Cell compositions comprising viral vectors and methods of treatment

Abstract

To provide, in part, improved compositions and methods for delivering viruses to tumor cells.SOLUTION: The present disclosure relates to cell compositions that have been modified to introduce recombinant viruses. Such compositions can be used to treat cancer by delivering the virus to cancer cells.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 62 / 882,840, filed August 5, 2019, which is incorporated herein by reference in its entirety.

[0002] References 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; Created: August 3, 2020) are incorporated herein by reference in accordance with 37 CFR §1.52(e)(5).

[0003] FIELD OF THE INVENTION 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. [Background technology]

[0004] Background of the Invention Cancer treatment typically involves surgical resection, standard chemotherapy and / or radiation therapy to remove or kill cancer cells. However, the effectiveness of these treatments is often limited due to the aggressiveness of tumors and / or collateral damage to healthy tissue. This situation indicates the need for new therapeutic strategies, and one such approach is the use of viruses.

[0005] Oncolytic viruses are viruses that can specifically replicate in and destroy cancer cells, either inherently 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 host immunity. [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 2014273211 [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 literature]

[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] F.M. Ausubel et al. (eds.), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience (1988, includes all current information) [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. (editors), Current Protocols in Immunology, John Wiley & Sons (contains all current information) [Non-patent document 8] Hopkins, (2013) Science., 6144:399~402 pages [Non-licensed 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) Summary of the Invention [Problem to be solved by the invention]

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

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

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

[0011] In another example, mesenchymal progenitor or stem cells are modified to transduce recombinant viruses, for example, mesenchymal progenitor or stem cells can be modified to transduce recombinant viruses comprising a herpes simplex virus (HSV) backbone.

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

[0013] The present inventors have also identified mesenchymal progenitor or stem cells as effective carriers of recombinant viruses containing a herpes simplex virus (HSV) backbone and expressing a PTEN transgene, and have described particularly high infection and replication rates with these viral constructs. Combined with the above-described capabilities of modified cells according to the present disclosure, the inventors' findings suggest that mesenchymal progenitor or stem cells containing the modifications discussed herein, particularly when modified to introduce recombinant viruses containing an HSV backbone, may represent novel and effective compositions for treating various cancers that can be commercially scaled up. Thus, in one example, the recombinant virus contains a herpes simplex virus (HSV) backbone.

[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 a population disclosed herein contain the virus. In another example, at least 20% of the cells in a population disclosed herein contain the virus. In another example, between 20% and 80% of the cells in a population disclosed herein contain the virus.

[0015] In one example, the polynucleotide encoding PTEN-alpha is operably linked to a tumor-specific promoter. In a further example, the tumor-specific promoter is a survivin promoter, a COX-2 promoter, a PSA promoter, a CXCR4 promoter, a STAT3 promoter, an hTERT promoter, an AFP promoter, a CCKAR promoter, a CEA promoter, an erbB2 promoter, an E2F1 promoter, a HE4 promoter, an LP promoter, a MUC-1 promoter, a TRP1 promoter, or a Tyr promoter.

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

[0017] In one example, the recombinant virus comprises a capsid protein that binds to a tumor-specific cell surface molecule. In a further example, the capsid protein is a fiber, penton, or hexon protein.

[0018] In one example, the recombinant virus comprises the nucleic acid sequence set forth in SEQ ID NO: 1, or a variant thereof, which is translated into a protein comprising the amino acid sequence set forth in SEQ ID NO: 2. In one example, the variant of SEQ ID NO: 1 shares at least 85%, 90%, 95%, 99% sequence identity with SEQ ID NO: 2.

[0019] In one example, the recombinant virus is HSV.

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

[0021] In one example, the mesenchymal progenitor 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 one example, the mesenchymal progenitor or stem cells express STRO-1. In another example, the mesenchymal progenitor or stem cells express substantially STRO-1. bri In one example, the mesenchymal precursor or stem cells express Ang1:VEGF at a ratio of at least 2:1 to 30:1. In another example, the mesenchymal precursor or stem cells express Ang1:VEGF at a ratio of at least about 10:1. In a further example, the mesenchymal precursor or stem cells express Ang1:VEGF at a ratio of at least about 20:1. In another example, the mesenchymal precursor or stem cells express Ang1:VEGF at a ratio of at least about 30:1.

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

[0023] In one example, the mesenchymal progenitor or stem cells express two or more markers selected from the group consisting of STRO-1 and alpha 1, alpha 2, alpha 3, alpha 4 and alpha 5, alpha v, beta 1 and beta 3.

[0024] In one example, the population of cells is expanded in culture.

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

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

[0027] The present disclosure also encompasses a method of treating cancer in a subject, comprising administering a population or composition according to any one of the examples provided above. In one example, the present disclosure also encompasses a method of killing cancer cells, comprising contacting a population of cancer cells with a population or composition according to any one of the examples provided above. In another example, the present disclosure also encompasses a method of delivering mesenchymal progenitor or stem cells to cancer cells in a subject, comprising administering a population or composition according to any 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 via intravenous, intraarterial, or intraperitoneal administration. In one example, the composition is administered directly to the target tumor.

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

[0029] Any example in this specification should be construed mutatis mutandis to apply to any other example unless specifically stated otherwise.

[0030] The present disclosure is not to be limited in scope by the specific examples described herein, which are intended for the purpose of illustration only. Functionally equivalent products, compositions and methods, as described herein, are clearly within the scope of the present disclosure.

[0031] Throughout this specification, unless otherwise specified or the content clearly indicates otherwise, reference to a single step, composition, group of steps or group of compositions should be interpreted to encompass one and more (i.e., one or more) of that step, composition, group of steps or group of compositions.

[0032] The present disclosure is herein below described by way of the following non-limiting examples and with reference to the accompanying figures. BRIEF DESCRIPTION OF THE ATTACHED DRAWINGS [Brief explanation of the drawings]

[0033] [Figure 1] (A and B) Schematic diagram showing the viral backbone of HSVQ (parent virus) and HSV-P10 (PTENα-expressing virus). [Figure 2] (A and B) HSV-P10 loading of mesenchymal stem cells (MSCs). [Figure 3A] FIG. 1 shows the viability of HSV-P10 and HSVQ loaded mesenchymal stem cells (MSCs). [Figure 3B] FIG. 1 shows the viability of HSV-P10 and HSVQ loaded mesenchymal stem cells (MSCs). [Figure 4](A and B) Effects of HSV-P10-loaded mesenchymal stem cells (MSCs) on PTENα expression and the PI3K / AKT signaling pathway. [Figure 5] FIG. 1 shows the migration of HSV-P10 and HSVQ-loaded mesenchymal stem cells (MSCs) into human breast cancer cells (MDA-468). [Figure 6] (A and B) Effect of HSV-P10-loaded mesenchymal stem cells (MSCs) on human glioma cells. [Figure 7] FIG. 1 shows induction of tumor cell death in DB7 mouse breast cancer cells co-cultured with HSV-P10 and HSVQ-loaded mesenchymal stem cells (MSCs). DETAILED DESCRIPTION OF THE INVENTION

[0034] Detailed Description of the Invention General Techniques and Selective Definitions Unless otherwise specified, all technical and scientific terms used herein should be understood to have the same meaning as commonly understood by one of ordinary skill 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 utilized 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 Harbor Laboratory Press (1989), T.A. Brown (editor), Essential Molecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press (1991), D.M. Glover and B.D. Hames (editors), DNA Cloning: A Practical Approach, Volumes 1-4, IRL Press (1995 and 1996), and F.M. Ausubel et al. (editors), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience (1988, including all current information), Ed. Harlow and David Lane (editors), Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, (1988), and J.E. Coligan et al. (editors), Current Protocols in It is described and explained throughout the literature in sources such as Immunology, John Wiley & Sons (including all current updates to date).

[0036] As used in this specification and the appended claims, singular words and the singular forms "a," "an," and "the" optionally include plural references unless the content clearly dictates otherwise, so that, for example, "an analyte" optionally includes one or more analytes.

[0037] As used herein, unless otherwise stated, the term "about" refers to + / -10%, more preferably + / -5%, more preferably + / -1% of the specified value.

[0038] The term "and / or," e.g., "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" will be understood to imply the inclusion of a stated element, integer or step or group of elements, integers or steps, 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) that arises from an alternative translation start site 519 base pairs upstream of the ATG start sequence, adding 173 N-terminal amino acids to the normal PTEN translational region. In one example, PTEN-alpha comprises the amino acid sequence set forth in SEQ ID NO: 2. In another example, PTEN-alpha is described in Hopkins et al. (2013) Science., 6144:399-402. The term "phosphatase and tensin homolog deleted on chromosome 10 (PTEN) alpha" is used in the context of the present disclosure to refer to the gene encoding PTEN-alpha. In one example, PTEN-alpha is encoded by a nucleic acid comprising SEQ ID NO: 1, or a variant thereof that encodes a protein comprising the amino acid sequence set forth in SEQ ID NO: 2. Thus, in one example, a population of cells disclosed herein can be modified to increase expression of a nucleic acid encoding a protein comprising the amino acid sequence set forth in SEQ ID NO: 2. In one example, the PTEN-alpha-expressing nucleic acid is modified to promote a higher level of PTEN-alpha translation than PTEN translation. In one example, the PTEN-alpha-expressing nucleic acid is not translated into PTEN in cancer cells.

