Cell composition containing a viral vector and treatment method

Abstract

The present disclosure relates to cell compositions modified to incorporate recombinant viruses. Such compositions can be used to treat cancer by delivering the viruses to cancer cells.

Description

[Technical Field]

[0001] Cross-reference of related patents This application claims priority to U.S. Provisional Patent Application No. 62 / 882,840, filed 5 August 2019, which is incorporated herein by reference in its entirety.

[0002] Reference to sequence listings submitted electronically via EFS-WEB The contents of the electronically submitted sequence listing (name: 3944_063PC01_SL_ST25.txt; size: 6,885 bytes; creation date: August 3, 2020) are incorporated herein by reference in accordance with 37 CFR §1.52(e)(5).

[0003] This disclosure relates to cell compositions modified to introduce recombinant viruses. Such compositions may be used to treat cancer by delivering viruses to cancer cells. [Background technology]

[0004] Cancer treatment typically includes surgical resection to remove or kill cancer cells, standard chemotherapy, and / or radiation therapy. However, the effectiveness of these treatments is often limited due to the invasiveness of the tumor and / or the resulting damage to healthy tissue. This situation highlights the need for novel therapeutic strategies, one such approach being the use of viruses.

[0005] Oncolytic viruses are viruses that can specifically replicate in cancer cells and destroy them; this property is either intrinsic or genetically engineered. Unfortunately, promising experimental results have not yet led to improved clinical outcomes, which are thought to depend on the complex interactions between the tumor and its microenvironment, the virus, and the host immune system. [Prior art documents] [Patent Documents]

[0006] [Patent Document 1] WO 2004 / 85630 [Patent Document 2] U.S. Patent No. 5,486,359 [Patent Document 3] US 7,615,374 [Patent Document 4] US 2014 / 273211 [Patent Document 5] US 9,453,203 [Patent Document 6] US 6,251,295 [Patent Document 7] WO 2003 / 082200 [Patent Document 8] WO 2003 / 080083 [Patent Document 9] WO 2005 / 086922 [Patent Document 10] WO 2007 / 088229 [Patent Document 11] WO 2008 / 110579 [Patent Document 12] WO 2010 / 108931 [Patent Document 13] WO 2010 / 128182 [Patent Document 14] WO 2013 / 112942 [Patent Document 15] WO 2013 / 116778 [Patent Document 16] WO 2014 / 204814 [Patent Document 17] WO 2015 / 077624 [Patent Document 18] WO 2015 / 166082 [Patent Document 19] WO 2015 / 089280 [Non-Patent Document]

[0007] [Non-Patent Document 1] J. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984) [Non-Patent Document 2] J. Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989) [Non-Patent Document 3] TA Brown (editor), Essential Molecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press (1991) [Non-Patent Document 4] DM Glover and BD Hames (editors), DNA Cloning: A Practical Approach, Volumes 1-4, IRL Press (1995 and 1996) [Non-Patent Document 5] FM Ausubel et al. (eds.), *Current Protocols in Molecular Biology*, Greene Pub. Associates and Wiley-Interscience (1988, includes all the latest information to date). [Non-Patent Document 6] Ed Harlow and David Lane (editors) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, (1988) [Non-Patent Document 7] JE Coligan et al. (eds.), *Current Protocols in Immunology*, John Wiley & Sons (includes all the latest information to date) [Non-Patent Document 8] Hopkins et al., (2013) Science., 6144: pp. 399-402. [Non-Patent Document 9] Barberi,; Plos medicine, Vol 2(6):0554~0559 pages (2005) [Non-licensed Document 10] Vodyanikら、Cell Stem cell, Vol 7:718~728 pages (2010) [Non-licensed Document 11] Obinata M., Cell, Vol 2: 235-244 (1997) [Non-licensed Document 12] Akimov, Stem Cells, Vol 23: 1423~1433 pages [Non-licensed Document 13] Kabara, Laboratory Investigation, Vol 94: Pages 1340~1354 (2014) [Non-licensed Document 14] Fuら, (2003) Molecular Therapy, 7:748~54 pages [Non-licensed Document 15] Guedan, (2012) Gene Therapy, 19:1048~1057 pages [Non-licensed Document 16] Russellら, (2018) Nat Comm., 9:5006 [Non-licensed Document 17] Nakashima, (2014) Journal of Virology, Vol 88:345~353 pages [Non-licensed Document 18] Shenら、(2016) PlosOne 11:e0147173 [Non-licensed Document 19] Justusら、2014 J Vis Exp., 88:51046 [Non-licensed Document 20] Rissら、(2013) Assay Guidance Manual、Last updated July 2016 [Non-licensed Document 21] Dulbecco and Vogt (1953) Cold Spring Harbor Symp. Quant. Biol., 18: pp. 273-279 [Non-Patent Document 22] Johnson et al. (1990) Quantitative Assays for Virus Infectivity. In: Aldovini A. [Non-Patent Document 23] Walker BD (ed.) Techniques in HIV Research. Palgrave Macmillan, London. [Non-Patent Document 24] Remington's Pharmaceutical Sciences, 16th edition, Mac Publishing Company (1980) [Overview of the Initiative] [Problems that the invention aims to solve]

[0008] Therefore, there is a need to improve compositions and methods for delivering viruses to tumor cells. [Means for solving the problem]

[0009] The inventors have identified that mesenchymal lineage progenitor cells or stem cells can be modified to enhance tumor cell death. For example, the inventors have identified that modified mesenchymal lineage progenitor cells or stem cells can deliver payloads to tumor cells in a manner that reduces tumor cell growth. The inventors have also identified modifications that can enhance the migration of modified mesenchymal lineage progenitor cells or stem cells to tumor cells. In one example, the inventors have identified that increasing the expression of a phosphatase deleted on chromosome 10 and tensin homolog alpha (PTENα) in mesenchymal lineage progenitor cells or stem cells can enhance the ability of these cells to migrate to and / or kill tumor cells. These findings suggest that the modified cells according to the present disclosure can advantageously home to tumor cells and deliver therapeutic payloads.

[0010] Accordingly, in a first aspect, the disclosure encompasses a population of mesenchymal progenitor cells or stem cells that have been modified to increase the expression of a phosphatase and tensin homolog alpha (PTENα) deleted on chromosome 10. In one example, increased PTENα expression is sufficient to reduce the level of phosphorylated AKT in the modified cells. In another example, increased PTENα expression is sufficient to enhance tumor cell death. In yet another example, increased PTENα expression is sufficient to enhance migration to tumor cells. In yet another example, increased PTENα expression is sufficient to enhance both migration to tumor cells and tumor cell death.

[0011] In another example, mesenchymal progenitor cells or stem cells are modified to introduce recombinant viruses. For example, mesenchymal progenitor cells or stem cells may be modified to introduce recombinant viruses containing the herpes simplex virus (HSV) skeleton.

[0012] In one example, mesenchymal progenitor cells or stem cells are modified to introduce a recombinant virus containing a polynucleotide encoding PTENα. In one example, the recombinant virus is an oncolytic virus.

[0013] The inventors have identified mesenchymal progenitor cells or stem cells as effective carriers for recombinant viruses containing a herpes simplex virus (HSV) skeleton and expressing a PTEN transgene, and describe particularly high infection rates and replication with these viral constructs. Combined with the capabilities described on the modified cells according to this disclosure, the inventors' findings suggest that mesenchymal progenitor cells or stem cells, including the modifications discussed herein, could become novel and effective compositions for treating various cancers that can be commercially produced on a large scale, especially when modified to introduce recombinant viruses containing an HSV skeleton. For example, the recombinant virus contains a herpes simplex virus (HSV) skeleton.

[0014] In one example, HSV has a high infection rate of mesenchymal progenitor cells or stem cells. In one example, at least 10% of the cells in the population disclosed herein contain the virus. In another example, at least 20% of the cells in the population disclosed herein contain the virus. In yet another example, between 20% and 80% of the cells in the population disclosed herein contain the virus.

[0015] In one example, a polynucleotide encoding PTEN-alpha is operably ligated to a tumor-specific promoter. In further examples, tumor-specific promoters include the Survivin promoter, COX-2 promoter, PSA promoter, CXCR4 promoter, STAT3 promoter, hTERT promoter, AFP promoter, CCKAR promoter, CEA promoter, erbB2 promoter, E2F1 promoter, HE4 promoter, LP promoter, MUC-1 promoter, TRP1 promoter, and Tyr promoter.

[0016] In one example, the polynucleotide encoding PTEN-alpha is operably ligated to an inducible promoter.

[0017] In one example, recombinant viruses contain capsid proteins that bind to tumor-specific cell surface molecules. In further examples, the capsid protein is a fiber, penton, or hexon protein.

[0018] In one example, a recombinant virus contains a variant of that virus that is translated into a protein containing the nucleic acid sequence shown in SEQ ID NO: 1, or the amino acid sequence shown in SEQ ID NO: 2. In one example, the variant of SEQ ID NO: 1 shares at least 85%, 90%, 95%, and 99% sequence identity with SEQ ID NO: 2.

[0019] One example of a recombinant virus is HSV.

[0020] In one example, the tumor cells are breast cancer or brain cancer cells.

[0021] In one example, mesenchymal progenitor cells or stem cells express one or more markers selected from the group consisting of α1, α2, α3, α4 and α5, αv, β1 and β3. In another example, mesenchymal progenitor cells or stem cells express STRO-1. In yet another example, mesenchymal progenitor cells or stem cells express STRO-1. bri In one example, mesenchymal progenitor cells or stem cells express Ang1:VEGF in a ratio of at least 2:1 to 30:1. In another example, mesenchymal progenitor cells or stem cells express Ang1:VEGF in a ratio of at least approximately 10:1. In yet another example, mesenchymal progenitor cells or stem cells express Ang1:VEGF in a ratio of at least approximately 20:1. In yet another example, mesenchymal progenitor cells or stem cells express Ang1:VEGF in a ratio of at least approximately 30:1.

[0022] In one example, the mesenchymal progenitor cells or stem cells are not genetically modified to express Ang1 or VEGF. In another example, the mesenchymal progenitor cells or stem cells are derived from pluripotent cells. In yet another example, the pluripotent cells are induced pluripotent stem (iPS) cells.

[0023] In one example, mesenchymal progenitor cells or stem cells express STRO-1 and two or more markers selected from the group consisting of α1, α2, α3, α4 and α5, αv, β1 and β3.

[0024] In one example, a population of cells is cultured and grown.

[0025] In one example, a pharmaceutical composition comprising the population disclosed herein is provided.

[0026] The inventors have also demonstrated that PTENα expression in cells can be increased by contacting cells with a population of modified mesenchymal progenitor cells or stem cells disclosed herein. In one example, the cells to be contacted are cancer cells. In another example, increased PTENα expression in the contacted cells reduces the level of phosphorylated AKT in the cells.

[0027] This disclosure also includes methods for treating cancer in a subject, comprising the step of administering a population or composition according to one of the examples provided above. In one example, this disclosure also includes methods for killing cancer cells, comprising the step of contacting a population of cancer cells with a population or composition according to one of the examples provided above. In another example, this disclosure also includes methods for delivering mesenchymal progenitor cells or stem cells to cancer cells in a subject, comprising the step of administering a population or composition according to one of the examples provided above. In one example, the cancer is selected from the group consisting of lung cancer, pancreatic cancer, colorectal cancer, liver cancer, cervical cancer, prostate cancer, breast cancer, endometrial cancer, thyroid cancer, kidney cancer, brain cancer, glioblastoma, osteosarcoma, and melanoma. In a further example, the cancer is breast cancer or brain cancer. In another example, the population or composition is administered to the subject by intravenous, intra-arterial, or intraperitoneal administration. In one example, the composition is administered directly to the tumor of the subject.

[0028] In another example, this disclosure relates to the use of the populations disclosed herein in the manufacture of a pharmaceutical product for treating cancer. In yet another example, this disclosure relates to the use of the populations disclosed herein in the manufacture of a pharmaceutical product for delivering mesenchymal progenitor cells or stem cells to cancer cells.

[0029] Any example in this specification should be interpreted as being modifiable and applicable to any other example unless otherwise specified.

[0030] This disclosure is not limited in scope by the specific examples described herein, which are intended for illustrative purposes only. Functionally equivalent products, compositions, and methods are clearly within the scope of this disclosure as described herein.

[0031] Throughout this specification, unless otherwise specifically designated or the content expressly indicates otherwise, any reference 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 these steps, compositions, groups of steps, or groups of compositions.

