Cellular compositions and therapeutic methods

Abstract

The present disclosure relates to cell compositions modified to transduce oncolytic viruses, which can be used to treat cancer by delivering the oncolytic viruses to cancer cells.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This international application claims the benefit of priority to U.S. Provisional Application No. 63 / 063,657, filed August 10, 2020, which is incorporated herein by reference in its entirety.

[0002] The present disclosure relates to cell compositions modified to transduce oncolytic viruses, which can be used to treat cancer by delivering the oncolytic viruses to cancer cells. [Background technology]

[0003] Cancer treatment typically involves surgical resection, standard chemotherapy, and / or radiation therapy to remove or kill tumor cells.However, the effectiveness of these treatments is often limited due to the invasiveness of tumors and / or collateral damage to healthy tissue.This situation means the need for new therapeutic strategies, and one such approach is the use of viruses.

[0004] Oncolytic viruses are viruses that can replicate specifically within and destroy tumor cells, either inherently or genetically engineered. Unfortunately, promising research results have not yet translated into improved clinical outcomes, which appear to be determined by the complex interplay between the tumor and its microenvironment, the virus, and host immunity. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] U.S. Provisional Application No. 63 / 063,657 Summary of the Invention [Problem to be solved by the invention]

[0006] Therefore, there is a need for improved methods of delivering oncolytic viruses to cancer cells. [Means for solving the problem]

[0007] The present inventors have identified that mesenchymal precursors or stem cells can deliver oncolytic viruses to cancer cells to reduce their proliferation, and have also identified that mesenchymal precursors or stem cells are better vehicles than mesenchymal stem cells for infecting target cells with oncolytic viruses.

[0008] One advantage of using mesenchymal precursors or stem cells for oncolytic virus delivery to cancer cells is the ability of mesenchymal precursors or stem cells to home to cancer cells. The migration and adhesion capabilities of mesenchymal precursors or stem cells make them particularly suitable for this purpose.

[0009] Another advantage of using mesenchymal precursors or stem cells for oncolytic virus delivery to cancer cells is their ability to suppress inflammatory mediators such as TNF-alpha and / or IL-6. Mesenchymal precursors or stem cells that express high levels of ANG1 and relatively low levels of VEGF may be particularly suitable for this purpose.

[0010] Thus, in a first example, the disclosure relates to a composition comprising mesenchymal precursor or stem cells, the cells modified to introduce an oncolytic virus. In one example, the mesenchymal precursor lineage or stem cells are STRO-1+. In one example, the mesenchymal precursor lineage or stem cells are STRO-3+. In one example, the mesenchymal precursor lineage or stem cells are TNAP+. In one example, the mesenchymal precursor lineage 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 precursor cells have not yet differentiated into mesenchymal stem cells.

[0011] In another example, the disclosure relates to a method of treating cancer in a subject, the method comprising administering a composition of the disclosure. In one example, the method comprises administering a composition comprising STRO-1+ mesenchymal progenitor or stem cells, the cells modified to transduce an oncolytic virus. In another example, the disclosure relates to a method of delivering an oncolytic virus to cancer cells, the method comprising contacting the cancer cells with mesenchymal progenitor cells modified to transduce an oncolytic virus. In one example, the mesenchymal progenitor lineage or stem cells express STRO-1+ and one or more markers selected from the group consisting of α1, α2, α3, α4, and α5, αv, β1, and β3. In one example, the contacting is performed under conditions that allow the mesenchymal progenitor or stem cells to form gap junctions with the cancer cells, whereby the oncolytic virus is delivered to the cancer cells by crossing the gap junctions. In one example, the gap junctions are formed by Cx40 or Cx43. In another example, the gap junctions are formed by Cx43. In another example, the delivery of the oncolytic virus is via a mechanism other than Cx43. In one example, the cancer cells are lung cancer, pancreatic cancer, colon cancer, liver cancer, cervical cancer, prostate cancer, osteosarcoma, breast cancer, or melanoma cells. In another example, the cancer cells are syncytial carcinoma cells. In another example, the oncolytic virus is modified to insert a nucleotide sequence that is complementary to an oligonucleotide expressed by mesenchymal precursors or stem cells and not expressed by the cancer cells. In one example, the oligonucleotide is an miRNA.

[0012] In one example, the mesenchymal progenitor or stem cell is substantially STRO-1 bri is.

[0013] In one example, the oncolytic virus comprises a capsid protein that binds to a tumor-specific promoter and / or a tumor-specific cell surface molecule. For example, the tumor-specific promoter can be 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.

[0014] In one example, the capsid protein is a fiber, penton, or hexon protein.

[0015] In another example, the oncolytic virus comprises a tumor-specific cell surface molecule for transductive targeting of tumor cells.

[0016] In one example, the tumor-specific cell surface molecule is selected from the group consisting of integrins, EGF receptor family members, proteoglycans, disialogangliosides, B7-H3, cancer antigen 125 (CA-125), epithelial cell adhesion molecule (EpCAM), vascular endothelial growth factor receptor 1, vascular endothelial growth factor receptor 2, carcinoembryonic antigen (CEA), tumor-associated glycoprotein, cluster of differentiation 19 (CD19), CD20, CD22, CD30, CD33, CD40, CD44, CD52, CD74, CD152, mucin 1 (MUC1), tumor necrosis factor receptor, insulin-like growth factor receptor, folate receptor a, transmembrane glycoprotein NMB, CC chemokine receptor, prostate-specific membrane antigen (PSMA), receptor d'origine Nantais (RON) receptor, and cytotoxic T lymphocyte antigen 4.

[0017] In one example, the oncolytic virus is respiratory syncytial virus (RSV), conditionally replicating adenovirus (CRAd), adenovirus, herpes simplex virus (HSV), vaccinia virus, lentivirus, reovirus, coxsackievirus, Seneca Valley virus, poliovirus, measles virus, Newcastle disease virus, or vesicular stomatitis virus (VSV), and parvovirus.

[0018] In another example, the mesenchymal precursor or stem cell expresses a connexin selected from the group consisting of Cx40, Cx43, Cx45, Cx32, and Cx37. In another example, the mesenchymal precursor or stem cell expresses an integrin selected from the group consisting of alpha 2, alpha 3, and alpha 5.

[0019] In another example, mesenchymal precursors or stem cells are modified to introduce an oncolytic virus that kills cancer cells but does not substantially affect the viability of the mesenchymal precursors or stem cells.

[0020] In another example, mesenchymal precursors or stem cells are modified to introduce an oncolytic virus that does not kill the mesenchymal precursors or stem cells before they can deliver the oncolytic virus to cancer cells.

[0021] In another example, oncolytic viruses express viral fusion membrane glycoproteins to mediate the induction of mesenchymal progenitor lineage or stem cell fusion to tumor cells. For example, the viral fusion membrane glycoproteins can be gibbon ape leukemia virus (GLAV) envelope glycoprotein, measles virus protein F (MV-F), and measles virus protein H (MV-H).

[0022] In one example, the mesenchymal precursor or stem cell is at least 0.1 μg / 10 6 In one example, the mesenchymal progenitor or stem cells express angiopoietin-1 (Ang1) in an amount of at least 0.3 μg / 10 cells. 6In one example, the mesenchymal progenitor or stem cells express angiopoietin-1 (Ang1) in an amount of at least 0.5 μg / 10 cells. 6 In one example, the mesenchymal progenitor or stem cells express angiopoietin-1 (Ang1) in an amount of at least 0.7 μg / 10 cells. 6 In one example, the mesenchymal progenitor or stem cells express angiopoietin-1 (Ang1) in an amount of at least 1.0 μg / 10 cells. 6 They express angiopoietin-1 (Ang1) in amounts of 1000 cells.

[0023] In another example, the mesenchymal progenitor or stem cell is about 0.05 μg / 10 6 In another example, the mesenchymal progenitor or stem cells express vascular endothelial growth factor (VEGF) in an amount of less than about 0.02 μg / 10 cells. 6 They express vascular endothelial growth factor (VEGF) in amounts less than one cell.

[0024] In another example, the mesenchymal precursor or stem cell expresses Ang1:VEGF at a ratio of at least about 2:1. In another example, the mesenchymal precursor or stem cell expresses Ang1:VEGF at a ratio of at least about 10:1. In another example, the mesenchymal precursor or stem cell expresses Ang1:VEGF at a ratio of at least about 20:1. In another example, the mesenchymal precursor or stem cell expresses Ang1:VEGF at a ratio of at least about 30:1. In another example, the mesenchymal precursor or stem cell expresses Ang1:VEGF at a ratio of at least about 50:1.

[0025] In another example, the mesenchymal precursor or stem cells are not genetically modified to express Ang1 or VEGF.

[0026] In another example, the mesenchymal precursor or stem cell is derived from a pluripotent cell. In one example, the pluripotent cell is an induced pluripotent stem (iPS) cell.

[0027] In another example, the mesenchymal precursor or stem cell expresses STRO-1+ and two or more of the markers selected from the group consisting of α1, α2, α3, α4, and α5, αv, β1, and β3.