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

[0042] As used herein, a "PTEN-mutated or -deficient cancer" is a cancer that has been identified by testing a cancer sample from an individual 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 level in normal cells. PTEN mutations or deficiencies have been observed in many cancers, including glioblastoma, endometrial cancer, colon cancer, lung cancer, breast cancer, prostate cancer, and ovarian cancer. In one example, the PTEN-mutated or -deficient cancer has a mutation in PTEN. In another example, the PTEN-mutated or -deficient cancer has a mutation in PTEN-alpha.

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

[0044] As used herein, the term "treatment" refers to a clinical intervention designed to alter the natural course of the individual or cell being treated during the course of clinical pathology. Desirable effects of treatment include reducing the rate of disease progression, ameliorating or alleviating the disease state, and remission or improved prognosis. For example, an individual is successfully "treated" if one or more symptoms associated with a disease are alleviated or eliminated.

[0045] "Effective amount" refers to the minimum amount effective, at the dosage and for the period of time necessary, to achieve the desired therapeutic or preventive result. An effective amount can be provided in one or more administrations. In some examples of the present disclosure, the term "effective amount" is used herein to refer to the amount necessary to effect treatment of the aforementioned disease or condition. The effective amount may vary depending on the disease or condition being treated, as well as the body weight, age, ethnic background, sex, health status and / or physical condition of the mammal being treated, and other relevant factors. Typically, the effective amount falls within a relatively broad range (e.g., a "dosage" range) that can be determined through routine testing and experimentation by a physician. The effective amount can be administered in a single dose or in one or more repeated doses over the treatment period.

[0046] A "therapeutically effective amount" is the minimum concentration required to produce a measurable improvement in a particular disorder (e.g., cancer). As used herein, a therapeutically effective amount 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 desired response in an individual. A therapeutically effective amount is also one in which the therapeutically beneficial effects of the composition outweigh any toxic or adverse effects. In the case of cancer, a therapeutically effective amount can reduce the number of cancer cells; reduce primary tumor size; prevent (i.e., slow to some extent, and in some cases, stop) cancer cell invasion into peripheral organs; prevent (i.e., slow to some extent, and in some cases stop) tumor metastasis; prevent or slow tumor growth or tumor progression to some extent; and / or alleviate to some extent one or more symptoms associated with the disorder. To the extent that the composition can prevent growth and / or kill existing cancer cells, it can be cytostatic and / or cytotoxic. For cancer therapy, in vivo efficacy can be measured, for example, by assessing survival, time to progression (TTP), response rate (RR), duration of response, and / or quality of life.

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

[0048] Culturing and expanding cells from cryopreserved intermediates involves thawing cryogenically frozen cells and culturing them in vitro under conditions suitable for cell growth.

[0049] Mesenchymal progenitor or stem cells As used herein, the term "mesenchymal progenitor or stem cell" refers to an undifferentiated, multipotent cell that has the ability to self-renew while maintaining multipotency and to differentiate into any of a number of cell types of mesenchymal origin, such as osteoblasts, chondrocytes, adipocytes, stromal cells, fibroblasts, and tendons, or into cells of non-mesodermal origin, such as hepatocytes, neurons, and epithelial cells. In various examples, the disclosure encompasses a population of mesenchymal progenitor or stem cells, wherein the cells are modified to enhance migration toward tumor cells. In one example, the disclosure encompasses a population of mesenchymal progenitor or stem cells comprising a recombinant virus, wherein the cells are modified to enhance migration toward tumor cells. For example, the disclosure encompasses a population of mesenchymal progenitor or stem cells, wherein the cells are modified to increase expression of phosphatase and tensin homolog deleted on chromosome 10 alpha (PTENα) sufficiently to enhance migration toward tumor cells. In another example, the disclosure encompasses a population of mesenchymal progenitor or stem cells, which are modified to introduce a recombinant virus containing a polynucleotide encoding PTENα. In this example, expression of PTENα from the virus and translation of the same into PTENα protein is sufficient to enhance migration to tumor cells.

[0050] The term "mesenchymal precursor or stem cell" includes both the parent cell and their undifferentiated progeny. The term also includes mesenchymal precursor or stem cells (MPCs), multipotent stromal cells, mesenchymal stem cells, perivascular mesenchymal precursor or stem cells, and their undifferentiated progeny. Thus, in one example, the mesenchymal precursor or stem cell is a mesenchymal stem cell.

[0051] Mesenchymal progenitor or stem cells can be autologous, allogeneic, xenogeneic, syngeneic, or isogeneic. Autologous cells are isolated from the same individual to be reimplanted. Allogeneic cells are isolated from a donor of the same species. Xenogeneic cells are isolated from a donor of another species. Syngenic or isogeneic cells are isolated from genetically identical organisms such as twins, clones, or highly inbred research animal models.

[0052] In one example, the mesenchymal progenitor or stem cells are allogeneic. In one example, the allogeneic mesenchymal progenitor or stem cells are expanded in culture and cryopreserved.

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

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

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

[0056] In another example, mesenchymal progenitor or stem cells according to the present disclosure express STRO-1 and an integrin having an alpha chain polypeptide selected from the group consisting of alpha 1 (CD49a), alpha 2 (CD49b), alpha 3 (CD49c), alpha 4 (CD49d), alpha 5 (CD49e), and alpha v (CD51). Thus, in one example, mesenchymal progenitor or stem cells according to the present disclosure express STRO-1 and alpha 1. In another example, mesenchymal progenitor or stem cells express STRO-1 and alpha 2. In another example, mesenchymal progenitor or stem cells express STRO-1 and alpha 3. In another example, mesenchymal progenitor or stem cells express STRO-1 and alpha 4. In another example, mesenchymal progenitor or stem cells express STRO-1 and alpha 5. In another example, mesenchymal progenitor or stem cells express STRO-1 and alpha v. In another example, the mesenchymal progenitor or stem cells express STRO-1, α2, and α3. In another example, the mesenchymal progenitor or stem cells express STRO-1, α2, and α5. In another example, the mesenchymal progenitor or stem cells express STRO-1, α3, and α5. In another example, the mesenchymal progenitor or stem cells express STRO-1, α2, α3, and α5.

[0057] In another example, the disclosure encompasses a population of mesenchymal progenitor 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 encompasses a population of mesenchymal progenitor or stem cells enriched for STRO-1 and α2+ cells. In this example, the population enriched for α2+ cells may comprise at least about 30%, 40%, or 50% α2+ cells.

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

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

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

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

[0063] In another example, the present disclosure encompasses a population of mesenchymal progenitor or stem cells enriched for STRO-1, α1+, α3+, α4+ and α5+ cells.

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

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

[0066] Identifying and / or enriching for mesenchymal progenitor or stem cells that express the above-referenced integrins can be accomplished using a variety of methods well known in the art. In one example, fluorescence activated cell sorting (FACS) using commercially available antibodies (e.g., Thermofisher; Pharmingen; Abcam) can be used to identify and select cells that express the desired integrin polypeptide chains or combinations thereof.

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

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

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

[0070] In one example, the mesenchymal progenitor or stem cells express STRO-1, a chimeric antigen receptor (CAR), for example, the mesenchymal progenitor or stem cells express STRO-1, a CAR, αvβ3 and αvβ5.

[0071] In one example, mesenchymal progenitor or stem cells expressing a CAR can induce a T cell-mediated immune response. In another example, a CAR acts as a means for attaching mesenchymal progenitor or stem cells to cancer cells. In another example, a CAR acts as a means for causing enhanced attachment of mesenchymal progenitor or stem cells to cancer cells.

[0072] In one example, the CAR is composed of an extracellular antigen-binding domain, a transmembrane domain, and an intracellular domain. In one 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, b-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, 4-IBB, etc., and in some cases the CAR may include any combination of CD3-zeta, CD28, 4-1BB, TLR-4.

[0074] Exemplary transmembrane domains can be derived from (i.e., comprise at least the transmembrane region of) the alpha, beta, or zeta chain of the T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CDS, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154. In another example, the transmembrane domain can be synthetic, in which case it comprises primarily hydrophobic residues such as leucine and valine.

[0075] Mesenchymal progenitor 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 to allow for the selection of mesenchymal progenitor or stem cells. In one example, mesenchymal progenitor or stem cells can be enriched using STRO-1 antibodies as described in Simmons & Torok-Storb, 1991.

