TW202138000A - Methods for mobilizing stem cells - Google Patents

Abstract

The present disclosure found that a soluble P-selectin (sP-sel) may interfere in the interaction between stem cells and niches and thus mobilize stem cells from bone marrow. Accordingly, the mobilization of stem cells with sP-sel can treat a subject in need of one or more of preservation, repair, or regeneration of a tissue, or revascularization in the subject.

Description

Methods of mobilizing stem cells

The present invention generally relates to a method of mobilizing stem cells by administration of soluble P-selectin.

Stem cells (SC) are defined as cells that have the unique ability to self-replicate and differentiate into various cell types of the body throughout the life cycle of an organism. Two well-known types of stem cells are embryonic stem cells and adult stem cells. Since stem cells can differentiate into a wide range of cell types, they play an important role in the healing and regeneration of various tissues and organs. Some stem cells (such as bone marrow stem cells and hematopoietic stem cells) are released from the source tissue and circulate in the individual's circulation or immune system to migrate to various organs and tissues to become mature terminally differentiated cells. Therefore, enhancing stem cell migration (ie, release, circulation, homing, and/or migration) can amplify these physiological processes and provide potential treatments for various pathologies.

Currently, stem cells are mobilized from bone marrow to peripheral blood before collection for use in a clinical setting where allogeneic stem cell transplantation is not bone marrow. The recently approved CXCR4 inhibitor AMD3100 for stem cell mobilization induces more specific mobilization of cells into the circulation through the interaction of CXCR4-SDF1, which destroys bone marrow cells and their microenvironment, compared with G-CSF. For example, US 20180142211 uses CXCR4 antagonist peptide to mobilize mesenchymal stem cells to peripheral blood and then obtain cells.

However, there is still a need to develop reagents for stem cell mobilization.

The present invention provides a method for mobilizing circulating stem cells in an individual, which comprises administering to the individual soluble P-selectin (sP-sel) in an amount that can effectively mobilize a population of hematopoietic stem cells or progenitor cells. In one embodiment, soluble P-selectin can interfere with the interaction between stem cells and niches.

The present invention also provides a method for treating an individual in need of one or more of tissue preservation, repair or regeneration, or vascular reformation in the individual, which comprises administering to the individual a method that can effectively mobilize hematopoietic stem or progenitor cell populations The amount of sP-sel.

The present invention also provides a method for performing allogeneic hematopoietic stem cell transplantation in a patient in need, the method comprising infusing a therapeutically effective amount of allogeneic hematopoietic stem cells to the patient, wherein the hematopoietic stem cell line comprises administering an effective amount to the donor The method of sP-sel is mobilized from the bone marrow of a human donor to the peripheral blood of the human donor.

In one embodiment, the stem cells are hematopoietic cells, progenitor cells or bone marrow stem cells.

In one embodiment, the method further comprises administering the second agent before, after or simultaneously with the administration of sP-sel. In another embodiment, the second reagent is selected from the group consisting of: G-CSF, GM-CSF, IL-3, GM-CSF/IL-3 fusion protein, FLK-2/FLT-3 ligand , Stem cell factor, IL-6, IL-11, TPO, VEGF, AMD3100 and combinations thereof.

In one embodiment, the amount of sP-sel is in the ^{range of about 10 -5} μg to about 1.5 mg per kilogram of body weight per administration.

In one embodiment, circulating stem cells (PselMSC) mobilized by sP-sel can produce stem cell-derived extracellular vesicles.

In one embodiment, PselMSC can improve tissue or organ damage, increase repair, improve glucose tolerance, and/or reduce inflammation. In another embodiment, the tissue damage is liver damage.

In one embodiment, PselMSC can be repopulated with bone marrow or hematopoietic stem cell populations.

In one embodiment, PselMSC can repopulate the bone marrow or hematopoietic stem cell population and save tissue damage, proliferative disorders, inflammatory diseases, immunodeficiency diseases, autoimmune disorders, and/or metabolic diseases.

In one embodiment, sP-sel is naturally occurring sP-sel or recombinant sP-sel.

In one embodiment, sP-sel can be further bound to vesicles or liposomes.