[0028] In another example, the disclosure relates to a method of treating cancer in a subject, the method comprising administering a composition disclosed herein. In one example, the composition comprises mesenchymal precursors or stem cells that express STRO-1 and one or more markers selected from the group consisting of α1, α2, α3, α4, and α5, αv, β1, and β3, and the cells are modified to transduce an oncolytic virus. In one example, the mesenchymal precursors or stem cells express a connexin that is also expressed by cancer cells, including cancer cells in the subject. For example, the connexin can be Cx40 or Cx43.

[0029] In one example, the cancer cells comprising the subject's cancer express Cx43. In one example, the cancer is selected from the group consisting of lung cancer, pancreatic cancer, colon cancer, liver cancer, cervical cancer, prostate cancer, breast cancer, osteosarcoma, and melanoma.

[0030] In another example, the modified mesenchymal progenitor or stem cell is treated to modify cell surface glycans on the mesenchymal progenitor or stem cell. In one example, the treatment involves exposing the mesenchymal progenitor or stem cell to a glycosyltransferase under conditions that modify cell surface glycans on the mesenchymal progenitor or stem cell. In one example, the glycosyltransferase is a fucosyltransferase, a galactosyltransferase, or a sialyltransferase. For example, the fucosyltransferase can be an α1,3 fucosyltransferase, such as α1,3 fucosyltransferase III, α1,3 fucosyltransferase IV, α1,3 fucosyltransferase VI, α1,3 fucosyltransferase VII, or α1,3 fucosyltransferase IX.

[0031] In one example, mesenchymal progenitor or stem cells are exposed to exogenous glycosyltransferases, where the exposure to the glycosyltransferases results in enhanced retention of the cells at sites of inflammation in vivo.

[0032] In another example, mesenchymal precursor or stem cells are modified to introduce a nucleic acid encoding a glycosyltransferase, and expression of the glycosyltransferase in the cells results in enhanced retention of the cells at sites of inflammation in vivo.

[0033] Any example in this specification shall apply mutatis mutandis to any other example unless otherwise stated.

[0034] The present disclosure is not to be limited in scope by the specific examples described herein, which are for illustrative purposes only. Functionally equivalent products, compositions, and methods are clearly within the scope of the present disclosure as described herein.

[0035] Throughout this specification, unless specifically stated otherwise or the context requires otherwise, references to a single step, composition of matter, group of steps, or group of compositions of matter should be taken to encompass one and more than one (i.e., one or more) of that step, composition of matter, group of steps, or group of compositions of matter.

[0036] The present disclosure will now be described by way of the following non-limiting examples and with reference to the accompanying drawings. [Brief explanation of the drawings]

[0037] [Figure 1] (A and B) Overview of viral delivery to MPCs. [Figure 2] (A and B) Lentiviral delivery of GFP [Figure 3] (A and B) Adenoviral delivery of GFP [Figure 4] (A and B) rAAV-2 delivery of GFP [Figure 5] (A and B) rAAV-DJ delivery of GFP [Figure 6] (A and B) Viral backbones of HSVQ (parent virus) and HSV-P10 (PTENα-expressing virus). [Figure 7] (A and B) HSV-P10 load of mesenchymal stem cells (MSCs). [Figure 8] (A and B) Viability of HSV-P10 and HSVQ-loaded mesenchymal stem cells (MSCs). [Figure 9] (A and B) Effects of HSV-P10-loaded mesenchymal stem cells (MSCs) on the expression of PTENα and the PI3K / AKT signaling pathway. [Figure 10] Migration of HSV-P10 and HSVQ-loaded mesenchymal stem cells (MSCs) toward human breast cancer cells (MDA-468). [Figure 11] (A and B) Effect of HSV-P10-loaded mesenchymal stem cells (MSCs) on human glioma cells. [Figure 12] Induction of tumor cell death in DB7 mouse breast cancer cells cocultured with HSV-P10 and HSVQ-loaded mesenchymal stem cells (MSCs). [Figure 13] (A and B) Oncolytic HSV replication in MSCs and MPCs. [Figure 14] MSC and MPC viability after oncolytic HSV infection. [Figure 15] RSV-infected A549. LHS - fluorescence microscopy; RHS - cell viability. [Figure 16] RSV-infected H1299. LHS - fluorescence microscopy; RHS - cell viability. [Figure 17] RSV-infected H1650. LHS - fluorescence microscopy; RHS - cell viability. [Figure 18] RSV-infected LLC. LHS - fluorescence microscopy; RHS - cell viability. [Figure 19] RSV-infected U2-OS. LHS - fluorescence microscopy; RHS - cell viability. [Figure 20] RSV-infected SK-ES1. LHS - fluorescence microscopy; RHS - cell viability. [Figure 21] RSV-infected 4T1. LHS - fluorescence microscopy; RHS - cell viability. [Figure 22] MPC fluorescence microscopy. [Figure 23] RSV-infected MPCs. LHS - fluorescence microscopy; RHS - cell viability. [Figure 24] MSC fluorescence microscopy. [Figure 25] RSV-infected MSCs. LHS - fluorescence microscopy; RHS - cell viability. [Figure 26] Fluorescence microscopy of A549 cells after contact with supernatant from RSV-infected MPCs or MSCs expressing the red fluorescent marker mKate2. [Figure 27] Fluorescence microscopy of H1299 cells after contact with supernatant from RSV-infected MPCs or MSCs expressing the red fluorescent marker mKate2. [Figure 28] Fluorescence microscopy of H1650 cells after contact with supernatant from RSV-infected MPCs or MSCs expressing the red fluorescent marker mKate2. [Figure 29] Fluorescence microscopy of LLC cells after contact with supernatant from RSV-infected MPC or MSC expressing the red fluorescent marker mKate2. [Figure 30] Fluorescence microscopy of U2-OS cells after contact with supernatant from RSV-infected MPC or MSC expressing the red fluorescent marker mKate2. [Figure 31] Fluorescence microscopy of 4T1 cells after contact with supernatant from RSV-infected MPC or MSC expressing the red fluorescent marker mKate2. DETAILED DESCRIPTION OF THE INVENTION

[0038] General Techniques and Selected Definitions Unless otherwise defined, all technical and scientific terms used herein 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, gene modification, virology, oncology, biochemistry, physiology, and clinical research).

[0039] 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), TABrown (editor), Essential Molecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press (1991), DMGlover and BDHames(editors),DNA Cloning:A Practical Approach,Volumes 1-4,IRL Press(1995 and 1996),and FMAusubel et al.(editors),Current Protocols in Molecular Biology,Greene Pub.Associates and Wiley-Interscience(1988,including all updates until present),Ed Harlow and David Lane(editors)Antibodies:A Laboratory Manual,Cold Spring Harbor Laboratory,(1988),and JEColigan et al. (editors) Current Protocols in Immunology, John Wiley & Sons (including all updates until present), and other source literature.

[0040] As used in this specification and the appended claims, terms in the singular and singular forms "a," "an," and "the" optionally include plural references unless the content clearly dictates otherwise. Thus, for example, reference to an "analyte" optionally includes one or more analytes.

[0041] As used herein, unless stated to the contrary, the term "about" refers to + / -10%, more preferably + / -5%, more preferably + / -1% of the specified value.

[0042] The term "and / or," e.g., "X and / or Y," is understood to mean either "X and Y" or "X or Y," and is interpreted as explicitly endorsing both meanings or either meaning.

[0043] 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.

[0044] As used herein, the term "connexin" refers to a large family of transmembrane proteins that assemble to form gap junctions, allowing cell-to-cell communication and the movement of ions and small signaling molecules. Connexins are four-transmembrane proteins with both C and N cytoplasmic ends, a cytoplasmic loop (CL), and two extracellular loops (EL-I) and (EL-2). Connexins assemble in groups of six to form hemichannels or connexons, and two hemichannels (one in each cell) combine to form gap junctions between two cells. The term connexin is abbreviated as Cx, the gene that encodes Cx.

[0045] As used herein, the term "gap junction" refers to a specialized intercellular connection between cell types. Gap junctions directly connect the cytoplasm of two cells, allowing various molecules, such as nucleic acids, ions, and electrical impulses, to pass directly through a regulated gate between the cells.

[0046] A variety of subjects can be administered the cell compositions according to the present disclosure. In one example, the subject is a mammal. The mammal can 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 can be used interchangeably in this disclosure, in context.

[0047] 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 a reduction in the rate of disease progression, an improvement or mitigation of the disease state, and remission or improved prognosis. An individual is successfully "treated," for example, if one or more symptoms associated with a disease are alleviated or eliminated.

[0048] An "effective amount" refers to at least an amount effective, at a dosage and for a 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 to refer to an amount necessary to bring about treatment of a disease or condition described herein. The effective amount may vary depending on the disease or condition being treated, as well as on the body weight, age, ethnic background, sex, health and / or physical condition, and other factors associated with the mammal being treated. Typically, an effective amount falls within a relatively broad range (e.g., a "dosage" range), which can be determined by routine testing and experimentation by a physician. An effective amount can be administered in a single dose or in one or several repeated doses over a treatment period.