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

[0077] As used herein, the term "enriched" describes a population of cells that has an increased proportion of one particular cell type or a number of particular cell types compared to an untreated cell population (e.g., cells in their natural environment). In one 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 expressly supporting the term "a population of cells comprising X% STRO-1+ cells," where X% is a percentage recited herein. STRO-1+ cells, in some instances, can form clonogenic colonies, such as CFU-F (fibroblasts), or a subset thereof (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 comprising 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 allows for the selection of STRO-1+ cells. The marker may, but is not necessarily, STRO-1. For example, as described and / or exemplified herein, cells (e.g., MPCs) that express STRO-2 and / or STRO-3 (TNAP) and / or STRO-4 and / or VCAM-1 and / or CD146 and / or 3G5 also express STRO-1 (and may be STRO-1 bright). Thus, the indication that cells are STRO-1+ does not mean that the cells have been selected by STRO-1 expression. In one example, the cells are selected based on at least STRO-3 expression, e.g., they are STRO-3+ (TNAP+).

[0079] Reference to the selection of a cell or population thereof does not necessarily require selection from a particular tissue source. As described herein, STRO-1+ cells may be selected or isolated or enriched from a variety of sources. Thus, in some instances, these terms support selection from any tissue, including STRO-1+ cells or vascularized tissue or tissue containing pericytes (e.g., STRO-1+ or 3G5+ pericytes), or any one or more of the tissues listed herein.

[0080] In one example, the mesenchymal progenitor 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+, 3G5+.

[0081] By "individually," it is meant that the present disclosure encompasses the listed markers or groups of markers separately, and that notwithstanding that individual markers or groups of markers may not be separately described herein, the appended claims can define such markers or groups of markers separately and separably from one another.

[0082] By "collectively," it is meant that the present disclosure encompasses any number or combination of the listed markers or groups of markers, and that notwithstanding that such number or combination of markers or groups of markers may not be specifically set forth herein, the appended claims may define such combinations or subcombinations separately and separably from any other combinations of markers or groups of markers.

[0083] A cell referred to as "positive" for a given marker can express low (lo, dim, or dull), moderate (median), or high (bri) levels of the marker, depending on the extent to which the marker is present on the cell surface, where the terms relate to the intensity of fluorescence or other markers used in the cell sorting process or flow cytometry analysis of cells. The distinction between low (lo, dim, or dull), moderate (median), or high (bri) is understood in the context of the marker used in the particular cell population being sorted or analyzed. A cell referred to as "negative" for a given marker does not necessarily mean that the marker is completely absent from the cell. This term means that the marker is expressed at a relatively very low level by the cell and, when detectably labeled, produces a very low signal or is not detectable above background levels, e.g., the level detected using an isotype control antibody.

[0084] As used herein, the term "bright" or "bri" refers to a marker on the cell surface that generates a relatively high signal when detectably labeled. While not wishing to be limited by theory, it is proposed that "bright" cells express more of the target marker protein (e.g., the antigen recognized by the STRO-1 antibody) than other cells in the sample. For example, STRO-1 bri cells, when labeled with a FITC-conjugated STRO-1 antibody, produce a greater fluorescent signal than non-brightly positive cells (STRO-1 low positive / weakly positive / faintly positive / moderate / intermediate), as determined by fluorescence activated cell sorting (FACS) analysis. In one example, mesenchymal progenitor or stem cells are isolated from bone marrow and enriched by selection for STRO-1+ cells. In this example, "bright" cells comprise 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 comprise 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 positively labeled bone marrow mononuclear cells contained in the starting sample. In one example, strongly STRO-1 positive cells have a 2 log magnitude higher expression of STRO-1 surface expression compared to "background," i.e., cells that are STRO-1-. In comparison, STRO-1 low / weakly / faintly positive and / or STRO-1 moderate / intermediate cells have a 2 log magnitude lower than high expression of STRO-1 surface expression, typically about 1 log or less of "background."

[0085] In one example, STRO-1+ cells are strongly STRO-1 positive. In one example, strongly STRO-1 positive cells are preferentially enriched compared to low / dim / dull STRO-1 positive or intermediate STRO-1 positive cells.

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

[0087] In one example, STRO-1 strongly positive cells are perivascular mesenchymal progenitor 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 non-specific alkaline phosphatase. For example, the term encompasses liver isoform (LAP), bone isoform (BAP) and kidney isoform (KAP). In one example, TNAP is BAP. In one example, TNAP refers to a molecule capable of binding to STRO-3 antibody produced by the hybridoma cell line deposited with ATCC under the provisions of the Budapest Treaty on December 19, 2005 under deposit accession number PTA-7282.

[0089] Furthermore, in one example, STRO-1+ cells are capable of giving rise to clonogenic CFU-F.

[0090] In one example, a significant proportion of STRO-1+ cells can differentiate into at least two different germ lineages. Non-limiting examples of cell lineages to which cells can commit include bone progenitors; hepatocyte progenitors that are multipotent for bile duct epithelial cells and hepatocytes; neural restricted cells that can generate glial progenitors that progress to oligodendrocytes and astrocytes; neural progenitors that progress to neurons; progenitors for cardiac muscle and cardiac myocytes; and glucose-responsive insulin-secreting pancreatic beta cell lines. Other cell lineages include, but are not limited to, odontoblasts, dentin-producing cells, and chondrocytes, as well as precursors for the following cells: retinal pigment epithelial cells, fibroblasts, skin cells such as keratinocytes, dendritic cells, hair follicle cells, renal duct epithelial cells, smooth and skeletal muscle cells, testicular progenitors, vascular endothelial cells, tendons, ligaments, cartilage, adipocytes, fibroblasts, bone marrow stroma, cardiac muscle, smooth muscle, skeletal muscle, pericytes, blood vessels, epithelium, glia, neurons, astrocytes, and oligodendrocytes.

[0091] In one example, the mesenchymal progenitor or stem cells are MSCs. The MSCs can be a homogeneous composition or a mixed cell population enriched for 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 unique monoclonal antibodies. Methods for obtaining MSC-enriched cell populations are described, for example, in U.S. Patent No. 5,486,359. MSCs prepared by conventional plastic adherence isolation rely on the nonspecific plastic adhesion properties of CFU-Fs. Mesenchymal progenitor or stem cells isolated from bone marrow by STRO-1-based immunoselection specifically isolate clonogenic mesenchymal progenitor cells from bone marrow populations in the absence of other plastic-adherent bone marrow populations. Alternative sources for MSCs include, but are not limited to, blood, skin, umbilical cord blood, muscle, fat, bone, and perichondrium. In one example, the MSCs are allogeneic. In one example, the MSCs are cryopreserved. In one example, the MSCs are culture-expanded and cryopreserved.

[0092] In one example, the mesenchymal progenitor 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. Suitable processes for generating mesenchymal progenitor 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):0554-0559 (2005), and Vodyanik et al., Cell Stem cell, Vol 7:718-728 (2010).

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

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

[0095] In another example, the mesenchymal precursor cells express Cx43. In another example, the mesenchymal precursor cells express Cx40. In another example, the mesenchymal precursor cells express Cx43 and Cx40. In another example, the mesenchymal precursor cells express Cx45, Cx32, and / or Cx37. In one example, the mesenchymal precursor cells are not engineered to express a particular connexin.

[0096] Isolated or enriched mesenchymal precursor cells can be expanded in vitro by culturing. Isolated or enriched mesenchymal precursor cells can be expanded in vitro by cryopreservation, thawing, and subsequent culturing.

[0097] In one example, isolated or enriched mesenchymal progenitor cells are 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 density of 50,000 viable cells / cm. 2 and incubated overnight at 37°C and 20% O 2 The culture medium is then replaced and / or changed as needed, and the cells are incubated for an additional 68 to 72 hours at 37°C, 5% O 2 It is cultivated in.

[0098] As will be appreciated by those skilled in the art, cultured mesenchymal precursor cells are phenotypically distinct 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 precursor cells are also biologically distinct from in vivo cells, having a higher proliferation rate compared to the largely non-cycling (quiescent) cells in vivo.

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

[0100] Mesenchymal precursor or stem cells encompassed by the present disclosure may be cryopreserved prior to administration to a subject, hi one example, the mesenchymal precursor or stem cells are expanded in culture and cryopreserved prior to administration to a subject.

[0101] In one example, the present disclosure encompasses mesenchymal progenitor cells or stem cells and their progeny, soluble factors derived therefrom, and / or extracellular vesicles isolated therefrom. In another example, the present disclosure encompasses mesenchymal progenitor cells or stem cells and extracellular vesicles isolated therefrom. For example, mesenchymal progenitor cell lineages or stem cells of the present disclosure can be cultured and grown for a period of time and under conditions suitable for secretion of extracellular vesicles into 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 that are naturally released from cells and range in size from about 30 nm to 10 microns, although typically they are less than 200 nm in size. They contain proteins, nucleic acids, lipids, metabolites, or other substances released from the cells (e.g., mesenchymal stem cells; STRO-1). + They may contain organelles from living cells.

[0103] As used herein, the term "exosome" refers to a type of extracellular vesicle that generally ranges in size from about 30 nm to about 150 nm and originates from the endosomal compartment of mammalian cells, from which it is transported to the plasma 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 other cells to deliver their cargo.

