Methods for treating inflammatory bowel disease I

Abstract

The therapeutic needs of patients with IBD and / or related conditions or symptoms remain unmet, and new treatment options are needed. [Solution] This disclosure relates to a method for treating or preventing inflammatory bowel disease (IBD) in a subject in need thereof, comprising administering a composition comprising mesenchymal lineage progenitor or stem cell (MLPSC) to the subject.

Description

[Technical Field]

[0001] This disclosure relates to a method for treating or preventing inflammatory bowel disease (IBD) in a population that needs it. [Background technology]

[0002] Inflammatory bowel disease (IBD) is a debilitating, relapsing condition that initially appears in young adults and can affect patients throughout their lives. IBD encompasses a group of disorders in which the gastrointestinal tract becomes inflamed. The main types of IBD include Crohn's disease, in which inflammation affects the entire thickness of the intestinal wall at various points along the gastrointestinal tract, and ulcerative colitis, in which inflammation affects the inner lining (mucosa) of the colon and rectum. Crohn's disease affects nearly 2 million people in the United States and another 1 million more worldwide, and the number of cases continues to increase for unknown reasons. Since the FDA approved infliximab in 2006, monoclonal antibodies have become a cornerstone of medical treatment for moderate to severe cases. However, their use is limited by the risk of primary and secondary non-response, as well as serious opportunistic infections. Furthermore, it can take a long time for biology to demonstrate clinical improvement. Therefore, non-response patients generally do not receive treatment during this period, and may become increasingly malnourished, anemic, and suffer from complications of their disease while waiting for evaluation of medical response. [Prior art documents] [Patent Documents]

[0003] [Patent Document 1] WO2018 / 182612 [Patent Document 2] WO2018 / 002930 [Overview of the Initiative] [Problems that the invention aims to solve]

[0004] Therefore, the therapeutic needs of patients with IBD and / or related conditions or symptoms remain unmet, and new treatment options are needed. [Means for solving the problem]

[0005] The inventors have surprisingly demonstrated that early disease remission (day 28) can be achieved in subjects with inflammatory bowel disease by injection of mesenchymal progenitor lines or stem cells. Thus, in the first example, the present disclosure relates to a method for treating or preventing inflammatory bowel disease in a human subject in need, comprising administering a composition comprising mesenchymal progenitor lines or stem cells (MLPSCs) to the subject, wherein the composition is administered to the gastrointestinal wall of the subject.

[0006] In one example, the composition is administered to the submucosa of the target gastrointestinal wall. In another example, the composition may be administered to the site of inflammation in the target gastrointestinal wall. In one example, the composition may be administered to the target colon and / or rectum.

[0007] In one example, the composition is administered by intraluminal injection. For example, the composition may be administered by endoscopy. In another example, the method further includes simultaneous or sequential intravenous administration of mesenchymal progenitor lines or stem cells to a subject.

[0008] In one example, the subject is refractory to at least one biological therapy. In another example, the subject is refractory to only one biological therapy. In yet another example, the subject is refractory to at least one anti-TNF therapy. In yet another example, the subject is refractory to steroid immunosuppressants and / or biological therapies.

[0009] In one example, inflammatory bowel disease is Crohn's disease or ulcerative colitis. For example, inflammatory bowel disease can be Crohn's disease. In one example, Crohn's disease manifests in the rectum and / or colon of the subject. In one example, Crohn's disease is Crohn's disease with fistulas. In another example, Crohn's disease is moderate to severe.

[0010] In one example, the subject has a partial clinical and / or endoscopic response at least 28 days after treatment. In another example, the subject has a partial clinical and / or endoscopic response at least 28 to 56 days after treatment. In one example, a partial clinical response is characterized by - a reduction greater than 25% in C-reactive protein (CRP); - a decrease of less than 100 points in the Crohn's Disease Activity Index (CDAI); - radiographic healing with improvement of inflammation as evaluated by MR enterography characterized by one or more or all of the above.

[0011] In one example, a partial endoscopic response is characterized by - a decrease in the Crohn's Disease Simple Endoscopic Score (SES-CD) of greater than 25% and SES-CD < 50%; - an SES-CD score of 10 to 15 characterized by one or both of the above.

[0012] In one example, the subject has a clinical and / or endoscopic response at least 28 days after treatment. In another example, the subject has a clinical and / or endoscopic response at least 28 to 56 days after treatment. In one example, a clinical response is characterized by - a reduction greater than 50% in CRP; - normalization of CRP; - a decrease of 100 points or more in CDAI; - radiographic healing with improvement of inflammation as evaluated by MR enterography characterized by one or more or all of the above.

[0013] In one example, an endoscopic response is characterized by - an SES-CD that has decreased by more than 25% but less than 50%; - an SES-CD score of 5 to 10 characterized by one or both of the above.

[0014] In one case, the subject is in a state of clinical and / or endoscopic remission at least 28 days after treatment. In another case, the subject is in a state of clinical and / or endoscopic remission at least 28 to 56 days after treatment. In one case, clinical remission was, - Normalization of CRP to less than 2.87 mg per liter; - Radiographic healing with improvement of inflammation as evaluated by MR enterography Characterized by one or both of the following:

[0015] In one example, endoscopic remission was achieved. - Absence of mucosal ulcers; - SES-CD scores from 0 to 5 Characterized by one or both of the following:

[0016] In one example, MLPSCs are administered into the submucosa of the target colon wall. In another example, MLPSCs are administered to multiple sites in the target gastrointestinal wall.

[0017] In one example, MLPSCs are mesenchymal stem cells (MSCs). In another example, MLPSCs are allogeneic. For example, MLPSCs can be allogeneic MSCs.

[0018] In one example, the target CDAI is greater than 300.

[0019] In one example, MLPSC is administered via endoscopy.

[0020] For example, the method disclosed herein is 1 × 10 7 ~2×10 8 This includes administering individual cells. In another example, the method of the present disclosure involves administering 1 × 10¹⁶ cells to 2, 3, 4, 5, 6 or more sites on the wall of the target gastrointestinal tract. 7 ~2×10 8 This includes administering individual cells. For example, MLPSCs may be administered to two, three, four, five, six or more sites in the colon and / or rectum of the subject.

[0021] In one example, the MLPSC composition of this disclosure further comprises Plasma-Lyte A, dimethyl sulfoxide (DMSO), and human serum albumin (HSA). For example, such a composition may further comprise a solution of Plasma-Lyte A (70%), DMSO (10%), and HSA (25%), where the HSA solution may contain 5% HSA and 15% buffer. In another example, such a composition may comprise 6.68 × 10⁻⁶ 6 It may contain more than one live cell / mL. [Brief explanation of the drawing]

[0022] [Figure 1] Percentage of patients achieving a CDAI score of 150 or less on day 28. FAS: Fully randomized, at least one procedure, at least one post-baseline evaluation; PP: All FAS except major protocol events. [Modes for carrying out the invention]

[0023] Throughout this specification, references to a single step, composition, group of steps, or group of compositions should be interpreted as encompassing one or more (i.e., one or more) of those steps, compositions, group of steps, or group of compositions, unless otherwise specified or required by the context.

[0024] Those skilled in the art will recognize that the disclosure described herein permits variations and modifications other than those specifically described herein. It should be understood that all such variations and modifications are included in the disclosure. The disclosure includes all the steps, features, compositions and compounds mentioned or indicated herein, individually or collectively, as well as any combination of the above steps or features, or any two or more of them.

[0025] This disclosure should not be limited in scope by the specific embodiments described herein, which are intended for illustrative purposes only. Functionally equivalent products, compositions, and methods are clearly included within the scope of this disclosure as described herein.

[0026] Unless otherwise specified, any examples disclosed herein should be construed as applicable to any other examples, with appropriate modifications.

[0027] Unless otherwise specified, all scientific and technical terms used herein should be interpreted as having the same meaning as those generally understood by those skilled in the art (e.g., cell culture, molecular genetics, stem cell differentiation, immunology, immunohistochemistry, protein chemistry, and biochemistry).

[0028] Unless otherwise specified, the surgical techniques used in this disclosure are standard procedures well known to those skilled in the art.

[0029] Methods for obtaining and enriching populations of mesenchymal stem or progenitor cells are known in the art. For example, enriched populations of mesenchymal stem or progenitor cells can be obtained by using flow cytometry and cell sorting techniques based on the use of cell surface markers expressed on mesenchymal stem or progenitor cells.

[0030] Together with any manufacturer's instructions, manuals, product specifications, and product sheets relating to any product mentioned herein or in any document incorporated herein by reference, all documents cited or mentioned herein, and all documents cited or mentioned in any document cited herein, are incorporated herein by reference in their entirety.

[0031] Excerpt from the definition The term "and / or," for example "X and / or Y," should be understood to mean either "X and Y" or "X or Y," and should be taken as providing explicit support for both meanings or either one of them.

[0032] As used herein, the term "about" means + / - 10%, more preferably + / - 5%, of the specified value, unless otherwise stated.

[0033] Throughout this specification, it will be understood that the word “comprise,” or its variations such as “comprises” or “contains,” implicitly means that it includes the element, integer, or step, or group of elements, integers, or steps, but does not exclude any other element, integer, or step, or group of elements, integers, or steps.