[0032] This disclosure is described herein hereafter by the means of the following non-limiting examples and with reference to the accompanying figures. [Brief explanation of the drawing]

[0033] [Figure 1] (A and B) These figures show the viral skeletons of HSVQ (parent virus) and HSV-P10 (PTENα-expressing virus). [Figure 2] (A and B) These figures show the HSV-P10 load of mesenchymal stem cells (MSCs). [Figure 3A] This figure shows the survival rates of mesenchymal stem cells (MSCs) loaded with HSV-P10 and HSVQ. [Figure 3B] This figure shows the survival rates of mesenchymal stem cells (MSCs) loaded with HSV-P10 and HSVQ. [Figure 4](A and B) These figures show the effects of HSV-P10-loaded mesenchymal stem cells (MSCs) on PTENα expression and the PI3K / AKT signaling pathway. [Figure 5] This figure shows the migration of HSV-P10 and HSVQ-loaded mesenchymal stem cells (MSCs) to human breast cancer cells (MDA-468). [Figure 6] (A and B) Figures showing the effects of HSV-P10-loaded mesenchymal stem cells (MSCs) on human glioma cells. [Figure 7] This figure shows the induction of tumor cell death in DB7 mouse mammary cancer cells co-cultured with HSV-P10 and HSVQ-loaded mesenchymal stem cells (MSCs). [Modes for carrying out the invention]

[0034] General technical and selective definitions Unless otherwise specified, all technical and scientific terms used herein should be construed to have the same meaning as those generally understood by those skilled in the art (e.g., molecular biology, cell culture, stem cell differentiation, cell therapy, genetic modification, virology, oncology, biochemistry, physiology, and clinical research).

[0035] Unless otherwise indicated, the molecular and statistical techniques used in this disclosure are standard procedures well known to those skilled in the art. Such techniques are described in J. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984), J. Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbour Laboratory Press (1989), TA Brown (ed.), Essential Molecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press (1991), DM Glover and BD Hames (eds.), DNA Cloning: A Practical Approach, Volumes 1-4, IRL Press (1995 and 1996), and FM Ausubel et al. (eds.), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience (1988, including all the latest information to date), Ed Harlow and David Lane (eds.), Antibodies: A Laboratory Manual, Cold Spring Harbour Laboratory, (1988), and JE Coligan et al. (eds.), Current Protocols in This is described and explained throughout the literature in sources such as Immunology, John Wiley & Sons (including all the latest information to date).

[0036] As used herein and in the appended claims, singular words and singular "a," "an," and "the" include, for example, multiple references, unless the content explicitly indicates otherwise. Thus, for example, "analyte" includes, for example, one or more analytes.

[0037] Where used herein, the term “about” means + / - 10%, more preferably + / - 5%, and more preferably + / - 1% of the specified value, unless otherwise stated.

[0038] 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 interpreted as providing clear support for both meanings or either meaning.

[0039] Throughout this specification, the word "comprise," or variations such as "comprises" or "comprising," are understood to mean the inclusion of the element, integer, or step or group of elements, integers, or steps mentioned, but not the exclusion of any other element, integer, or step or group of elements, integers, or steps.

[0040] The term “Phosphatase and Tensin Homolog Deleted on Chromosome 10 (PTEN)” is used in the context of this disclosure to refer to the gene encoding phosphatidylinositol-3,4,5-trisphosphate 3-phosphatase (PTEN; gene ID: 5728; UniProtKB# P60484). The term “PTEN-alpha” or “PTEN-α” is used to refer to a 576-amino acid translational variant of PTEN (P60484-2; also known as PTEN-Long) arising from an alternative translational initiation site 519 base pairs upstream of the ATG start sequence, with 173 N-terminal amino acids added to the normal PTEN translational region. For example, PTEN-alpha includes the amino acid sequence shown in SEQ ID NO: 2. For another example, PTEN-alpha is described by Hopkins et al., (2013) Science., 6144:399-402. The term “phosphatase and tensin homolog (PTEN) alpha deleted on chromosome 10” is used in the context of this disclosure to refer to the gene encoding PTEN-alpha. For example, PTEN-alpha is encoded by a nucleic acid containing a variant thereof that encodes a protein having the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2. Thus, for example, a population of cells disclosed herein may be modified to increase the expression of the nucleic acid encoding the protein having the amino acid sequence shown in SEQ ID NO: 2. For example, a nucleic acid expressing PTEN-alpha is modified to promote a higher level of PTEN-alpha translation than PTEN translation. For example, a nucleic acid expressing PTEN-alpha is not translated to PTEN in cancer cells.

[0041] In another example, PTEN-alpha is encoded by a nucleic acid containing a sequence corresponding to the PTEN gene (gene ID: 5728), where the PTEN-alpha CUG start codon is mutated to AUG. In this example, the PTEN AUG start codon may also be mutated; for example, the PTEN AUG start codon is mutated to AUA. In these examples, the cell populations disclosed herein may be modified to increase the expression of such nucleic acids.

[0042] As used herein, “PTEN mutation or deficiency cancer” refers to cancer identified by examination of an individual-derived cancer sample as having one or more mutations in the PTEN protein, or in which the PTEN gene is absent or reduced compared to the protein / gene levels in normal cells. PTEN mutations or deficiencies have been observed in numerous cancers, including glioblastoma, endometrial cancer, colon cancer, lung cancer, breast cancer, prostate cancer, and ovarian cancer. In one example, PTEN mutation or deficiency cancer has a mutation in PTEN. In another example, PTEN mutation or deficiency cancer has a mutation in PTEN-alpha.

[0043] Various subjects may be administered the cell compositions according to this disclosure. In one example, the subject is a mammal. The mammal may be a companion animal such as a dog or a cat, or a domestic animal such as a horse or a cow. In another example, the subject is a human. The terms “subject,” “patient,” or “individual” are terms that may be used interchangeably in the context of this disclosure.

[0044] As used herein, the term “treatment” refers to a clinical intervention designed to alter the natural course of an individual or cell being treated in the course of a clinicopathological condition. Desired effects of treatment include reducing the rate of disease progression, restoring or mitigating the disease state, and achieving remission or improving prognosis. For example, an individual is “treated” well if one or more symptoms associated with the disease are alleviated or eliminated.

[0045] The “effective dose” refers to the minimum effective amount in terms of dosage and duration required to achieve the desired therapeutic or prophylactic outcome. The effective dose may be provided in one or more doses. In some examples of this disclosure, the term “effective dose” is used herein to refer to the amount required to bring about treatment for the disease or condition described herein. The effective dose may vary depending on the disease or condition being treated, as well as the body weight, age, racial background, sex, health status and / or physical condition of the mammal being treated, and other related factors. Typically, the effective dose falls within a relatively broad range (e.g., “dosage” range) that can be determined through routine examination and experimentation by a physician. The effective dose may be administered in a single dose, or in doses repeated once or multiple times over a period of treatment.

[0046] The “therapeutic dose” is the minimum concentration required to produce a measurable improvement in a particular disorder (e.g., cancer). In this specification, the therapeutic dose may vary depending on factors such as the patient’s disease state, age, sex, and weight, as well as the ability of the cell composition to produce a desirable response in the individual. The therapeutic dose is also the amount at which the therapeutically beneficial effects of the composition outweigh any toxic or adverse effects. In the case of cancer, the therapeutic dose can reduce the number of cancer cells; reduce the size of the primary tumor; prevent (i.e., slow to some extent, and in some cases stop) the invasion of cancer cells into peripheral organs; prevent (i.e., slow to some extent, and in some cases stop) tumor metastasis; to some extent prevent or slow tumor growth or progression; and / or alleviate to some extent one or more symptoms associated with the disorder. The compositions according to this disclosure may be cell proliferation inhibitory and / or cytotoxic to the extent that they can prevent the growth of existing cancer cells and / or kill them. The in vivo effectiveness of cancer treatment can be measured, for example, by evaluating survival time, time to progression (TTP), response rate (RR), duration of response, and / or quality of life.

[0047] In one example, the level of a specific marker is determined under culture conditions. The term “culture conditions” is used to refer to cells that are growing in culture. In one example, culture conditions refer to a population of actively dividing cells. Such cells may, in one example, be in the logarithmic growth phase. For example, the level of a specific marker may be determined by taking a sample of the cell culture medium and measuring the level of the marker in the sample. In another example, the level of a specific marker may be determined by taking a sample of cells and measuring the level of the marker in the cell lysate. Those skilled in the art will measure secretory markers by taking a sample of the culture medium, while markers expressed on the surface of cells may be measured by evaluating a sample of cell lysate. In one example, the sample is taken when the cells are in the logarithmic growth phase. In another example, the sample is taken after being cultured for at least two days.

[0048] Culturing and growing cells from cryopreserved intermediates means thawing the cryopreserved cells and culturing them in vitro under conditions suitable for cell growth.

[0049] Mesenchymal progenitor cells or stem cells As used herein, the term “mesenchymal progenitor cells or stem cells” refers to undifferentiated pluripotent cells that have the ability to self-replicate while maintaining multipotency and the ability to differentiate into any number of mesenchymal cell types, such as osteoblasts, chondrocytes, adipocytes, stromal cells, fibroblasts, and tendons, or non-mesoderm-derived cells, such as hepatocytes, nerve cells, and epithelial cells. In various examples, the disclosure encompasses a population of mesenchymal progenitor cells or stem cells, said cells modified to enhance migration to tumor cells. In one example, the disclosure encompasses a population of mesenchymal progenitor cells or stem cells containing recombinant viruses, said cells modified to enhance migration to tumor cells. For example, the disclosure encompasses a population of mesenchymal progenitor cells or stem cells, said cells modified to increase the expression of a phosphatase deleted on chromosome 10 and tensin homolog alpha (PTENα) to a sufficient degree to enhance migration to tumor cells. In another example, the disclosure encompasses a population of mesenchymal progenitor cells or stem cells that are modified to introduce a recombinant virus containing a polynucleotide encoding PTENα. In this example, the expression of PTENα from the virus and its translation into the PTENα protein are sufficient to enhance migration to tumor cells.

[0050] The term "mesenchymal progenitor cells or stem cells" includes both parent cells and their undifferentiated progeny. The term also includes mesenchymal progenitor cells or stem cells (MPCs), pluripotent stromal cells, mesenchymal stem cells, perivascular mesenchymal progenitor cells or stem cells, and their undifferentiated progeny. Therefore, in one example, mesenchymal progenitor cells or stem cells are mesenchymal stem cells.

[0051] Mesenchymal progenitor cells or stem cells can be autologous, allogeneic, xenogeneic, syngeneic, or isogeneic. Autologous cells are isolated from the same individual being re-transplanted. Allogeneic cells are isolated from a donor of the same species. Xenogeneic cells are isolated from a donor of a different species. Syngeneic or isogeneic cells are isolated from genetically identical organisms, such as twins, clones, or highly purebred research animal models.

[0052] In one example, the mesenchymal progenitor cells or stem cells are allogeneic. In another example, the allogeneic mesenchymal progenitor cells or stem cells are cultured, grown, and cryopreserved.

[0053] Mesenchymal progenitor cells or stem cells are primarily found in the bone marrow, but have also been shown to be present in a variety of host tissues, including umbilical cord blood and umbilical cord, adult peripheral blood, adipose tissue, cancellous bone, and dental pulp.

[0054] For example, mesenchymal progenitor cells or stem cells express STRO-1 and one or more integrins. Integrins are a type of cell adhesion receptor that mediates both cell-to-cell and cell-to-extracellular matrix adhesion events. Integrins consist of heterodimeric polypeptides, where a single α-chain polypeptide non-covalently associates with a single β-chain. Currently, there are about 16 different α-chain polypeptides and at least about 8 different β-chain polypeptides that constitute the integrin family of cell adhesion receptors. Generally, different binding specificities and tissue distributions are derived from unique combinations of α and β-chain polypeptides or integrin subunits. The family to which a particular integrin associates is usually characterized by its β-subunit. However, the ligand-binding activity of an integrin is greatly influenced by its α-subunit.

[0055] In one example, the mesenchymal progenitor cells or stem cells according to this disclosure express integrins having STRO-1 and β1(CD29) chain polypeptides.

[0056] In another example, the mesenchymal progenitor cells or stem cells according to this disclosure express STRO-1 and an integrin having an α-chain polypeptide selected from the group consisting of α1 (CD49a), α2 (CD49b), α3 (CD49c), α4 (CD49d), α5 (CD49e), and αv (CD51). Therefore, in one example, the mesenchymal progenitor cells or stem cells according to this disclosure express STRO-1 and α1. In another example, the mesenchymal progenitor cells or stem cells express STRO-1 and α2. In another example, the mesenchymal progenitor cells or stem cells express STRO-1 and α3. In another example, the mesenchymal progenitor cells or stem cells express STRO-1 and α4. In another example, the mesenchymal progenitor cells or stem cells express STRO-1 and α5. In another example, the mesenchymal progenitor cells or stem cells express STRO-1 and αv. In another example, mesenchymal progenitor cells or stem cells express STRO-1, α2, and α3. In yet another example, mesenchymal progenitor cells or stem cells express STRO-1, α2, and α5. In yet another example, mesenchymal progenitor cells or stem cells express STRO-1, α3, and α5. In yet another example, mesenchymal progenitor cells or stem cells express STRO-1, α2, α3, and α5.