[0104] Cell culture growth In one example, mesenchymal progenitor cells or stem cells are culture-expanded. "Culture-expanded" mesenchymal progenitor cells or stem cells are distinguished from freshly isolated cells in that they have been cultured in a cell culture medium and passaged (i.e., subcultured). In one example, the culture-expanded mesenchymal progenitor cells or stem cells are passage-expanded approximately 4 to 10 times. In one example, the mesenchymal progenitor cells or stem cells are passage-expanded at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, or at least 10 times. For example, the mesenchymal progenitor cells or stem cells may be passage-expanded at least 5 times. In one example, the mesenchymal progenitor cells or stem cells may be passage-expanded at least 5 to 10 times. In one example, the mesenchymal progenitor cells or stem cells may be passage-expanded at least 5 to 8 times. In one example, the mesenchymal progenitor cells or stem cells may be passage-expanded at least 5 to 7 times. In one example, the mesenchymal progenitor cells or stem cells may be passage-expanded more than 10 times. In another example, the mesenchymal progenitor or stem cells may be expanded in culture for more than seven passages. In these examples, the stem cells may be expanded in culture before being cryopreserved to produce an intermediate cryopreserved MLPSC population. In one example, the compositions of the present disclosure are produced by culturing cells from an intermediate cryopreserved MLPSC population, or in other words, a cryopreservation intermediate.

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

[0106] In one example, mesenchymal progenitor or stem cells cultured and expanded from cryopreserved intermediates can be cultured and expanded in a medium free of animal proteins. In one example, mesenchymal progenitor or stem cells cultured and expanded from cryopreserved intermediates can be cultured and expanded in a xeno-free medium. In one example, mesenchymal progenitor or stem cells cultured and expanded from cryopreserved intermediates can be cultured and expanded in a medium free of fetal bovine serum.

[0107] In one embodiment, the mesenchymal progenitor or stem cells can be obtained from a single donor, or from multiple donors, where the donor samples or mesenchymal progenitor or stem cells are subsequently pooled and then culture expanded. In one example, the culture expansion process comprises: i. expanding the number of viable cells by serial propagation to provide a preparation of at least about 1 billion viable cells, the serial propagation comprising establishing a primary culture of the isolated mesenchymal progenitor or stem cells and then sequentially establishing a first non-primary (P1) culture of the mesenchymal progenitor or stem cells isolated from the previous culture; ii. Expanding the P1 culture of isolated mesenchymal progenitor or stem cells by passage expansion into a second, non-primary (P2) culture of mesenchymal progenitor or stem cells; and iii. Preparing and cryopreserving an in-process intermediate mesenchymal progenitor or stem cell preparation obtained from a P2 culture of mesenchymal progenitor or stem cells; and iv. Thawing a cryopreserved in-process intermediate mesenchymal progenitor or stem cell preparation and expanding the in-process intermediate mesenchymal progenitor or stem cell preparation by serial expansion. Includes:

[0108] In one example, the expanded mesenchymal progenitor or stem cell preparation comprises: i. less than about 0.75% CD45+ cells; ii. at least about 95% CD105+ cells; iii. At least about 95% CD166+ cells and having an antigenic and activity profile comprising:

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

[0110] In one example, the cultured expanded mesenchymal progenitor or stem cells have been cultured and expanded for approximately 4-10 passages, wherein the mesenchymal progenitor or stem cells have been cultured and expanded at least 2 or 3 times and then cryopreserved before being cultured further. In one example, the mesenchymal progenitor or stem cells have been cultured and expanded for at least 1, at least 2, at least 3, at least 4, or at least 5 passages, cryopreserved, and then cultured and expanded for at least 1, at least 2, at least 3, at least 4, or at least 5 more passages before being cultured according to the disclosed methods.

[0111] The process of mesenchymal progenitor or stem cell isolation and ex vivo expansion can be carried out using any device and cell handling method known in the art. Various culture expansion embodiments of the present disclosure use steps that require cell manipulation, such as seeding, feeding, dissociation of adherent cultures, or washing. Any step that manipulates cells has the potential to damage the cells. While mesenchymal progenitor or stem cells can generally tolerate a certain degree of damage during preparation, cells are preferably manipulated using handling procedures and / or devices that appropriately perform a given step while minimizing damage to the cells.

[0112] In one example, mesenchymal progenitor or stem cells are washed in an apparatus comprising a cell source bag, a wash solution bag, a recirculating wash bag, a spinning membrane filter with inlet and outlet ports, a filter bag, a mixing area, a final product bag for the washed cells, and appropriate tubing, for example, as described in U.S. Pat. No. 6,251,295, which is incorporated herein by reference.

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

[0114] In one example, mesenchymal progenitor cells or stem cells of the present disclosure are cultured and expanded in 3D culture. For example, mesenchymal progenitor cells or stem cells of the present disclosure can be cultured and expanded in a bioreactor. In one example, mesenchymal progenitor cells or stem cells of the present disclosure are first cultured and expanded in 2D culture before being further expanded in 3D culture. In one example, mesenchymal progenitor cells or stem cells of the present disclosure are cultured and expanded from a master cell bank. In one example, mesenchymal progenitor cells or stem cells of the present disclosure are cultured and expanded from a master cell bank in 2D culture before seeding in 3D culture. In one example, mesenchymal progenitor cells or stem cells of the present disclosure are cultured and expanded from a master cell bank in 2D culture for at least three days before seeding in 3D culture in a bioreactor. In one example, mesenchymal progenitor cells or stem cells of the present disclosure are cultured and expanded from a master cell bank in 2D culture for at least four days before seeding in 3D culture in a bioreactor. In one example, mesenchymal progenitor or stem cells of the present disclosure are culture-expanded from a master cell bank in 2D culture for 3 to 5 days prior to seeding in 3D culture in a bioreactor. In these examples, the 2D culture may be performed in a cell factory. Various cell factory products are commercially available (e.g., Thermofisher, Sigma).

[0115] Ang1 and VEGF levels In another aspect, mesenchymal precursor or stem cells according to the present disclosure express Ang1:VEGF in a ratio of at least about 2: 1. However, in other examples, mesenchymal precursor 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, 50:1.

[0116] The amount of cellular Ang1 and / or VEGF expressed in a mesenchymal progenitor or stem cell composition or culture 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 a quantitative ELISA assay. In this example, cell lysates from a mesenchymal progenitor or stem cell culture are added to wells of an ELISA plate. The wells may be coated with a primary antibody, either monoclonal or polyclonal, against Ang1 or VEGF. The wells are then washed and then contacted with a secondary antibody, either monoclonal or polyclonal, against the primary antibody. The secondary antibody is conjugated to a suitable enzyme, such as horseradish peroxidase. The wells may then be incubated and then washed after the incubation period. The wells are then contacted with an appropriate substrate for the enzyme conjugated to the secondary antibody, such as one or more chromogens. Chromogens that may be used include, but are not limited to, hydrogen peroxide and tetramethylbenzidine. After the substrate is added, the wells are incubated for an appropriate period of time. Upon completion of the incubation, a "stop" solution is added to the wells to stop the reaction between the enzyme and the substrate. The optical density (OD) of the sample is then measured. The optical density of the sample is correlated with the optical density of samples containing known amounts of Ang1 or VEGF to determine the amount of Ang1 or VEGF expressed by the stem cell culture being tested.

[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] In one example, mesenchymal precursor or stem cells of the present 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 have not been modified by transfection with a nucleic acid that expresses or encodes Ang1 and / or VEGF. For the avoidance of doubt, in the context of the present disclosure, mesenchymal precursor or stem cells that have been transfected with a nucleic acid encoding Ang1 and / or VEGF are considered to be genetically modified. In the context of the present disclosure, cells that are not genetically modified to express Ang1 and / or VEGF naturally express some level of Ang1 and / or VEGF without being transfected with a nucleic acid encoding Ang1 and / or VEGF1.

[0119] Recombinant viruses In one embodiment, a cell as defined herein is modified to introduce a recombinant virus. The term "recombinant virus" is used in the context of the present disclosure to refer to a virus that expresses a transgene of interest in a cell (or population thereof) as defined herein. In one example, the recombinant virus expresses a transgene that increases the migration of mesenchymal progenitor or stem cells into cancer cells. In one example, the recombinant virus comprises a herpes simplex virus backbone. In one example, the recombinant virus is a herpes simplex virus.

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

[0121] In one example, oncolytic viruses can naturally infect and reduce the growth of cancer cells. Examples of such viruses include Newcastle disease virus, vesicular stomatitis virus, myxoma virus, reovirus, Sindbis virus, measles virus, and coxsackie virus. Oncolytic viruses naturally infect and reduce the growth of cancer cells, typically target cancer cells, by exploiting cellular abnormalities that occur in these cells. For example, oncolytic viruses can exploit surface attachment receptors, activated oncogenes such as Ras, Akt, and p53, and / or defective interferon (IFN) pathways.