The present invention also provides a method for cell therapy in an individual, which comprises administering to the individual an amount of sP-sel that can effectively mobilize stem cells and an amount of stem cells effective for cell therapy. In one embodiment, sP-sel and stem cells are administered simultaneously, separately or intermittently.

Unless otherwise specified, when the definition of a term deviates from the usual meaning of the term, the applicant intends to use the definition provided below.

Unless the content clearly indicates otherwise, the singular form "一 (a/an)" and "the (the)" used in the scope of this specification and the attached application include plural indicators.

As used herein, the use of "or" means "and/or" unless otherwise stated. In the case of multiple ancillary items, the use of "or" merely refers to more than one of the aforementioned independent items or ancillary items as an alternative.

As used herein, the term "one or more" is easily understood by those skilled in the art, especially when read in the context in which it is used.

As used interchangeably herein, the terms "individual", "individual", "host" and "patient" refer to mammals, including but not limited to murines (rats, mice), non-human primates, Humans, canines, cats, ungulates (e.g. equines, bovines, sheep, swines, goats), etc.

As used herein, the term "soluble P-selectin" refers to the naturally-occurring soluble form of P-selectin and its recombinant form, or its polytype or allele variant or other isoforms. The term also includes modified or unmodified soluble P-selectins, such as glycosylated or unglycosylated forms.

As used herein, the terms "mobilize" and "mobilization" refer to the process by which hematopoietic stem or progenitor cell populations are released from the stem cell niche.

As used herein, the term "niche" refers to the cellular and molecular microenvironment in vivo or in vitro that regulates stem cell functions along with the spontaneous mechanism of stem cells. This includes controlling the balance between stillness, self-renewal and differentiation, and participating in specific programs in response to stress.

As used herein, the term "hematopoietic stem cell" or "HSC" refers to the ability to differentiate into the bone marrow lineage (ie monocytes, macrophages, neutrophils, basophils, eosinophils, red blood cells, megakaryocytes/ Platelets and some dendritic cells) and stem cells of the lymphoid lineage (ie T cells, B cells, NK cells and some dendritic cells).

As used herein, the term "individual" refers to any animal, including mammals, birds, reptiles, and amphibians, and in preferred embodiments refers to mammals, including humans, companion animals, food-producing animals, and wild animals .

As used herein, the term "donor" refers to an individual from which one or more cells have been isolated before the cells or their progeny are administered to the recipient.

As used herein, the term "effective amount" refers to a certain amount of one or more reagents, such as a certain amount of soluble P-selectin and/or the second reagent described herein, which mobilizes hematopoietic stem cells or Progenitor cell population.

Stem cells can generate new cells to repair damage to tissues and therefore have great potential for regenerative drugs. However, stem cells are present in tissues and especially in peripheral blood in small amounts, making it difficult to collect stem cells or use stem cells clinically. The mobilization of stem cells is a way of collecting stem cells from the bone marrow into the blood. The present invention has carefully discovered that soluble P-selectin (sP-sel) can interfere with the interaction between stem cells and niches, and therefore mobilize stem cells from the bone marrow. Therefore, the use of sP-sel to mobilize stem cells can treat individuals who need one or more of tissue preservation, repair or regeneration, or blood vessel reformation in individuals.

P-selectin is a member of the selectin family located in the membrane of platelet α-granules and the Weibel-Palade body (WP body) of endothelial cells. P-selectin appears in two different forms; one is the "cell surface" form and the other is the "soluble" form. The former is manifested on activated platelets or endothelial cells involved in white blood cell inflammation and HSC homing. The latter (ie, sP-sel) is almost exclusively expressed in plasma in animals/humans under stress (e.g., hypoxic) ( Chang, HH and Sun, DS Methods of reducing hypoxic stress in a mammal by adminstring soluble P-selectin. US Patent US 8377887 B1 (2012) ). The soluble P-selectin molecule in the form of a monomer in the blood is 3 kDa smaller than the P-selectin molecule in the form of an oligomer on the membrane. The soluble P-selectin in healthy individuals is derived from the alternate splicing form that exists in endothelial cells and platelets. The present invention unexpectedly discovered that the treatment of soluble P-selectin can interfere with the interaction between stem cells and niches, and thus mobilize stem cells. Therefore, the mobilized hematopoietic stem cells and progenitor cells can be subsequently extracted from the donor and administered to the patient, where the cells can home to the hematopoietic stem cell niche and reconstitute the damaged or insufficient cell population in the patient.