[0034] As used herein, the singular forms "a," "an," and "the" include both singular and plural meanings, unless the context indicates otherwise.

[0035] "Isolated" or "purified" means that cells have been separated from at least some components of their natural environment. This term includes macroscopic physical separation of cells from their natural environment (e.g., removal from a donor). The term "isolated" includes changes in the relationship between a cell and its immediate neighboring cells, such as by dissociation. The term "isolated" does not mean cells present in a tissue section. When used in reference to a population of cells, the term "isolated" includes a population of cells obtained by proliferation of isolated cells as described in this disclosure.

[0036] In the context of this disclosure, the terms “subculturing,” “subculturing,” or “subculturing” are used to refer to known cell culture techniques used to keep cells alive and growing over extended periods under culture conditions so that the cell number can continuously increase. The degree to which a cell line has undergone subculturing is often expressed as “passage number,” which generally refers to the number of times the cells have been subculturised. In one example, one passage includes removing non-adherent cells and leaving adherent mesenchymal line precursors or stem cells. Such mesenchymal line precursors or stem cells may then be dissociated from the substrate or flask (e.g., by using a protease such as trypsin or collagenase), culture medium may be added, optional washing (e.g., by centrifugation) may be performed, and then the mesenchymal line precursors or stem cells may be reseeded or re-seeded into one or more culture vessels having a larger total surface area. The mesenchymal line precursors or stem cells may then continue to grow in the culture. In another example, the method for removing non-adherent cells includes a non-enzymatic treatment step (e.g., with EDTA). In one example, mesenchymal progenitor or stem cells are passaged to confluence or near confluence (e.g., approximately 75-95% confluence). In another example, mesenchymal progenitor or stem cells are seeded at a cell / ml culture medium concentration of approximately 10%, 15%, or 20%.

[0037] As used in the context of this disclosure, the terms “medium” or “media” include the components of the environment surrounding cells in a culture. The medium is expected to provide conditions that are helpful for and / or suitable for cell growth. The medium may be solid, liquid, gaseous, or a mixture of phases and substances. Mediums may include liquid growth media and liquid media that do not sustain cell growth. An example of a gaseous medium is the gas phase to which cells growing on a Petri dish or other solid or semi-solid support are exposed.

[0038] The term “gastrointestinal tract” or “GI tract” encompasses the human organ system from mouth to anus. The gastrointestinal tract includes the mouth, esophagus, stomach, and intestines. Therefore, references to the walls of the gastrointestinal tract in this disclosure include the walls of the mouth, esophagus, stomach, and intestines. To avoid misunderstanding, the term “intestines” includes the colon and rectum.

[0039] As used herein, the terms “to treat,” “to heal,” or “to cure” include administering a population of mesenchymal stem or progenitor cells, and / or their offspring, and / or soluble factors derived therefrom, thereby reducing or eliminating at least one symptom of inflammatory bowel disease. For example, treatment includes administering a cultured population of mesenchymal stem or progenitor cells. For example, treatment results in a partial clinical and / or endoscopic response. For example, the partial clinical and / or endoscopic response occurs at least 25 days after treatment. For example, the partial clinical and / or endoscopic response occurs at least 28 days after treatment. For example, the partial clinical and / or endoscopic response occurs at least 30 days after treatment. For example, the partial clinical and / or endoscopic response occurs at least 35 days after treatment. For another example, the partial clinical and / or endoscopic response occurs at least 28–65 days after treatment. In another example, partial clinical and / or endoscopic responses are achieved at least 28–56 days after treatment.

[0040] In one example, a partial clinical response was observed. - A reduction of more than 25% in C-reactive protein (CRP); - A decrease of less than 100 points in the CDA Activity Index (CDAI); - Radiographic healing with improvement of inflammation as assessed by MR enterography; - A reduction of less than 3 points in the Mayo Clinic score, accompanied by a decrease of at least 2 points in the rectal bleeding subscale, resulting in an absolute rectal bleeding score of 1 or 2, and a reduction of less than 30% from baseline. Characterized by one or more or all of the following.

[0041] In one example, a partial endoscopic response was observed. - Crohn's disease plain endoscopy score (SES-CD) decreased by more than 25%, and SES-CD < 50%; - SES-CD scores for 10-15; - Mayo Clinic Scale Endoscopy Subscores remain the same or decrease, showing no improvement. Characterized by one or both of the following:

[0042] In one case, the treatment results in a clinical and / or endoscopic response. In another case, the clinical and / or endoscopic response occurs at least 25 days after treatment. In yet another case, the clinical and / or endoscopic response occurs at least 28 days after treatment. In yet another case, the clinical and / or endoscopic response occurs at least 30 days after treatment. In yet another case, the clinical and / or endoscopic response occurs at least 35 days after treatment. In yet another case, the clinical and / or endoscopic response occurs at least 28 to 65 days after treatment. In yet another case, the clinical and / or endoscopic response occurs at least 28 to 56 days after treatment.

[0043] In one example, the clinical response was: - A reduction of more than 50% in CRP; - Normalization of CRP levels; - A drop of 100 points or more on the CDAI; - Radiographic healing with improvement of inflammation as assessed by MR enterography; - A reduction of 3 points in the Mayo Clinic score, accompanied by a decrease of at least 2 points in the rectal bleeding subscale, resulting in an absolute rectal bleeding score of 1 or 2, and a reduction of at least 30% from baseline. Characterized by one or more or all of the following.

[0044] In one example, the endoscopic response was, - SES-CD decreased by more than 25% but less than 50%; - SES-CD scores of 5-10; - A decrease of at least one point in the Mayo Clinic Scale Endoscopy subscore. Characterized by one or both of the following:

[0045] In one case, treatment results in clinical and / or endoscopic remission. In another case, clinical and / or endoscopic remission is achieved at least 25 days after treatment. In one case, clinical and / or endoscopic remission is achieved at least 28 days after treatment. In one case, clinical and / or endoscopic remission is achieved at least 30 days after treatment. In one case, clinical and / or endoscopic remission is achieved at least 35 days after treatment. In yet another case, clinical and / or endoscopic remission is achieved at least 28–65 days after treatment. In yet another case, clinical and / or endoscopic remission is achieved at least 28–56 days after treatment.

[0046] In one example, clinical remission was, - Normalization of CRP to less than 2.87 mg per liter; - Radiographic healing with improvement of inflammation as evaluated by MR enterography Characterized by one or more or all of the following.

[0047] In one example, endoscopic remission was achieved. - Absence of mucosal ulcers; - SES-CD scores from 0 to 5 Characterized by one or both of the following:

[0048] In one example, treatment may result in reduced C-reactive protein (CRP); decreased CDA activity index (CDAI); radiographic cure as assessed by MR enterography; and reduced Crohn's disease plain endoscopic score (SES-CD) compared to baseline values ​​(i.e., control, or before administration of mesenchymal stem or progenitor cells and / or their offspring and / or derived soluble factors).

[0049] As used herein, the terms “prevent” or “prevent” include administering a population of mesenchymal stems or progenitor cells, and / or their offspring, and / or soluble factors derived thereby halting or suppressing the progression of at least one symptom of inflammatory bowel disease.

[0050] In the context of this disclosure, the term “inflammatory bowel disease” (IBD) is used to refer to inflammatory diseases of the gastrointestinal tract, such as ulcerative colitis (UC), irritable bowel syndrome, irritable colon syndrome, Crohn’s colitis, and Crohn’s disease (CD).

[0051] The term "ulcerative colitis (UC)" can encompass mild to moderate ulcerative colitis. Mild to moderate ulcerative colitis is characterized by one or more of the following: - Ulcerative colitis - Disease Activity Index (UC-DAI) score of 4-10; - Sigmoidoscopy score greater than 4; - A General Physician Assessment (PGA) score greater than 2.

[0052] The Crohn's Disease Activity Index (CDAI score) is a research tool developed in 1976 by WR Best and colleagues at the Midwest Regional Health Center in Illinois to quantify the symptoms of Crohn's disease. This index is the most widely used method for assessing Crohn's disease activity (Sandbom et al. (2002) Gastroenterology., 122:512-530) and consists of eight coefficients / variables. These eight variables are summed after adjustment with weighting factors. The components of the CDAI and their weighting factors are shown in the table below: [Table 1]

[0053] The total CDAI score ranges from 0 to approximately 600, with higher scores indicating more active disease. For example, a CDAI score of less than 150 represents "clinical remission" of Crohn's disease. For example, 150-219 represents "mildly active Crohn's disease." For example, 220-450 represents "moderately active Crohn's disease." For example, a score above 450 represents "severely active Crohn's disease."

[0054] In one example, treatment results in a CDAI score reduction of 50 points or more. In another example, treatment results in a CDAI score reduction of 75 points or more. In yet another example, treatment results in a CDAI score reduction of 90 points or more. In yet another example, treatment results in a CDAI score reduction of 100 points or more. In yet another example, treatment results in a CDAI score reduction of 150 points or more. In yet another example, treatment results in a CDAI score reduction of 50 to 150 points. In yet another example, treatment results in a CDAI score reduction of 75 to 125 points. In yet another example, treatment results in a CDAI score reduction of 90 to 110 points.

