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WO2017036925A1 - Cellules souches mésenchymateuses destinées au traitement d'une maladie du foie métastatique - Google Patents

Cellules souches mésenchymateuses destinées au traitement d'une maladie du foie métastatique Download PDF

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Publication number
WO2017036925A1
WO2017036925A1 PCT/EP2016/070112 EP2016070112W WO2017036925A1 WO 2017036925 A1 WO2017036925 A1 WO 2017036925A1 EP 2016070112 W EP2016070112 W EP 2016070112W WO 2017036925 A1 WO2017036925 A1 WO 2017036925A1
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liver
mesenchymal stem
stem cell
treatment
medicament according
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PCT/EP2016/070112
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English (en)
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Christine Günther
Sylvia Peter
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Apceth Gmbh & Co. Kg
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Publication of WO2017036925A1 publication Critical patent/WO2017036925A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0662—Stem cells
    • C12N5/0663—Bone marrow mesenchymal stem cells (BM-MSC)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2502/00—Coculture with; Conditioned medium produced by
    • C12N2502/99—Coculture with; Conditioned medium produced by genetically modified cells

Definitions

  • 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).
  • MSC mesenchymal stem cells
  • MSCs 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.
  • 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.
  • liver metastases 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.
  • liver metastases 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.
  • 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 131 1 via NIS expression under the RANTES promoter.
  • 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.
  • HCC Hepatocellular carcinoma
  • 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).
  • MSC mesenchymal stem cell
  • 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.
  • 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.
  • 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.
  • 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.
  • LFT liver function tests
  • 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.
  • 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.
  • 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.
  • LFT liver function tests
  • 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 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.
  • PT/INR prothrombin time
  • aPTT activated partial thromboplastin time
  • albumin albumin
  • bilirubin direct and indirect
  • 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)
  • gammaGT or G-GT aspartate transaminase
  • AST serum glutamate-oxaloacetate transaminase
  • ALT alanine transaminase
  • GTT serum glutamate-pyruvate transaminase
  • LFTs serum glutamate-pyruvate transaminase
  • the preferred tested values according to the present invention are the transaminases gamma glutamyl transpeptidase (GGT), glutamate- pyruvate transaminase (GPT) and glutamate-oxaloacetate transaminase (GOT), and alkaline phosphatase and bilirubin.
  • GGT glutamyl transpeptidase
  • GGT glutamate- pyruvate transaminase
  • GOT glutamate-oxaloacetate transaminase
  • alkaline phosphatase and bilirubin 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
  • 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
  • GPT glutamate-oxaloacetate transaminase
  • GOT glutamate-oxaloacetate transaminase
  • Increased levels refers preferably to an elevated level over a healthy person or population average.
  • 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.
  • LFT liver function tests
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • gamma-GT levels may be increased to about 300 U/L in diseased individuals and reduced to about 150 U/L after treatment.
  • gamma-GT levels may be increased to about 400 U/L in diseased individuals and reduced to about 250 U/L after treatment.
  • 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.
  • gamma-GT levels may be reduced to or below typical values for healthy patients after treatment.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • liver values showed clear improvement immediately after administration and continued improvement over two weeks of treatment comprising only three rounds of administration.
  • 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.
  • 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.
  • 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.).
  • 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.
  • liver metastasis Various cancers may lead to liver metastasis.
  • 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 is enabled by the present invention for various cancer types.
  • 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.
  • 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.
  • the MSCs described herein exhibit beneficial homing or migratory properties, thereby allowing the targeting of multiple tumor types by the MSCs
  • 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.
  • 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.
  • 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.
  • 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
  • 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.
  • 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.
  • liver metastasis is a major product of metastatic breast cancer, novel means are for combatting this condition are required.
  • 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.
  • 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.
  • the mesenchymal stem cell (MSC) for use in restoring liver function as described herein is characterized in that the MSC is not genetically modified.
  • 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.
  • the therapeutic transgene is a cytotoxic transgene, preferably HSV-TK.
  • the promoter is induced in conditions of inflammation, and is preferably the RANTES promoter.
  • the invention relates to the use of the cells described in WO
  • 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.
  • mesenchymal stem cells 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.
  • mesenchymal stem cells include, without limitation, CD34-negative stem cells.
  • the mesenchymal stem cells are plastic-adherent cells, also known as multipotent mesenchymal stromal cells, and include CD34-negative cells.
  • plastic-adherent cells also known as multipotent mesenchymal stromal cells
  • CD34-negative cells include CD34-negative cells.
  • 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.
  • 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 stem cells can be distinguished from hematopoietic stem cells (HSCs) by a number of indicators.
  • HSCs hematopoietic stem cells
  • mesenchymal stem cells adhere to plastic surfaces.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the genetically modified mesenchymal stem cell as described herein is characterized in that the exogenous nucleic acid is a non-viral expression construct.
  • 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.
  • the 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.
  • MMV Murine Leukemia Virus
  • 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.
  • 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.
  • 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.
  • the promoter is an EF1 alpha promoter, for example the EFI alphaS promoter, PGK promoter, or CMV or SV40 viral promoters.
  • 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.
  • mesenchymal stem cells can show a selective migration to different tissue
  • tissue-specific promoters 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • promoters activated by anti-inflammatory mediators e.g. TGF-beta
  • 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.
  • promoters which are inducible by inflammation mediators, enables a selective treatment of tumors, which have not yet undergone angiogenesis.
  • 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.
  • 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).
  • 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.
  • pancreatic cancer 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.
  • CCC cholangiocellular carcinoma
  • 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).
  • 90 % of all CRC are resectable of which 50 % end up in a complete healing.
  • a resection of metastases liver or lung
  • a preoperative therapy may be useful.
  • Another therapeutic option for liver metastases is the radiofrequency ablation.
  • 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.
  • 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.
  • 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.
  • a "therapeutically effective number of cells” includes, without limitation, the following amounts and ranges of amounts: (i) from about 1 x 10 2 to about 1 x 10 8 cells/kg body weight; (ii) from about 1 x 10 3 to about 1 x 10 7 cells/kg body weight; (iii) from about 1 x 10 4 to about 1 x 10 6 cells/kg body weight; (iv) from about 1 x 10 4 to about 1 x 10 5 cells/kg body weight; (v) from about 1 x 10 5 to about 1 x 10 6 cells/kg body weight; (vi) from about 5 x 10 4 to about 0.5 x 10 5 cells/kg body weight; (vii) about 1 x 10 3 cells/kg body weight; (viii) about 1 x 10 4 cells/kg body weight; (ix) about 5 x 10 4 cells/kg body weight; (x) about 1 x 10 5 cells/kg body weight; (xi) about 5 x 10 5 cells/kg body weight; (xii) about 5 x 10
  • 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 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.
  • 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.
  • treating a subject afflicted with a disorder means reversing the disorder's progression, ideally to the point of eliminating the disorder itself.
  • 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.
  • 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.
  • 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).
  • LFTs liver function tests
  • Liver function tests 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).
  • kits may be obtained for assessing LFTs, for example MAK089 - ⁇ -Glutamyltransferase (GGT) Activity Colorimetric Assay Kit, MAK055 - AST Activity Assay Kit, or MAK052 - ALT Activity Assay.
  • GTT ⁇ -Glutamyltransferase
  • stroma 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.
  • ECM extracellular matrix
  • stroma is distinct from the parenchyma, which consists of the key functional elements of that organ.
  • Stromal cells in the dermis layer
  • epidermis the very top layer of the skin
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • paramagnetic compounds e.g., iron oxide nanoparticles
  • 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 ⁇ -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.
  • 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
  • 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.
  • 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.
  • 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;
  • 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
  • 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.
  • PBC primary sclerosing cholangitis
  • cholangiopathy of unknown origin or for the improvement of liver regeneration after surgical interventions.
  • the present invention relates to the treatment of disease of the liver in patients suffering from a tumor disease.
  • FIG. 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.
  • FIG. 1 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.
  • FIG. 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.
  • FIG. 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.
  • FIG. 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.
  • FIG. 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • MSCs autologous bone-marrow derived mesenchymal stromal/stem cells
  • HSV-TK HSV-Thymidine Kinase
  • 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.
  • GCV tlhe prodrug Ganciclovir
  • the study consists of a phase I and a phase II.
  • phase I run-in phase
  • 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.
  • 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
  • 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.
  • 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:
  • liver values in patients with initially elevated liver values represents a surprising and unexpected technical effect.
  • 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.
  • 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.
  • the observance of improved liver values despite continued .umor presence in the liver also represents a surprising finding of the present invention.
  • 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.
  • Hepatocyte growth factor HGF
  • FGF-7 Keratinocyte growth factor