[0057] In another example, the disclosure includes a population of mesenchymal progenitor cells or stem cells enriched for STRO-1 and α1+ cells. In this example, the population enriched for α1+ cells may contain at least about 3%, 4%, or 5% α1+ cells.

[0058] In another example, the disclosure includes a population of mesenchymal progenitor cells or stem cells enriched for STRO-1 and α2+ cells. In this example, the population enriched for α2+ cells may contain at least about 30%, 40%, or 50% α2+ cells.

[0059] In another example, the disclosure includes a population of mesenchymal progenitor cells or stem cells enriched for STRO-1 and α3+ cells. In this example, the population enriched for α3+ cells contains at least about 40%, 45%, or 50% α3+ cells.

[0060] In another example, the disclosure includes a population of mesenchymal progenitor cells or stem cells enriched for STRO-1 and α4+ cells. In this example, the population enriched for α4+ cells contains at least about 5%, 6%, or 7% α4+ cells.

[0061] In another example, the disclosure encompasses a population of mesenchymal progenitor cells or stem cells enriched for STRO-1 and α5+ cells. In this example, the population enriched for α5+ cells contains at least about 45%, 50%, or 55% α5+ cells.

[0062] In another example, the disclosure includes a population of mesenchymal progenitor cells or stem cells enriched for STRO-1 and αv+ cells. In this example, the population enriched for αv+ cells contains at least about 5%, 6%, or 7% αv+ cells.

[0063] In another example, the present disclosure includes populations of mesenchymal progenitor cells or stem cells enriched with STRO-1, α1+, α3+, α4+, and α5+ cells.

[0064] In the above example, the mesenchymal progenitor cells or stem cells may have a β1 chain polypeptide. For example, the mesenchymal progenitor cells or stem cells according to this disclosure can express an integrin selected from the group consisting of α1β1, α2β1, α3β1, α4β1, and α5β1. Thus, in one example, the mesenchymal progenitor cells or stem cells according to this disclosure express STRO-1 and α1β1. In another example, the mesenchymal progenitor cells or stem cells express STRO-1 and α2β1. In yet another example, the mesenchymal progenitor cells or stem cells express STRO-1 and α4β1. In yet another example, the mesenchymal progenitor cells or stem cells express STRO-1 and α5β1.

[0065] In another example, the mesenchymal progenitor cells or stem cells according to this disclosure express STRO-1 and an integrin having a β3 (CD61) chain polypeptide. In one example, this disclosure encompasses a population of mesenchymal progenitor cells or stem cells enriched for STRO-1 and β3+ cells. In this example, the population enriched for β3+ cells contains at least about 8%, 10%, or 15% β3+ cells. In another example, the mesenchymal progenitor cells or stem cells express STRO-1 and αvβ3. In another example, the mesenchymal progenitor cells or stem cells according to this disclosure express STRO-1 and an integrin having a β5 (ITGB5) chain polypeptide. In one example, the mesenchymal progenitor cells or stem cells express STRO-1 and αvβ5. In another example, the mesenchymal progenitor cells or stem cells express STRO-1 and αvβ6.

[0066] Identifying and / or enriching mesenchymal progenitor cells or stem cells expressing the integrins referenced above can be achieved using various methods well known in the art. For example, fluorescent cell sorting (FACS) using commercially available antibodies (e.g., Thermofisher; Pharmingen; Abcam) can be used to identify and select cells expressing a desired integrin polypeptide chain or combination thereof.

[0067] In one example, mesenchymal progenitor cells or stem cells express STRO-1 and coxsackievirus and adenovirus receptors. In another example, mesenchymal progenitor cells or stem cells express STRO-1, coxsackievirus and adenovirus receptors and one or more of the integrins referenced above.

[0068] In another example, mesenchymal progenitor cells or stem cells express STRO-1, coxsackievirus and adenovirus receptors, αvβ3, and αvβ5.

[0069] In one example, mesenchymal progenitor cells or stem cells are genetically modified to express one or more of the integrins referenced above, or coxsackievirus and adenovirus receptors, on their cell surface.

[0070] For example, mesenchymal progenitor cells or stem cells express STRO-1 and chimeric antigen receptors (CARs). For instance, mesenchymal progenitor cells or stem cells express STRO-1, CAR, αvβ3, and αvβ5.

[0071] In one example, mesenchymal progenitor cells or stem cells expressing CARs may trigger a T cell-mediated immune response. In another example, CARs act as a means to cause mesenchymal progenitor cells or stem cells to adhere to cancer cells. In yet another example, CARs act as a means to enhance the adhesion of mesenchymal progenitor cells or stem cells to cancer cells.

[0072] In one example, a CAR consists of an extracellular antigen-binding domain, a transmembrane domain, and an intracellular domain. In another example, the antigen-binding domain has affinity for one or more tumor antigens. Exemplary tumor antigens include HER2, CLPP, 707-AP, AFP, ART-4, BAGE, MAGE, GAGE, SAGE, β-catenin / m, bcr-abl, CAMEL, CAP-1, CEA, CASP-8, CDK / 4, CDC-27, Cyp-B, DAM-8, DAM-10, ELV-M2, ETV6, G250, Gp100, HAGE, HER-2 / neu, EPV-E6, LAGE, hTERT, Survivin, iCE, MART-1, tyrosinase, MUC-1, MC1-R, TEL / AML, and WT-1.

[0073] Exemplary intracellular domains include CD3-zeta, CD28, and 4-IBB, and in some cases, CAR may contain any combination of CD3-zeta, CD28, 4-1BB, and TLR-4.

[0074] Exemplary transmembrane domains may be derived from the alpha, beta, or zeta chains of T cell receptors, CD28, CD3 epsilon, CD45, CD4, CD5, CDDS, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD154 (i.e., including at least their transmembrane regions). In another example, the transmembrane domain may be synthesized and in which case it contains mainly hydrophobic residues such as leucine and valine.

[0075] Mesenchymal progenitor cells or stem cells can be isolated from host tissues, such as those referenced above, and enriched by immunoselection. For example, bone marrow aspirate from a subject can be further treated with antibodies against STRO-1 or TNAP that allow for the selection of mesenchymal progenitor cells or stem cells. In one example, mesenchymal progenitor cells or stem cells can be enriched using the STRO-1 antibody described in Simmons & Torok-Storb, 1991.

[0076] STRO-1+ cells are found in bone marrow, blood, dental pulp cells, adipose tissue, skin, spleen, pancreas, brain, kidney, liver, heart, retina, hair follicles, intestines, lungs, lymph nodes, thymus, bone, ligaments, tendons, skeletal muscle, dermis, and periosteum; they can differentiate into germline cells such as mesoderm and / or endoderm and / or ectoderm. Thus, STRO-1+ cells can differentiate into numerous cell types, including, but not limited to, fat, bone (osseous), cartilage, elastic, muscle, and fibrous connective tissue. The specific differentiation lineage and differentiation pathways followed by these cells depend on mechanical influences and / or various influences from endogenous bioactive factors such as growth factors, cytokines, and / or the local microenvironmental conditions established by the host tissue.

[0077] As used herein, the term “enriched” describes a population of cells in which the proportion of one particular cell type or a number of particular cell types is increased compared to an untreated cell population (e.g., cells in their natural environment). For example, a population enriched for STRO-1+ cells contains at least about 0.1%, 0.5%, 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 50%, or 75% STRO-1+ cells. In this regard, the term “population of cells enriched for STRO-1+ cells” is interpreted as explicitly supporting the term “population of cells containing X% STRO-1+ cells,” where X% is a percentage enumerated herein. STRO-1+ cells can, in some examples, form clonal colonies, e.g., CFU-F (fibroblasts), or a subset of them (e.g., 50%, 60%, 70%, 70%, 90%, or 95%) may have this activity.

[0078] In one example, a population of cells is enriched from a cell preparation containing STRO-1+ cells in a selectable form. In this context, the term “selectable form” is understood to mean that the cells express a marker (e.g., a cell surface marker) that enables the selection of STRO-1+ cells. The marker may, 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., MPCs), as described and / or illustrated herein, also express STRO-1 (and may be brightly STRO-1 positive). Therefore, the indication that cells are STRO-1+ does not mean that the cells are selected 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] Referencing the selection of cells or populations thereof does not necessarily require selection from a specific tissue source. As described herein, STRO-1+ cells can be selected, isolated, or enriched from a variety of sources. Therefore, in some examples, these terms support the selection from any tissue containing STRO-1+ cells or vascularized tissue or pericytes (e.g., STRO-1+ or 3G5+ pericytes) or any one or more of the tissues enumerated herein.

[0080] In one example, the mesenchymal progenitor cells or stem cells of the present 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+, and 3G5+.

[0081] The phrase "individually" means that the present disclosure may separately encompass the enumerated markers or groups of markers, and even though individual markers or groups of markers may not be described separately herein, the appended claims may define such markers or groups of markers in a manner that allows them to be defined separately and distinct from one another.

[0082] The term "collectively" means that the present disclosure encompasses any number or combination of the enumerated markers or groups of markers, and that such a number or combination of markers or groups of markers may not be specifically described herein, but that such a combination or subcombination can be defined in such a way that it can be separated from and apart from any other combination of markers or groups of markers.

[0083] Cells referred to as "positive" for a given marker may express the marker at low (low-positive (lo), weak-positive (dim), or dull), moderate (median), or high (strongly positive, bri) levels, depending on the extent to which the marker is present on the cell surface, where the terminology relates to the intensity of fluorescence or other markers used in cell sorting processes or flow cytometry analysis of cells. The distinction between low (low-positive, weak-positive, or dull), moderate (median), and high (strongly positive, bri) is understood in the context of the marker used in a particular population of cells being sorted or analyzed. Cells referred to as "negative" for a given marker do not necessarily have to be completely absent in the cell. This term means that the marker is expressed by the cell at a relatively very low level and, if detectably labeled, produces a very low signal or is undetectable above background levels, for example, levels detectable using an isotype control antibody.

[0084] As used herein, the terms “bright” or “bri” refer to cell surface markers that, when detectably labeled, produce a relatively high signal. While we do not wish to be limited by theory, it is proposed that “bright” cells express more target marker proteins (e.g., antigens recognized by STRO-1 antibody) than other cells in the sample. For example, STRO-1bri cells, when labeled with FITC-conjugated STRO-1 antibody, produce a greater fluorescence signal than non-brightly positive cells (STRO-1 low-positive / weakly positive / slightly positive / moderate / intermediate) as determined by fluorescent cell sorting (FACS) analysis. In one example, mesenchymal progenitor cells or stem cells are isolated from bone marrow and enriched by selection of STRO-1+ cells. In this example, “brightly positive” cells constitute at least about 0.1% of the most strongly labeled bone marrow mononuclear cells contained in the starting sample. In other examples, "strongly positive" cells constitute at least about 0.1%, at least about 0.5%, at least about 1%, at least about 1.5%, or at least about 2% of the most strongly labeled bone marrow mononuclear cells contained in the starting sample. In one example, STRO-1 strongly positive cells have STRO-1 surface expression that is 2 log magnitude higher than "background" cells, i.e., STRO-1-. In comparison, STRO-1 low-positive / weakly positive / slightly positive and / or STRO-1 moderate / intermediate cells have STRO-1 surface expression that is 2 log magnitude lower than high expression, typically about 1 log or less than "background".

[0085] In one example, STRO-1+ cells are strongly positive for STRO-1. In another example, strongly positive STRO-1 cells are preferentially enriched compared to low-positive (lo) / weakly positive (dim) / slightly positive (dull) or moderately / intermediate STRO-1 cells.

[0086] In one example, additionally, STRO-1 strongly positive cells may be one or more of TNAP+, VCAM-1+, THY-1+, STRO-2+, STRO-4+ (HSP-90β), and / or CD146+. For example, cells are selected for one or more of the aforementioned markers and / or are shown to express one or more of the aforementioned markers. In this regard, cells shown to express a marker do not need to be specifically tested; rather, pre-enriched or isolated cells may be tested, and it can be reasonably inferred that subsequently used, isolated, or enriched cells also express the same marker.

[0087] For example, STRO-1 strongly positive cells are perivascular mesenchymal progenitor cells or stem cells as defined in WO 2004 / 85630, characterized by the presence of the perivascular marker 3G5.