[0055] C-reactive protein, or CRP, is an inflammation-mediating factor whose levels rise under conditions of acute inflammatory recurrence and rapidly normalize as inflammation subsides. In one case, treatment reduces CRP by more than 20% from baseline. In another case, treatment reduces CRP by more than 30% from baseline. In yet another case, treatment reduces CRP by more than 40% from baseline. In yet another case, treatment reduces CRP by more than 50% from baseline. In yet another case, treatment reduces CRP by more than 60% from baseline. In yet another case, treatment reduces CRP by 20–60% from baseline. In yet another case, treatment reduces CRP by 30–50% from baseline. In yet another case, treatment reduces CRP to less than 2.95 mg per liter. In yet another case, treatment reduces CRP to less than 2.87 mg per liter. In yet another case, treatment normalizes CRP levels in the subject.

[0056] In one example, treatment results in an SES-CD score of 0-5. In another example, treatment results in an SES-CD score of 5-10. In yet another example, treatment results in an SES-CD score of 10-15. In yet another example, treatment results in an SES-CD score of 0-15.

[0057] For example, the method disclosed herein suppresses disease progression or disease complications in a subject. “Suppression” of disease progression or disease complications in a subject means preventing or mitigating disease progression and / or disease complications in a subject.

[0058] As used herein, the term “subject” refers to a human subject. For example, a subject may be an adult. In another example, a subject may be a child. In yet another example, a subject may be a young adult. Terms such as “subject,” “patient,” or “individual” are interchangeable terms within the context of this disclosure.

[0059] Subjects treated in accordance with this disclosure may have symptoms suggestive of inflammatory bowel disease. For example, subjects may have gastrointestinal symptoms suggestive of inflammatory bowel disease. Exemplary gastrointestinal symptoms include diarrhea, constipation, nausea, vomiting, flatulence, cramps, bloating, abdominal pain, steatorrhea, and rectal bleeding. In one example, subjects treated in accordance with this disclosure may present with one or more symptoms selected from the group consisting of fatigue, weakness and lethargy, iron deficiency, anemia, vitamin and mineral deficiencies, developmental delay, delayed puberty, weight loss, bone and arthralgia, recurrent oral ulcers and / or swelling of the mouth or tongue, changes in mental attention and excitability, skin rashes, such as herpetic dermatitis, susceptibility to skin bruising, and routine reflux. In one example, subjects may have previously failed at least one anti-TNF therapy. In one example, subjects may have contraindications to biological therapies.

[0060] In another example, the subjects are between 18 and 75 years old. In yet another example, the subjects have Crohn's disease. In yet another example, the subjects have ulcerative colitis. In yet another example, the subjects have both Crohn's disease and ulcerative colitis. In yet another example, the subjects' Crohn's disease manifests in the subjects' intestines. In yet another example, the subjects' Crohn's disease manifests in the subjects' rectum and / or colon.

[0061] In other cases, the subject had Crohn's disease for a duration of at least six months. In other cases, the subject's Crohn's disease was moderate to severe. In other cases, the subject's Crohn's disease was Crohn's disease with fistulas (see, for example, Geese et al. 2013 United European Gastroenterol J., 1:206-213).

[0062] In another example, the target CDAI is greater than 200. In yet another example, the target CDAI is greater than 250. In yet another example, the target CDAI is greater than 300. In yet another example, the target CDAI is between 200 and 450. In yet another example, the target CDAI is between 250 and 400. In yet another example, the target CDAI is between 300 and 400.

[0063] In another example, the method of the Disclosure prevents or treats subjects with active, mild Crohn's disease. In yet another example, the method of the Disclosure prevents or treats subjects with active, moderate Crohn's disease. In yet another example, the method of the Disclosure prevents or treats subjects with active, severe Crohn's disease. In yet another example, the method of the Disclosure prevents or treats subjects with active, moderate or severe Crohn's disease. For example, the method of the Disclosure may be used to prevent or treat subjects with active, moderate Crohn's disease who are refractory to immunosuppressants and / or biological therapies. For example, the Crohn's disease of a subject may be refractory to TNF-alpha antagonists and / or steroids.

[0064] Where used herein, the term “refractory” is used in the context of this disclosure to refer to a subject who has failed to receive or is resistant to a particular treatment, such as a “biological therapy,” or a biological agent such as infliximab or adalimumab. For example, a subject is refractory to a biological therapy if the next step in medical management is expansion of medical management. For example, a subject is refractory to a biological therapy if the next step in medical management is alternative biological therapy. For example, a subject is refractory to a biological therapy if the next step in medical management is less than a total colectomy. For example, a subject is refractory to a single biological therapy. For example, a subject has not been treated with more than one biological therapy.

[0065] In the context of this disclosure, the term "biological therapy" is used to refer to recombinant proteins derived from or synthesized from living biological organisms. For example, a biological therapy is used to treat inflammatory conditions, such as inflammatory bowel disease, such as Crohn's colitis. For example, a biological therapy is an antibody; for example, a monoclonal antibody. For another example, a biological therapy is anti-TNF therapy. Examples of biological therapies included in this disclosure include infliximab, adalimumab, certolizumab pegol, vedolizumab, or ustekinumab. Thus, for example, a subject included by this disclosure may be refractory to infliximab, adalimumab, certolizumab pegol, vedolizumab, or ustekinumab.

[0066] As used herein, the term “non-genetically modified” means that the cells have not been modified by nucleic acid transfection. To avoid misunderstanding, in the context of this disclosure, mesenchymal lineage progenitors or stem cells transfected with the nucleic acid encoding Ang1 will be considered genetically modified.

[0067] Mesenchymal progenitor cells As used herein, the term “mesenchymal progenitor or stem cell (MLPSC)” refers to an undifferentiated pluripotent cell that has the ability to regenerate while maintaining pluripotency, and the ability to differentiate into multiple cell types, either of mesenchymal origin, such as osteoblasts, chondrocytes, adipocytes, stromal cells, fibroblasts, and tendons, or of non-mesoderm origin, such as hepatocytes, nerve cells, and epithelial cells. To avoid misunderstanding, “mesenchymal progenitor cell” refers to a cell that can differentiate into mesenchymal cells, such as bone, cartilage, muscle, and adipocytes, as well as fibrous connective tissue.

[0068] The term “mesenchymal progenitor or stem cell” includes both parent cells and their undifferentiated offspring. The term also includes mesenchymal progenitor cells, pluripotent stromal cells, mesenchymal stem cells (MSCs), perivascular mesenchymal progenitor cells and their undifferentiated offspring.

[0069] Mesenchymal progenitor or stem cells can be autologous, allogeneic, heterogeneous, syngeneic, or homogeneous. Autologous cells are isolated from the same individual from which they will be re-transplanted. Allogeneic cells are isolated from a homogeneous donor. Heterogeneous cells are isolated from a different species donor. Syngeneic or allogeneic cells are isolated from genetically identical organisms, such as twins, clones, or highly related research animal models.

[0070] In one example, the mesenchymal progenitor or stem cells are allogeneic. In another example, the allogeneic mesenchymal progenitor or stem cells were cultured, grown, and then cryopreserved.

[0071] Mesenchymal progenitor or stem cells are primarily found in bone marrow, but have also been shown to exist in a variety of host tissues, including, for example, umbilical cord blood and cord, adult peripheral blood, adipose tissue, trabeculae, and dental pulp. They are found in the skin, spleen, pancreas, brain, kidney, liver, heart, retina, hair follicles, intestines, lungs, lymph nodes, thymus, ligaments, tendons, skeletal muscle, dermis, and periosteum, and can differentiate into germline cells, such as mesoderm and / or endoderm and / or ectoderm. Thus, mesenchymal progenitor or stem cells can differentiate into a multitude of cell types, including but not limited to adipose tissue, bone, cartilage, elastic tissue, muscle, and fibrous connective tissue. The specific phylogenetic specialization and differentiation pathways into which these cells enter are determined by various influences from mechanistic influencing factors and / or endogenous bioactive factors, such as growth factors, cytokines, and / or local microenvironmental conditions established by the host tissue.

[0072] The terms “enriched,” “enriched,” or variations thereof are used herein to describe a population of cells in which the proportion of one particular cell type or a combination of particular cell types is increased compared to a population of untreated cells (e.g., cells in their natural environment). For example, a population enriched with mesenchymal progenitor or stem cells contains at least about 0.1%, 0.5%, 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 50%, or 75% mesenchymal progenitor or stem cells. In this regard, the term “population of cells enriched with mesenchymal progenitor or stem cells” would be interpreted as providing explicit support to the term “population of cells containing X% mesenchymal progenitor or stem cells,” where X% is one of the percentages enumerated herein. Mesenchymal progenitor or stem cells may, in some examples, form clonal colonies, e.g., CFU-F (fibroblasts), or a subset thereof (e.g., 50%, 60%, 70%, 70%, 90%, or 95%) may possess this activity.

[0073] In one example of this disclosure, the mesenchymal lineage progenitor or stem cell is a mesenchymal stem cell (MSC). The MSC may be a homogeneous composition or a mixed cell population enriched with MSCs. A homogeneous MSC composition may be obtained by culturing adherent bone marrow or periosteal cells, and the MSC may be identified by a unique cell surface marker identified by a distinctive monoclonal antibody. A method for obtaining a cell population enriched with MSCs is described, for example, in U.S. Patent No. 5,486,359. Alternative sources of MSCs include, but are not limited to, blood, skin, umbilical cord blood, muscle, fat, bone, and perichondrium. In one example, the MSCs are allogeneic. In one example, the MSCs are cryopreserved. In another example, the MSCs are cultured, grown, and then cryopreserved.