[0049] A "therapeutically effective amount" is at least the minimum concentration required to produce a measurable improvement in a particular disorder (e.g., cancer). The therapeutically effective amount herein may vary according to factors such as the patient's disease state, age, sex, and weight, as well as the ability of the cell composition to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the composition are outweighed by the therapeutically beneficial effects. In the case of cancer, a therapeutically effective amount may reduce the number of cancer cells, reduce the size of the primary tumor, inhibit (i.e., slow to some extent, and in some instances, halt) cancer cell invasion into peripheral organs, inhibit (i.e., slow to some extent, and in some instances, halt) tumor metastasis, inhibit or slow to some extent tumor growth or tumor progression, and / or alleviate to some extent one or more symptoms associated with the disorder. To the extent a composition according to the present disclosure may prevent the proliferation and / or killing of existing cancer cells, it may be cytostatic and / or cytotoxic. For cancer therapy, in vivo efficacy can be measured, for example, by assessing survival, time to disease progression (TTP), response rate (RR), duration of response, and / or quality of life.

[0050] 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 a population of actively dividing cells. Such cells may be in an exponential growth phase, for example. For example, the level of a particular marker can 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 can be determined by taking a sample of cells and measuring the level of the marker in a cell lysate. Those skilled in the art will recognize that secreted markers are measured by sampling the culture medium, while markers expressed on the surface of cells can be measured by evaluating a sample of the cell lysate. In one example, the sample is taken when the cells are in the exponential growth phase. In one example, the sample is taken after at least two days of culture.

[0051] Culturing to expand cells from a cryopreserved intermediate refers to thawing cells that have undergone cryogenic freezing and in vitro culture under conditions suitable for cell expansion.

[0052] Mesenchymal precursor (MPC) or stem cells As used herein, the term "mesenchymal lineage precursor or stem cell" refers to an undifferentiated pluripotent cell that has the capacity to self-renew while maintaining pluripotency and the ability to differentiate into several cell types, either of mesenchymal origin, e.g., osteoblasts, chondrocytes, adipocytes, stromal cells, fibroblasts and tendons, or of non-mesodermal origin, e.g., hepatocytes, neurons and epithelial cells.

[0053] The term "mesenchymal precursor or stem cell" includes both parent cells 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.

[0054] Mesenchymal precursors or stem cells can be autologous, allogeneic, xenogeneic, syngeneic, or allogeneic. Autologous cells are isolated from the same individual into whom they will be reimplanted. Allogeneic cells are isolated from a donor of the same species. Xenogeneic cells are isolated from a donor of another species. Syngeneic or allogeneic cells are isolated from genetically identical organisms, such as twins, clones, or highly inbred research animal models.

[0055] In one example, the mesenchymal precursors or stem cells are allogeneic. In one example, the allogeneic mesenchymal precursors or stem cells are culture expanded and cryopreserved.

[0056] Mesenchymal precursors or stem cells reside primarily in bone marrow, but have also been shown to reside in a variety of host tissues, including, for example, umbilical cord blood and cord, adult peripheral blood, adipose tissue, cancellous bone, and dental pulp.

[0057] In one example, the mesenchymal precursors or stem cells express STRO-1. In one example, the mesenchymal precursors or stem cells of the present disclosure are culture expanded from a population of mesenchymal precursors or stem cells that express STRO-1+ before being modified to introduce an oncolytic virus as disclosed herein. Culture expansion and methods therefor are discussed further below.

[0058] In one example, mesenchymal precursors or stem cells express STRO-1 and one or more integrins. Integrins are a class of cell adhesion receptors that mediate both cell-cell and extracellular matrix adhesion events. Integrins consist of heterodimeric polypeptides in which a single α-chain polypeptide noncovalently associates with a single β-chain. Currently, there are approximately 16 different α-chain polypeptides and at least about 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 families to which particular integrins are associated are usually characterized by the β-subunit. However, the ligand-binding activity of integrins is primarily influenced by the α-subunit.

[0059] In one example, mesenchymal precursor or stem cells according to the present disclosure express integrins having STRO-1 and β1 (CD29) chain polypeptides.

[0060] In another example, mesenchymal precursors 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 precursors or stem cells according to the present disclosure express STRO-1+ and alpha 1. In another example, mesenchymal precursors or stem cells express STRO-1+ and alpha 2. In another example, mesenchymal precursors or stem cells express STRO-1+ and alpha 3. In another example, mesenchymal precursors or stem cells express STRO-1+ and alpha 4. In another example, mesenchymal precursors or stem cells express STRO-1+ and alpha 5. In another example, mesenchymal precursors or stem cells express STRO-1+ and alpha v. In another example, the mesenchymal precursor or stem cell expresses STRO-1+, α2 and α3. In another example, the mesenchymal precursor or stem cell expresses STRO-1+, α2 and α5. In another example, the mesenchymal precursor or stem cell expresses STRO-1+, α3 and α5. In another example, the mesenchymal precursor or stem cell expresses STRO-1+, α2, α3 and α5.

[0061] In another example, the disclosure encompasses a population of mesenchymal progenitors or stem cells enriched for STRO-1 and α1+ cells. In this example, the population enriched for α1+ cells can comprise at least about 3%, 4%, or 5% α1+ cells.

[0062] In another example, the disclosure encompasses a population of mesenchymal progenitors or stem cells enriched for STRO-1 and α2+ cells. In this example, the population enriched for α2+ cells can comprise at least about 30%, 40%, or 50% α2+ cells.

[0063] In another example, the disclosure encompasses a population of mesenchymal progenitor or stem cells enriched for STRO-1 and α3+ cells, in which the enriched population comprises at least about 40%, 45%, or 50% α3+ cells.

[0064] 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.

[0065] 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 45%, 50%, or 55% α5+ cells.

[0066] 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.

[0067] 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.

[0068] In the above examples, the mesenchymal precursors or stem cells may have a β1 chain polypeptide. For example, mesenchymal precursors or stem cells according to the present disclosure may express an integrin selected from the group consisting of α1β1, α2β1, α3β1, α4β1, and α5β1. Thus, in one example, mesenchymal precursors or stem cells according to the present disclosure express STRO-1+ and α1β1. In another example, mesenchymal precursors or stem cells express STRO-1+ and α2β1. In another example, mesenchymal precursors or stem cells express STRO-1+ and α4β1. In another example, mesenchymal precursors or stem cells express STRO-1+ and α5β1.

[0069] In another example, mesenchymal precursors 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 precursors or stem cells enriched for STRO-1 and β3+ cells. In this example, the population enriched for β3+ cells comprises at least 8%, 10%, or 15% β3+ cells. In another example, mesenchymal precursors or stem cells express STRO-1+ and αvβ3. In another example, mesenchymal precursors or stem cells according to the present disclosure express STRO-1 and an integrin having a β5 (ITGB5) chain polypeptide. In one example, mesenchymal precursors or stem cells express STRO-1 and αvβ5. In another example, mesenchymal precursors or stem cells express STRO-1+ and αvβ6.

[0070] In another example, a mesenchymal precursor or stem cell according to the present disclosure expresses CD271.

[0071] Identification and / or enrichment of mesenchymal precursors or stem cells expressing the above-referenced integrins can be achieved using a variety of methods known in the art. In one example, fluorescence-activated cell sorting (FACS) can be used to identify and select cells expressing the desired integrin polypeptide chains or combinations thereof using commercially available antibodies (e.g., Thermofisher, Pharmingen, Abcam).

[0072] In one example, the mesenchymal precursor or stem cell expresses STRO-1 and coxsackievirus and adenovirus receptors, hi another example, the mesenchymal precursor or stem cell expresses STRO-1, coxsackievirus and adenovirus receptors, and one or more of the integrins referenced above.

[0073] In another example, the mesenchymal precursor or stem cells express STRO-1+, coxsackievirus and adenovirus receptors, αvβ3 and αvβ5.

[0074] In one example, mesenchymal precursors or stem cells are genetically modified to express one or more of the above-referenced integrins or coxsackievirus and adenovirus receptors on the cell surface.

[0075] In one example, the mesenchymal precursor or stem cell expresses the chimeric antigen receptor (CAR), STRO-1. For example, the mesenchymal precursor or stem cell expresses STRO-1+, CAR, αvβ3 and αvβ5.

[0076] In one example, mesenchymal precursors or stem cells expressing a CAR can induce a T cell-mediated immune response. In another example, a CAR serves as a means for attaching mesenchymal precursors or stem cells to cancer cells. In another example, a CAR serves as a means for inducing enhanced attachment of mesenchymal precursors or stem cells to cancer cells.

[0077] In one example, a CAR consists 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.

[0078] Exemplary intracellular domains include CD3-zeta, CD28, 4-IBB, and the like; in some cases, the CAR can include any combination of CD3-zeta, CD28, 4-1BB, TLR-4.

[0079] 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, CD3ε, 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.

[0080] Mesenchymal precursors or stem cells can be isolated from host tissues such as those referred to 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 precursors or stem cells. In one example, mesenchymal precursors or stem cells can be enriched by using STRO-1 antibodies as described in Simmons & Torok-Storb, 1991.

[0081] STRO-1+ cells are 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, and can differentiate into germline cells such as mesoderm and / or endoderm and / or ectoderm. Thus, STRO-1+ cells can differentiate into numerous cell types, including, but not limited to, adipose, bony, cartilaginous, elastic, muscular, and fibrous connective tissue. The specific lineage commitment and differentiation pathways these cells enter depend on various influences from mechanical and / or endogenous bioactive factors, such as growth factors, cytokines, and / or local microenvironmental conditions established by the host tissue.

