro- and antiangiogenic factors. The differences between signal intensities from conditioned to unconditioned media were calculated after normalization of signals from the different microarray membranes.

Of the multiple proteins assessed, reproducibly increased levels of protein secretion of

Hepatocyte growth factor (HGF) and Keratinocyte growth factor (FGF-7) were detected in the culture medium in which the MSCs were cultivated in comparison to the culture media alone. HGF is a paracrine cellular growth, motility and morphogenic factcr, known to play a role in liver regeneration. FGF-7 is a potent epithelial cell-specific growth factor, which is also known to play a role in liver regeneration. To the knowledge of the inventors, it was unknown at the time the present data was obtained that the MSCs employed in the clinical study showed increased secretion of these growth factors and that these factors could play a therapeutic role in restoring liver function after systemic MSC administration.

Claims

Mesenchymal stem cell (MSC) for use as a medicament in restoring liver function in subjects with metastatic cancer of the liver (metastatic liver disease).

Mesenchymal stem cell (MSC) for the use according to claim 1 , wherein the subject exhibits, prior to treatment with said MSC, increased values in liver function tests (LFT) compared to a control, such as healthy subjects.

Mesenchymal stem cell (MSC) for use as a medicament according to the preceding claim, wherein the increased values in liver function tests (LFT) evident in a subject prior to treatment are selected from increased levels of gamma glutamyl transpeptidase (GGT), glutamate-pyruvate transaminase (GPT) and/or glutamate-oxaloacetate transaminase (GOT).

Mesenchymal stem cell for use as a medicament according to any one of the preceding claims, wherein restoring liver function comprises reducing the values in liver function tests (LFT) compared to elevated levels as in diseased subjects.

Mesenchymal stem cell (MSC) for use as a medicament according to the preceding claim, wherein said LFT values that are reduced are gamma glutamyl transpeptidase (GGT), glutamate-pyruvate transaminase (GPT) and/or glutamate-oxaloacetate transaminase (GOT) levels.

Mesenchymal stem cell for use as a medicament according to any one of the preceding claims comprising the stimulation and/or support of liver regeneration.

Mesenchymal stem cell for use as a medicament according to any one of the preceding claims comprising an inhibition in metastatic liver tumor growth and/or migration.

Mesenchymal stem cell for use as a medicament according to any one of the preceding claims, wherein said cells secrete Hepatocyte growth factor (HGF).

Mesenchymal stem cell for use as a medicament according to any one of the preceding claims, wherein said cells secrete Keratinocyte growth factor (FGF-7).

Mesenchymal stem cell for use as a medicament according to any one of the preceding claims, wherein the subject of treatment has metastatic gastrointestinal cancer.

Mesenchymal stem cell for use as a medicament according to the preceding claim, wherein the metastatic gastrointestinal cancer is colon, pancreatic or colorectal cancer, or cholangiocarcinoma.

Mesenchymal stem cell for use as a medicament according to any one of the preceding claims, wherein the subject of treatment has metastatic breast cancer.

Mesenchymal stem cell for use as a medicament according to any one of the preceding claims, wherein the subject of treatment has metastatic lung cancer.

14. Mesenchymal stem cell for use as a medicament according to any one of the preceding claims, wherein said MSCs comprise one or more exogenous nucleic acid molecule(s), wherein said exogenous nucleic acid molecule(s) comprise a region encoding a therapeutic transgene operably linked to a promoter or promoter/enhancer combination.

15. Mesenchymal stem cell for use as a medicament according to the preceding claim, wherein the therapeutic transgene is a cytotoxic transgene.

16. Mesenchymal stem cell for use as a medicament according to the preceding claim, wherein the therapeutic transgene is HSV-TK.

17. Mesenchymal stem cell for use as a medicament according to the preceding claim, wherein Ganciclovir or other guanosine nucleoside analogues (e.g. Valaciclovir) is administered in combination with the mesenchymal stem cell.

18. Mesenchymal stem cell for use as a medicament according to claim 13 to 16, wherein the promoter is induced under conditions of inflammation, such as in a tumor.

19. Mesenchymal stem cell for use as a medicament according to the preceding claim, wherein the promoter is the RANTES promoter.