[0088] As used herein, the term "TNAP" is intended to encompass all isoforms of tissue-nonspecific alkaline phosphatase. For example, the term encompasses the liver isoform (LAP), bone isoform (BAP), and kidney isoform (KAP). In one example, TNAP is BAP. In one example, TNAP refers to a molecule that can bind to STRO-3 antibody produced by a fusion cell line deposited with ATCC on December 19, 2005, under deposit acceptance number PTA-7282, in accordance with the provisions of the Budapest Convention.

[0089] Furthermore, in one example, STRO-1+ cells can produce clonal CFU-F.

[0090] In one example, a significant proportion of STRO-1+ cells can differentiate into at least two different germlines. Non-limiting examples of cell lines in which cells may be deeply involved include bone progenitor cells; pluripotent hepatocyte progenitor cells for cholangioepithelial cells and hepatocytes; neural restricted cells that can generate glial progenitor cells that progress to oligodendrocytes and astrocytes; neural progenitor cells that progress to neurons; progenitor cells for cardiomyocytes and cardiomyocytes; and glucose-responsive insulin-secreting pancreatic beta cell lines. Other cell lines, though not limited to these, include odontoblasts, dentin-producing cells and chondrocytes, as well as the following cells: retinal pigment epithelial cells, fibroblasts, keratinocytes and other skin cells, dendritic cells, hair follicle cells, renal duct epithelial cells, smooth and skeletal muscle cells, testicular progenitor cells, vascular endothelial cells, tendons, ligaments, cartilage, adipocytes, fibroblasts, bone marrow stroma, cardiac muscle, smooth muscle, skeletal muscle, pericytes, blood vessels, epithelium, glia, nerve cells, astrocytes, and oligodendrocyte progenitor cells.

[0091] In one example, mesenchymal progenitor cells or stem cells are MSCs. MSCs can be a homogeneous composition or a mixed cell population enriched with MSCs. A homogeneous MSC composition can be obtained by culturing adherent bone marrow or periosteal cells, and MSCs can be identified by specific cell surface markers identified using specific monoclonal antibodies. A method for obtaining a cell population enriched with MSCs is described, for example, in U.S. Patent No. 5,486,359. MSCs prepared by conventional plastic adhesion isolation depend on the nonspecific plastic adhesion properties of CFU-F. Mesenchymal progenitor cells or stem cells isolated from bone marrow by immunoselection based on STRO-1 specifically isolate clonal mesenchymal progenitor cells from a bone marrow population in the absence of other plastic-adhering bone marrow populations. Alternative sources for MSCs, but not limited to, include blood, skin, umbilical cord blood, muscle, fat, bone, and perichondrium. In one example, MSCs are homogeneous. In one example, MSCs are cryopreserved. In another example, MSCs are cultured, grown, and cryopreserved.

[0092] In one example, mesenchymal progenitor cells or stem cells are derived from pluripotent cells such as induced pluripotent stem cells (iPS cells). In one embodiment, the pluripotent cells are human pluripotent cells. Preferred processes for generating mesenchymal progenitor cells or stem cells from pluripotent cells are described, for example, in US 7,615,374 and US 2014273211, Barberi et al., Plos medicine, Vol 2(6):pp. 0554-0559 (2005), and Vodyanik et al., Cell Stem cell, Vol 7:pp. 718-728 (2010).

[0093] In another example, mesenchymal progenitor cells or stem cells are immortalized. Exemplary processes for generating immortalized mesenchymal progenitor cells or stem cells are described, for example, in Obinata M., Cell, Vol 2: pp. 235-244 (1997), US 9, 453, 203, Akimov et al., Stem Cells, Vol 23: pp. 1423-1433, and Kabara et al., Laboratory Investigation, Vol 94: pp. 1340-1354 (2014).

[0094] In preferred embodiments of the present disclosure, mesenchymal progenitor cells or stem cells are obtained from a master cell bank derived from mesenchymal progenitor cells or stem cells enriched from the bone marrow of healthy individuals. The use of mesenchymal progenitor cells or stem cells from such sources is particularly advantageous for subjects who do not have suitable family members who can serve as mesenchymal progenitor cell or stem cell donors, or who require urgent treatment and are at high risk of relapse, disease-related decline, or death in the time required to generate mesenchymal progenitor cells or stem cells.

[0095] In another example, the mesenchymal progenitor cells express Cx43. In yet another example, the mesenchymal progenitor cells express Cx40. In yet another example, the mesenchymal progenitor cells express both Cx43 and Cx40. In yet another example, the mesenchymal progenitor cells express Cx45, Cx32, and / or Cx37. In one example, the mesenchymal progenitor cells are not modified to express a specific connexin.

[0096] Isolated or concentrated mesenchymal progenitor cells can be grown in vitro by culture. Isolated or concentrated mesenchymal progenitor cells can be grown in vitro by cryopreservation, thawing, and subsequent culture.

[0097] In one example, isolated or concentrated mesenchymal progenitor cells were cultured in a culture medium (serum-free or serum-supplemented), such as alpha minimal essential medium (αMEM) supplemented with 5% fetal bovine serum (FBS) and glutamine, at a concentration of 50,000 live cells / cm². 2 The seeds were sown and left overnight at 37°C and 20% O2. 2 The cells are then attached to the culture vessel. The culture medium is then replaced and / or changed as needed, and the cells are left for a further 68 to 72 hours at 37°C, 5% O 2 It is cultured in [a specific location].

[0098] As will be understood by those skilled in the art, cultured mesenchymal progenitor cells are phenotypically different 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 cells are also biologically different from in vivo cells, having a higher proliferation rate compared to most in vivo non-periodic (quiescent) cells.

[0099] In one example, mesenchymal progenitor cells or stem cells are obtained from a single donor or multiple donors, where the donor samples or mesenchymal progenitor cells or stem cells are subsequently pooled and then cultured and grown.

[0100] The mesenchymal progenitor cells or stem cells contained herein may be cryopreserved before administration to a subject. In one example, the mesenchymal progenitor cells or stem cells are cultured, grown, and cryopreserved before administration to a subject.

[0101] In one example, the disclosure includes mesenchymal progenitor cells or stem cells and their progeny, soluble factors derived therefrom, and / or extracellular vesicles isolated therefrom. In another example, the disclosure includes mesenchymal progenitor cells or stem cells and extracellular vesicles isolated therefrom. For example, it is possible to culture and grow the mesenchymal progenitor cell lineage or stem cells of the disclosure for a period and under conditions suitable for the secretion of extracellular vesicles into a cell culture medium. The secreted extracellular vesicles can then be obtained from the culture medium for use in therapy.

[0102] As used herein, the term “extracellular vesicles” refers to lipid particles spontaneously released from cells, typically less than 200 nm in size, but ranging in size from approximately 30 nm to 10 microns. They may contain proteins, nucleic acids, lipids, metabolites, or released cells (e.g., mesenchymal stem cells; STRO-1). + May contain organelles from cells.

[0103] As used herein, the term “exosome” generally refers to a type of extracellular vesicle arising from the endosomal compartment of mammalian cells, typically ranging in size from about 30 nm to about 150 nm, which are then transported to the cell membrane and released. They can contain nucleic acids (e.g., RNA; microRNA), proteins, lipids, and metabolites, and function in intercellular communication by being secreted from one cell and taken up by another cell to deliver their cargo.

[0104] Cell culture and proliferation In one example, mesenchymal progenitor cells or stem cells are cultured and grown. "Cultured" mesenchymal progenitor cells or stem cells are distinguished from freshly isolated cells by being cultured in cell culture medium and subcultured (i.e., subcultured). In one example, cultured mesenchymal progenitor cells or stem cells are subcultured approximately 4 to 10 times. In one example, mesenchymal progenitor cells or stem cells are subcultured at least 5, 6, 7, 8, 9, or 10 times. For example, mesenchymal progenitor cells or stem cells can be subcultured at least 5 times. In one example, mesenchymal progenitor cells or stem cells can be subcultured at least 5 to 10 times. In one example, mesenchymal progenitor cells or stem cells can be subcultured at least 5 to 8 times. In one example, mesenchymal progenitor cells or stem cells can be subcultured at least 5 to 7 times. In one example, mesenchymal progenitor cells or stem cells can be subcultured more than 10 times. In another example, mesenchymal progenitor cells or stem cells may be cultured and grown more than seven times. In these examples, stem cells may be cultured and grown before being cryopreserved to yield an intermediate cryopreserved MLPSC population. In one example, the compositions of this disclosure are produced by culturing cells from an intermediate cryopreserved MLPSC population, or in other words, a cryopreserved intermediate.

[0105] In one example, the composition of the present disclosure includes mesenchymal progenitor cells or stem cells cultured from a cryopreserved intermediate. In one example, cells cultured from a cryopreserved intermediate are passaged at least 5, 6, 7, 8, 9, or 10 times. For example, mesenchymal progenitor cells or stem cells can be passaged at least 5 times. In one example, mesenchymal progenitor cells or stem cells can be passaged at least 5 to 10 times. In one example, mesenchymal progenitor cells or stem cells can be passaged at least 5 to 8 times. In one example, mesenchymal progenitor cells or stem cells can be passaged at least 5 to 7 times. In one example, mesenchymal progenitor cells or stem cells can be passaged more than 10 times. In another example, mesenchymal progenitor cells or stem cells can be passaged more than 7 times.

[0106] In one example, mesenchymal progenitor cells or stem cells cultured from cryopreserved intermediates can be cultured in a medium free of animal proteins. In another example, mesenchymal progenitor cells or stem cells cultured from cryopreserved intermediates can be cultured in a medium free of heterogeneous components. In yet another example, mesenchymal progenitor cells or stem cells cultured from cryopreserved intermediates can be cultured in a medium free of fetal bovine serum.

[0107] In one embodiment, mesenchymal progenitor cells or stem cells can be obtained from a single donor or multiple donors, where the donor samples or mesenchymal progenitor cells or stem cells are subsequently pooled and then cultured and grown. In one example, the culture and growth process is as follows: i. Proliferating the number of viable cells by subculturing to provide a preparation of at least approximately 1 billion viable cells, wherein subculturing includes establishing a primary culture of isolated mesenchymal progenitor cells or stem cells and then sequentially establishing a first non-primary (P1) culture of mesenchymal progenitor cells or stem cells isolated from the first culture; ii. Propagating isolated mesenchymal progenitor cells or stem cells into a second non-primary (P2) culture of mesenchymal progenitor cells or stem cells by subculturing; and, iii. Preparing and cryopreserving in-process intermediate mesenchymal progenitor cell or stem cell preparations obtained from P2 cultures of mesenchymal progenitor cells or stem cells; and, iv. Thaw the cryopreserved in-process mesenchymal progenitor cells or stem cell preparations and proliferate the in-process mesenchymal progenitor cells or stem cell preparations by subculturing. Includes.

[0108] In one example, the proliferated mesenchymal progenitor cells or stem cell preparations were: i. Less than approximately 0.75% of CD45+ cells; ii. At least approximately 95% CD105+ cells; iii. At least approximately 95% of CD166+ cells It has an antigen profile and an activity profile that include these features.

[0109] In one example, proliferated mesenchymal progenitor cells or stem cell preparations can inhibit IL2Ra expression by CD3 / CD28-activated PBMCs by at least approximately 30% compared to the control.

[0110] In one example, cultured mesenchymal progenitor cells or stem cells were passed through approximately 4 to 10 times, where they were cryopreserved after being passed through at least 2 or 3 times before being further cultured. In another example, mesenchymal progenitor cells or stem cells were passed through at least 1, at least 2, at least 3, at least 4, and at least 5 times, cryopreserved, and then further passed through at least 1, at least 2, at least 3, at least 4, and at least 5 times before being cultured by the method of the present disclosure.

[0111] The processes of isolating and ex vivo growing mesenchymal progenitor cells or stem cells can be carried out using any apparatus and cell handling methods known in the art. Various culture and growth embodiments of this disclosure utilize steps that require cell manipulation, such as seeding, feeding, dissociation of adherent cultures, or washing. Any step that manipulates cells may damage them. While mesenchymal progenitor cells or stem cells can generally tolerate a certain degree of damage during preparation, cells are preferably manipulated by handling procedures and / or apparatus that appropriately carry out a given step while minimizing damage to the cells.

[0112] In one example, mesenchymal progenitor cells or stem cells are washed in an apparatus comprising, for example, a cell source bag, a washing solution bag, a recirculation washing bag, a rotating membrane filter with inlet and outlet ports, a filter bag, a mixing area, a final product bag for the washed cells, and appropriate tubing, as described in US 6,251,295 incorporated herein by reference.