[0082] The term "enriched" as used herein describes a cell population in which the proportion of one particular cell type or the proportion of multiple particular cell types is increased 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 used to expressly support the term "population of cells comprising X% STRO-1+ cells," where X% is a percentage as recited herein. STRO-1+ cells, in some instances, can form clonogenic colonies; for example, CFU-F (fibroblasts) or a subset thereof (e.g., 50%, 60%, 70%, 70%, 90%, or 95%) can have this activity.

[0083] In one example, a cell population is enriched from a cell preparation that contains STRO-1+ cells in a selectable morphology. In this regard, the term "selectable morphology" will be understood to mean that the cells express a marker (e.g., a cell surface marker) that allows for the selection of STRO-1+ cells. The marker may, but need not be, 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, indicating that cells are STRO-1+ does not mean that the cells are selected by STRO-1 expression. In one example, cells are selected based on at least STRO-3 expression, e.g., they are STRO-3+ (TNAP+).

[0084] Reference to the selection of cells or populations thereof does not necessarily require selection from a particular tissue source. As described herein, STRO-1+ cells can be selected, isolated, or enriched from a wide variety of sources. That said, in some instances, these terms provide support for selection from any tissue containing STRO-1+ cells, or vascularized tissues or tissues containing peripheral cells (e.g., STRO-1+ or 3G5+ peripheral cells), or any one or more of the tissues listed herein.

[0085] 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+.

[0086] "Individually" means that the present disclosure encompasses the listed markers or groups of markers separately; notwithstanding that individual markers or groups of markers may not be separately recited herein, the appended claims may define such markers or groups of markers separately and divisibly from one another.

[0087] "Collectively" means 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 be specifically recited herein, the appended claims may define such combination or subcombination separately and divisibly from any other combination of markers or groups of markers.

[0088] Cells referred to as "positive" for a given marker may express either low (lo, dim, or dull), intermediate (median), or high (bright, bri) levels of that marker, depending on the extent to which the marker is present on the cell surface; this term refers 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), intermediate (median), and high (bright, bri) is understood in the context of the marker used in the particular cell population being sorted or analyzed. Cells referred to as "negative" for a given marker are not necessarily completely absent from the cell. This term means that the marker is expressed at a relatively very low level by the cell, producing a very low signal when detectably labeled, or is undetectable below background levels, e.g., levels detected using an isotype control antibody.

[0089] As used herein, the term "bright" or "bri" refers to a marker on a cell surface that generates a relatively high signal when detectably labeled. Without 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 generate a greater fluorescent signal when labeled with a FITC-conjugated STRO-1 antibody than non-bright cells (STRO-1 lo / dim / dull / intermediate / median) as determined by fluorescence-activated cell sorting (FACS) analysis. In one example, mesenchymal precursor 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 brightly labeled bone marrow mononuclear cells contained in the starting sample. In other examples, "bright" 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 brightly labeled bone marrow mononuclear cells contained in the starting sample. In one example, STRO-1 bright cells have a 2 log increase in STRO-1 surface expression compared to "background," i.e., cells that are STRO-1-. In comparison, STRO-1 lo / dim / dull and / or STRO-1 intermediate / median cells have less than 2 logs of STRO-1 surface expression, typically about 1 log, or expression below "background."

[0090] In one example, STRO-1+ cells are STRO-1bright. In one example, STRO-1bright cells are preferentially enriched relative to STRO-1lo / dim / dull or STRO-1 intermediate / median cells.

[0091] In one example, STRO-1 bright 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 aforementioned markers. In this regard, cells shown to express a marker need not be specifically tested; rather, previously enriched or isolated cells can be tested and then reasonably assumed to also express the same marker.

[0092] In one example, STRO-1bright cells are perivascular mesenchymal precursor or stem cells as defined in WO2004 / 85630, characterized by the presence of the perivascular marker 3G5.

[0093] As used herein, the term "TNAP" is intended to encompass all isoforms of tissue-nonspecific alkaline phosphatase. For example, this term encompasses the liver isoform (LAP), bone isoform (BAP), and kidney isoform (KAP). In one example, TNAP is BAP. In one example, TNAP refers to a molecule capable of binding to the STRO-3 antibody produced by the hybridoma cell line deposited with the American Type Culture Collection (ATCC) on December 19, 2005 under the provisions of the Budapest Treaty under deposit accession number PTA-7282.

[0094] Furthermore, in one example, STRO-1+ cells are capable of giving rise to clonogenic CFU-F.

[0095] In one example, a significant proportion of STRO-1+ cells can differentiate into at least two different germ cell lineages. Non-limiting examples of lineages that can be committed include bone precursor cells, bile duct epithelial cells, and hepatocyte progenitors that are multipotent for hepatocytes, neural-restricted cells that can generate glial precursors that progress to oligodendrocytes and astrocytes, neural precursors that progress to neurons, cardiac muscle and cardiomyocyte precursors, and glucose-responsive insulin secreting pancreatic beta cell lines. Other lineages include, but are not limited to, odontoblasts, dentin-producing cells, and chondrocytes, as well as precursor cells for retinal pigment epithelial cells, fibroblasts, and skin cells, such as keratinocytes, dendritic cells, hair follicle cells, renal ductal epithelial cells, smooth and skeletal muscle cells, testicular progenitor cells, vascular endothelial cells, tendons, ligaments, cartilage, adipocytes, fibroblasts, bone marrow stroma, cardiac muscle, smooth muscle, skeletal muscle, pericytes, blood vessels, epithelium, glia, neurons, astrocytes, and oligodendrocytes.

[0096] In one example, the mesenchymal precursors or stem cells are MSCs. The MSCs may be a homogenous composition or a mixed cell population enriched for MSCs. Homogeneous MSC compositions can be obtained by culturing adherent bone marrow or periosteal cells, and MSCs can be identified by specific cell surface markers identified with 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-adherent isolation rely on the nonspecific plastic-adherence properties of CFU-Fs. Mesenchymal precursors or stem cells isolated from bone marrow by STRO-1-based immunoselection specifically isolate clonogenic mesenchymal precursors from the bone marrow population in the absence of other plastic-adherent bone marrow populations. Alternative sources of MSCs include, but are not limited to, blood, skin, umbilical cord blood, muscle, fat, bone, and perichondrium. In one example, the MSCs are allogeneic. In one example, the MSCs are cryopreserved. In one example, the MSCs are culture-expanded and cryopreserved.

[0097] In one example, the mesenchymal precursor or stem cell is derived from a pluripotent cell, such as an induced pluripotent stem cell (iPS cell). In one embodiment, the pluripotent cell is a human pluripotent cell. Suitable processes for generating mesenchymal precursor or stem cells from pluripotent cells are described, for example, in US 7,615,374 and US 2014 / 273211, Barberi et al; Plos medicine, Vol 2(6):0554-0559 (2005), and Vodyanik et al. Cell Stem cell, Vol 7:718-728 (2010).

[0098] In another example, the mesenchymal precursor or stem cell is immortalized. Exemplary processes for generating immortalized mesenchymal precursor or stem cells are described, for example, in Obinata M., Cell, Vol 2:235-244 (1997), US9,453,203, Akimov et al. Stem Cells, Vol 23:1423-1433 and Kabara et al. Laboratory Investigation, Vol 94:1340-1354 (2014).

[0099] In a preferred embodiment of the present disclosure, the mesenchymal precursors or stem cells are obtained from a master cell bank derived from mesenchymal precursors or stem cells enriched from the bone marrow of healthy volunteers. The use of mesenchymal precursors or stem cells derived from such a source is particularly advantageous for subjects who do not have suitable family members that can serve as mesenchymal precursor or stem cell donors, or who require immediate treatment and are at high risk of relapse, disease-related decline, or death during the time required to generate mesenchymal precursors or stem cells.

[0100] 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 modified to express a particular connexin.

[0101] Isolated or enriched mesenchymal precursor cells can be expanded in vitro by culture. Isolated or enriched mesenchymal precursor cells can be cryopreserved, thawed, and then expanded in vitro by culture.

[0102] In one example, isolated or enriched mesenchymal lineage precursor cells are cultured at 50,000 viable cells / cm in culture medium (serum-free or serum-supplemented), for example, alpha minimal essential medium (αMEM) supplemented with 5% fetal bovine serum (FBS) and glutamine. 2 Cells are seeded at 100°C and allowed to attach to the culture vessel overnight at 37°C and 20% O. The culture medium is then replaced and / or modified as needed, and the cells are cultured for an additional 68-72 hours at 37°C and 5% O.

[0103] 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.

[0104] In one example, mesenchymal precursors or stem cells are obtained from a single donor, or from multiple donors, and the donor samples or mesenchymal precursors or stem cells are then pooled and then expanded in culture.

[0105] Mesenchymal precursors or stem cells encompassed by the present disclosure may also be cryopreserved prior to administration to a subject, hi one example, the mesenchymal precursors or stem cells are culture expanded and cryopreserved prior to administration to a subject.

[0106] In one example, the present disclosure encompasses mesenchymal precursor or stem cells, their progeny, soluble factors derived therefrom, and / or extracellular vesicles isolated therefrom. In another example, the present disclosure encompasses mesenchymal precursor or stem cells, and extracellular vesicles isolated therefrom. For example, mesenchymal precursor or stem cells of the present disclosure can be cultured and grown for a period of time under conditions suitable for secreting extracellular vesicles into cell culture medium. The secreted extracellular vesicles can then be obtained from the culture medium for use in therapy.