[0122] In another example, oncolytic viruses encompassed by the present disclosure are engineered to infect and reduce the growth of cancer cells. Exemplary viruses suitable for such engineering include oncolytic DNA viruses, such as adenoviruses, herpes simplex viruses (HSV), and vaccinia viruses; and oncolytic RNA viruses, such as lentiviruses, reoviruses, coxsackieviruses, Seneca Valley viruses, polioviruses, measles viruses, Newcastle disease viruses, vesicular stomatitis viruses (VSV), and parvoviruses, such as rodent protoparvovirus H-1PV. In one example, the oncolytic virus comprises the backbone of the viruses referenced above. For example, the oncolytic virus can comprise an HSV backbone. 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 grow in cancer cells but mutate or delete a gene required for viral survival in normal cells. For the avoidance of doubt, an oncolytic virus with a mutated or deleted gene can survive in mesenchymal progenitor cells or stem cells for a sufficient period of time to allow its migration to cancer cells. For example, an oncolytic virus can be engineered by mutating or deleting a gene encoding thymidine kinase, an enzyme required for nucleic acid metabolism. In this example, the virus depends on cellular thymidine kinase expression, which is high in proliferating cancer cells but suppressed in normal cells. In another example, an oncolytic virus can be engineered to contain a capsid protein that binds to a tumor-specific cell surface molecule. In one example, the capsid protein is a fiber, penton, or hexon protein. In another example, an oncolytic virus can be engineered to contain a tumor-specific cell surface molecule to target cancer cells for transduction. Exemplary tumor-specific cell surface molecules include integrins, EGF receptor family members, proteoglycans, disialogangliosides, 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 glycoprotein, CD19, CD20, CD22, CD30, CD33, CD40, CD44, CD52, CD74, CD152, CD155, MUC1, tumor necrosis factor receptor, insulin-like growth factor receptor, folate receptor a, 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 the ability of infected mesenchymal progenitor cells or stem cells to deliver the viral payload to cancer cells. For example, oncolytic viruses can be engineered to express viral fusogenic membrane glycoproteins to mediate the induction of mesenchymal progenitor cell lineage or stem cell fusion to cancer cells. Examples of viral fusogenic membrane glycoproteins include gibbon ape leukemia virus (GLAV) envelope glycoprotein, measles virus protein F (MV-F), and measles virus protein H (MV-H).

[0125] In one example, the viral fusogenic membrane glycoprotein is under the control of a late promoter, such as the adenovirus major late promoter. In another example, the viral fusogenic membrane glycoprotein is under the control of a strict late promoter, such as UL38p (WO 2003 / 082200), which is active only after the initiation of viral DNA replication. Examples of such promoters and engineered viruses are disclosed in Fu et al. (2003) Molecular Therapy, 7:748-54 and Guedan et al. (2012) Gene Therapy, 19:1048-1057.

[0126] In one example, the oncolytic virus is replication competent. In one example, the oncolytic virus selectively replicates in cancer cells when compared to corresponding normal cells and / or mesenchymal progenitor or stem cells. In one example, the tumor specificity of the oncolytic virus can be engineered to restrict 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 another example, the oncolytic virus is a conditionally replicating adenovirus, reovirus, measles, herpes simplex virus, Newcastle disease virus, or vaccinia.

[0127] In one example, conditional replication is achieved by inserting a tumor-specific promoter that drives the expression of a key gene. Such promoters can be identified based on differences in gene expression between tumors, corresponding surrounding tissues, and / or mesenchymal progenitor or stem cells. For example, one method for identifying suitable tumor-specific promoters is to compare gene expression levels between tumors, corresponding normal tissues, and mesenchymal progenitor or stem cells to identify genes that are expressed at high levels in tumors and at low levels in corresponding healthy tissues and / or mesenchymal progenitor or stem cells. Tumor-specific promoters can be natural or composite. Exemplary natural promoters include AFP, CCKAR, CEA, erbB2, Cerb2, COX2, CXCR4, E2F1, HE4, LP, MUC1, PSA, survivin, TRP1, STAT3, hTERT, and Tyr. Exemplary composite promoters include AFP / hAFP, SV40 / AFP, CEA / CEA, PSA / PSA, SV40 / Tyr, and Tyr / Tyr. Those skilled in the art will appreciate that the appropriate tumor-specific promoter will depend in part on 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 can 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 suitable tumor-specific promoters is to compare promoter activity between cancer cells and corresponding normal cells and / or mesenchymal progenitor cells or stem cells to identify promoters with high activity in cancer cells and low activity in corresponding normal cells and / or mesenchymal progenitor cells or stem cells. In one example, a tumor-specific promoter can be a late or strict late viral promoter. The terms "late" and "strict late" are used to refer to promoters whose activity depends on the initiation of viral DNA replication. Thus, late and strict late promoters are suitable for inclusion in oncolytic viruses that can replicate in cancer cells but have limited replication ability in non-dividing normal cells. Exemplary late or strict late promoters include the major late promoter (MLP) and UL38p.

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

[0130] In another example, the tumor specificity of an oncolytic virus can be engineered to take advantage of tumor-specific tropism. In another example, an oncolytic virus is sensitive to an oligonucleotide or binding protein that is expressed in normal cells and / or mesenchymal progenitor or stem cells, but is expressed at low levels or absent in cancer cells. For example, an oncolytic virus can be engineered to insert a nucleotide sequence that is complementary to an oligonucleotide expressed by mesenchymal progenitor or stem cells and / or normal cells, but not expressed by cancer cells. For example, an oncolytic virus can be sensitive to an inhibitory oligonucleotide, such as an miRNA.Exemplary miRNAs that are 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, 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, the virus can be engineered to express a gene in infected cancer cells. For example, the virus can be engineered to express a gene such as PTEN. In one example, the virus expresses PTEN-alpha (PTENα). In one example, the virus includes the nucleic acid sequence set forth in SEQ ID NO: 1 or a variant thereof that translates into a functional PTEN protein (e.g., SEQ ID NO: 2). In one example, the virus expresses a transgene that is expressed and translated into a protein having the amino acid sequence set forth in SEQ ID NO: 2. In these examples, the oncolytic virus can include an HSV backbone. In one example, the virus is HSV. In one example, HSV is described in Russell et al. (2018) Nat Comm., 9:5006. In these examples, the virus can increase the level of a protein having the amino acid sequence set forth in SEQ ID NO: 2 in infected cancer cells.

[0132] In one example, the gene enhances an immune response to infected tumor cells. For example, the gene can be GM-CSF, FLT3L, CCL3, CCL5, IL2, IL4, IL6, IL12, IL15, IL18, IFNA1, IFNB1, IFNG, CD80, 4-1BBL, CD40L, a heat shock protein (HSP), or a combination thereof.

[0133] Various viruses can be engineered as outlined in the above references. In one example, the oncolytic virus is a modified HSV, lentivirus, baculovirus, retrovirus, adenovirus (AdV), adeno-associated virus (AAV), or a recombinant such as recombinant adeno-associated virus (rAAV), and its derivatives such as self-complementary AAV (scAAV) and non-integrating AV. For example, the oncolytic virus can be a modified HSV. For example, the oncolytic virus can be a modified lentivirus. Other exemplary viruses include vaccinia virus, vesicular stomatitis virus (VSV), measles virus, and Maraba virus.

[0134] In other examples, the oncolytic virus can be one of various AV or AAV serotypes. In one example, the oncolytic virus is serotype 1. In another example, the oncolytic virus is serotype 2. In another example, the oncolytic virus is serotype 3, 4, 7, 8, 9, 10, 11, 12, or 13. In another example, the oncolytic virus is serotype 5. In another example, the oncolytic virus is serotype 6.

[0135] Exemplary oncolytic viruses that can be introduced into mesenchymal progenitor or stem cells according to the present 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, the oncolytic virus is replication-deficient. For example, the replication gene can be mutated, deleted, or replaced with an expression cassette comprising a tumor-specific promoter. In one example, the E1 / E3 genes can be mutated, deleted, or replaced. In another example, the E1A / E1B genes can be 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 another example, the oncolytic virus can contain a mutated E1, E3, E1A, or E1B gene. For example, the E1A gene can be mutated in the region encoding the retinoblastoma protein (RB) binding site. In another example, the E3 gene can be mutated in the region encoding the endoplasmic reticulum retention domain. In another example, the oncolytic virus can contain a mutation in the gamma-34.5 gene and / or the alpha-47 gene.

[0138] In one example, an oncolytic virus is replication-deficient in mesenchymal progenitor or stem cells and 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 88:345-353. Other exemplary viruses of this type include RGD mutants, such as those described in Shen et al. (2016) PlosOne 11:e0147173, that replicate in pRb- or p53-inactive cancer cells, and / or viruses containing a delta24 mutation in E1 that allows for controlled expression of E1 under the control of tumor cell-specific promoters, such as the α-chemokine SDF-1 receptor (CXCR4), survivin, cyclooxygenase-2 (COX-2), and midkine.