[0113] In one example, the mesenchymal progenitor cell or stem cell composition cultured according to this disclosure is 95% homogeneous in that it is CD105-positive and CD166-positive, and CD45-negative. In one example, this homogeneity persists through ex vivo proliferation; i.e., through multiple population doublings.

[0114] In one example, the mesenchymal progenitor cells or stem cells of the Disclosure are cultured and grown in 3D culture. For example, the mesenchymal progenitor cells or stem cells of the Disclosure may be cultured and grown in a bioreactor. In one example, the mesenchymal progenitor cells or stem cells of the Disclosure are initially cultured and grown in 2D culture before being further cultured in 3D culture. In one example, the mesenchymal progenitor cells or stem cells of the Disclosure are cultured and grown from a master cell bank. In one example, the mesenchymal progenitor cells or stem cells of the Disclosure are cultured and grown from a master cell bank in 2D culture before seeding in 3D culture. In one example, the mesenchymal progenitor cells or stem cells of the Disclosure are cultured and grown from a master cell bank in 2D culture for at least 3 days before seeding in 3D culture in a bioreactor. In one example, the mesenchymal progenitor cells or stem cells of the Disclosure are cultured and grown from a master cell bank in 2D culture for at least 4 days before seeding in 3D culture in a bioreactor. In one example, mesenchymal progenitor cells or stem cells in this disclosure are cultured and grown from a master cell bank in 2D culture for 3 to 5 days before seeding in 3D culture in a bioreactor. In these examples, 2D culture may be carried out in a cell factory. Various cell factory products are commercially available (e.g., Thermofisher, Sigma).

[0115] Ang1 and VEGF levels In another embodiment, the mesenchymal progenitor cells or stem cells according to this disclosure express Ang1:VEGF in a ratio of at least about 2:1. However, in other examples, the mesenchymal progenitor cells or stem cells express Ang1:VEGF in a ratio of at least about 10:1, 15:1, 20:1, 21:1, 22:1, 23:1, 24:1, 25:1, 26:1, 27:1, 28:1, 29:1, 30:1, 31:1, 32:1, 33:1, 34:1, 35:1, or 50:1.

[0116] The amount of cellular Ang1 and / or VEGF expressed in a composition or culture of mesenchymal progenitor cells or stem cells can be determined by methods well known to those skilled in the art. Such methods include, but are not limited to, quantitative assays such as quantitative ELISA assays. In this example, cell lysates from a culture of mesenchymal progenitor cells or stem cells are added to wells of an ELISA plate. The wells may be coated with a primary antibody, monoclonal or polyclonal antibody, against Ang1 or VEGF. The wells are then washed and then contacted with a secondary antibody, monoclonal or polyclonal antibody, against the primary antibody. The secondary antibody is conjugated with a suitable enzyme, such as horseradish peroxidase. The wells may then be incubated and washed after the incubation period. The wells are then contacted with a suitable substrate for the enzyme conjugated with the secondary antibody, such as one or more chromogens. Examples of chromogens used include, but are not limited to, hydrogen peroxide and tetramethylbenzidine. After the substrate is added, the wells are incubated for an appropriate amount of time. At the completion of incubation, a “stop” solution is added to the wells to halt the reaction between the enzyme and the substrate. The optical density (OD) of the sample is then measured. The optical density of the sample correlates with the optical density of a sample containing a known amount of Ang1 or VEGF to determine the amount of Ang1 or VEGF expressed by the stem cell culture being examined.

[0117] Methods for determining the Ang1:VEGF expression ratio are also apparent to those skilled in the art. For example, the expression levels of Ang1 and VEGF can be quantified via quantitative ELISA as discussed above. After quantifying the levels of Ang1 and VEGF, the ratio based on the quantified levels of Ang1 and VEGF can be expressed as: (Ang1 level / VEGF level) = Ang1:VEGF ratio.

[0118] For example, the mesenchymal progenitor cells or stem cells in this disclosure are not genetically modified to express Ang1 and / or VEGF at the exemplary levels or ratios described above. Cells that are not genetically modified to express Ang1 and / or VEGF are not modified by transfection with nucleic acids that express or encode Ang1 and / or VEGF. To avoid misunderstanding, in the context of this disclosure, mesenchymal progenitor cells or stem cells transfected with nucleic acids encoding Ang1 and / or VEGF are considered genetically modified. In the context of this disclosure, cells that are not genetically modified to express Ang1 and / or VEGF spontaneously express some degree of Ang1 and / or VEGF without being transfected with nucleic acids encoding Ang1 and / or VEGF.

[0119] Recombinant viruses In one embodiment, the cells as defined herein are modified to introduce a recombinant virus. The term “recombinant virus” is used in the context of this disclosure to mean a virus that expresses a desired transgene in the cells (or population thereof) as defined herein. In one example, the recombinant virus expresses a transgene that increases the migration of mesenchymal progenitor cells or stem cells to cancer cells. In one example, the recombinant virus contains a herpes simplex virus skeleton. In another example, the recombinant virus is a herpes simplex virus.

[0120] In the context of this disclosure, the term "oncolytic virus" is used to refer to a virus that can infect cancer cells and reduce their growth. For example, an oncolytic virus can suppress cell proliferation. In another example, an oncolytic virus can kill cancer cells. In one example, an oncolytic virus preferentially infects cancer cells compared to corresponding normal cells and suppresses their growth. In yet another example, an oncolytic virus preferentially replicates in cancer cells compared to corresponding normal cells and suppresses their growth.

[0121] For example, oncolytic viruses can infect cancer cells in a natural state and reduce their growth. Examples of such viruses include Newcastle disease virus, vesicular stomatitis, myxoma, reovirus, Sindbis, measles, and coxsackievirus. Oncolytic viruses can infect cancer cells, generally target cancer cells, in a natural state and reduce their growth by exploiting cellular abnormalities that occur in these cells. For example, oncolytic viruses can exploit attachment receptors, activated oncogenes such as Ras, Akt, and p53, and / or interferon (IFN) pathway deficiencies.

[0122] In another example, the oncolytic viruses contained herein are engineered to infect cancer cells and inhibit their growth. Suitable exemplary viruses for such engineering include oncolytic DNA viruses, e.g., adenoviruses, herpes simplex virus (HSV), and vaccinia virus; as well as oncolytic RNA viruses, e.g., lentiviruses, reoviruses, coxsackieviruses, Seneca Valley virus, poliovirus, measles virus, Newcastle disease virus, varicella stomatitis virus (VSV); and parvoviruses, e.g., rodent protoparvovirus H-1PV. In one example, the oncolytic virus contains the viral skeleton of the viruses referenced above. For example, the oncolytic virus may contain the HSV skeleton. In one example, the oncolytic virus is HSV.

[0123] In one example, the tumor specificity of an oncolytic virus can be engineered to allow it to proliferate in cancer cells, but to mutate or delete genes necessary for the virus's survival in normal cells. To avoid misunderstanding, an oncolytic virus with mutated or deleted genes can survive in mesenchymal progenitor cells or stem cells for a sufficient period to enable migration to cancer cells. For example, an oncolytic virus can be engineered by mutating or deleting a gene encoding thymidine kinase, an enzyme necessary for nucleic acid metabolism. In this example, the virus relies on cellular thymidine kinase expression, which is high in proliferating cancer cells but suppressed in normal cells. In another example, an oncolytic virus is engineered to contain a capsid protein that binds to tumor-specific cell surface molecules. In one example, the capsid protein is a fiber, penton, or hexon protein. In yet another example, an oncolytic virus is engineered to contain tumor-specific cell surface molecules for targeting cancer cells by transduction. Exemplary tumor-specific cell surface molecules include integrins, EGF receptor family members, proteoglycans, disiarogangliosides, B7-H3, CA-125, EpCAM, ICAM-1, DAF, A21, integrin-α2β1, vascular endothelial growth factor receptor 1, vascular endothelial growth factor receptor 2, CEA, tumor-associated glycoproteins, CD19, CD20, CD22, CD30, CD33, CD40, CD44, CD52, CD74, CD152, CD155, MUC1, tumor necrosis factor receptor, insulin-like growth factor receptor, folate receptor α, transmembrane glycoprotein NMB, CC chemokine receptor, PSMA, RON- receptor, and cytotoxic T-lymphocyte antigen 4.

[0124] In another example, oncolytic viruses are engineered to increase their ability to deliver the viral payload of infected mesenchymal progenitor cells or stem cells to cancer cells. For example, oncolytic viruses may be engineered to express viral membrane fusion glycoproteins to mediate the induction of mesenchymal progenitor cell lineage or stem cell fusion into cancer cells. Examples of viral membrane fusion glycoproteins include gibbon leukemia virus (GLAV) envelope glycoprotein, measles virus protein F (MV-F), and measles virus protein H (MV-H).

[0125] In one example, viral membrane-fusion glycoproteins are regulated by late promoters such as the major late promoter of adenoviruses. In another example, viral membrane-fusion glycoproteins are regulated by strict late promoters such as UL38p (WO 2003 / 082200), which is active only after the initiation of viral DNA replication. Examples of such promoters and manipulated viruses are disclosed in Fu et al., (2003) Molecular Therapy, 7:748-754 and Guedan et al., (2012) Gene Therapy, 19:1048-1057.

[0126] In one example, an oncolytic virus is replication competent. In another example, an oncolytic virus selectively replicates in cancer cells compared to corresponding normal cells and / or mesenchymal progenitor cells or stem cells. In one example, the tumor specificity of an oncolytic virus can be manipulated to limit viral replication by relying on transcriptional activity that is constitutively activated in cancer cells (i.e., conditional replication). In one example, the oncolytic virus is a conditionally replicating lentivirus. In yet another example, the oncolytic virus is a conditionally replicating adenovirus, reovirus, measles virus, herpes simplex virus, Newcastle disease virus, or vaccinia.

[0127] In one example, conditional replication is achieved by the insertion of tumor-specific promoters that drive the expression of important genes. Such promoters can be identified based on differences in gene expression between the tumor, the corresponding surrounding tissue, and / or mesenchymal progenitor cells or stem cells. For example, one method for identifying appropriate tumor-specific promoters is to compare gene expression levels between the tumor, the corresponding normal tissue, and mesenchymal progenitor cells or stem cells to identify genes that are expressed at high levels in the tumor and at low levels in the corresponding healthy tissue and / or mesenchymal progenitor cells or stem cells. Tumor-specific promoters may be native or compound. Exemplary native promoters include AFP, CCKAR, CEA, erbB2, Cerb2, COX2, CXCR4, E2F1, HE4, LP, MUC1, PSA, Survivin, TRP1, STAT3, hTERT, and Tyr. Exemplary compound promoters include AFP / hAFP, SV40 / AFP, CEA / CEA, PSA / PSA, SV40 / Tyr, and Tyr / Tyr. Those skilled in the art will understand that the appropriate tumor-specific promoter is, in some cases, determined by the target tumor. For example, the cerb2 promoter may be appropriate for breast and pancreatic cancer, while the PSA promoter may be appropriate for prostate cancer.

[0128] In another example, tumor-specific promoters may be identified based on differences in promoter activity in cancer cells compared to corresponding normal cells and / or mesenchymal progenitor cells or stem cells. For example, one method for identifying a suitable tumor-specific promoter is to compare promoter activity between cancer cells, corresponding normal cells, and / or mesenchymal progenitor cells or stem cells to identify promoters that are highly active in cancer cells and less active in corresponding normal cells and / or mesenchymal progenitor cells or stem cells. In one example, tumor-specific promoters may be late or strictly late viral promoters. The terms “late” and “strictly late” are used to refer to promoters whose activity depends on the initiation of viral DNA replication. Thus, late and strictly late promoters are suitable for oncolytic viruses that can replicate in cancer cells but have limited replication ability in non-dividing normal cells. Exemplary late or strictly late promoters include major late promoters (MLPs) and UL38p.

[0129] In one example, an oncolytic virus is a herpes simplex virus or an adenovirus containing a late or strictly late promoter. For example, an oncolytic virus is a herpes simplex virus containing the UL38p promoter. In another example, an oncolytic virus is an adenovirus containing MLP.