[0074] In another example, mesenchymal lineage progenitor or stem cells are CD29+, CD54+, CD73+, CD90+, CD102+, CD105+, CD106+, CD166+, MHC1+ MSCs.

[0075] Isolated or concentrated mesenchymal lineage precursors or stem cells can be grown by culture in vitro. Isolated or concentrated mesenchymal lineage precursors or stem cells can be cryopreserved, thawed, and then grown by culture in vitro.

[0076] In one example, isolated or concentrated mesenchymal lineage progenitor or stem cells are divided into 50,000 live cells / cm³. 2 Seeds are then seeded onto culture medium (serum-free or serum-supplemented), such as alpha minimal essential medium (αMEM) supplemented with 5% fetal bovine serum (FBS) and glutamine, and allowed to adhere to the culture vessel overnight at 37°C and 20% O2. Subsequently, the culture medium is changed and / or modified as needed, and the cells are cultured for a further 68–72 hours at 37°C and 5% O2.

[0077] As those skilled in the art will understand, cultured mesenchymal progenitor or stem cells have a different phenotype from in vivo cells. For example, in one embodiment, they express one or more of the following markers: CD44, NG2, DC146, and CD140b. Cultured mesenchymal progenitor or stem cells are also biologically different from in vivo cells, and their proliferation rate is faster than that of generally acyclic (quiescent) cells in vivo.

[0078] In one example, a population of cells is enriched from a cell preparation containing selectable forms of STRO-1+ cells. In this context, the term “selectable forms” would be understood to mean that the cells express a marker (e.g., a cell surface marker) that allows for the selection of STRO-1+ cells. The marker can, but not necessarily, be STRO-1. For example, cells expressing STRO-2 and / or STRO-3(TNAP) and / or STRO-4 and / or VCAM-1 and / or CD146 and / or 3G5 (e.g., mesenchymal progenitor cells), as described and / or illustrated herein, also express STRO-1 (and may be STRO-1bright). Therefore, even if cells are indicated as STRO-1+, this does not mean that the cells are selected solely by STRO-1 expression. In one example, cells are selected based on at least STRO-3 expression, for example, they are STRO-3+(TNAP+).

[0079] References to the selection of cells or populations do not necessarily require selection from a specific tissue source. As described herein, STRO-1+ cells can be selected, isolated, or enriched from a wide variety of sources. Thus, in some examples, these terms provide support for selection from any tissue containing STRO-1+ cells (e.g., mesenchymal progenitor cells), or angiogenic tissue, or pericytes (e.g., STRO-1+ pericytes), or any one or more of the tissues enumerated herein.

[0080] For example, the cells used in this disclosure express one or more markers individually or collectively selected from the group consisting of TNAP+, VCAM-1+, THY-1+, STRO-2+, STRO-4+(HSP-90β), CD45+, CD146+, 3G5+, or any combination thereof.

[0081] "Individually" means that the listed markers or groups of markers are individually covered by this disclosure, and that even if individual markers or groups of markers are not separately listed herein, the claims described herein may define such markers or groups of markers separately and divisibly from one another.

[0082] "Collectively" means that the Disclosure encompasses any number or combination of the enumerated markers or groups of markers, and that even if such multiple or combined markers or groups of markers are not specifically enumerated herein, the claims described herein may define such combinations or partial combinations separately and divisibly from any other markers or groups of markers.

[0083] As used herein, the term "TNAP" is intended to encompass all isoforms of tissue-nonspecific alkaline phosphatase. For example, the term includes the liver isoform (LAP), bone isoform (BAP), and kidney isoform (KAP). In one example, TNAP is BAP. In one example, as used herein, TNAP refers to a molecule capable of binding to STRO-3 antibody produced by a hybridoma cell line deposited with ATCC on December 19, 2005, under deposit acceptance number PTA-7282, in accordance with the provisions of the Baptist Convention.

[0084] Furthermore, in one example, STRO-1+ cells can induce clonal CFU-F.

[0085] For example, the majority of STRO-1+ cells can differentiate into at least two different germ cell lines. Non-limiting examples of lineages that can specialize into STRO-1+ cells include bone progenitor cells; hepatocyte progenitor cells that are pluripotent to bile duct epithelial cells and hepatocytes; neuronally specialized cells that can produce glial cell precursors that evolve into oligodendrocytes and astrocytes; neuronal precursors that evolve into neurons; cardiomyocyte and cardiomyocyte precursors; glucose-responsive insulin-secreting pancreatic beta cell lines. Other lineages, though not limiting, include odontoblasts, dentin-producing cells and chondrocytes, as well as progenitor cells of the following: retinal pigment epithelial cells, fibroblasts, skin cells such as keratinocytes, dendritic cells, hair follicle cells, tubular epithelial cells, smooth and skeletal muscle cells, testicular progenitor cells, vascular endothelial cells, tendons, ligaments, cartilage, adipocytes, fibroblasts, bone marrow stroma, cardiomyocytes, smooth muscle, skeletal muscle, pericytes, vascular cells, epithelial cells, glial cells, neuroadipocytes, astrocytes and oligodendrocytes.

[0086] For example, mesenchymal progenitor or stem cells may be obtained from a single donor, or from multiple donors if the donor samples or mesenchymal progenitor or stem cells are later pooled and cultured.

[0087] The mesenchymal progenitor or stem cells contained herein may be cryopreserved before administration to a subject. For example, the mesenchymal progenitor or stem cells may be cultured, cryopreserved, and then administered to a subject.

[0088] In one example, the disclosure includes mesenchymal progenitor or stem cells, as well as their offspring, soluble factors derived therefrom, and / or extracellular vesicles isolated therefrom. In another example, the disclosure includes mesenchymal progenitor or stem cells and extracellular vesicles isolated therefrom. For example, the mesenchymal progenitor cell lineage or stem cells of the disclosure can be cultured and grown for a period of time under conditions suitable for the secretion of extracellular vesicles into the cell culture medium. The secreted extracellular vesicles can then be obtained from the culture medium for use in therapy.

[0089] As used herein, the term “extracellular vesicles” refers to liquid particles spontaneously released from cells, ranging in size from approximately 30 nm to 10 microns, although their size is typically less than 200 nm. They are released by cells (e.g., mesenchymal stem cells, STRO-1 + It may contain proteins, nucleic acids, lipids, metabolites, or organelles from cells.

[0090] As used herein, the term “exosome” refers to a type of extracellular vesicle, typically ranging in size from approximately 30 nm to 150 nm, derived from the endosomal compartment of mammalian cells, which migrates from that compartment to the cell membrane and is released. It may contain nucleic acids (e.g., RNA, microRNA), proteins, lipids, and metabolites, and plays a role in intercellular communication by being secreted from one cell and taken up by other cells to deliver its cargo.

[0091] Cell culture and proliferation In one example, mesenchymal progenitor or stem cells are cultured and grown. The culture medium of "cultured" mesenchymal progenitor or stem cells is distinguished from recently isolated cells in that they have been cultured and subcultured (i.e., passed through) in cell culture medium. In one example, the cultured mesenchymal progenitor or stem cells have been cultured for approximately 4 to 10 subcultures. In another example, the mesenchymal progenitor or stem cells have been cultured for at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 subcultures. For example, the mesenchymal progenitor or stem cells may have been cultured for at least 5 subcultures. In one example, the mesenchymal progenitor or stem cells may have been cultured for at least 5 to 10 subcultures. In another example, the mesenchymal progenitor or stem cells may have been cultured for at least 5 to 8 subcultures. In another example, the mesenchymal progenitor or stem cells may have been cultured for at least 5 to 7 subcultures. In another example, the mesenchymal progenitor or stem cells may have been cultured for more than 10 subcultures. In another example, the mesenchymal progenitor or stem cells may have been cultured and grown for more than seven passages. In these examples, the stem cells may be cultured and then cryopreserved to obtain an intermediate cryopreserved MLPSC population. In one example, the composition of this disclosure is prepared from the intermediate cryopreserved MLPSC population. For example, the intermediate cryopreserved MLPSC population may be further cultured and then administered as further described below. Thus, in one example, the mesenchymal progenitor or stem cells are cultured and cryopreserved. In one embodiment of these examples, the mesenchymal progenitor or stem cells may be obtained from a single donor, or from multiple donors if the donor samples or mesenchymal progenitor or stem cells are subsequently pooled and cultured. In one example, the culture and growth process is -i. Increase the number of living cells by subculturing to obtain at least approximately 1 billion living cells. This includes, and the subculturing involves establishing a primary culture of an isolated mesenchymal progenitor or stem cell, and then stepwise establishing a first non-primary (P1) culture of the isolated mesenchymal progenitor or stem cell from the previous culture; -ii. Propagating isolated mesenchymal progenitor or stem cell P1 cultures by subculturing to obtain second nonprimary (P2) cultures of mesenchymal progenitor or stem cells; and -iii. Preparing and cryopreserving mesenchymal progenitor or stem cell intermediate preparations obtained from P2 cultures of mesenchymal progenitor or stem cells; and -iv. Thaw the frozen mesenchymal lineage precursor or stem cell intermediate preparation and propagate the mesenchymal lineage precursor or stem cell intermediate preparation by subculturing. Includes.