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Abstract

La présente invention concerne des cellules souches mésenchymateuses (MSC) et leur utilisation comme médicament dans la restauration de la fonction du foie chez des sujets atteints d'un cancer métastatique du foie (maladie du foie métastatique)
PCT/EP2016/070112 2015-08-28 2016-08-25 Cellules souches mésenchymateuses destinées au traitement d'une maladie du foie métastatique WO2017036925A1 (fr)

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Publication number Priority date Publication date Assignee Title
WO2010119039A1 (fr) * 2009-04-13 2010-10-21 Apceth Gmbh & Co. Kg Cellules souches mésenchymateuses obtenues par génie génétique, et procédé d'utilisation de celles-ci pour traiter des tumeurs

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010119039A1 (fr) * 2009-04-13 2010-10-21 Apceth Gmbh & Co. Kg Cellules souches mésenchymateuses obtenues par génie génétique, et procédé d'utilisation de celles-ci pour traiter des tumeurs

Non-Patent Citations (2)

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Title
K. KNOOP ET AL: "Mesenchymal Stem Cell-Mediated, Tumor Stroma-Targeted Radioiodine Therapy of Metastatic Colon Cancer Using the Sodium Iodide Symporter as Theranostic Gene", THE JOURNAL OF NUCLEAR MEDICINE, vol. 56, no. 4, 1 April 2015 (2015-04-01), US, pages 600 - 606, XP055266264, ISSN: 0161-5505, DOI: 10.2967/jnumed.114.146662 *
KEUN-YEONG JEONG ET AL: "Irradiation-induced localization of IL-12-expressing mesenchymal stem cells to enhance the curative effect in murine metastatic hepatoma", INTERNATIONAL JOURNAL OF CANCER, vol. 137, no. 3, 5 February 2015 (2015-02-05), US, pages 721 - 730, XP055266423, ISSN: 0020-7136, DOI: 10.1002/ijc.29428 *

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