[0107] As used herein, the term "extracellular vesicles" refers to lipid particles that are typically less than 200 nm in size but are naturally released from cells and range in size from about 30 nm to 10 microns. These are released by the releasing cells (e.g., mesenchymal stem cells; STRO-1). + The sample may contain proteins, nucleic acids, lipids, metabolites, or organelles from a living cell.

[0108] As used herein, the term "exosome" generally refers to a type of extracellular vesicle that ranges in size from about 30 nm to about 150 nm and originates from the endosomal compartment of mammalian cells, where it is transported to and released from the plasma membrane. They may 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.

[0109] Cell culture growth In one example, mesenchymal precursors or stem cells are culture-expanded. "Culture-expanded" mesenchymal precursors or stem cells are distinguished from freshly isolated cells in that they have been cultured in cell culture medium and passaged (i.e., subcultured). In one example, the culture-expanded mesenchymal precursors or stem cells have been culture-expanded for approximately 4 to 10 passages. In one example, the mesenchymal precursors or stem cells are culture-expanded for at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 passages. For example, the mesenchymal precursors or stem cells can be culture-expanded for at least 5 passages. In one example, the mesenchymal precursors or stem cells can be culture-expanded for at least 5 to 10 passages. In one example, the mesenchymal precursors or stem cells can be culture-expanded for at least 5 to 8 passages. In one example, the mesenchymal precursors or stem cells can be culture-expanded for at least 5 to 7 passages. In one example, the mesenchymal precursors or stem cells can be culture-expanded for more than 10 passages. In another example, the mesenchymal precursors or stem cells can be expanded in culture for more than seven passages. In these examples, the stem cells can be expanded in culture before being cryopreserved to provide 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 stated alternatively as a cryopreserved intermediate.

[0110] In one example, the composition of the present disclosure includes mesenchymal precursors or stem cells cultured and expanded from a cryopreserved intermediate. In one example, the cells cultured and expanded from a cryopreserved intermediate are cultured and expanded for at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 passages. For example, the mesenchymal precursors or stem cells can be cultured and expanded for at least 5 passages. In one example, the mesenchymal precursors or stem cells can be cultured and expanded for at least 5 to 10 passages. In one example, the mesenchymal precursors or stem cells can be cultured and expanded for at least 5 to 8 passages. In one example, the mesenchymal precursors or stem cells can be cultured and expanded for at least 5 to 7 passages. In one example, the mesenchymal precursors or stem cells can be cultured and expanded for more than 10 passages. In another example, the mesenchymal precursors or stem cells can be cultured and expanded for more than 7 passages.

[0111] In one example, mesenchymal precursor or stem cell cultures expanded from cryopreserved intermediates can be expanded in culture in an animal protein-free medium. In one example, mesenchymal precursor or stem cell cultures expanded from cryopreserved intermediates can be expanded in culture in a xeno-free medium. In one example, mesenchymal precursor or stem cell cultures expanded from cryopreserved intermediates can be expanded in culture in a fetal bovine serum-free medium.

[0112] In one embodiment, the mesenchymal precursors or stem cells can be obtained from a single donor, or from multiple donors, and the donor samples or mesenchymal precursors or stem cells are then pooled and then culture expanded. In one example, the culture expansion process includes: i. expanding the number of viable cells by serial propagation to provide a preparation of at least about 1 billion viable cells, wherein serial propagation comprises establishing a primary culture of isolated mesenchymal precursor or stem cells and then serially establishing first non-primary (P1) cultures of isolated mesenchymal precursor or stem cells from the previous culture; ii. Expanding the P1 culture of isolated mesenchymal precursors or stem cells by sub-expansion into a second, non-primary (P2) culture of mesenchymal precursors or stem cells; iii. Preparing and cryopreserving an in-process intermediate mesenchymal precursor or stem cell preparation obtained from the P2 culture of mesenchymal precursor or stem cells; iv. thawing the cryopreserved in-process intermediate mesenchymal precursor or stem cell preparation and expanding the in-process intermediate mesenchymal precursor or stem cell preparation by serial expansion.

[0113] 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. have an antigenic profile and activity profile comprising at least about 95% CD166+ cells.

[0114] In one example, the expanded mesenchymal progenitor or stem cell preparation can inhibit IL2Rα expression by CD3 / CD28-activated PBMCs by at least about 30% compared to a control.

[0115] In one example, the cultured expanded mesenchymal precursors or stem cells are cultured and expanded for about 4-10 passages, and the mesenchymal precursors or stem cells are cryopreserved after at least 2 or 3 passages before further culture expansion. In one example, the mesenchymal precursors or stem cells are cultured and expanded for at least 1, at least 2, at least 3, at least 4, or at least 5 passages, cryopreserved, and then further cultured and expanded for at least 1, at least 2, at least 3, at least 4, or at least 5 passages before being cultured according to the methods of the present disclosure.

[0116] The process of mesenchymal precursor or stem cell isolation and ex vivo expansion can be carried out using any equipment and cell handling methods known in the art. Various culture expansion embodiments of the present disclosure employ steps that require cell manipulation, such as seeding, feeding, dissociation of adherent cultures, or washing. Any step that manipulates cells has the potential to inflict damage on the cells. While mesenchymal precursor or stem cells can generally tolerate some damage during preparation, it is preferable to manipulate the cells using handling procedures and / or equipment that appropriately perform a given step while minimizing damage to the cells.

[0117] In one example, mesenchymal precursors 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 filtrate bag, a mixing zone, a final product bag for washed cells, and appropriate tubing, for example, as described in U.S. Pat. No. 6,251,295, incorporated herein by reference.

[0118] In one example, mesenchymal progenitor or stem cell compositions cultured according to the present disclosure are 95% homogeneous with respect to being CD105-positive and CD166-positive and CD45-negative. In one example, this homogeneity persists through ex vivo expansion, i.e., multiple population doublings.

[0119] In one example, mesenchymal precursors or stem cells of the present disclosure are cultured and expanded in 3D culture. For example, mesenchymal precursors or stem cells of the present disclosure can be cultured and expanded in a bioreactor. In one example, mesenchymal precursors 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 precursors or stem cells of the present disclosure are cultured and expanded from a master cell bank. In one example, mesenchymal precursors or stem cells of the present disclosure are cultured and expanded from a master cell bank in 2D culture before being seeded into 3D culture. In one example, mesenchymal precursors 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 being seeded into 3D culture in a bioreactor. In one example, mesenchymal precursors 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 being seeded into 3D culture in a bioreactor. In one example, mesenchymal precursors or stem cells of the present disclosure are culture-expanded from a master cell bank in 2D culture for 3-5 days before seeding into 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).

[0120] Ang1 and VEGF levels In one example, the mesenchymal precursor or stem cell is at least 0.1 μg / 10 6 However, in other examples, the mesenchymal progenitor or stem cell expresses Ang1 in an amount of at least 0.2 μg / 10 cells. 6 cells, 0.3μg / 10 6 cells, 0.4μg / 10 6 cells, 0.5μg / 10 6 cells, 0.6μg / 10 6 cells, 0.7μg / 10 6 cells, 0.8μg / 10 6 cells, 0.9μg / 10 6 cells, 1μg / 10 6 cells, 1.1μg / 10 6 cells, 1.2μg / 10 6cells, 1.3μg / 10 6 cells, 1.4μg / 10 6 cells, 1.5μg / 10 6 They express Ang1 in amounts of cells.

[0121] In another example, the mesenchymal progenitor or stem cell is administered with VEGF at about 0.05 μg / 10 6 However, in other instances, mesenchymal progenitor or stem cells are expressed in amounts of less than about 0.05 μg / 10 cells. 6 cells, 0.04μg / 10 6 cells, 0.03μg / 10 6 cells, 0.02μg / 10 6 cells, 0.01μg / 10 6 cells, 0.009μg / 10 6 cells, 0.008μg / 10 6 cells, 0.007μg / 10 6 cells, 0.006μg / 10 6 cells, 0.005μg / 10 6 cells, 0.004μg / 10 6 cells, 0.003μg / 10 6 cells, 0.002μg / 10 6 cells, 0.001μg / 10 6 VEGF is expressed in amounts less than 100 cells.

[0122] The amount of cellular Ang1 and / or VEGF expressed in a mesenchymal precursor or stem cell composition or culture can be determined by methods known to those skilled in the art. Such methods include, but are not limited to, quantitative assays such as quantitative ELISA assays. In this example, cell lysates from a mesenchymal precursor or stem cell culture are added to wells of an ELISA plate. The wells can 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 can then be incubated and then washed after an 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 can 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. Once incubation is complete, 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.

[0123] In another embodiment, the mesenchymal precursor or stem cell expresses Ang1:VEGF at a ratio of at least about 2: 1. However, in other instances, the mesenchymal precursor or stem cell expresses Ang1:VEGF at 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.

[0124] Methods for determining the Ang1:VEGF expression ratio will be apparent to those skilled in the art. For example, Ang1 and VEGF expression levels 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.