[0092] In one example, the amplified mesenchymal lineage precursor or stem cell preparation is -i. Less than approximately 0.75% of CD45+ cells; -ii. At least approximately 95% of CD105+ cells; -iii. At least approximately 95% of CD166+ cells It has an antigen profile and activity profile that include the following:

[0093] For example, proliferated mesenchymal lineage precursors or stem cell preparations can inhibit IL2Ra expression by CD3 / CD28-activated PBMCs by at least approximately 30% compared to the control.

[0094] In one example, the cultured mesenchymal progenitor or stem cells were cultured for approximately 4 to 10 passages, and the mesenchymal progenitor or stem cells were cryopreserved after at least 2 or 3 passages and then further cultured. In another example, the mesenchymal progenitor or stem cells were cultured for at least 1, at least 2, at least 3, at least 4, or at least 5 passages, cryopreserved, and then further cultured for at least 1, at least 2, at least 3, at least 4, or at least 5 passages, after which they were administered or further cryopreserved.

[0095] For example, the majority of mesenchymal lineage progenitors or stem cells in the compositions of the present disclosure are of roughly the same generation number (i.e., they are approximately the first, second, third, or fourth division products of each other). For example, the average number of divisions in the compositions of the present invention is approximately 20 to 25 divisions. For example, the average number of divisions in the compositions of the present invention is approximately 9 to 13 divisions (e.g., approximately 11 or 11.2 divisions) resulting from the primary culture, plus approximately 1, 2, 3, or 4 divisions per passage (e.g., approximately 2.5 divisions per passage). In the compositions of the present invention, the exemplary mean cell divisions, when occurring over approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 passages, are approximately 13.5, 16, 18.5, 21, 23.5, 26, 28.5, 31, 33.5, and 36, respectively.

[0096] The processes of isolating and in vitro proliferation of mesenchymal lineage precursors or stem cells can be carried out using any equipment and cell handling methods known in the art. Various culture proliferation embodiments of this disclosure employ steps that require cell manipulation, such as seeding, nutrient supply, dissociation of adherent cultures, or washing. Any step that manipulates cells has the potential to damage them. Although mesenchymal lineage precursors or stem cells can generally withstand a certain amount of damage during preparation, it is preferable to manipulate the cells with handling procedures and / or equipment that appropriately carry out a given step(s) while minimizing damage to the cells.

[0097] For example, mesenchymal lineage precursors or stem cells are washed in an apparatus comprising a cell source bag, a wash solution bag, a recirculation wash bag, a swirling membrane filter with inlet and outlet ports, a filtrate bag, a mixing area, a final product bag for washed cells, and appropriate tubing, such as that described in US6,251,295, which is incorporated herein by reference.

[0098] In one example, the mesenchymal progenitor or stem cell composition according to the present disclosure is 95% homogeneous with respect to being CD105 positive and CD166 positive, and being CD45 negative. In one example, this homogeneity persists through in vitro proliferation, i.e., over multiple population doublings. In one example, the composition comprises at least one therapeutic dose of mesenchymal progenitor or stem cells, and the mesenchymal progenitor or stem cells comprise less than about 1.25% CD45+ cells, at least about 95% CD105+ cells, and at least about 95% CD166+ cells. In one example, this homogeneity persists even after cryopreservation and thawing, and the cells also have an overall viability of about 70% or more.

[0099] In one example, the composition of the present disclosure comprises mesenchymal progenitor or stem cells that express a significant level of TNFR1, e.g., more than 13 pg per million mesenchymal progenitor or stem cells. In one example, this phenotype is stable throughout in vitro proliferation and cryopreservation. In one example, the expression of TNFR1 at a level of about 13 to about 179 pg (e.g., about 13 pg to about 44 pg) per million mesenchymal progenitor or stem cells is associated with desirable therapeutic potential that persists over in vitro proliferation and cryopreservation.

[0100] In one example, tumor necrosis factor receptor 1 (TNFR1) is expressed in cultured and expanded mesenchymal progenitor or stem cells in an amount of at least 110 pg / ml. For example, TNFR1 can be expressed in mesenchymal progenitor or stem cells in an amount of at least 150 pg / ml, or at least 200 pg / ml, or at least 250 pg / ml, or at least 300 pg / ml, or at least 320 pg / ml, or at least 330 pg / ml, or at least 340 pg / ml, or at least 350 pg / ml.

[0101] In one example, TNFR1 is expressed in mesenchymal progenitor or stem cells in an amount of at least 13 pg / 10 6 cells. For example, TNFR1 can be expressed in mesenchymal progenitor or stem cells in an amount of at least 15 pg / 10 6 cells, or at least 20 pg / 106 Cells, or at least 25 pg / 10 6 Cells, or at least 30 pg / 10 6 Cells, or at least 35 pg / 10 6 Cells, or at least 40 pg / 10 6 Cells, or at least 45 pg / 10 6 Cells, or at least 50 pg / 10 6 Expression is determined by the amount of cells.

[0102] In another example, the mesenchymal lineage progenitors or stem cells disclosed herein inhibit IL-2Rα expression on T cells. In one example, the mesenchymal lineage progenitors or stem cells may inhibit IL-2Rα expression by at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, or at least about 60%.

[0103] For example, the composition of the present disclosure comprises at least one therapeutic dose of mesenchymal lineage progenitor or stem cells, which may contain, for example, at least about 100 million cells or about 125 million cells.

[0104] Cell modification The mesenchymal progenitor or stem cells disclosed herein may be modified to suppress cell lysis upon administration. Antigen modification may induce immunological non-responsiveness or intolerance, thereby preventing the induction of effector stages of the immune response (e.g., cytotoxic T cell generation, antibody production, etc.) that ultimately carry out the rejection of foreign cells in a normal immune response. Antigens that may be modified to achieve this goal include, for example, MHC class I antigens, MHC class II antigens, LFA-3, and ICAM-1.

[0105] Mesenchymal progenitor or stem cells may be genetically modified to express proteins important for the differentiation and / or maintenance of rhabdomyomuscular cells. Exemplary proteins include growth factors (TGF-β, insulin-like growth factor 1 (IGF-1), FGF), myogenic factors (e.g., myoD, myogenin, myogenic factor 5 (Myf5), myogenic regulator (MRF)), transcription factors (e.g., GATA-4), cytokines (e.g., cardiotropin-1), members of the neuregulin family (e.g., neuregulin 1, 2, and 3), and homeobox genes (e.g., the Csx, tinman, and NKx families).

[0106] Composition of the present disclosure In one example of this disclosure, mesenchymal progenitor or stem cells and / or their offspring and / or soluble factors derived therefrom are administered in the form of a composition. In one example, such a composition comprises a pharmaceutically acceptable carrier and / or excipient. Thus, in one example, the composition of this disclosure may comprise cultured mesenchymal progenitor or stem cells.

[0107] The terms “carrier” and “excipient” refer to compositions conventionally used in the art to facilitate the storage, administration, and / or biological activity of active compounds (see, for example, Remington's Pharmaceutical Sciences, 16th Ed., Mac Publishing Company (1980)). A carrier may also mitigate any undesirable side effects of the active compound. A suitable carrier is, for example, stable, and, for example, non-reactive with other components in the carrier. In one example, a carrier does not cause significant local or systemic side effects in the recipient at the dosage and concentration used for therapeutic purposes.

[0108] Suitable carriers for this disclosure include conventionally used ones such as water, physiological saline, aqueous dextrose, lactose, Ringer's solution, and buffer solutions, while hyaluronan and glycol are exemplary liquid carriers, particularly for solutions (when isotonic). Suitable pharmaceutical carriers and excipients include starch, cellulose, glucose, lactose, sucrose, gelatin, malt, rice, wheat flour, chalk, silica gel, magnesium stearate, sodium stearate, glycerol monostearate, sodium chloride, glycerol, propylene glycol, water, and ethanol.

[0109] In another example, the carrier is, for example, a culture medium composition in which cells are grown or suspended. For example, such a culture medium composition does not induce any side effects in the subject to which it is administered.

[0110] The exemplary carriers and excipients do not adversely affect the viability of cells and / or their ability to reduce, prevent, or delay metabolic syndromes and / or obesity.

[0111] In one example, the carrier or excipient provides buffering activity to maintain cells and / or soluble factors at a suitable pH, thereby enabling biological activity, for example, the carrier or excipient is phosphate-buffered saline (PBS). PBS is an attractive carrier or excipient because it interacts minimally with cells and factors and allows for their rapid release, in which case the compositions of the present disclosure can be prepared as liquids for direct application, for example, by injection, into the bloodstream, or into tissue, or into areas surrounding or adjacent to tissue.

[0112] Mesenchymal lineage precursors or stem cells and / or their offspring and / or soluble factors derived therefrom may be incorporated or embedded in scaffolds that are recipient-compatible and degrade into products that are harmless to the recipient. These scaffolds provide support and protection to cells that will be transplanted into the recipient. Natural and / or synthetic biodegradable scaffolds are examples of such scaffolds.