Any form of sP-sel suitable for stem cell mobilization can be used in the present invention. Examples of sP-sel of the present invention include, but are not limited to, naturally occurring sP-sel and recombinant sP-sel. sP-sel can be easily obtained by general techniques, such as isolation from natural sources, purchase from commercial sources, or synthesis by molecular biotechnology.

sP-sel can be used in combination with a second reagent to mobilize stem cells. Examples of the second reagent include but are not limited to G-CSF, GM-CSF, IL-3, GM-CSF/IL-3 fusion protein, FLK-2/FLT-3 ligand, stem cell factor, IL-6, IL -11, TPO, VEGF, AMD3100 and combinations thereof. Preferably, the second reagent is G-CSF. The sP-sel and the second reagent can be used simultaneously or sequentially.

sP-sel can be used in combination with stem cells. Administer sP-sel and stem cells simultaneously, individually or intermittently.

Hematopoietic stem cell transplantation therapy can be administered to individuals in need of treatment to fill or refill one or more blood cell types, such as blood cell lineages that are insufficient or lacking in patients with stem cell disorders. Hematopoietic stem cells and progenitor cells exhibit versatility, and therefore can differentiate into a variety of different blood lineages. Hematopoietic stem cells produce different types of blood cells in the line called bone marrow and lymph. Both bone marrow and lymphatic lineages are involved in the formation of dendritic cells. Bone marrow cells include monocytes, macrophages, neutrophils, basophils, eosinophils, red blood cells and megakaryocytes to platelets. Lymphocytes include T cells, B cells, natural killer cells and innate lymphocytes.

The hematopoietic stem cells or progenitor cells mobilized to the peripheral blood of the individual can be drawn from the individual (for example, collection or collection) by any suitable technique. For example, hematopoietic stem cells or progenitor cells can be extracted by drawing blood. In some embodiments, hematopoietic cell separation can be used to collect (ie collect) hematopoietic stem cells or progenitor cells mobilized to the individual's peripheral blood as covered herein. In some embodiments, hemocytometry can be used to enrich the donor's blood with mobilized hematopoietic stem or progenitor cells.

The sP-sel disclosed herein can be administered according to various routes, usually by injection, such as local or systemic injection. However, other routes of administration such as intramuscular, intravenous, intradermal, subcutaneous, etc. can also be used. For administration, sP-sel is usually combined with one or more adjuvants suitable for the indicated route of administration. In addition, if necessary, repeated injections can be performed. sP-sel is administered in the range of ^{about 10 -5} μg to 1.5 mg per kilogram of body weight.

Circulating stem cells (PselMSC) mobilized by sP-sel can improve tissue or organ damage, increase repair, improve glucose tolerance and/or reduce inflammation. PselMSC can also be repopulated into bone marrow or hematopoietic stem cell populations and can save tissue damage, proliferative disorders, inflammatory diseases, immunodeficiency diseases, genetic disorders, degenerative disorders, autoimmune disorders, and/or metabolic diseases. Examples of proliferative disorders include, but are not limited to, blood cancer and myeloproliferative diseases. Examples of immune deficiency diseases include, but are not limited to, congenital immune deficiency diseases and acquired immune deficiency diseases. Examples of autoimmune disorders include, but are not limited to, juvenile arthritis, ulcerative colitis, type 1 diabetes (Type 1 diabetes mellitus/Type 1 diabetes), multiple sclerosis (MS), inflammatory bowel disease (IBD), Psoriasis, psoriatic arthritis, rheumatoid arthritis (RA), human systemic lupus (SLE), autoimmune lymphoproliferative syndrome (ALPS), and lymphocytic colitis. Examples of metabolic diseases include, but are not limited to, glycosidosis, mucopolysaccharidosis, Gaucher's Disease, Hurlers Disease, sphingolipidosis, and metachromatic white matter nutrition bad.

The recovered hematopoietic stem cells or progenitor cells can be re-infused into the patient, so that the cells will then return to the hematopoietic tissue and establish a productive hematopoietic effect, thereby filling or refilling cell lines that are lacking or insufficient in the patient.