[0130] In another example, the tumor specificity of oncolytic viruses can be manipulated to utilize tumor-specific affinity. In yet another example, oncolytic viruses are sensitive to oligonucleotides or binding proteins that are expressed in normal cells and / or mesenchymal progenitor cells or stem cells, but are expressed at low levels or absent in cancer cells. For example, oncolytic viruses can be manipulated to insert complementary nucleotide sequences into oligonucleotides that are expressed by mesenchymal progenitor cells or stem cells and / or normal cells, but not by cancer cells. For example, oncolytic viruses may be sensitive to repressive oligonucleotides such as miRNAs.Exemplary miRNAs expressed at low levels in some cancer cells and at high levels in corresponding normal cells include let-7a-5p, miR-122-5p, miR-125b-5p, miR-141-3p, miR-143-3p, miR-15a-5p, miR-16-5p, miR-181a-5p, miR-181b-5p, miR-192-5p, miR-195-5p, miR-200b-3p, miR-200c-3p, miR-211-5p, miR-215-5p, miR-22-3p, and miR-29a- 3p, miR-29b-3p, miR-29c-3p, miR-30a-5p, miR-30c-5p, miR-34a-5p, miR-34c-5p, miR-424-5p, miR-497-5p, miR-7-5p, miR-101-3p, miR-124-3 p, miR-126-3p, miR-137, miR-138-5p, miR-140-5p, miR-152-3p, miR-185-5p, miR-214-3p, miR-25-3p, miR-26a-5p, miR-26b-5p, miR-372-3p, mi R-517a-3p, miR-520c-3p, miR-128-3p, miR-145-5p, miR-200a-3p, miR-502-5p, let-7d-5p, let-7e-5p, let-7f-5p, miR-155-5p, miR-98-5p, le t-7b-5p, miR-1, miR-100-5p, miR-125a-5p, miR-133a-3p, miR-133b, miR-146a-5p, miR-150-5p, miR-193a-3p, miR-193b-3p, miR-196b-5p, miR- 206, miR-218-5p, miR-223-3p, miR-23b-3p, miR-24-3p, miR-34b-3p, miR-449a, miR-542-5p, miR-99a-5p, let-7c-5p, let-7g-5p, let-7i-5p, m Examples include iR-142-3p, miR-216b-5p, miR-622, miR-96-5p, miR-1291, miR-370-3p, miR-296-5p, miR-335-5p, miR-483-3p, miR-483-5p, and miR-486-5p.

[0131] In another example, a virus may be engineered to express a gene in infected cancer cells. For example, a virus may be engineered to express a gene such as PTEN. In one example, the virus expresses PTEN-alpha (PTENα). In another example, the virus contains a nucleic acid sequence shown in SEQ ID NO: 1 or a variant thereof that is translated into a functional PTEN protein (e.g., SEQ ID NO: 2). In yet another example, the virus expresses a transgene that is expressed and translated into a protein having the amino acids shown in SEQ ID NO: 2. In these examples, the oncolytic virus may contain an HSV backbone. In one example, the virus is HSV. In another example, HSV is described by Russell et al., (2018) Nat Comm., 9:5006. In these examples, the virus can increase the level of a protein having the amino acids shown in SEQ ID NO: 2 in infected cancer cells.

[0132] In one example, a gene enhances the immune response against infected tumor cells. For example, the gene could be GM-CSF, FLT3L, CCL3, CCL5, IL2, IL4, IL6, IL12, IL15, IL18, IFNA1, IFNB1, IFNG, CD80, 4-1BBL, CD40L, heat shock proteins (HSPs), or a combination of these.

[0133] Various viruses can be manipulated as outlined in the references above. For example, oncolytic viruses include modified HSV, lentiviruses, baculoviruses, retroviruses, adenoviruses (AdV), adeno-associated viruses (AAV), or recombinant adeno-associated viruses (rAAV), as well as their derivatives such as self-complementary AAV (scAAV) and non-integrating AV. For example, an oncolytic virus may be a modified HSV. For example, an oncolytic virus may be a modified lentivirus. Other exemplary viruses include vaccinia virus, varicella stomatitis virus (VSV), measles virus, and maraba virus.

[0134] In other cases, the oncolytic virus may be one of several AV or AAV serotypes. In one case, the oncolytic virus is serotype 1. In another case, the oncolytic virus is serotype 2. In yet another case, the oncolytic virus is serotype 3, 4, 7, 8, 9, 10, 11, 12, or 13. In yet another case, the oncolytic virus is serotype 5. In yet another case, the oncolytic virus is serotype 6.

[0135] Exemplary oncolytic viruses that can be introduced into mesenchymal progenitor cells or stem cells according to this disclosure include T-Vec (HSV-1; Amgen), JX-594 (Vaccinia; Sillajen), JX-594 (AdV; Cold Genesys), and Reolysin (Reovirus; Oncolytics Biotech). Other examples of oncolytic viruses are disclosed in WO 2003 / 080083, WO 2005 / 086922, WO 2007 / 088229, WO 2008 / 110579, WO 2010 / 108931, WO 2010 / 128182, WO 2013 / 112942, WO 2013 / 116778, WO 2014 / 204814, WO 2015 / 077624 and WO 2015 / 166082, WO 2015 / 089280.

[0136] In one example, oncolytic viruses have replication defects. For example, replication genes can be replaced using expression cassettes containing mutations, deletions, or tumor-specific promoters. In one example, the E1 / E3 genes are mutated, deleted, or replaced. In another example, the E1A / E1B genes are mutated, deleted, or replaced. For example, in the context of AV, the E1 / E3 genes can be mutated, deleted, or replaced. In the context of AAV, the E1A and E1B genes can be mutated, deleted, or replaced. Various examples of suitable tumor-specific promoters are discussed above.

[0137] In other examples, oncolytic viruses may contain mutated E1, E3, E1A, or E1B genes. For example, the E1A gene may be mutated in the region encoding the retinoblastoma protein (RB) binding site. In another example, the E3 gene may be mutated in the region encoding the endoplasmic reticulum-retaining domain. In yet another example, oncolytic viruses may contain mutations in the gamma-34.5 gene and / or alpha-47 gene.

[0138] For example, oncolytic viruses are replication-deficient in mesenchymal progenitor cells or stem cells, but replication-competent in tumor cells. An example of a replication-deficient virus switching to a replication-competent virus is described in Nakashima et al., (2014) Journal of Virology, Vol 88: pp. 345-353. Other exemplary viruses of this type include RGD variants such as those described in Shen et al., (2016) PlosOne 11:e0147173, viruses that replicate in pRb or p53-inactive cancer cells, and / or viruses containing the delta 24 mutation in E1 that enable E1 expression regulation under the control of tumor cell-specific promoters such as the α-chemokine SDF-1 receptor (CXCR4), survivorbin, cyclooxygenase-2 (COX-2), and midkine.

[0139] For example, the viruses disclosed herein include polynucleotides operably linked to a tumor-specific promoter. For instance, a virus may include a polynucleotide encoding PTENα operably linked to a tumor-specific promoter.

[0140] In another example, the viruses disclosed herein include polynucleotides operably linked to a constitutive promoter. For example, a virus may include a polynucleotide encoding PTENα operably linked to a constitutive promoter.

[0141] Modification The mesenchymal progenitor cells or stem cells disclosed herein may be modified to enhance the killing and / or migration of cancer cells. For example, such modifications include increasing the expression of PTENα. Various examples of modifications that increase gene expression are well known in the art. For example, the cells disclosed herein may be modified using a vector that expresses a transgene, such as a viral vector. Thus, for example, the mesenchymal progenitor cells or stem cells disclosed herein may be modified to introduce a recombinant virus that expresses a transgene. For example, the mesenchymal progenitor cells or stem cells disclosed herein may be modified to introduce a recombinant virus, such as a virus that contains an HSV backbone and expresses a polynucleotide encoding PTENα. For example, the mesenchymal progenitor cells or stem cells disclosed are considered “modified” if the virus is transferred to the cell by any preferred means of artificial manipulation, or if the cell is a progeny of the first modified cell that harbors the virus. For example, cells transfected with a “naked” nucleic acid molecule encoding a transgene are not considered “modified.” For example, cells transfected with a “naked” mRNA molecule encoding a transgene are not considered “modified.”

[0142] In other examples, cell populations modified to introduce recombinant viruses may also be modified to express binding proteins such as antibodies or fragments thereof on their cell surface. For example, a cell population may be modified to express anti-epidermal growth factor receptor (EGFR; ErbB1) binding protein.

[0143] The enhanced migration of modified cells disclosed herein can be evaluated using various migration assays well known in the art, such as Transwell cell migration and invasion assays (see, for example, Justus et al., 2014 J Vis Exp., 88:51046 for an overview; commercially available from suppliers such as Sigma and Merck; live cell analysis systems are also commercially available and suitable for tracking migration in real time). The enhanced cancer cell death disclosed herein can also be evaluated using various assays well known in the art, such as cell viability / cytotoxicity assays exemplified below (e.g., evaluation of cytosolic activity (aqua live / dead dye) by flow cytometry; see, for example, Riss et al., (2013) Assay Guidance Manual, last updated July 2016; commercially available kits from suppliers such as Promega; live cell analysis systems are also commercially available and suitable for tracking cell viability in real time). Similarly, increased gene expression can be quantified using various methods, such as detection techniques based on routine amplification, including real-time polymerase chain reactions. In some cases, the increase in protein expression corresponding to increased gene expression can also be quantified using routine methods such as Western blotting.

[0144] Mesenchymal progenitor cells or stem cells can be modified using various methods known in the art. In one example, mesenchymal progenitor cells or stem cells are contacted with a virus in vitro. For example, the virus may be added to the culture medium of the mesenchymal progenitor cells or stem cells. In another example, the mesenchymal progenitor cells or stem cells are centrifuged together with the virus.

[0145] The efficiency of infection is rarely 100%, and it is usually desirable to enrich a population of well-modified cells. In one example, modified cells can be enriched by taking advantage of the functional properties of a novel genotype. One exemplary method for enriching modified cells is positive selection using resistance to drugs such as neomycin, or colorimetric selection based on lacZ expression. We have found that HSV expressing the PTEN-alpha transgene has a high infectivity rate in mesenchymal progenitor cells or stem cells. For example, HSV according to this disclosure has at least 15% infectivity. In another example, HSV according to this disclosure has at least 20% infectivity. In yet another example, HSV according to this disclosure has at least 25% infectivity. In yet another example, HSV according to this disclosure has at least 30% infectivity. In yet another example, HSV according to this disclosure has at least 40% infectivity. In yet another example, HSV according to this disclosure has at least 50% infectivity. In yet another example, HSV according to this disclosure has infectivity between 15% and 80%. In another example, HSV according to this disclosure has an infectivity of between 20 and 80%. In another example, HSV according to this disclosure has an infectivity of between 30 and 80%. In another example, HSV according to this disclosure has an infectivity of between 35 and 80%. In another example, HSV according to this disclosure has an infectivity of between 45 and 80%. In yet another example, HSV according to this disclosure has an infectivity of between 55 and 80%.

[0146] Viral infectivity can be determined using various routine methods such as plaque assays (Dulbecco and Vogt (1953) Cold Spring Harbor Symp. Quant. Biol., 18: pp. 273-279; exemplary assays described in Johnson et al., (1990) Quantitative Assays for Virus Infectivity. In: Aldovini A., Walker BD (eds.) Techniques in HIV Research. Palgrave Macmillan, London).

[0147] The inventors have found that HSV expressing the PTEN-alpha transgene replicates at a high rate in mesenchymal progenitor cells or stem cells. For example, HSV expressing the PTEN-alpha transgene can replicate at least 10% more than the corresponding HSV control. In one example, HSV expressing the PTEN-alpha transgene can replicate at least 20% more than the corresponding HSV control. In another example, HSV expressing the PTEN-alpha transgene can replicate at least 30% more than the corresponding HSV control. In yet another example, HSV expressing the PTEN-alpha transgene can replicate at a rate between 20% and 40% more than the corresponding HSV control.

[0148] Delivery to cancer cells The inventors have identified that mesenchymal progenitor cells or stem cells migrate toward cancer cells and transfer payloads such as viruses or transgenes expressed by them. Therefore, in one example, this disclosure includes a method for delivering the oncolytic virus referenced above to cancer cells by administration to a subject of the mesenchymal progenitor cells or stem cells disclosed herein. In one example, the viral payload may be transferred by contacting cancer cells with mesenchymal progenitor cells or stem cells modified to introduce the oncolytic virus referenced above. To avoid misunderstanding, the oncolytic virus delivered to cancer cells is the oncolytic virus introduced into the mesenchymal progenitor cells or stem cells. In another example, this disclosure includes a method for increasing PTENα expression in cells, comprising contacting the cells with the population disclosed herein. In this example, the cells may be cancer cells. In one example, increasing PTENα expression in cells reduces the level of phosphorylated AKT in the cells. In one example, the method is carried out in vivo. For example, the population disclosed herein may be administered to a subject.

[0149] The term “contact” is used in the context of this disclosure to mean “direct” or “indirect” contact. “Direct contact” is used in the context of this disclosure to mean physical contact between cancer cells and modified mesenchymal progenitor cells or stem cells that facilitate the transfer of payloads, such as oncolytic viruses and / or transgenes, expressed by them. For example, cancer cells and modified mesenchymal progenitor cells or stem cells may come into direct contact via a common connexin (i.e., a connexin expressed by both cancer cells and modified mesenchymal progenitor cells or stem cells). In this example, the common connexin facilitates the transfer of payloads from mesenchymal progenitor cells or stem cells to cancer cells via gap connections.