[0113] A wide variety of scaffolds can be successfully used in the practice of this disclosure. Exemplary scaffolds include, but are not limited to, biodegradable biological scaffolds. Examples of naturally occurring biodegradable scaffolds include collagen, fibronectin, and laminin scaffolds. Suitable synthetic materials for cell transplantation scaffolds must be able to broadly support cell growth and cellular function. Such scaffolds may also be absorbable. Suitable scaffolds include polyglycolic acid scaffolds (such as those described by Vacanti, et al. J. Ped. Surg. 23:3-9 1988, Cima, et al. Biotechnol. Bioeng. 38:145 1991, and Vacanti, et al. Plast. Reconstr. Surg. 88:753-9 1991); or synthetic polymers, such as polyacid anhydrides, polyorthoesters, and polylactic acid.

[0114] In another example, mesenchymal progenitor or stem cells and / or their offspring and / or soluble factors derived therefrom may be administered within a gel scaffold (e.g., Gelfoam from Upjohn Company).

[0115] The compositions described herein may be administered alone or in mixtures with other cells. Different types of cells may be mixed with the compositions of this disclosure immediately before or shortly before administration, or they may be co-cultured together for a period of time prior to administration.

[0116] For example, the composition contains an effective amount or a therapeutic or prophylactic effective amount of mesenchymal lineage precursor or stem cells and / or their offspring and / or soluble factors derived therefrom. For example, the composition contains about 1 × 10⁻⁶ 5 Individual stem cells ~ approximately 1 x 10⁻⁶ 9 A single stem cell, or approximately 1.25 × 10⁶ 3 Individual stem cells ~ approximately 1.25 × 10⁻¹⁴ 7 Contains 1 stem cell / kg (for an 80kg subject). The exact amount of cells administered depends on various factors, including the subject's age, weight, and sex, as well as the degree and severity of the disorder being treated.

[0117] For example, 50 x 10 6 ~200×10 7 Administer individual cells. In other examples, 60 × 10 6 ~200×10 6 Individual cells, or 75 × 10 6 ~150×10 6 Individual cells are administered. For example, 75 × 10 6 Administer individual cells. In another example, 150 × 10 6 Individual cells are administered.

[0118] For example, the composition is 5.00 × 10 6 It contains more than 10 live cells / mL. In another example, the composition contains 5.50 × 10 6 It contains more than 10 live cells / mL. In another example, the composition contains 6.00 × 10 6 It contains more than 10 live cells / mL. In another example, the composition contains 6.50 × 10 6 It contains more than 10 live cells / mL. In another example, the composition contains 6.68 × 10 6 Contains more than one live cell / mL.

[0119] For example, mesenchymal lineage progenitors or stem cells constitute at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, and at least about 99% of the cell population of the composition.

[0120] The compositions of this disclosure can be cryopreserved. Cryopreservation of mesenchymal lineage precursors or stem cells can be carried out using slow-freezing methods or “fast” freezing protocols known in the art. Preferably, the cryopreservation method maintains similar phenotypes, cell surface markers, and growth rates of the cryopreserved cells compared to unfrozen cells.

[0121] Frozen compositions may contain a cryopreservation solution. The pH of the cryopreservation solution is typically 6.5 to 8, preferably 7.4.

[0122] The cryopreservation solution may contain a sterile, non-pyrogenic isotonic solution, such as PlasmaLyte A®. 100 mL of PlasmaLyte A® contains 526 mg of sodium chloride, USP (NaCl); 502 mg of sodium gluconate (C6H) 11 It contains 368 mg of sodium acetate trihydrate (USP(C2H3NaO2·3H2O)), 37 mg of potassium chloride (USP(KCl)), and 30 mg of magnesium chloride (USP(MgCl2·6H2O)). It does not contain antibacterial agents. The pH is adjusted with sodium hydroxide. The pH is 7.4 (6.5-8.0).

[0123] The cryopreservation solution may contain Profreeze®. The cryopreservation solution may further, or otherwise, contain a culture medium, such as αMEM.

[0124] Typically, cryoprotective agents, such as dimethyl sulfoxide (DMSO), are added to cryopreservation solutions to facilitate freezing. Ideally, the cryoprotective agent should be non-toxic to cells and patients, non-antigenic, chemically inactive, provide high viability after thawing, and allow transplantation without washing. However, DMSO, the most commonly used cryoprotective agent, exhibits some cytotoxicity. Hydroxyethyl starch (HES) may be used as an alternative or in combination with DMSO to mitigate the cytotoxicity of the cryopreservation solution.

[0125] The cryopreservation solution may contain one or more of DMSO, hydroxyethyl starch, human serum components, and other protein extenders. In one example, the cryopreservation solution contains about 5% human serum albumin (HSA) and about 10% DMSO. The cryopreservation solution may further contain one or more of methylcellulose, polyvinylpyrrolidone (PVP), and trehalose.

[0126] In one embodiment, cells are suspended in 42.5% Profreeze® / 50% αMEM / 7.5% DMSO and cooled in a speed-controlled freezer.

[0127] Frozen compositions can be thawed and administered directly to the subject, or added to another solution, such as a solution containing HA. Alternatively, frozen compositions can be thawed and the mesenchymal lineage precursor or stem cells can be resuspended in an alternative carrier before administration.

[0128] For example, the cell composition of the Disclosure may comprise Plasma-Lyte A, dimethyl sulfoxide (DMSO), and human serum albumin (HSA). For instance, the composition of the Disclosure may comprise a solution of Plasma-Lyte A (70%), DMSO (10%), and HSA (25%), wherein the HSA solution may comprise 5% HSA and 15% buffer.

[0129] In one example, the compositions described herein may be administered as a single dose.

[0130] In some cases, the compositions described herein may be administered in multiple doses. For example, doses of at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten times.

[0131] In one example, mesenchymal progenitor or stem cells may be cultured and grown before being administered to a subject. Various cell culture methods are known in the art. In one example, mesenchymal progenitor or stem cells are cultured and grown for approximately 4 to 10 passages. In another example, mesenchymal progenitor or stem cells are cultured and grown for at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 passages. In yet another example, mesenchymal progenitor or stem cells are cultured and grown for at least 5 passages. In these examples, stem cells may be cultured and grown before being cryopreserved.

[0132] In one example, mesenchymal progenitor or stem cells are cultured and grown in serum-free medium before administration.

[0133] In some cases, cells are housed in containers that do not allow cells to exit into the target's circulatory system, but allow factors secreted by the cells to enter the circulatory system. In this way, soluble factors can be administered to the target by allowing cells to secrete the factors into the target's circulatory system. Such containers can also be implanted at the site of the target to increase local levels of soluble factors, for example, in or near the gastrointestinal wall.

[0134] In one example, mesenchymal progenitor or stem cells may be administered to the wall of the target gastrointestinal tract. In another example, mesenchymal progenitor or stem cells are administered locally to the luminal wall of the gastrointestinal tract. In yet another example, mesenchymal progenitor or stem cells may be administered locally. For example, mesenchymal progenitor or stem cells may be administered within the wall of the target gastrointestinal tract. For example, mesenchymal progenitor or stem cells may be administered submucosa to the wall of the target gastrointestinal tract. In one example, mesenchymal progenitor or stem cells may be administered to the site of inflammation in the wall of the target gastrointestinal tract. For example, mesenchymal progenitor or stem cells may be administered within the site of inflammation in the wall of the target gastrointestinal tract. In these examples, the site of inflammation may be confirmed endoscopically before administration. For example, endoscopic confirmation may be based on visual inspection by a trained physician and / or histological analysis of endoscopic biopsy material. In one example, the wall of the gastrointestinal tract is the intestinal wall. For example, mesenchymal progenitor or stem cells may be administered to the colon wall and / or intestinal wall of the target. In one example, mesenchymal progenitor or stem cells may be administered directly submucosa to the colon wall and / or intestinal wall of the target. In one example, the mesenchymal progenitor or stem cells may be administered directly submucosa to the colon and / or rectal wall of the subject. In another example, the compositions of this disclosure may be administered by intraluminal injection.

[0135] In various cases, the dose of cells may need to be administered to multiple sites in the target gastrointestinal tract. The number of administration sites required per dose may depend on the number of cells being administered. For example, a dose of approximately 75 million cells may need to be administered to 5 sites in the gastrointestinal tract. In another example, approximately 150 million cells may need to be administered to 15 sites in the gastrointestinal tract. In yet another example, the dose may need to be administered to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more sites in the target gastrointestinal tract. In one embodiment of these examples, the dose is administered to the walls of the target cecum, proximal transverse colon, distal transverse colon, descending colon, sigmoid colon, and rectum. In this embodiment, the dose may be administered to one, two, three, four, five, or more sites on the walls of the target cecum, proximal transverse colon, distal transverse colon, descending colon, sigmoid colon, and rectum.

[0136] In one example, mesenchymal progenitor or stem cells are administered via endoscopy. For instance, mesenchymal progenitor or stem cells may be injected submucosa into the target gastrointestinal wall via endoscopy. In another example, endoscopy is used to visually identify the site of inflammation before directly administering the mesenchymal progenitor or stem cells to the site of inflammation.

[0137] Those skilled in the art will recognize that numerous variations and / or modifications can be made to the above embodiments without departing from the broad overall scope of this disclosure. Accordingly, the embodiments of the present invention should be considered illustrative and not restrictive in all respects.