Although the present invention has been described with reference to the preferred embodiments and examples of the present invention, the scope of the present invention is not limited to their described embodiments. It will be obvious to those who are familiar with the technology that modifications and changes can be made to the above-mentioned present invention without departing from the spirit and scope of the present invention defined and limited by the scope of the attached patent application. The following examples are provided to illustrate the intent of the embodiments and advantages of the present invention, and are not intended to limit its scope. Instance

Instance 1 Solubility P- Selectin mediated Of CD34 ⁺ Mobilization of cells

According to previous reports ( Tajima, F., Sato, T., Laver, JH and Ogawa, M. CD34 expression by murine hematopoieti c stem cells mobilized by granulocyte colony-stimulating factor. Blood 96 , 1989-1993 (2000)), G -CSF-mediated ^{mobilization of mouse CD34+} cells requires 5 injection doses. Here we show that on day 3 (after 2 injections of G-CSF), the mice still did not induce the induction of ^{circulating CD34+ cells.}

In each treatment, recombinant mouse P-selectin (rmP-sel) and pellet-community stimulating factor (G-CSF; Filgrastim®) were injected intravenously into mice (0.1 mg/kg body weight). Compared with not inducing circulating CD34 ⁺ stem cells, rmP-sel treatment triggered a large number of CD34 ⁺ cells with only 2 injections (see Figure 1A for experimental overview, 1B). These results show that P-selectin is much more effective than G-CSF when ^{mobilizing circulating CD34+ cells.}

Instance 2 Solubility P- Selectin improves thioacetamide (TAA) Induced thrombocytopenia and liver damage

C57BL/6J mice were injected with TAA to induce acute liver injury, and then rescued with or without rm-Psel (Figure 2A, timeline). We have found that pretreatment with twice rmP-sel can rescue the reduced platelet count in the peripheral blood to a normal level (Figure 2B). Aspartate transaminase (AST) enzyme activity is a standard marker for measuring liver injury. The data also revealed that rmP-sel can improve liver injury (Figure 2C). Our data show that rmP-sel treatment exerts a tissue protective effect to reduce TAA-induced damage (n=4; a statistically significant result, P<0.05 rmP-sel vs. normal saline).

It has previously been shown that stem cell treatment may have a beneficial effect on the improvement of liver damage. ^{In order to characterize whether the CD34 +} cells induced by rmP-Sel have a tissue protective effect, we conducted a grant-acceptance transfer experiment. ^{Our data show that accepting transfer of CD34+} stem cells mobilized by P-selectin instead of peripheral blood mononuclear cells (PBMC) can rescue TAA-induced hepatitis in mice (Figure 3).

In humans, G-CSF treatment mobilizes CD34 ⁺ stem cells, which can repopulate gamma-irradiated bone marrow. LSK hematopoietic stem cells in mice are a cell lineage ^{equivalent to CD34+ stem cells in humans.} To study the potential role of LSK cells mobilized by P-selectin in the repopulation of bone marrow, C57Bl/6 recipient mice that had been lethal irradiated with gamma rays were transplanted with 1×10 ⁵ LSK cells. These cells were mobilized with soluble P-selectin (Figure 4) and G-CSF (Figure 5). As shown by the rescue of 100% lethal γ-irradiation in mice, the graft was successfully transplanted (Figure 5; 100% lethality in the non-transplant group compared with LSK mobilized by soluble P-selectin and mobilization by G-CSF About 83% survival rate of both in the LSK group).

The TAA hepatitis mouse model was used to prove the protective effect of PselMSC and PselSCMV, soluble P-selectin and soluble P-selectin combined liposomes. The treatment of PselMSC and PselSCMV, soluble P-selectin and soluble P-selectin-conjugated liposomes significantly rescued the TAA-induced increase in the content of high circulating alanine aminotransferase (ALT) in mice, indicating that these reagents It can improve the liver injury induced by TAA.