[0150] In the context of this disclosure, “indirect contact” is used to refer to the delivery of oncolytic viruses from modified mesenchymal progenitor cells or stem cells to cancer cells without direct contact. For example, modified mesenchymal progenitor cells or stem cells in close proximity to cancer cells may indirectly contact the cancer cells. In one example, modified mesenchymal progenitor cells or stem cells in indirect contact with cancer cells may deliver a payload to the cancer cells via exosomes. In another example, modified mesenchymal progenitor cells or stem cells in indirect contact with cancer cells may deliver a payload to the cancer cells via secretion into the surrounding environment.

[0151] In one example, both direct and indirect contact may be mediated by administration to the population disclosed herein.

[0152] In another example, modified mesenchymal progenitor cells or stem cells that come into direct contact with cancer cells can deliver payloads to the cancer cells via common connexins and indirectly via exosomes.

[0153] Cancer cells that receive a payload from modified mesenchymal progenitor cells or stem cells are not specifically limited insofar as they can come into direct or indirect contact with the modified mesenchymal progenitor cells or stem cells to facilitate the transfer of the oncolytic virus. In one example, the cancer cells are brain cancer cells. For example, the cancer cells may be derived from glioblastoma. In one example, the cancer cells are glioma cells. In one example, the cancer cells are pancreatic cancer cells. In another example, the cancer cells are lung cancer cells. In another example, the cancer cells are cervical cancer cells. In another example, the cancer cells are colorectal cancer cells. In another example, the cancer cells are liver cancer cells. In another example, the cancer cells are osteosarcoma cells. In another example, the cancer cells are prostate cancer cells. In another example, the cancer cells are melanoma cells. In one example, the cancer cells are breast cancer cells. In one example, the cancer cells are PTEN-deficient cancer cells.

[0154] In another example, cancer cells are syncytium cancer cells. In the context of this disclosure, the term “syncytium” is used to refer to cancerous tissue or tumors consisting of cells interconnected by specialized membranes containing electrically synchronized gap connections in action potentials.

[0155] The delivery of oncolytic viruses from modified mesenchymal progenitor cells or stem cells to cancer cells can be facilitated in vivo via various exemplary routes. For example, mesenchymal progenitor cells or stem cells can be administered systemically, for example, by intravenous, intra-arterial, or intraperitoneal administration. In other examples, mesenchymal progenitor cells or stem cells can be administered intranasally or intramuscularly. In one example, mesenchymal progenitor cells or stem cells are administered to a site close to the cancer cells, such as in surrounding tissue. In another example, mesenchymal progenitor cells or stem cells are administered directly to the cancer.

[0156] Treatment method For example, the cell populations and compositions comprising them as disclosed herein may be administered for the treatment of cancer. The term “cancer” refers to or describes a physiological condition in mammals typically characterized by uncontrolled cell growth. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid neoplasms. Further detailed examples of such cancers, though not limited to these, include: squamous cell carcinoma (e.g., epithelial squamous cell carcinoma), small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma and squamous cell carcinoma of the lung, lung cancer, peritoneal cancer, hepatocellular carcinoma, gastrointestinal cancer and gastrointestinal stromal cancer, gastric or stomach cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, urinary tract cancer, hepatocellular carcinoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney or renal cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, anal cancer, penile cancer, melanoma, superficial spreading melanoma, melanoma derived from lentigo malignant melanoma, acral lentiginous melanoma, nodular melanoma, and multiple bone cancers. Myeloma and B-cell lymphoma (including low-grade / follicular non-Hodgkin lymphoma (NHL); small lymphocytic (SL) NHL; intermediate-grade / follicular NHL; intermediate-grade diffuse NHL; high-grade immunoblastic NHL; high-grade lymphoblastic NHL; high-grade small non-incisional nuclear cell NHL; giant tumor NHL; mantle cell lymphoma; AIDS-associated lymphoma; and Waldenström hypergammaglobulinemia); chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); hairy cell leukemia; chronic myeloblastic leukemia; and post-transplant lymphoproliferative disorders (PTLD), as well as abnormal angiogenesis associated with nevus (phakomatoses), edema (such as that associated with brain tumors), Meggs syndrome, brain and head and neck cancers, and associated metastases.

[0157] In one example, the cancer is brain cancer. In another example, the cancer is glioblastoma. In another example, the cancer is pancreatic cancer. In yet another example, the cancer is lung cancer. In yet another example, the cancer is cervical cancer. In yet another example, the cancer is colorectal cancer. In yet another example, the cancer is liver cancer. In yet another example, the cancer is osteosarcoma. In yet another example, the cancer is prostate cancer. In yet another example, the cancer is melanoma.

[0158] In one example, the cancer is PTEN-mutated or deficient cancer. In another example, the cancer is PTEN-mutated or deficient glioblastoma, endometrial cancer, colon cancer, lung cancer, breast cancer, prostate cancer, and ovarian cancer. In yet another example, the cancer is PTEN-mutated or deficient breast cancer. In yet another example, the cancer is PTEN-mutated or deficient brain cancer. In yet another example, the cell population and compositions comprising the cell population and thereof according to this disclosure may be used in a method for killing cancer cells. In yet another example, the cancer cells killed by such a method may be derived from the types of cancer referenced above.

[0159] cell composition This disclosure encompasses a population of mesenchymal progenitor cell lines or stem cells. Such populations may be provided in compositions. For example, in carrying out the methods of this disclosure, mesenchymal progenitor cells or stem cells may be provided in compositions suitable for administration to a subject.

[0160] The exemplary compositions of this disclosure may include mesenchymal progenitor cells or stem cells modified to introduce HSV. Exemplary HSVs are described above. In one example, the compositions of this disclosure may include mesenchymal progenitor cells or stem cells modified to introduce oncolytic viruses or combinations thereof as referenced above. For example, mesenchymal progenitor cells or stem cells may be modified to introduce HSV containing a PTEN-alpha transgene. In one example, the PTEN-alpha transgene contains the nucleic acid sequence shown in SEQ ID NO: 1.

[0161] In another example, the composition according to this disclosure may include mesenchymal progenitor cells or stem cells modified to introduce highly infectious HSV in the mesenchymal progenitor cells or stem cells. In one example, the level of infectivity exceeds 15% of the mesenchymal progenitor cells or stem cells. In another example, the level of infectivity exceeds 25% of the mesenchymal progenitor cells or stem cells. In yet another example, the level of infectivity exceeds 35% of the mesenchymal progenitor cells or stem cells. In yet another example, the level of infectivity exceeds 45% of the mesenchymal progenitor cells or stem cells.

[0162] In another example, the composition according to the present disclosure may include mesenchymal progenitor cells or stem cells that have been modified to introduce HSVs that do not substantially affect the viability of the mesenchymal progenitor cells or stem cells.

[0163] In another example, the compositions according to the present disclosure may include mesenchymal progenitor cells or stem cells that have been modified to introduce HSV in a way that does not kill the mesenchymal progenitor cells or stem cells before delivering the oncolytic virus to cancer cells.

[0164] For example, such a composition may include a pharmaceutically acceptable carrier and / or excipient.

[0165] The terms “carrier” and “excipient” refer to compositions conventionally used in the art to preserve, administer, and / or enhance the biological activity of active compounds (see, for example, Remington's Pharmaceutical Sciences, 16th edition, Mac Publishing Company (1980)). Carriers may also reduce any undesirable side effects of the active compound. A suitable carrier is, for example, stable, and does not react with other components in the carrier. In one example, the carrier does not produce significant local or systemic adverse effects in the recipient at the dosage and concentration used for treatment.

[0166] Suitable carriers for this disclosure include conventionally used ones such as water, physiological saline, water-soluble glucose, lactose, Ringer's solution, buffer solutions, and hyaluronic acid, while glycol is an exemplary liquid carrier, particularly for solutions (in the case of isotonic solutions). 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, glycerin, propylene glycol, water, and ethanol.

[0167] In another example, the carrier is a culture medium composition, for example, on which cells grow or are suspended. Such a culture medium composition does not induce any adverse effects on the subject to which it is administered.

[0168] The exemplary carriers and excipients do not adversely affect cell viability and / or the ability of cells to treat or prevent disease.

[0169] In one example, the carrier or excipient provides buffering activity to maintain cells and / or soluble factors at a suitable pH for them to exert 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 may be produced as a liquid for direct application, for example, by injection, into the bloodstream or into tissue or the surrounding or adjacent area of ​​tissue.

[0170] The cell 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.

[0171] For example, the composition contains an effective or therapeutically effective amount of cells. For instance, the composition contains approximately 1 x 10⁶ cells. 5 From a single cell, approximately 1 x 10⁶ cells9 Cells or about 1.25 x 10 3 cells to about 1.25 x 10 7 cells are included. The exact amount of cells administered depends on various factors including the age, weight and gender of the subject and the degree and severity of the disorder being treated.

[0172] Exemplary dosages are at least about 1.2 x 10 8 to about 8 x 10 10 cells, for example between about 1.3 x 10 8 and about 8 x 10 9 cells, between about 1.4 x 10 8 and about 8 x 10 8 cells, between about 1.5 x 10 8 and about 7.2 x 10 8 cells, between about 1.6 x 10 8 and about 6.4 x 10 8 cells, between about 1.7 x 10 8 and about 5.6 x 10 8 cells, between about 1.8 x 10 8 and about 4.8 x 10 8 cells, between about 1.9 x 10 8 and about 4.0 x 10 8 cells, between about 2.0 x 10 8 and about 3.2 x 10 8 cells, between about 2.1 x 10 8 and about 2.4 x 10 8 cells are included. For example, the dosage can include at least about 1.5 x 10 8 cells. For example, the dosage can include at least about 2.0 x 10 8 cells.

[0173] In other words, exemplary dosages include at least about 1.5 x 10 6 cells / kg (subject 80 kg). In one example, the dosage can include at least about 2.5 x 10 6 cells / kg. In other examples, the dosage is between about 1.5 x 10 6 and about 1 x 10 9cells / kg, cells approximately 1.6 x 10 6 From approximately 1 x 10 8 cells / kg, cells approximately 1.8 x 10 6 From approximately 1 x 10 7 cells / kg, cells approximately 1.9 x 10 6 From approximately 9 x 10 6 cells / kg, approximately 2.0 x 10 cells 6 From approximately 8 x 10 6 cells / kg, approximately 2.1 x 10 cells 6 From approximately 7 x 10 6 cells / kg, approximately 2.3 x 10 cells 6 From approximately 6 x 10 6 cells / kg, approximately 2.4 x 10 cells 6 From approximately 5 x 10 6 cells / kg, approximately 2.5 x 10 cells 6 From approximately 4 x 10 6 cells / kg, approximately 2.6 x 10 cells 6 From approximately 3 x 10 6 May contain pieces / kg.

[0174] For example, modified mesenchymal progenitor cells 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.

[0175] The compositions of this disclosure may be cryopreserved. Cryopreservation of mesenchymal progenitor cells or stem cells may 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 the unfrozen cells.

[0176] The cryopreserved composition may contain a cryopreservation solution. The pH of the cryopreservation solution is typically 6.5 to 8, preferably 7.4.

[0177] The cryopreservation solution may include, for example, 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 (C6H11NaO7); 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). No antimicrobial agents are contained. The pH is adjusted using sodium hydroxide. The pH is 7.4 (6.5 to 8.0).

[0178] The cryopreservation solution may contain Profreeze®. The cryopreservation solution may additionally or alternatively contain a culture medium, such as αMEM.

[0179] To facilitate freezing, cryoprotective substances, such as dimethyl sulfoxide (DMSO), are usually added to the cryopreservation solution. Ideally, the cryoprotective substance should be non-toxic, non-antigenic, and chemically inactive to cells and patients, resulting in high viability after thawing and enabling transplantation without washing. However, the most commonly used cryoprotector, DMSO, exhibits some cytotoxicity. Hydroxyethyl starch (HES) can be used as an alternative or in combination with DMSO to reduce the cytotoxicity of the cryopreservation solution.

[0180] The cryopreservation solution may contain one or more of DMSO, hydroxyethyl starch, human serum components, and other protein bulk agents. In one example, the cryopreserved 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.

[0181] In one embodiment, the cells are suspended in 42.5% Profreeze® / 50% αMEM / 7.5% DMSO and cooled in a controlled-rate freezer.