[0125] In one example, mesenchymal precursors 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 precursors or stem cells 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 Ang1 and / or VEGF to some extent without transfection with a nucleic acid encoding Ang1 and / or VEGF1.

[0126] Oncolytic viruses 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 tumor cells. For example, an oncolytic virus can inhibit cell proliferation. In another example, an oncolytic virus can kill tumor cells. In one example, an oncolytic virus preferentially infects and inhibits the growth of tumor cells compared to corresponding normal cells. In another example, an oncolytic virus preferentially replicates in tumor cells and inhibits the growth of tumor cells compared to corresponding normal cells.

[0127] In one example, oncolytic viruses can naturally infect tumor cells and reduce their growth. Examples of such viruses include Newcastle disease virus, vesicular stomatitis virus, myxoma virus, reovirus, Sindbis virus, measles virus, and coxsackie virus. Oncolytic viruses that naturally infect and reduce the growth of tumor cells generally target tumor cells by exploiting the cellular aberrations that occur in these cells. For example, oncolytic viruses can exploit surface-attached receptors that are activated oncogenes, such as Ras, Akt, p53, and / or interferon (IFN) pathway defects.

[0128] In another example, oncolytic viruses encompassed by the present disclosure are engineered to infect and reduce tumor cell growth. Exemplary viruses suitable for such engineering include oncolytic DNA viruses, such as respiratory syncytial virus (RSV), adenovirus, herpes simplex virus (HSV), and vaccinia virus, as well as oncolytic RNA viruses, such as lentivirus, reovirus, coxsackievirus, Seneca Valley virus, poliovirus, measles virus, Newcastle disease virus, vesicular stomatitis virus (VSV), and parvoviruses, such as the rodent protoparvovirus H-1PV.

[0129] In one example, the tumor specificity of an oncolytic virus can be enhanced by mutating or deleting a gene that is necessary for viral survival in normal cells but consumable in cancer cells. For the avoidance of doubt, an oncolytic virus with a mutated or deleted gene can survive in mesenchymal precursors or stem cells long enough to allow metastasis 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 is 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 is engineered to contain a tumor-specific cell surface molecule for transductive targeting of tumor cells. 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, transmembrane glycoprotein NMB, CC-cocaine receptor, PSMA, RON receptor, and cytotoxic T-lymphocyte antigen 4.

[0130] In another example, oncolytic viruses are engineered to increase the ability of infected mesenchymal precursors or stem cells to deliver the viral payload to cancer cells. For example, oncolytic viruses can be engineered to express virus-induced membrane glycoproteins to mediate the induction of mesenchymal precursor or stem cell fusion to tumor 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).

[0131] In one example, the viral fusogenic membrane glycoprotein is under the control of a late promoter, such as the adenovirus major late promoter. In one example, the virally induced membrane glycoprotein is under the control of a strict late promoter, such as UL38p (WO2003 / 082200), which is active only after the onset of viral DNA replication. Examples of such promoters and genetically engineered viruses are disclosed in Fu et al. (2003) Molecular Therapy, 7:748-54 and Guedan et al. (2012) Gene Therapy, 19:1048-1057.

[0132] In one example, the oncolytic virus is replication competent. In one example, the oncolytic virus selectively replicates in tumor cells compared to corresponding normal cells and / or mesenchymal precursors or stem cells. In one example, the tumor specificity of the oncolytic virus can be engineered to restrict viral replication by relying on constitutively activated transcriptional activity (i.e., conditional replication) in tumor cells. 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, New Cattle disease virus, or vaccinia.

[0133] 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 precursors 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 precursors 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 precursors or stem cells. Tumor-specific promoters can be native or composite. Exemplary native 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, in some instances, be dictated by the target tumor, for example, the cerb2 promoter may be suitable for breast and pancreatic cancer, and the PSA promoter may be suitable for prostate cancer.

[0134] In another example, tumor-specific promoters can be identified based on differences in promoter activity in tumor cells compared to corresponding normal cells and / or mesenchymal precursors or stem cells. For example, one method for identifying suitable tumor-specific promoters is to compare promoter activity between tumor cells and corresponding normal cells and / or mesenchymal precursors or stem cells to identify promoters with high activity in tumor cells and low activity in corresponding normal cells and / or mesenchymal precursors or stem cells. In one example, a tumor-specific promoter can be a late or strictly late viral promoter. The terms "late" and "strictly late" are used to refer to promoters whose activity depends on the initiation of viral DNA replication. Thus, late and strictly late promoters are suitable for inclusion in oncolytic viruses that can replicate in tumor cells but have limited ability to replicate in non-dividing normal cells. Exemplary late or strictly late promoters include major late promoter (MLP) and UL38p.

[0135] In one example, the oncolytic virus is a respiratory syncytial virus (RSV), herpes simplex virus, or adenovirus containing a late or severe late promoter. For example, the oncolytic virus is a herpes simplex virus containing the UL38p promoter. In another example, the oncolytic virus is an adenovirus containing an MLP.

[0136] In another example, the tumor specificity of oncolytic viruses can be engineered to take advantage of tumor-specific tropism. In another example, oncolytic viruses are sensitive to oligonucleotides or binding proteins expressed in normal cells and / or mesenchymal precursors or stem cells that are expressed at low levels or are not present in tumor cells. For example, oncolytic viruses can be engineered to insert nucleotide sequences that are complementary to oligonucleotides expressed by mesenchymal precursors or stem cells and / or normal cells but not expressed by cancer cells. For example, oncolytic viruses can be sensitive to inhibitory oligonucleotides such as miRNAs.Exemplary miRNAs that are expressed at low levels in several tumor cells and at high levels in corresponding normal cells may 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-�p, miR-424-5p, miR-497-5p, miR-7-5p, miR-101-3p, miR-124-3p, miR-126-3p, miR-137, miR-138-5p, miR-140-5p, miR-152-3p, miR-185-5p, miR-214-꜆p, miR-25-3p, miR-26a-5p, miR-26b-5p, miR-372-3p, miR-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, let-7b-5p, miR-1, miR-100-5p, miR-125a-5p, miR-133a-3p, miR-133b, miR-14꜆a-5p, miR-150-5p, miR-193a-3p, miR-193b-3p, miR-196b-5p, miR-206, miR-218-5p, miR-22꜆-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, miR-142-3p, miR-이16b-5p, miR-622, miR-96-5p, miR-1291, miR-370-3p, miR-296-5p, miR-335-5p, miR-483-3p, miR-483-5p, miR-486-5p.\n

[0137] In another example, the oncolytic virus can be engineered to express a gene in infected tumor cells. In one example, the expression of the gene is suppressed in mesenchymal precursors or stem cells. In one example, the gene enhances the immune response against the 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, heat shock protein (HSP), or a combination thereof.

[0138] As outlined in the above examples, various viruses can be engineered.In one example, oncolytic virus is modified respiratory syncytial virus (RSV), lentivirus, baculovirus, retrovirus, adenovirus (AdV), adeno-associated virus (AAV) or recombinant form such as recombinant adeno-associated virus (rAAV), and its derivatives such as self-complementary AAV (scAAV) and non-integrated AV.For example, oncolytic virus can be modified lentivirus.In one example, oncolytic virus can be modified RSV.

[0139] 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.

[0140] 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 (Vaccina, Sillajen), JX-594 (AdV, Cold Genesys), and Reolysin (Reovirus, Oncolytics Biotech). Other examples of oncolytic viruses are disclosed in WO2003 / 080083, WO2005 / 086922, WO2007 / 088229, WO2008 / 110579, WO2010 / 108931, WO2010 / 128182, WO2013 / 112942, WO2013 / 116778, WO2014 / 204814, WO2015 / 077624, and WO2015 / 166082, WO2015 / 089280.

[0141] In one example, the oncolytic virus is replication-deficient. For example, the replication gene can be mutated, deleted, or replaced with an expression cassette having a tumor-specific promoter. In one example, the E1 / E3 genes are mutated, deleted, or replaced. In another example, the E1A / E1B genes are mutated, deleted, or replaced. For example, in the context of AV, the E1 / E3 genes can be mutated, deleted, or replaced. In the context of AAV, the E1A and E1B genes can be mutated, deleted, or replaced. Various examples of suitable tumor-specific promoters are discussed above.

[0142] In another example, the oncolytic virus can comprise a mutated E1, E3, E1A, or E1B gene. For example, the E1A gene can be mutated in a region encoding a retinoblastoma protein (RB) binding site. In another example, the E3 gene can be mutated in a region encoding an endoplasmic reticulum retention domain. In another example, the oncolytic virus can comprise a mutation in the gamma-34.5 gene and / or the alpha-47 gene.

[0143] In one example, an oncolytic virus is replication-deficient in mesenchymal precursors or stem cells and replication-competent in tumor cells. An example of switching a replication-deficient virus 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, δ24 mutations that enable replication in pRb or p53-inactive tumor cells, and / or viruses containing regulated expression of E1 under the control of tumor cell-specific promoters such as α-chemokine SDF-1 receptor (CXCR4), survivin, cyclooxygenase-2 (COX-2), and midcan.

[0144] qualification The mesenchymal precursors or stem cells of the present disclosure can be modified to introduce the oncolytic viruses referenced above. A mesenchymal precursor or stem cell is considered "modified" if the oncolytic virus has been transferred into the cell by any suitable means of artificial manipulation, or if the cell is the progeny of an original modified cell that harbors the oncolytic virus.