It was previously shown that adipocyte-derived leaf stem cells improve glucose homeostasis in obese mice induced by a high-fat ^diet2,3 . However, it is still difficult to realize whether PselMSC, PselSCMV, soluble P-selectin and soluble P-selectin are combined with liposomes. The results of glucose tolerance (OGTT) analysis revealed that PselMSC, PselSCMV, soluble P-selectin, and soluble P-selectin-conjugated liposomes all improved high-fat diet (HFD)-induced glucose resistance, thereby reducing HFD- The blood glucose level of mice (Figure 7).

Analyze the circulating TNF-α content showing the degree of inflammation in TAA-induced hepatitis mice. Data show that PselMSC, PselSCMV, soluble P-selectin, and soluble P-selectin-conjugated liposomes all have anti-inflammatory properties, because the treatment of these agents significantly reduces the plasma TNF-α content in TAA-induced mice Induction (Figure 8).

Figure 1. Soluble P-selectin but not G-CSF treatment induces ^{the mobilization of CD34+} stem cells in mice. Recombinant mouse soluble P-selectin and G-CSF (Filgrastim®) (both 0.1 mg/kg body weight) were injected intravascularly twice within 24 h intervals into 8-week-old experimental male C57Bl/6J mice for another 24 hours h Collect blood samples (A, overview). Flow cytometry (FC) was used to measure ^{the content of circulating CD34 +} monocytes (A), and the vehicle (normal saline) control group was standardized to 100% (B).

Figure 2. Soluble P-selectin instead of G-CSF treatment improves thioacetamide (TAA)-induced thrombocytopenia and liver damage in mice. Recombinant mouse soluble P-selectin and G-CSF (Filgrastim®) (both 0.1 mg/kg body weight) were injected intravascularly into 8-week-old experimental male C57Bl/6J mice twice within 24 hours; and additional administration With hepatotoxic drugs thrombocytopenia (TAA) for 24 hours (A, overview). According to the method ¹ described previously, the platelet (PLT) count and the circulating liver-specific enzyme aspartate transaminase (AST) content (B) were analyzed 48 h after TAA treatment.

^{Figure 3. CD34+} cells mobilized by soluble P-selectin can rescue the liver injury induced by thioacetamide (TAA). After the aforementioned method, soluble P-selectin mobilized CD34 ⁺ cells and peripheral blood mononuclear cells (PBMC; monocytes) (both 5×10 ⁶ /mouse injection) were injected together with TAA challenge (n=4 ; A statistically significant result, P <0.05 rm P-sel compared to the "no cell metastasis" and "monocyte sphere" groups).

Figure 4. The level of circulating Lin ^- Sca-1 ⁺ c-Kit ⁺ (LSK) stem cells after stimulation with soluble P-selectin. LSK hematopoietic stem cells are stem cell lineages that can be repopulated with bone marrow stem cells after lethal γ-irradiation. The summary of the experiment is shown (A). C57BL/6J mice were injected intravenously with soluble P-selectin (0.1 mg/kg) twice a day (n=5). Peripheral blood (PB) was collected and analyzed immediately before the first injection and 24 hours after the first injection using flow cytometry (FC) analysis. Quantify the absolute number of LSK cells in peripheral blood (B). Data reported as mean ± SD. *P<0.05, compared with the group before the experiment.

Figure 5. LSK stem cells mobilized by P-selectin rescued the mortality of γ-irradiated mice, showing the repopulation of bone marrow hematopoietic stem cells. The C57BL/6J mouse line has or has not received a lethal dose of γ-irradiation (which is commonly used for bone marrow transplantation). Their γ-irradiated mice were further treated with vehicle, LSK stem cells, and these stem cells were obtained by mobilization mediated by P-selectin or G-CSF treatment. Because both G-CSF and LSK stem cells mobilized by P-selectin can save the mortality of mice irradiated with -γ, these results show the repopulation of bone marrow hematopoietic stem cells using two methods of preparing LSK stem cells.

Figure 6. P-selectin-mobilized CD34 ⁺ stem cells (PselMSC), P-selectin-mobilized CD34 ⁺ stem cell-derived vesicles (PselSCMV), soluble P-selectin and soluble P-selectin-conjugated liposomes , To improve the liver injury mediated by thioacetamide (TAA). The treatment of PselMSC and PselSCMV, soluble P-selectin and soluble P-selectin-conjugated liposomes can rescue TAA-mediated liver damage, as indicated by circulating ALT and enzyme content specific to hepatocytes. **P＜0.01, compared with normal/vehicle group; #P＜0.05, ##P＜0.01, compared with TAA group. n=6.