[0139] In one example, the viruses disclosed herein include a polynucleotide operably linked to a tumor-specific promoter. For example, the virus can include a polynucleotide encoding PTEN alpha operably linked to a tumor-specific promoter.

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

[0141] Modification The mesenchymal progenitor or stem cells of the present disclosure may be modified to enhance cancer cell killing and / or migration toward cancer cells. In one example, such a modification includes increasing the expression of PTENα. Examples of various 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, in one example, the mesenchymal progenitor or stem cells of the present disclosure may be modified to introduce a recombinant virus that expresses a transgene. For example, the mesenchymal progenitor or stem cells of the present disclosure may be modified to introduce a recombinant virus, such as a virus comprising an HSV backbone and expressing a polynucleotide encoding PTENα. In one example, a mesenchymal progenitor or stem cell is considered "modified" if the virus has been transferred to the cell by any suitable means of artificial manipulation, or if the cell is the progeny of an initially modified cell that harbors the virus. In one example, a cell transfected with a "naked" nucleic acid molecule encoding a transgene is not considered "modified." For example, a cell transfected with a "naked" mRNA molecule encoding a transgene is not considered "modified."

[0142] In another example, a cell population engineered to transduce a recombinant virus can also be engineered to express a binding protein, such as an antibody or fragment thereof, on the cell surface. For example, a cell population can be engineered to express an anti-epidermal growth factor receptor (EGFR; ErbB1) binding protein.

[0143] Enhanced migration of the modified cells disclosed herein can be assessed using various migration assays known in the art, such as transwell cell migration and invasion assays (see, e.g., 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). Enhanced cancer cell killing disclosed herein can also be assessed using various assays known in the art, such as the cell viability / cytotoxicity assays exemplified below (e.g., assessment of cytosolic activity (aqua live / dead dye) by flow cytometry; see also, e.g., 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, increases in gene expression can be quantified using a variety of methods, such as routine amplification-based detection techniques, including, for example, real-time polymerase chain reaction. In some cases, increases in protein expression corresponding to increases in gene expression can also be quantified using routine methods, such as Western blot.

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

[0145] Infection efficiency is rarely 100%, and enriching for a population of successfully modified cells is usually desirable. In one example, modified cells can be enriched by taking advantage of functional properties of the 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. The inventors have found that HSV expressing a PTEN-alpha transgene has a high rate of infectivity in mesenchymal progenitor or stem cells. For example, HSV according to the present disclosure is at least 15% infective. In another example, HSV according to the present disclosure is at least 20% infective. In another example, HSV according to the present disclosure is at least 25% infective. In another example, HSV according to the present disclosure is at least 30% infective. In another example, HSV according to the present disclosure is at least 40% infective. In another example, HSV according to the present disclosure is at least 50% infective. In another example, HSV according to the present disclosure is between 15 and 80% infective. In another example, the HSV of the present disclosure has an infectivity between 20 and 80%. In another example, the HSV of the present disclosure has an infectivity between 30 and 80%. In another example, the HSV of the present disclosure has an infectivity between 35 and 80%. In another example, the HSV of the present disclosure has an infectivity between 45 and 80%. In another example, the HSV of the present disclosure has an infectivity between 55 and 80%.

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

[0147] The present inventors have found that HSV expressing a PTEN-alpha transgene replicates at a high rate in mesenchymal progenitor or stem cells. For example, HSV expressing a PTEN-alpha transgene may exhibit at least a 10% increase in replication compared to a matched HSV control. In one example, HSV expressing a PTEN-alpha transgene exhibits at least a 20% increase in replication compared to a matched HSV control. In another example, HSV expressing a PTEN-alpha transgene exhibits at least a 30% increase in replication compared to a matched HSV control. In another example, HSV expressing a PTEN-alpha transgene exhibits between a 20% and 40% increase in replication compared to a matched HSV control.

[0148] Delivery to cancer cells The inventors have identified that mesenchymal progenitor cells or stem cells migrate toward cancer cells and transfer a payload, such as a virus or transgene, expressed by the same. Accordingly, in one example, the present disclosure encompasses a method of delivering the above-referenced oncolytic virus to cancer cells by administering the mesenchymal progenitor cells or stem cells disclosed herein to a subject. In one example, the viral payload can be transferred by contacting the cancer cells with mesenchymal progenitor cells or stem cells modified to introduce the above-referenced oncolytic virus. For the avoidance of doubt, the oncolytic virus delivered to the cancer cells is an oncolytic virus introduced into mesenchymal progenitor cells or stem cells. In another example, the present disclosure encompasses a method of increasing PTENα expression in a cell, comprising contacting the cell with a population disclosed herein. In this example, the cell can be a cancer cell. In one example, increasing PTENα expression in the cell reduces the level of phosphorylated AKT in the cell. In one example, the method is performed in vivo. For example, the population disclosed herein can be administered to a subject.

[0149] The term "contacting" is used in the context of the present disclosure to refer to "direct" or "indirect" contact. "Direct contact" is used in the context of the present disclosure to refer to physical contact between cancer cells and modified mesenchymal progenitor or stem cells that facilitates the transfer of a payload, such as an oncolytic virus and / or a transgene, expressed by the same. For example, cancer cells and modified mesenchymal progenitor or stem cells may be in direct contact via a common connexin (i.e., a connexin expressed by both the cancer cells and the modified mesenchymal progenitor or stem cells). In this example, the common connexin facilitates the transfer of the payload from the mesenchymal progenitor or stem cells to the cancer cells via a gap junction.

[0150] "Indirect contact," in the context of the present disclosure, is used to refer to delivery of an oncolytic virus from an engineered mesenchymal precursor cell or stem cell to a cancer cell without direct contact. For example, engineered mesenchymal precursor cells or stem cells in close proximity to a cancer cell may indirectly contact the cancer cell. In one example, engineered mesenchymal precursor cells or stem cells in indirect contact with a cancer cell can deliver a payload to the cancer cell via exosomes. In another example, engineered mesenchymal precursor cells or stem cells in indirect contact with a cancer cell can deliver a payload to the cancer cell via secretion into the surrounding environment.

[0151] In one example, both direct and indirect contact can be mediated by administering the populations disclosed herein to a subject.

[0152] In another example, engineered mesenchymal progenitor or stem cells in direct contact with cancer cells can deliver payloads to cancer cells via common connexins and indirectly via exosomes.

[0153] The cancer cells that receive the payload from the modified mesenchymal progenitor cells or stem cells are not particularly limited, as long as they can be directly or indirectly contacted with the modified mesenchymal progenitor cells or stem cells to promote 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, the cancer cells are syncytial cancer cells. In the context of this disclosure, the term "syncytium" is used to refer to a cancerous tissue or mass consisting of cells interconnected by specialized membranes containing gap junctions that are electrically synchronized in action potentials.

[0155] Delivery of oncolytic viruses from engineered mesenchymal progenitor or stem cells to cancer cells can be facilitated in vivo via various exemplary routes. For example, mesenchymal progenitor or stem cells can be administered systemically, such as by intravenous, intraarterial, or intraperitoneal administration. In another example, mesenchymal progenitor or stem cells can be administered intranasally or intramuscularly. In one example, mesenchymal progenitor or stem cells are administered to a site in close proximity to cancer cells, such as surrounding tissue. In another example, mesenchymal progenitor or stem cells are administered directly to the cancer.

[0156] Treatment method In one example, cell populations according to the present disclosure and compositions comprising the same can be administered to treat cancer. The term "cancer" refers to or describes a physiological condition in mammals that is typically characterized by uncontrolled cell growth. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies. More particular examples of such cancers include, but are not limited to, squamous cell carcinoma (e.g., epithelial squamous cell carcinoma), lung cancer, including small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and squamous cell carcinoma of the lung, cancer of the peritoneum, hepatocellular carcinoma, gastric or stomach cancer, including gastrointestinal cancer and gastrointestinal stromal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, cancer of the urinary tract, hepatocellular carcinoma, breast cancer, colon cancer, endorectal 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, lentigo maligna melanoma, acral lentiginous melanoma, nodular melanoma, multiple bone marrow cancer, Myeloma and B-cell lymphomas (including low-grade / follicular non-Hodgkin's 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-cleaved cell NHL; bulky mass disease NHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom's hypergammaglobulinemia); chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); hairy cell leukemia; chronic myeloblastic leukemia; and post-transplant lymphoproliferative disorder (PTLD), as well as abnormal blood vessel growth associated with phakomatoses, edema (such as associated with brain tumors), Meigs' syndrome, brain and head and neck cancers and associated metastases.

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

[0158] In one example, the cancer is a PTEN-mutated or -deficient cancer. In one example, the cancer is a PTEN-mutated or -deficient glioblastoma, endometrial cancer, colon cancer, lung cancer, breast cancer, prostate cancer, and ovarian cancer. In one example, the cancer is a PTEN-mutated or -deficient breast cancer. In another example, the cancer is a PTEN-mutated or -deficient brain cancer. In one example, the cell populations and compositions comprising the same according to the present disclosure can be used in methods of killing cancer cells. In one example, the cancer cells killed using such methods can be from the cancer types referenced above.