[0138] The following of specific examples should be interpreted as merely illustrative and in no way as limiting the remainder of this disclosure. Those skilled in the art will likely be able to make the most of the present invention based on the description herein without further effort.

[0139] This application claims priority to Australian Provisional Patent Application No. 2020900742, filed on 11 March 2020, the entire contents of which are incorporated herein by reference. [Examples]

[0140] Adult allogeneic bone marrow-derived stem cells (MSCs) cultured and grown in vitro for the treatment of medically refractory Crohn's colitis. composition The composition contains cultured mesenchymal stromal cells (ceMSCs) isolated from the bone marrow of a healthy adult donor. The final composition is 6.68 × 10⁻¹⁶ of Plasma-Lyte A (70%), dimethyl sulfoxide (DMSO, 10%), and human serum albumin (HSA) (25%) solution (20%, containing 5% HSA and 15% buffer). 6 Contains ceMSCs formulated at a viable cell concentration of cells / mL or higher. Each dose vial contains 3.8 mL of cryopreserved cell suspension (total cells per vial ≥ 25 × 10⁶). 6 pieces).

[0141] the purpose 1st purpose To determine the safety of endoscopic delivery of MSCs (Medical Stem Cells) that are allogeneic, allogeneic, bone marrow-derived, and grown in vitro for the treatment of medically refractory Crohn's colitis.

[0142] Secondary purpose To preliminarily evaluate the efficacy of luminal healing induced by endoscopic delivery of MSCs (Methodoxylated Myeloid Stem Cells) grown in vitro from allogeneic bone marrow, for the treatment of medically refractory Crohn's colitis.

[0143] Clinically - A decrease in the number of bowel movements in a 24-hour period; - Decreased blood in the stool; - Decreased serum levels of C-reactive protein; - Decrease in Crohn's disease activity index (CDAI score).

[0144] radiographically - Cross-sectional imaging using magnetic resonance (MR) enterography.

[0145] Endoscopy and Histopathology - Improvement of the Crohn's disease plain endoscopy score (SES-CD) in colonoscopy; - Improved histological healing in endoscopic biopsy or surgical pathology compared to endoscopic biopsy performed before MSC delivery.

[0146] subject Twenty-four patients will be treated for luminal disease with doses of either 75 million MSCs (n=12; 8 treated, 4 controls) or 150 million MSCs (n=12; 8 treated, 4 controls). MSCs will be delivered into the submucosa of the colon wall by targeted endoscopic delivery in the operating room. MSC dose escalation will be performed for two dose groups of patients, who will be assigned four times in a 2:1 treatment:control ratio (8:4 ratio for each dose group). Twelve patients will receive 75 million cells, and twelve patients will receive 150 million cells.

[0147] Inclusion criteria: - Men and women aged 18-75; - Crohn's colitis with medically refractory symptoms that has persisted for at least 6 months after one anti-TNF therapy failed, and the next step is less than a complete colectomy or expansion in medical management; Exposure to corticosteroids, 5-ASA drugs, thiopurines, methotrexate, anti-TNF therapy, anti-integrins, and anti-interleukins is acceptable, however, a two-week washout period will be implemented for corticosteroids, 5-ASA, thiopurines, and methotrexate, and a four-week washout period for any biological therapy. - No colonic dysplasia or malignant tumors within 30 days of MSC delivery, as ruled out by colonoscopy; - At least one anti-TNF treatment must have failed or there must be a contraindication to biological therapy.

[0148] Key evaluation criteria The primary endpoint of this study is to determine the safety and feasibility of endoscopic injection of mesenchymal stem cells (MSCs) for the treatment of Crohn's colitis.

[0149] Secondary evaluation items Clinical and endoscopic remission: - Clinical cure: ○ Normalization of CRP to less than 2.87 mg per liter; ○ CDAI levels decreased to below 150. - Radiographic healing: ○ MR enterography accompanied by improvement of inflammation. - Endoscopic healing: ○ Absence of mucosal ulcers and an SES-CD score of 0-5.

[0150] Clinical and endoscopic responses: - Clinical cure: ○ A reduction or normalization of CRP by more than 50%; ○ A greater decline than a perfect score of 100 on the CDAI. - Radiographic healing: ○ MR enterography accompanied by improvement of inflammation. - Endoscopic healing: ○ SES-CD scores that decreased by more than 50% or by 5-10.

[0151] Partial clinical and endoscopic responses: - Clinical cure: ○ A reduction of more than 25% in CRP; ○ Decrease in CDAI scores below 100. - Radiographic healing: ○ MR enterography accompanied by improvement of inflammation. - Endoscopic healing: ○ SES-CD scores decreased by more than 25% but less than 50%, or decreased to a score of 10-15.

[0152] Lack of effectiveness: - Clinical cure: ○ No improvement. - Radiographic healing: ○ MR enterography without the resolution of inflammation. - Endoscopic healing: ○ No improvement has been made to SES-CD.

[0153] Treatment plan Participants will be randomized to receive either 75 million cells or 150 million cells (25 million cells per 3.8 mL of Plasma-Lyte® A supplemented with human serum albumin (5%) and dimethyl sulfoxide (10%)) after being compared to a control receiving saline. The first cohort will receive a dose of 75 million cells from MSCs or saline, and the next cohort of 12 patients will receive 150 million cells from MSCs or saline. For the 75 million cell dose, 75 million cells will be suspended in 11.4 mL and delivered at 1.9 mL each to the cecum, proximal transverse colon, distal transverse colon, descending colon, sigmoid colon, and rectum. For a dosage of 150 million cells, 22.8 mL will be delivered as three injections (1.3 mL each) at the 12, 6, and 9 o'clock positions of the colon / rectal wall at the locations mentioned above. An adult colonoscope will be used during the procedure. A 23-gauge disposable sclerotherapy needle will be used to deliver the cells into the submucosa, which will be revealed by small, raised blisters within the submucosa. At each injection site, after the MSC injection, 0.5 mL of Plasma-Lyte A will be injected to flush out any remaining MSCs from the sclerotherapy needle.

[0154] First visit (screening / baseline): MSC treatment target The patient will complete the following tests and procedures during this visit: - Eligibility for a gastrointestinal or surgical consultation regarding a change in biological therapy or partial colectomy for medically refractory Crohn's colitis (inclusion / exclusion checklist); - Written informed consent; - Wash-off periods for the following drug therapies: For immunomodulatory therapy including 5-ASA, corticosteroids, azathioprine, methotrexate, and 6-mercaptopurine, the treatment period is 2 weeks. ○ For biological agents, 4 weeks: anti-TNF, anti-integrin, and interleukin. - Medical and surgical history; - Abdominal examination and a full body examination including vital signs (BP, pulse rate, respiratory rate, and body temperature); - Crohn's disease activity index (CDAI) score; - You will obtain an Inflammatory Bowel Disease Questionnaire (IBDQ) score; - Magnetic resonance enterography (MRE) if it has not been performed previously within the past 30 days; - Colonoscopy with biopsy if it has not been performed previously within the past 30 days; - Cytomegalovirus enteritis (CMV enteritis) should be ruled out; - Crohn's disease simple endoscopic score (SES-CD); - Laboratory tests including the following: ○ Urine pregnancy tests will only be performed on women who are pregnant or have a chance of becoming pregnant (WOCBP). ○ Liver function tests, AST / ALT ○ Acute hepatitis panel ○ Human immunodeficiency virus (HIV) ○ Complete blood cell count (CBC) ○ Comprehensive Metabolic Panel (CMP) ○ Prealbumin ○ C-reactive protein (CRP) ○ Erythrocyte sedimentation rate (ESR) ○ Clostridium difficile (C.diff) in stool ○ Calprotectin in feces ○ Concomitant medications - Adverse events (including changes in medical or surgical management; any side effects of drug therapy and any postoperative complications should be reported).

[0155] Second visit (Day 0 - Treatment) Within seven days prior to the second visit, the patient will complete the following tests and procedures during this visit: - Abdominal examination and a full body examination including vital signs; - Inflammatory Bowel Disease Questionnaire (IBDQ) score; - CDAI score; - Randomization to either a treatment group or a control group; - Colonoscopy (to be used for administering MSCs); - Concomitant medications; - Adverse events; - Delivery of MSC or physiological saline.

[0156] Third visit (Day 1) The following tests and procedures will be completed the following day: - Abdominal examination and a full body examination including vital signs; - Medical and surgical history since the last visit; - CDAI score; - Concomitant medications; - Adverse events.

[0157] Fourth visit (week 4 + / - 3 days) The following inspections and procedures will be completed during this visit: - Abdominal examination and a full body examination including vital signs; - Medical and surgical history since the last visit; - Flexible sigmoid colon endoscopy with biopsy; - Inflammatory Bowel Disease Questionnaire (IBDQ) score; - CDAI score; - Concomitant medications; - Adverse events.

[0158] Fifth visit (6th week + / - 3 days) The following inspections and procedures will be completed during this visit: - Abdominal examination and a full body examination including vital signs; - Medical and surgical history since the last visit; - Flexible sigmoid colon endoscopy with biopsy; - Inflammatory Bowel Disease Questionnaire (IBDQ) score; - CDAI score; - Concomitant medications; - Adverse events.