[0182] The cryopreserved composition may be thawed and administered directly to the subject or added to another solution, such as one containing hyaluronic acid. Alternatively, the cryopreserved composition may be thawed and the mesenchymal progenitor cells or stem cells may be resuspended in an alternative carrier before administration.

[0183] In one example, the cell composition described herein may be administered as a single dose. In another example, the cell composition may be administered as multiple doses, for example, 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 doses.

[0184] In one example, mesenchymal progenitor cells or stem cells may be cultured and grown before administration. Various methods for culturing mesenchymal progenitor cells or stem cells are well known in the art. In one example, mesenchymal progenitor cells or stem cells are cultured and grown in serum-free medium before administration. For example, mesenchymal progenitor cells or stem cells may be passaged at least once, twice, three times, four times, five times, six times, seven times, eight times, nine times, ten times or more before administration.

[0185] Mesenchymal progenitor cells or stem cells can be administered systemically, for example, by intravenous, intra-arterial, or intraperitoneal administration. Mesenchymal progenitor cells or stem cells can also be administered intranasally, intramuscularly, or intracardiacly. In one example, mesenchymal progenitor cells or stem cells are administered directly to the target tumor. [Examples]

[0186] (Example 1) HSV-P10 loading of mesenchymal stem cells (MSCs) PTENα-expressing herpes simplex virus (HSV-P10), an oncolytic virus, was generated using a modified PTENα gene sequence. The PTENα CUG start codon was mutated to AUG to enhance translation of the full-length protein with an extended N-terminus, and the internal canonical PTEN AUG start codon was mutated to AUA to suppress canonical PTEN expression from the construct. PTENα was incorporated into an oncolytic HSV1 scaffold in which both copies of γ34.5 within the viral ICP6 gene locus were deleted. Figure 1 shows the structures of the genetic manipulations performed within the ICP6 locus of the control (HSVQ) and the HSV-P10 virus used in the study.

[0187] Mesenchymal stem cells were loaded with either HSVQ or HSV-P10 at multiples of infection (MOI) of 0.025, 0.05, 0.1, 0.2, and 0.5, and infection was determined over time by detecting GFP in the cells (Figures 2A and 2E). GFP was monitored over time using a Cytation 5 Cell Imaging Multi-Mode Reader in conjunction with a BioSpa 8 Automated Incubator (Biotek Instruments, INC.). The number of GFP targets was quantified and graphed as the mean ± SEM of 4 wells per treatment group. Cell replication rate correlated with the MOI of HSVQ or HSV-P10 used to infect the mesenchymal stem cells.

[0188] To determine the dynamics of HSV-P10 and HSVQ virus replication in mesenchymal stem cells, a comparison was performed between mesenchymal stem cells loaded with HSV-P10 and HSVQ (Figure 2A). Mesenchymal stem cells were divided into 3x10 cells. 6 Individual cells were seeded in 6-well plates and cultured for 24 hours. The seeded mesenchymal stem cells were infected with 1 MOI of HSVQ or HSV-P10 for 1 hour. After incubation, the culture medium was removed and replaced with fresh DMEM, and cultured for another 24 hours. The mesenchymal stem cells loaded with HSVQ or HSV-P10 and the controlled culture medium were collected, and titration studies were performed using vero cells.

[0189] Compared to HSVQ, HSV-P10 was thought to have superior viral replication dynamics (Figure 2A). However, the viral titer of mesenchymal stem cells loaded with HSV-P10 was comparable to that of mesenchymal stem cells loaded with HSVQ (Figure 2B). Viral replication of both HSV-P10 and HSVQ was observed in loaded mesenchymal stem cells even after 5 passages in vitro.

[0190] To determine the effect of viral loading on the survival rate of mesenchymal stem cells, cytosolic activity (aqua live / dead pigment) and GFP expression were evaluated by flow cytometry in loaded mesenchymal stem cells, determined and quantified, and represented as a histogram (Figure 3). The data demonstrate that mesenchymal stem cells loaded with HSV-10 and HSVQ were viable for 24 hours after infection (Figure 3A). The four quadrants of the flow cytometry are shown in Figure 3B.

[0191] (Example 2) Evaluation of functional PTENα expressed by mesenchymal stem cells (MSCs) loaded with HSV-P10. To evaluate the functionality of PTENα expressed by HSV-P10, we determined the effect of HSV-P10 on the PI3K / AKT signaling pathway in HSV-P10-loaded mesenchymal stem cells. Western blot analysis revealed increased AKT in HSVQ-loaded mesenchymal stem cells, while mesenchymal stem cells loaded with HSV-P10 expressing PTENα showed decreased phosphorylated AKT compared to control virus-loaded cells (Figure 4A). PTENα was detected in the conditional medium of HSV-P10-loaded mesenchymal stem cells, suggesting PTENα secretion by HSV-P10-loaded mesenchymal stem cells (Figure 4B).

[0192] (Example 3) Effect of HSV-P10-loaded mesenchymal stem cells on tumor cells To determine the ability of HSV-P10-loaded mesenchymal stem cells to deliver HSV-P10 to cancer cells, a Boyden chamber assay was performed, and migration was monitored over time using a Cytation 5 Cell Imaging Multi-Mode Reader in conjunction with a BioSpa 8 Automated Incubator (Biotek Instruments, INC.). However, analysis of the migration of HSVQ and HSV-P10-loaded mesenchymal stem cells surprisingly revealed increased kinetics of HSV-P10-loaded mesenchymal stem cells to human breast cancer cells (MDA-468) compared to HSVQ-loaded mesenchymal stem cells (Figure 5).

[0193] (Example 4) Effect of HSV-P10-loaded mesenchymal stem cells on primary human glioma cells Mesenchymal stem cells loaded with HSVQ and HSV-P10 were co-cultured with RPF-expressing GMB12-primary human glioma cells (Figure 6A). The function of PTENα expressed by HSV-P10-loaded mesenchymal stem cells on the PI3K / AKT signaling pathway was determined. Western blot analysis revealed an increase in PTENα and a decrease in phosphorylated AKT in glioma cells after co-culture with MSCs (Figure 6B).

[0194] (Example 5) The effect of HSV-P10-loaded mesenchymal stem cells on breast cancer cells. Co-culture of HSV-P10-loaded mesenchymal stem cells with DB7 mouse mammary cancer cells was determined by cytosolic activity (aqua live / dead pigment) and GFP expression, resulting in the transfer of HSV-P10 to cancer cells and the induction of cell death in these cancer cells. An increase in the total number of dead DB7 mouse mammary cancer cells was observed after co-culture with HSV-Q-loaded mesenchymal stem cells compared to unloaded mesenchymal stem cells (control) (Figure 7). A further increase in the total number of dead DB7 mouse mammary cancer cells was observed after co-culture with HSV-P10-loaded mesenchymal stem cells compared to unloaded mesenchymal stem cells (control) and cells loaded with HSV-Q (Figure 7).

[0195] (Example 6) Cancer treatment Prior to administration to subjects diagnosed with PTEN mutation or deficiency cancer, a recombinant virus containing a polynucleotide encoding the phosphatase and tensin homolog alpha (PTENα) deleted on chromosome 10 is loaded into mesenchymal progenitor cells or stem cells. Approximately 200 million loaded mesenchymal progenitor cells are then administered.

[0196] The treated subjects will be evaluated for safety and efficacy of the treatment over approximately 2 to 6 weeks. Additional doses of loaded mesenchymal progenitor cells or stem cells will be administered as needed.

[0197] (Example 7) Cancer treatment Before administration to subjects diagnosed with cancer, a recombinant virus containing the herpes simplex virus (HSV) skeleton and polynucleotides encoding the phosphatase and tensin homolog alpha (PTENα) deleted on chromosome 10 is loaded into mesenchymal progenitor cells or stem cells. The virus is then added to the culture medium of the mesenchymal progenitor cells or stem cells to load them with approximately 10-50 infection units (iu) / MPC. The subjects are then administered approximately 200 million loaded mesenchymal progenitor cells or stem cells.

[0198] The treated subjects will be evaluated for safety and efficacy of the treatment over approximately 2 to 6 weeks. Additional doses of loaded mesenchymal progenitor cells or stem cells will be administered as needed.

[0199] It will be understood by those skilled in the art that numerous changes and / or modifications can be made to the disclosure as shown in the specific embodiments without departing from the spirit or scope of the extensively described disclosure. Therefore, these embodiments are considered illustrative and not limiting in all respects.

[0200] All publications discussed above are incorporated in their entirety into this specification.

[0201] This application claims priority to Filing No. 62 / 882840, dated 5 August 2019, the entire disclosure of which is incorporated herein by reference.

[0202] Any consideration of documents, actions, materials, devices, articles, etc., included herein is solely for the purpose of providing context for this disclosure. Any or all of these matters, by existing prior to the priority date of each claim of this application, shall not be construed as an acknowledgment of forming part of the prior art or being common general knowledge in the art relating to this disclosure. (References) Ausubel et al. (editors) (1988, including all updates until present) Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience. Bader et al. (2011) Gene Ther. 18:1121-6. Brown TA (editor) (1991) Essential Molecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press. Coligan et al. (editors) (including all updates until present) Current Protocols in Immunology, John Wiley & Sons. Glover and Hames (editors) (1995 & 1996) DNA Cloning: A Practical Approach, Volumes 1-4, IRL Press. Griffiths-Jones, S. 2004 Nucl Acids Res, 32, D109-D111. Harlow and Lane (editors) (1988) Antibodies: A Laboratory Manual, Cold Spring Harbour Laboratory. Kozomara et al. 2013; Nucl Acids Res, 42, D68-D73. Lennox and Behlke (2011) Gene Ther. 18”1111-20. Perbal J (1984) A Practical Guide to Molecular Cloning, John Wiley and Sons. Sambrook et al., (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbour Laboratory Press. Simmons & Torok-Storb (1991) Blood. 78:55-62.

Claims

1. A population of mesenchymal progenitor cells (MPCs) or mesenchymal stem cells (MSCs) wherein the MPCs or MSCs are modified to increase the expression of a phosphatase and tensin homolog alpha (PTENα) deleted on chromosome 10, wherein the modified MPCs or MSCs contain recombinant oncolytic herpes simplex virus (HSV) comprising a polynucleotide encoding PTENα, and the increase in PTENα expression is sufficient to reduce the level of phosphorylated AKT in the modified MPCs or MSCs, to enhance tumor cell death, and / or to enhance the migration of the modified MPCs or MSCs to tumor cells.

2. Between 20% and 80% of the cells contain the recombinant oncolytic HSV, and - The polynucleotide encoding PTENα is operably linked to a tumor-specific promoter or an inducible promoter; and / or - The recombinant oncolytic HSV contains a capsid protein that binds to tumor-specific cell surface molecules. The group described in claim 1.

3. The population according to claim 2, wherein the tumor-specific promoter is the Survivin promoter, COX-2 promoter, PSA promoter, CXCR4 promoter, STAT3 promoter, hTERT promoter, AFP promoter, CCKAR promoter, CEA promoter, erbB2 promoter, E2F1 promoter, HE4 promoter, LP promoter, MUC-1 promoter, TRP1 promoter, or Tyr promoter.

4. The population according to claim 2, wherein the capsid protein is a fiber, penton, or hexon protein.

5. The population according to any one of claims 1 to 4, wherein the recombinant oncolytic HSV comprises a variant thereof encoding a protein having the nucleic acid sequence shown in SEQ ID NO: 1 or the amino acid sequence shown in SEQ ID NO:

2.

6. The population according to any one of claims 1 to 5, wherein the tumor cells are breast cancer or brain cancer cells.

7. The group according to any one of claims 1 to 6, wherein the group is a group of MPCs.

8. The group according to any one of claims 1 to 6, wherein the group is a group of MSCs.

9. The population according to any one of claims 1 to 5, wherein the MPCs or MSCs in the population of MPCs or MSCs are cultured and grown from a population of cells containing 0.1% to 75% STRO-1+ cells.

10. A pharmaceutical composition comprising the group described in any one of claims 1 to 9.

11. A group according to any one of claims 1 to 9 for use in the treatment of cancer.

12. Use of the population according to any one of claims 1 to 9 in the manufacture of a pharmaceutical product for treating cancer in a subject.

13. The use according to claim 12, wherein the cancer is breast cancer or brain cancer.

14. The use according to claim 12, wherein the cancer is selected from the group consisting of lung cancer, pancreatic cancer, colorectal cancer, liver cancer, cervical cancer, prostate cancer, breast cancer, endometrial cancer, thyroid cancer, kidney cancer, brain cancer, glioblastoma, osteosarcoma, and melanoma.

15. The use according to claim 12, 13, or 14, wherein the pharmacopoeia is prescribed for intravenous, intra-arterial, intratumorial, or intraperitoneal administration.

Images