[0159] cell composition The present disclosure encompasses populations of mesenchymal progenitor cell lineages or stem cells. Such populations can be provided in compositions. For example, in practicing the methods of the present disclosure, the mesenchymal progenitor or stem cells can be provided in a composition suitable for administration to a subject.

[0160] Exemplary compositions according to the present disclosure may include mesenchymal progenitor cells or stem cells modified to introduce HSV. Exemplary HSVs are described above. In one example, a composition according to the present disclosure may include mesenchymal progenitor cells or stem cells modified to introduce the oncolytic viruses referenced above, or combinations thereof. 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 comprises the nucleic acid sequence set forth in SEQ ID NO: 1.

[0161] In another example, a composition according to the present disclosure can include mesenchymal progenitor or stem cells modified to introduce HSV with a high level of infectivity in mesenchymal progenitor or stem cells. In one example, the level of infectivity is greater than 15% of the mesenchymal progenitor or stem cells. In another example, the level of infectivity is greater than 25% of the mesenchymal progenitor or stem cells. In another example, the level of infectivity is greater than 35% of the mesenchymal progenitor or stem cells. In another example, the level of infectivity is greater than 45% of the mesenchymal progenitor or stem cells.

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

[0163] In another example, a composition according to the present disclosure may include mesenchymal progenitor or stem cells that have been modified to transduce HSV that does not kill the mesenchymal progenitor or stem cells prior to delivery of the oncolytic virus to the cancer cells.

[0164] In one example, such a composition comprises a pharmaceutically acceptable carrier and / or excipient.

[0165] The terms "carrier" and "excipient" refer to compositions conventionally used in the art to facilitate the storage, administration, and / or biological activity of an active compound (see, e.g., Remington's Pharmaceutical Sciences, 16th ed., Mac Publishing Company (1980)). A carrier may also reduce any undesirable side effects of the active compound. Suitable carriers are, for example, stable, e.g., incapable of reacting with other ingredients in the carrier. In one example, a carrier does not cause significant local or systemic adverse effects in a recipient at the dosages and concentrations used for treatment.

[0166] Suitable carriers for the present disclosure include those conventionally used, such as water, physiological saline, aqueous glucose, lactose, Ringer's solution, buffer solutions, hyaluronic acid, and glycols are exemplary liquid carriers, especially for isotonic solutions. Suitable pharmaceutical carriers and excipients include starch, cellulose, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, magnesium stearate, sodium stearate, glycerol monostearate, sodium chloride, glycerin, propylene glycol, water, ethanol, etc.

[0167] In another example, the carrier is a media composition, e.g., in which the cells are grown or suspended, such that the media composition does not induce any adverse effects in the subject to which it is administered.

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

[0169] In one example, the carrier or excipient provides buffering activity to maintain the cells and / or soluble factors at a suitable pH for biological activity, e.g., the carrier or excipient is phosphate buffered saline (PBS). PBS is an attractive carrier or excipient because it interacts minimally with the cells and factors, allowing for rapid release of the cells and factors; in such cases, the compositions of the present disclosure can be formulated as a liquid for direct application to the bloodstream or to tissues or areas surrounding or adjacent to tissues, e.g., by injection.

[0170] The cell compositions described herein can be administered alone or as a mixture with other cells. Different types of cells can be mixed with the compositions of the present disclosure immediately or shortly before administration, or they can be co-cultured together for a period of time before administration.

[0171] In one example, the composition comprises an effective or therapeutically effective amount of cells, e.g., about 1 x 10 cells. 5 Approximately 1x10 cells9 cells or approximately 1.25 x 10 cells 3 Approximately 1.25 x 10 cells 7 The exact amount of cells to be administered will depend on a variety of factors, including the age, weight, and sex of the subject, as well as the extent and severity of the disorder being treated.

[0172] An exemplary dosage is at least about 1.2 x 10 cells 8 From approximately 8 x 10 10 cells, e.g., approximately 1.3 x 10 8 From approximately 8 x 10 9 Between the cells, approximately 1.4 x 10 8 From approximately 8 x 10 8 Between the cells, approximately 1.5 x 10 8 From approximately 7.2 x 10 8 Between the cells, approximately 1.6 x 10 8 From approximately 6.4 x 10 8 Between the cells, approximately 1.7 x 10 8 From approximately 5.6 x 10 8 Between the cells, approximately 1.8 x 10 8 From approximately 4.8 x 10 8 Between the cells, approximately 1.9 x 10 8 From approximately 4.0 x 10 8 Between the cells, approximately 2.0 x 10 8 From approximately 3.2 x 10 8 Approximately 2.1 x 10 cells 8 From approximately 2.4 x 10 8 For example, the dose may be at least about 1.5 x 10 cells. 8 For example, the dose may comprise at least about 2.0 x 10 cells. 8 It may include pieces.

[0173] In other words, an exemplary dose is at least about 1.5 x 10 cells. 6 In one example, the dose comprises at least about 2.5 x 10 cells / kg (80 kg subject). 6 In other examples, the dose may include about 1.5 x 10 cells / kg. 6 From about 1x10 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 about 7 x 10 6 cells / kg, approximately 2.3 x 10 cells 6 From about 6 x 10 6 cells / kg, approximately 2.4 x 10 cells 6 From about 5 x 10 6 cells / kg, approximately 2.5 x 10 cells 6 From about 4 x 10 6 cells / kg, approximately 2.6 x 10 cells 6 From about 3 x 10 6 It may include pieces / kg.

[0174] In one example, the modified mesenchymal precursor or stem cells comprise 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%, or at least about 99% of the cell population of the composition.

[0175] The compositions of the present disclosure can be cryopreserved. Cryopreservation of mesenchymal progenitor or stem cells can be performed using slow-cooling or "rapid" freezing protocols well known in the art. Preferably, the cryopreservation method maintains similar phenotype, cell surface markers, and growth rate of the cryopreserved cells compared to unfrozen cells.

[0176] The cryopreserved composition may comprise a cryopreservation solution, the pH of which is typically between 6.5 and 8, preferably 7.4.

[0177] Cryopreservation solutions can include, for example, sterile, non-pyrogenic, isotonic solutions such as PlasmaLyte A™. 100 mL of PlasmaLyte A™ contains 526 mg sodium chloride, USP (NaCl); 502 mg sodium gluconate (C6H11NaO7); 368 mg sodium acetate trihydrate, USP (C2H3NaO2·3H2O); 37 mg potassium chloride, USP (KCl); and 30 mg magnesium chloride, USP (MgCl2·6H2O). No antimicrobial agents are included. The pH is adjusted with sodium hydroxide. The pH is 7.4 (6.5 to 8.0).

[0178] The cryopreservation solution may include Profreeze™. The cryopreservation solution may additionally or alternatively include culture medium, such as αMEM.

[0179] To facilitate freezing, cryoprotectants, such as dimethyl sulfoxide (DMSO), are typically added to cryopreservation solutions. Ideally, cryoprotectants should be nontoxic, nonantigenic, and chemically inert to cells and patients, resulting in high survival rates after thawing and enabling transplantation without irrigation. However, the most commonly used cryoprotectant, DMSO, exhibits some cytotoxicity. Hydroxyethyl starch (HES) can be used instead or in combination with DMSO to reduce the cytotoxicity of cryopreservation solutions.

[0180] The cryopreservation solution may include one or more of DMSO, hydroxyethyl starch, human serum components, and other protein bulking agents. In one example, the cryopreserved solution includes about 5% human serum albumin (HSA) and about 10% DMSO. The cryopreservation solution may further include one or more of methycellulose, polyvinylpyrrolidone (PVP), and trehalose.

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

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

[0183] In one example, the cell compositions described herein can be administered as a single dose. In another example, the cell compositions are administered as multiple doses. For example, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 doses.

[0184] In one example, mesenchymal precursor cells or stem cells can be cultured and expanded before administration. Various methods for culturing mesenchymal precursor cells or stem cells are well known in the art. In one example, mesenchymal precursor cells or stem cells can be cultured and expanded in a serum-free medium before administration. For example, mesenchymal precursor cells or stem cells can 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] The mesenchymal precursor or stem cells may be administered systemically, for example, by intravenous, intraarterial, or intraperitoneal administration. The mesenchymal precursor or stem cells may also be administered intranasally, intramuscularly, or intracardially. In one example, the mesenchymal precursor or stem cells are administered directly to the subject's tumor. [Example]

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

[0187] Mesenchymal stem cells were loaded with either HSVQ or HSV-P10 at multiplicities of infection (MOIs) 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 objects was quantified and graphed as the mean ± SEM of four wells per treatment group. The rate of replication in the cells correlated with the MOI of HSVQ or HSV-P10 used to infect the mesenchymal stem cells.