[0159] 6th visit (3 months + / - 7 days) The following inspections and procedures will be completed during this visit: - Abdominal examination and a full body examination including vital signs; - Medical and surgical history since the last visit; - Colonoscopy with SES-CD and biopsy; - Inflammatory Bowel Disease Questionnaire (IBDQ) score; - CDAI score; - MRE; - Detailed examination at the laboratory: ○ CBC; ○ CMP; ○ Prealbumin; ○ CRP; ○ ESR; ○ Calprotectin in feces. - Concomitant medications; - Adverse events.

[0160] 7th visit (6th month; + / - 7 days) The following inspections and procedures will be completed during this visit: - Abdominal examination and a full body examination including vital signs; - Medical and surgical history since the last visit; - Inflammatory Bowel Disease Questionnaire (IBDQ) score; - CDAI score; - MRE; - Detailed examination at the laboratory: ○ CBC; ○ CMP; ○ Prealbumin; ○ CRP; ○ ESR; ○ Calprotectin in feces. - Concomitant medications; - Adverse events.

[0161] 8th visit (9th month, + / - 14 days) The following inspections and procedures will be completed during this visit: - Abdominal examination and a full body examination including vital signs; - Medical and surgical history since the last visit; - Inflammatory Bowel Disease Questionnaire (IBDQ) score; - CDAI score; - MRE; - Detailed examination at the laboratory: ○ CBC; ○ CMP; ○ Prealbumin; ○ CRP; ○ ESR; ○ Calprotectin in feces. - Concomitant medications; - Adverse events.

[0162] 9th visit (12th month, + / - 14 days) The following inspections and procedures will be completed during this visit: - Abdominal examination and a full body examination including vital signs; - Medical and surgical history since the last visit; - Inflammatory Bowel Disease Questionnaire (IBDQ) score; - CDAI score; - MRE; - Colonoscopy with biopsy (only in patients undergoing treatment). - Detailed examination at the laboratory: ○ CBC; ○ CMP; ○ Prealbumin; ○ CRP; ○ ESR; ○ Calprotectin in feces. - Concomitant medications; - Adverse events.

[0163] 10th visit (15th month, + / - 14 days) The following inspections and procedures will be completed during this visit: - Abdominal examination and a full body examination including vital signs; - Medical and surgical history since the last visit; - Inflammatory Bowel Disease Questionnaire (IBDQ) score; - CDAI score; - MRE; - Colonoscopy with biopsy of the control arm (12 months after MSC treatment); - Detailed examination at the laboratory: ○ CBC; ○ CMP; ○ Prealbumin; ○ CRP; ○ ESR; ○ Calprotectin in feces. - Concomitant medications; - Adverse events.

[0164] Initial results In a male patient with Crohn's colitis, improved endoscopic and clinical resolution was observed 6 weeks after administration of 75 million MSCs. Three-month follow-up data from this patient were not yet available. The patient's baseline SES-CD was 16 and baseline CDAI was 294.

[0165] In a female patient with Crohn's colitis, improved endoscopic and clinical remission was observed 6 weeks after administration of 150 million MSCs. Endoscopic and clinical evaluations 3 months after the start of therapy revealed that the patient's disease was in remission. The patient's baseline SES-CD was 22.

[0166] In a male patient with ulcerative colitis, improved clinical remission was observed 6 weeks after administration of 150 million MSCs. The patient's baseline Mayo score was 7. After 6 weeks of MSC administration, the patient's Mayo score decreased to 3. Three-month follow-up data from this patient were not yet available.

[0167] Preliminary analysis Small-group analyses were conducted to explore possible identification of the most therapy-responsive patient groups, including Crohn's disease refractory to a single biological agent and refractory to multiple biological agents, as well as diseases with fistulas. These data are summarized in the table and Figure 1 below.

[0168] In patients with moderate to severe active Crohn's disease who had failed conventional therapies of steroids and TNF-alpha inhibitors, early (day 28) remission was clearly demonstrated, and the response rate was statistically significant compared to the control group (p=0.02, Figure 1). When the response rate was compared from day 28 to day 56, evidence of sustained remission was demonstrated.

[0169] The primary endpoint at day 28 in a population treated with a single biological agent. [Table 2]

[0170] The success rate from day 28 to day 56. [Table 3]

Claims

1. A method for treating or preventing inflammatory bowel disease in human subjects who require it, Administering a composition containing mesenchymal lineage progenitor or stem cells (MLPSCs) to the subject. The method comprising the composition being administered to the gastrointestinal wall of the target.

2. The method according to claim 1, wherein the composition is administered to the submucosal layer of the gastrointestinal wall of the target.

3. The method according to claim 1 or 2, wherein the composition is administered to the site of inflammation in the wall of the gastrointestinal tract of the target.

4. The method according to any one of claims 1 to 3, wherein the composition is administered to the colon and / or rectum of the subject.

5. The method according to any one of claims 1 to 4, wherein the composition is administered by intraluminal injection.

6. The method according to any one of claims 1 to 5, wherein the subject is refractory to at least one anti-TNF therapy.

7. The method according to any one of claims 1 to 6, wherein the subject is refractory to steroid immunosuppressants and / or biological therapies.

8. The method according to any one of claims 1 to 7, wherein the inflammatory bowel disease is Crohn's disease or ulcerative colitis.

9. The method according to claim 8, wherein the inflammatory bowel disease is Crohn's disease.

10. The method according to claim 9, wherein Crohn's disease manifests in the rectum and / or colon of the subject.

11. The method according to any one of claims 1 to 10, wherein the subject has a partial clinical and / or endoscopic response at least 28 days after treatment.

12. The method according to any one of claims 1 to 10, wherein the subject has a partial clinical and / or endoscopic response at least 28 to 56 days after treatment.

13. The aforementioned partial clinical response, - A reduction of more than 25% in C-reactive protein (CRP); - A decrease of less than 100 points in the CDA Activity Index (CDAI); - Radiographic healing with improvement of inflammation as evaluated by MR enterography The method according to claim 11 or claim 12, characterized by one or more or all of the above.

14. The aforementioned partial endoscopic response, - Crohn's disease plain endoscopic score (SES-CD) decreased by more than 25%, and SES-CD < 50%; - SES-CD scores for 10-15 The method according to claim 11 or claim 12, characterized by one or both of the above.

15. The method according to any one of claims 1 to 10, wherein the subject has a clinical and / or endoscopic response at least 28 days after treatment.

16. The method according to any one of claims 1 to 10, wherein the subject has a clinical and / or endoscopic response at least 28 to 56 days after treatment.

17. The aforementioned clinical response, - A reduction of more than 50% in CRP; - Normalization of CRP levels; - A decrease of 100 points or more in the CDAI score; - Radiographic healing with improvement of inflammation as evaluated by MR enterography The method according to claim 15 or claim 16, characterized by one or more or all of the above.

18. The aforementioned endoscopic response, - SES-CD decreased by more than 25% but less than 50%; - SES-CD scores for 5-10 The method according to claim 15 or claim 16, characterized by one or both of the above.

19. The method according to any one of claims 1 to 10, wherein the subject is in a state of clinical and / or endoscopic remission at least 28 days after treatment.

20. The method according to any one of claims 1 to 10, wherein the subject is in a state of clinical and / or endoscopic remission at least 28 to 56 days after treatment.

21. The aforementioned clinical remission, - Normalization of CRP to less than 2.87 mg per liter; - Radiographic healing with improvement of inflammation as evaluated by MR enterography The method according to claim 19 or claim 20, characterized by one or both of the above.

22. The aforementioned endoscopic remission, - Absence of mucosal ulcers; - SES-CD score (0-5) The method according to claim 19 or claim 20, characterized by one or both of the above.

23. The method according to any one of claims 1 to 22, wherein the MLPSC is administered into the submucosa of the colon wall of the subject.

24. The method according to any one of claims 1 to 23, wherein the MLPSC is administered to multiple sites on the wall of the target gastrointestinal tract.

25. The method according to any one of claims 1 to 24, wherein the MLPSC is a mesenchymal stem cell (MSC).

26. The method according to any one of claims 1 to 25, wherein the MLPSCs are of the same type but different in nature.

27. The method according to any one of claims 8 to 26, wherein the Crohn's disease is moderate to severe.

28. The method according to any one of claims 1 to 27, wherein the CDAI of the target is greater than 300.

29. The method according to any one of claims 8 to 28, wherein the Crohn's disease is Crohn's disease accompanied by a fistula.

30. The method according to any one of claims 1 to 29, wherein the mesenchymal progenitor or stem cell (MLPSC) is administered by endoscopy.

31. 1 x 10 ７ ~2 x 10 ８ The method according to any one of claims 1 to 29, comprising administering individual cells.

32. 1 × 10 ７ ~2 x 10 ８ The method according to any one of claims 1 to 29, comprising administering individual cells.

33. The method according to any one of claims 1 to 31, wherein the composition further comprises Plasma-Lyte A, dimethyl sulfoxide (DMSO), and human serum albumin (HSA).

34. The method according to any one of claims 1 to 32, wherein the composition further comprises a solution of Plasma-Lyte A (70%), DMSO (10%), and HSA (25%), the HSA solution comprising 5% HSA and 15% buffering agent.

35. The above composition is 6.68 × 10 ６ The method according to any one of claims 1 to 33, comprising more than 1 live cells / mL.

Images