[0145] Mesenchymal precursors or stem cells can be modified using various methods known in the art. In one example, mesenchymal precursors or stem cells are contacted with an oncolytic virus in vitro. For example, the oncolytic virus can be added to the mesenchymal precursor or stem cell culture medium. In another example, the mesenchymal precursors or stem cells are centrifuged with the oncolytic virus.

[0146] Infection efficiency is rarely 100%, and it is usually desirable to enrich for a population of successfully modified cells. In one example, modified cells can be enriched by taking advantage of functional characteristics of the new genotype. One exemplary method for enriching for modified cells is positive selection using resistance to drugs such as neomycin, or colorimetric selection based on expression of lacZ.

[0147] In another example, mesenchymal precursors or stem cells are modified to introduce an oncolytic virus that kills cancer cells but does not substantially affect the viability of the mesenchymal precursors or stem cells.

[0148] In another example, mesenchymal precursors or stem cells are modified to introduce an oncolytic virus that preferentially kills cancer cells compared to mesenchymal precursors or stem cells.

[0149] In another example, mesenchymal precursors or stem cells are modified to introduce an oncolytic virus that does not kill the mesenchymal precursors or stem cells before they can deliver the oncolytic virus to cancer cells.

[0150] Delivery to cancer cells The present inventors have identified that mesenchymal precursors or stem cells can transfer oncolytic viruses to cancer cells. Accordingly, in one example, the present disclosure encompasses a method of delivering the referenced oncolytic viruses to cancer cells by contacting them with mesenchymal precursors or stem cells that have been modified to transduce the referenced oncolytic viruses. For the avoidance of doubt, the oncolytic viruses delivered to cancer cells are oncolytic viruses introduced into mesenchymal precursors or stem cells.

[0151] The term "contacting" is used in the context of this disclosure to refer to "direct" or "indirect" contact. "Direct contact" is used in the context of this disclosure to refer to physical contact between cancer cells and modified mesenchymal precursors or stem cells that facilitates the transfer of oncolytic viruses. For example, cancer cells and modified mesenchymal precursors 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 precursors or stem cells). In this example, the common connexin facilitates the transfer of oncolytic viruses from the mesenchymal precursors or stem cells to the cancer cells via gap junctions. Thus, in one example, contact occurs under conditions that allow the mesenchymal precursors or stem cells to form gap junctions with the cancer cells, thereby delivering the oncolytic viruses to the cancer cells by crossing the gap junctions. In one example, the gap junctions are formed by Cx40. In another example, the gap junctions are formed by Cx43. In another example, the gap junctions are formed by Cx45, Cx32, and / or Cx37.

[0152] "Indirect contact" is used in the context of the present disclosure to refer to delivery of an oncolytic virus to a cancer cell without direct contact from a modified mesenchymal precursor or stem cell. For example, a modified mesenchymal precursor or stem cell in close proximity to a cancer cell may be in indirect contact with the cancer cell. In one example, a modified mesenchymal precursor or stem cell in indirect contact with a cancer cell can deliver an oncolytic virus to the cancer cell via an exosome.

[0153] In another example, modified mesenchymal precursors or stem cells in direct contact with cancer cells can deliver oncolytic viruses to cancer cells via common connexins and indirectly via exosomes.

[0154] The cancer cells that received the oncolytic virus from the modified mesenchymal precursors or stem cells are not particularly limited, as long as they can be directly or indirectly contacted by the mesenchymal precursors or stem cells that have been modified to facilitate the transfer of the oncolytic virus. 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 breast cancer cells. In another example, the cancer cells are prostate cancer cells. In another example, the cancer cells are melanoma cells.

[0155] In another example, the cancer cells share a common connexin with modified mesenchymal progenitor or stem cells. In one example, the cancer cells express Cx40. In another example, the cancer cells express Cx43. In another example, the cancer cells express Cx45, Cx32, and / or Cx37.

[0156] In another example, the cancer cells are syncytial cancer cells. The term "syncytial" is used in the context of this disclosure to refer to a cancerous tissue or mass composed of cells interconnected by specialized membranes with gap junctions that are electrically synchronized in their action potential.

[0157] The delivery of oncolytic viruses from modified mesenchymal precursors or stem cells to cancer cells can be enhanced in vitro or in vivo. In one example, the delivery of oncolytic viruses from modified mesenchymal precursors or stem cells to cancer cells can be enhanced in vitro by co-culturing the modified mesenchymal precursors or stem cells with cancer cells. In one example, the delivery of oncolytic viruses from modified mesenchymal precursors or stem cells to cancer cells can be enhanced in vivo by administering the modified mesenchymal precursors or stem cells to a subject. For example, the mesenchymal precursors or stem cells can be administered systemically, for example, intravenously, intraarterially, or intraperitoneally. In another example, the mesenchymal precursors or stem cells can be administered intranasally or intramuscularly. In one example, the mesenchymal precursors or stem cells are administered to a site proximal to cancer cells, such as to surrounding tissue. In another example, the mesenchymal precursors or stem cells are administered directly to the cancer.

[0158] Improved preservation and / or homing of modified mesenchymal precursor or stem cells In one aspect, mesenchymal precursors or stem cells as defined herein are treated to modify their cell surface glycans. Modification of glycans on cell surface proteins such as CD44 has been shown to create E-selectin ligands that can bind to E-selectin molecules expressed in vivo on microvessels at sites of inflammation. In this way, modification of cell surface glycans on mesenchymal precursors or stem cells improves the homing of mesenchymal precursors or stem cells to sites of tissue injury in vivo.

[0159] The inventors have also identified that glycosyltransferase-mediated modification of cell surface glycans improves cell viability after cryopreservation (i.e., more cells are viable after freeze-thaw cycles). Thus, in one example, the disclosure encompasses a cryopreserved population of mesenchymal progenitors or stem cells treated with a glycosyltransferase (E.C2.4) under conditions that modify cell surface glycans on the cells. In another example, the disclosure encompasses a method of cryopreserving mesenchymal progenitors or stem cells, the method comprising treating a population of mesenchymal progenitors or stem cells with a glycosyltransferase under conditions that result in modification of cell surface glycans on the cells, and cryopreserving the cells in a composition. In another example, the disclosure encompasses a method of producing therapeutic cells, the method comprising treating a population of mesenchymal progenitors or stem cells with a glycosyltransferase under conditions that result in modification of cell surface glycans on the cells, and cryopreserving the cells in a composition.

[0160] In one example, "treating" a mesenchymal progenitor or stem cell comprises contacting the cell with a glycosyltransferase under conditions in which the glycosyltransferase has enzymatic activity. In this example, the glycosyltransferase modifies cell surface glycans on the mesenchymal progenitor or stem cell. An example of a cell surface glycan modification is fucosylation. In one example, CD44 is modified. In another example, CD14 is modified. In another example, one or more of CD44, CD14, CD3, and CD19 are modified.

[0161] In one example, surface glycan modifications are identified using flow cytometry. In this example, the modified mesenchymal precursors or stem cells express 1 log greater fucosylated cell surface glycans than untreated mesenchymal precursor cells. In another example, the modified mesenchymal precursors or stem cells express 2 log greater fucosylated cell surface glycans than untreated mesenchymal precursor cells. In another example, the modified mesenchymal precursors or stem cells express 3 log greater fucosylated cell surface glycans than untreated mesenchymal precursor cells. For example, the modified mesenchymal precursors or stem cells can express 1 log greater fucosylated CD14 than untreated mesenchymal precursor cells. In another example, the modified mesenchymal precursors or stem cells express 2 log greater fucosylated CD14 than untreated mesenchymal precursor cells. In another example, the modified mesenchymal precursors or stem cells express 3 log greater fucosylated CD14 than untreated mesenchymal precursor cells.

[0162] In one example, "treating" includes contacting the mesenchymal progenitor or stem cell with a glycosyltransferase in the presence of a nucleotide sugar donor substrate. Suitable donor substrates include fucose, galactose, sialic acid, or N-acetylglucosamine. For example, the substrate can be GDP-fucose.

[0163] For example, the treatment can include contacting a population of mesenchymal precursors or stem cells with an exogenous glycosyltransferase, such as a fucosyltransferase. In this example, the glycosyltransferase can be added to a cell culture medium or other physiologically acceptable solution containing the mesenchymal precursors or stem cells. For example, the mesenchymal precursors or stem cells can be cultured in a medium containing the glycosyltransferase. In another example, the mesenchymal precursors or stem cells can be suspended in a culture medium containing the glycosyltransferase. For example, the mesenchymal precursors or stem cells can be dissociated from the culture and resuspended in a suitable medium containing the glycosyltransferase. In one example, the cells can be dissociated using ethylenediaminetetraacetic acid (EDTA). In another example, the cells can be dissociated using a protease, such as trypsin alone, in combination with EDTA.