[0188] To determine the kinetics of HSV-P10 and HSVQ viral 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 cultured at a concentration of 3 x 10 cells. 6 The cells were seeded into 6-well plates at 100 μg / well and cultured for 24 hours. The plated mesenchymal stem cells were infected with HSVQ or HSV-P10 at an MOI of 1 for 1 hour. After incubation, the medium was removed and replaced with fresh DMEM, and the cells were cultured for an additional 24 hours. The HSVQ- or HSV-P10-loaded mesenchymal stem cells and conditioned medium were collected, and titration studies were performed on Vero cells.

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

[0190] To determine the effect of viral loading on mesenchymal stem cell viability, cytosolic activity (aqua live / dead dye) and GFP expression were assessed by flow cytometry, determined, quantified, and presented as histograms in the loaded mesenchymal stem cells (Figure 3). The data demonstrate that mesenchymal stem cells loaded with HSV-10 and HSVQ were viable 24 hours post-infection (Figure 3A). The flow cytometry quadrants are shown in Figure 3B.

[0191] Example 2 Evaluation of functional PTENα expressed by HSV-P10-loaded mesenchymal stem cells (MSCs) To evaluate the functionality of PTENα expressed by HSV-P10, we determined the effect of HSV-P10 on the PI3K / AKT signaling pathway in mesenchymal stem cells loaded with HSV-P10. Western blot analysis revealed increased AKT in mesenchymal stem cells loaded with HSVQ, whereas mesenchymal stem cells loaded with HSV-P10, which express PTENα, showed reduced phosphorylated AKT compared to control virus-loaded cells (Figure 4A). PTENα was detected in the conditioned medium of mesenchymal stem cells loaded with HSV-P10, suggesting secretion of PTENα by mesenchymal stem cells loaded with HSV-P10 (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 viral GFP using a Cytation 5 Cell Imaging Multi-Mode Reader in conjunction with a BioSpa 8 Automated Incubator (Biotek Instruments, Inc.). However, analysis of HSVQ- and HSV-P10-loaded mesenchymal stem cell migration surprisingly revealed increased kinetics of HSV-P10-loaded mesenchymal stem cells toward human breast cancer cells (MDA-468) compared to HSVQ-loaded mesenchymal stem cells (Figure 5).

[0193] Example 4 Effects of HSV-P10-loaded mesenchymal stem cells on primary human glioma cells HSVQ- and HSV-P10-loaded mesenchymal stem cells were cocultured with RPF-expressing GMB12 primary human glioma cells (Figure 6A). The function of PTENα expressed by HSV-P10-loaded mesenchymal stem cells in the PI3K / AKT signaling pathway was determined. Western blot analysis revealed increased PTENα and decreased phosphorylated AKT in glioma cells after coculture with MSCs (Figure 6B).

[0194] Example 5 Effect of HSV-P10-loaded mesenchymal stem cells on breast cancer cells Coculture of DB7 mouse breast cancer cells with HSV-P10-loaded mesenchymal stem cells resulted in the transfer of HSV-P10 to cancer cells and the induction of cell death in these cancer cells, as determined by cytosolic activity (aqua Live / Dead dye) and GFP expression. An increase in the total number of dead DB7 mouse breast cancer cells was observed after coculture 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 breast cancer cells was observed after coculture with HSV-P10-loaded mesenchymal stem cells compared to unloaded mesenchymal stem cells (control) and HSV-Q-loaded cells (Figure 7).

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

[0196] Treated subjects are evaluated for safety and efficacy of treatment over a period of approximately 2-6 weeks, and additional doses of loaded mesenchymal progenitor or stem cells are administered as needed.

[0197] Example 7 Cancer treatment Prior to administration to a subject diagnosed with cancer, mesenchymal progenitor or stem cells are loaded with a recombinant virus containing a herpes simplex virus (HSV) backbone and a polynucleotide encoding phosphatase and tensin homolog alpha (PTENα) deleted on chromosome 10. By adding the virus to the mesenchymal progenitor or stem cell culture medium, the mesenchymal progenitor or stem cells are loaded at approximately 10-50 infectious units (iu) / MPC. Approximately 200 million loaded mesenchymal progenitor or stem cells are administered to the subject.

[0198] Treated subjects are evaluated for safety and efficacy of treatment over a period of approximately 2-6 weeks, and additional doses of loaded mesenchymal progenitor or stem cells are administered as needed.

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

[0200] All publications discussed above are incorporated herein in their entirety.

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

[0202] Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is solely for the purpose of providing a context for the present disclosure and is not to be construed as an admission that any or all of such matter existed prior to the priority date of each claim of this application and therefore formed part of the prior art document or was common general knowledge in the art relevant to the present disclosure.

[0203] (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 or stem cells that have been modified to increase expression of phosphatase and tensin homolog deleted on chromosome 10 alpha (PTENα).

2. The population of claim 1, wherein increased expression of PTENα is sufficient to decrease phosphorylated AKT levels in the modified cells.

3. 3. The population of claim 1 or 2, wherein increased PTENα expression is sufficient to enhance tumor cell killing.

4. 4. The population of any one of claims 1 to 3, wherein increased PTENα expression is sufficient to enhance migration into tumor cells.

5. 5. The population of any one of claims 1 to 4, wherein the mesenchymal progenitor or stem cells have been modified to introduce a recombinant virus comprising a polynucleotide encoding PTENα.

6. 6. The population of claim 5, wherein the recombinant virus is an oncolytic virus.

7. 7. The population of claim 5 or 6, wherein the virus comprises a herpes simplex virus (HSV) backbone.

8. 8. The population of any one of claims 4 to 7, wherein between 20% and 80% of the cells contain the recombinant virus.

9. 9. The population of any one of claims 4 to 8, wherein the polynucleotide encoding PTENα is operably linked to a tumor-specific promoter or an inducible promoter.

10. The population of claim 9, wherein the tumor-specific promoter is a survivin promoter, a COX-2 promoter, a PSA promoter, a CXCR4 promoter, a STAT3 promoter, an hTERT promoter, an AFP promoter, a CCKAR promoter, a CEA promoter, an erbB2 promoter, an E2F1 promoter, a HE4 promoter, an LP promoter, a MUC-1 promoter, a TRP1 promoter, or a Tyr promoter.

11. 11. The population of any one of claims 4 to 10, wherein the recombinant virus comprises a capsid protein that binds to a tumor-specific cell surface molecule.

12. 12. The population of claim 11, wherein the capsid protein is a fiber, penton, or hexon protein.

13. 13. The population of any one of claims 4 to 12, wherein the recombinant virus comprises the nucleic acid sequence shown in SEQ ID NO:

1.

14. 14. The population of any one of claims 4 to 13, wherein the recombinant virus is HSV.

15. 15. The population of any one of claims 3 to 14, wherein the tumor cells are breast cancer or brain cancer cells.

16. 16. The population of any one of claims 1 to 15, wherein the mesenchymal progenitor or stem cells are MSCs.

17. 17. The population of any one of claims 1 to 16, wherein the mesenchymal progenitor or stem cells are purified by immunoselection.

18. 16. The population of any one of claims 1 to 15, wherein the mesenchymal progenitor or stem cells express STRO-1.

19. 19. The population of any one of claims 1 to 18, wherein the mesenchymal progenitor or stem cells are derived from pluripotent cells.

20. 20. The population of claim 19, wherein the pluripotent cells are induced pluripotent stem (iPS) cells.

21. 21. The population of cells of any one of claims 1 to 20, wherein the population of cells is culture-expanded.

22. 22. A method of increasing PTENα expression in a cell, comprising contacting the cell with the population of any one of claims 1 to 21.

23. 23. The method of claim 22, wherein the contacted cells are cancer cells.

24. 24. The method of claim 22 or 23, wherein increasing PTENα expression in the cell reduces the level of phosphorylated AKT in the cell.

25. 25. The method of any one of claims 22 to 24, which is carried out in vivo.

26. 22. A pharmaceutical composition comprising a population according to any one of claims 1 to 21.

27. 27. A method of treating cancer in a subject, comprising administering the population of any one of claims 1 to 21 or the composition of claim 26.

28. 27. A method of killing cancer cells comprising contacting a population of cancer cells with a population according to any one of claims 1 to 21 or a composition according to claim 26.

29. 27. A method of delivering mesenchymal progenitor or stem cells to cancer cells in a subject, comprising administering a population described in any one of claims 1 to 21 or a composition described in claim 26.

30. 30. The method of any one of claims 27 to 29, 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.

31. 31. The method of claim 30, wherein the cancer is breast cancer or brain cancer.

32. 31. The method of any one of claims 27, 29 or 30, wherein the population or composition is administered to the subject by intravenous, intraarterial, intratumoral or intraperitoneal administration.

33. 22. Use of a population according to any one of claims 1 to 21 in the manufacture of a medicament for treating cancer.

34. 22. Use of the population of any one of claims 1 to 21 in the manufacture of a medicament for delivering mesenchymal precursor or stem cells to cancer cells.

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