^{Figure 7. CD34+} stem cells mobilized by P-selectin (PselMSC), CD34 ⁺ stem cell-derived vesicles (PselSCMV) mobilized by P-selectin, soluble P-selectin and soluble P-selectin-conjugated liposomes , Improves glucose tolerance in diabetic mice induced by high-fat diet (HFD). A glucose tolerance test (OGTT) experiment was performed, and the plasma glucose content was measured in different experimental mouse groups. *P＜0.05; **P＜0.01, compared with HFD groups. n = 6.

Figure 8. P-selectin-mobilized CD34 ⁺ stem cells (PselMSC), P-selectin-mobilized CD34 ⁺ stem cell-derived vesicles (PselSCMV), soluble P-selectin and soluble P-selectin-conjugated liposomes Anti-inflammatory effect on TAA-induced hepatitis in mice. The degree of TAA-induced inflammation was indicated by circulating TNF-α content in mice; the average TNF-α content in the normal/vehicle group was normalized to 100%. **P<0.01, compared to the normal/vehicle group; *P<0.05, **P<0.01, compared to the TAA group. n = 6.

Claims (20)

A method for mobilizing circulating stem cells in an individual, the method comprising administering to the individual soluble P-selectin (sP-sel) in an amount effective to mobilize the stem cells. The method of claim 1, wherein the soluble P-selectin can interfere with the interaction between the stem cell and the niche. The method of claim 1, wherein the stem cells are hematopoietic cells, progenitor cells or bone marrow stem cells. Such as the method of claim 1, which further comprises administering the second agent before, after or at the same time as administering the sP-sel. Such as the method of claim 4, wherein the second reagent is selected from the group consisting of G-CSF, GM-CSF, IL-3, GM-CSF/IL-3 fusion protein, FLK-2/FLT-3 Position, stem cell factor, IL-6, IL-11, TPO, VEGF, AMD3100 and combinations thereof. Such as the method of claim 4, wherein the amount of sP-sel is in the range of about 10 ^-5 μg to 1.5 mg per kilogram of body weight per administration. The method of claim 1, wherein circulating stem cells (PselMSC) mobilized by sP-sel can produce stem cell-derived extracellular vesicles. The method of claim 7, wherein the PselMSC and PselMSC extracellular vesicles can improve tissue or organ damage, increase repair, improve glucose tolerance, and/or reduce inflammation. The method of claim 8, wherein the tissue damage is liver damage. Such as the method of claim 7, wherein the PselMSCs can be refilled with bone marrow or hematopoietic stem cell populations. The method of claim 1, wherein the PselMSC and PselMSC extracellular vesicles can rescue tissue damage, proliferative disorders, inflammatory diseases, immunodeficiency diseases, genetic disorders, degenerative disorders, autoimmune disorders and/or metabolic diseases . The method of claim 1, wherein the sP-sel is naturally-occurring sP-sel or recombinant sP-sel. The method of claim 1, wherein the sP-sel can be further bound to vesicles and liposomes. A method for treating an individual in need of one or more of tissue preservation, repair, or regeneration, or revascularization in the individual, the method comprising administering to the individual an amount of sP-sel that can effectively mobilize stem cells in the individual . Such as the method of claim 14, which further comprises the step of administering a certain amount of stem cells. The method of claim 14, wherein the sP-sel and the stem cell lines are administered simultaneously, separately or intermittently. A method for performing allogeneic hematopoietic stem cell transplantation in a patient in need, the method comprising infusing a therapeutically effective amount of allogeneic hematopoietic stem cells to the patient, wherein the hematopoietic stem cell lines are administered to the donor in an effective amount by a method comprising The method of sP-sel is to mobilize the bone marrow of a human donor into the peripheral blood of the human donor. Such as the method of claim 17, wherein the amount of sP-sel is in the range of about 10 ^-5 μg to 1.5 mg per kilogram of body weight per administration. The method of claim 17, wherein the sP-sel is naturally-occurring sP-sel or recombinant sP-sel. The method of claim 17, wherein the sP-sel can be further bound to vesicles and liposomes.

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