[0164] In one example, the cell culture medium comprises at least 1.8 μg of glycosyltransferase. In another example, the cell culture medium comprises at least 2.0 μg of glycosyltransferase. In another example, the cell culture medium comprises at least 2.5 μg of glycosyltransferase. In another example, the cell culture medium comprises 2 to 15 μg of glycosyltransferase. In another example, the cell culture medium comprises 2 to 10 μg of glycosyltransferase. In another example, the cell culture medium comprises 2 to 5 μg of glycosyltransferase. In one example, the cell culture medium comprises at least 1.8 μg of fucosyltransferase. In another example, the cell culture medium comprises at least 2.0 μg of fucosyltransferase. In another example, the cell culture medium comprises at least 2.5 μg of fucosyltransferase. In another example, the cell culture medium comprises 2 to 15 μg of fucosyltransferase. In another example, the cell culture medium contains 2-10 μg of fucosyltransferase. In another example, 2-5 μg of fucosyltransferase is added to the cell culture medium. In these examples, the glycosyltransferase is added at a concentration of about 5×10 5 Each mesenchymal precursor or stem cell can be provided in a 30 μl reaction volume.

[0165] For example, mesenchymal precursors or stem cells can be treated with exogenous glycosyltransferases in a process known as exofucosylation. In this embodiment, the glycosyltransferases can be provided in a physiologically acceptable solution with low levels of divalent metal cofactors. In various embodiments, the physiologically acceptable solution is buffered. The physiologically acceptable solution can be, for example, Hank's Balanced Salt Solution, Dulbecco's Modified Eagle Medium, a Good's buffer, such as HEPES buffer, 2-morpholinoethanesulfonic acid (MES) buffer, or phosphate-buffered saline (PBS) (see N.E. Good, G.D. Winget, W. Winter, T. N. Conolly, S. Izawa, and R.M.M. Singh, Biochemistry 5, 467 (1966); N.E. Good, S. Izawa, Methods Enzymol. 24, 62 (1972)).

[0166] In one example, the physiologically acceptable solution is substantially free of glycerol.

[0167] In another example, mesenchymal progenitors or stem cells are treated with glycosyltransferases by modifying the cells to express the glycosyltransferases. For example, glycosyltransferases can be produced intracellularly by mesenchymal progenitors or stem cells. In this embodiment, a nucleic acid molecule encoding the glycosyltransferase is introduced into the mesenchymal progenitors or stem cells. The glycosyltransferases are then expressed by the mesenchymal progenitors or stem cells, resulting in the modification of their surface glycans.

[0168] A mesenchymal progenitor or stem cell is considered to be "genetically modified to express a glycosyltransferase" if a nucleic acid encoding a glycosyltransferase has been transferred into the cell by any suitable means of artificial manipulation, or if the cell is the progeny of an originally altered cell carrying a nucleic acid encoding a glycosyltransferase. Cells can be stably or transiently modified to express a glycosyltransferase.

[0169] In one example, expression of glycosyltransferases in genetically modified mesenchymal precursors or stem cells results in enhanced retention of the cells at sites of inflammation in vivo. For example, genetically modified mesenchymal precursors or stem cells can be retained in tumors or their metastases. In another example, genetically modified mesenchymal precursors or stem cells can be retained at sites of organ transplant rejection. In another example, genetically modified mesenchymal precursors or stem cells can be retained at sites of injury, such as an infarcted heart. Various methods are available for determining whether genetically modified mesenchymal precursors or stem cells are retained at sites of inflammation in vivo. In one example, cells are imaged in vivo using a radiotracer or other suitable label.

[0170] Mesenchymal precursors or stem cells can be genetically modified using various methods known in the art. In one example, mesenchymal precursors or stem cells are treated in vitro with a viral vector. Genetically modified viruses have been widely applied to deliver nucleic acids to cells. Exemplary viral vectors for the genetic modification of cells described herein include retroviral vectors such as gammaretroviral vectors, lentiviruses, murine leukemia viruses (MLV or MuLV), and adenoviruses. For example, viruses can be added to mesenchymal precursor or stem cell culture media. Non-viral methods can also be used. Examples include the application of plasmid transfer and targeted gene integration using integrase or transposase technology, liposome- or protein transduction domain-mediated delivery, and physical methods such as electroporation.

[0171] The efficiency of genetic modification is rarely 100%, and it is usually desirable to enrich for populations of successfully modified cells. In one example, modified cells can be enriched by taking advantage of functional characteristics of the new genotype. One exemplary method for enriching for modified cells is positive selection using resistance to drugs such as neomycin, or colorimetric selection based on expression of lacZ.

[0172] In various embodiments, mesenchymal progenitor or stem cells are contacted with two or more glycosyltransferases and their appropriate donor substrates (e.g., sugars). For example, cells are contacted simultaneously or sequentially with two glycosyltransferases, each of which adds a different monosaccharide in the appropriate linkage to the (extending) core glycan structure. In another example, the genetically modified cells express two glycosyltransferases.

[0173] In one embodiment, the treated mesenchymal precursors or stem cells express CD44, e.g., alpha(2,3) sialylated CD44. In another embodiment, the mesenchymal precursors or stem cells do not express CD34 or PSGL-1. In one example, the treated mesenchymal precursors or stem cells bind E-selectin and / or L-selectin. In one example, the modified mesenchymal precursors or stem cells do not bind P-selectin.

[0174] In another example, CD14 is fucosylated on the treated mesenchymal progenitor or stem cells. In another example, CD14 and CD3 are fucosylated on the treated mesenchymal progenitor or stem cells.

[0175] In one embodiment, the glycosyltransferase is capable of transferring 1.0 mmol of sugar per minute at 37° C. and pH 6.5.

[0176] In one example, the glycosyltransferase is a fucosyltransferase (catalyzing the transfer of L-fucose sugars). In another example, the glycosyltransferase is an α1,3 fucosyltransferase, such as α1,3 fucosyltransferase III, α1,3 fucosyltransferase IV, α1,3 fucosyltransferase VI, α1,3 fucosyltransferase VII, α1,3 fucosyltransferase IX, α1,3 fucosyltransferase X, or α1,3 fucosyltransferase XI. For example, cells can be treated with α1,3 fucosyltransferase VII. In another example, cells can be treated with α1,3 fucosyltransferase VI. In these examples, fucosylation of mesenchymal progenitor or stem cells can be identified by detecting an increased ability of the treated cells to bind to a selectin, such as E-selectin, and / or an increased reactivity of the treated cells with antibodies that bind to sLeX known in the art, including, but not limited to, HECA-452.

[0177] In another example, the glycosyltransferase is a galactosyltransferase (catalyzes the transfer of galactose), hi another example, the glycosyltransferase is a sialyltransferase (catalyzes the transfer of sialic acid).

[0178] Treatment methods In one example, a composition according to the present disclosure 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 specific examples of such cancers include squamous cell carcinoma (e.g., epithelial squamous cell carcinoma), lung cancer including small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung and squamous cell carcinoma of the lung, cancer of the peritoneum, hepatocellular carcinoma, stomach or gastric cancer including gastrointestinal cancer and gastrointestinal stromal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, urinary tract cancer, hepatoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney or renal cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, anal cancer, penile cancer, melanoma, shallow spreading melanoma, malignant melanoma, acral squamous melanoma, nodular melanoma, multiple myeloma, and B-cell lymphoma (low-grade / follicular) These include, but are not limited to, non-Hodgkin's lymphoma (NHL), small lymphocytic (SL) NHL, intermediate-grade / follicular NHL, intermediate-grade vasoactive NHL, high-grade immunoblastic NHL, high-grade lymphoblastic NHL, high-grade non-neoplastic NHL, bulky disease NHL, mantle cell lymphoma, AIDS-related lymphoma, and Waldenstrom's macroglobulinemia), 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 phacomatosis, edema (such as that associated with brain tumors), Meg-Gibber syndrome, brain, and head and neck cancers and associated metastases.

[0179] 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.

[0180] In another example, a cancer treated according to the present disclosure comprises cells that share a common connexin with mesenchymal precursors or stem cells according to the present disclosure, in this example, the common connexin facilitates the transfer of nucleic acids from the mesenchymal precursors or stem cells to the cancer cells.

[0181] In one example, the cancer contains cells that express Cx40. In another example, the cancer contains cells that express Cx43. In another example, the cancer contains cells that express Cx40 and Cx43.

[0182] cell composition In practicing the methods of the present disclosure, the mesenchymal precursors or stem cells may be administered in the form of a composition.

[0183] An exemplary composition according to the present disclosure can include mesenchymal precursors or stem cells modified to introduce an oncolytic virus. Exemplary oncolytic viruses are described above. In one example, a composition according to the present disclosure can include mesenchymal precursors or stem cells modified to introduce the above-mentioned oncolytic viruses or a combination thereof. For example, mesenchymal precursors or stem cells can be modified to introduce an oncolytic virus characterized as a parvovirus, such as a conditionally replicating adenovirus (CRAd), herpes simplex virus (HSV), lentivirus, vaccinia virus, vesicular stomatitis virus (VSV), sinbis virus, RSV, measles, and rodent protoparvovirus H-1PV. In one example, mesenchymal precursors or stem cells can be modified to introduce a conditionally replicating lentivirus.

[0184] In another example, a composition according to the present disclosure may include mesenchymal precursors or stem cells that have been modified to introduce an oncolytic virus that does not substantially affect the viability of the mesenchymal precursors or stem cells.

[0185] In another example, a composition according to the present disclosure can include mesenchymal precursors or stem cells that have been modified to introduce an oncolytic virus that does not kill the mesenchymal precursors or stem cells prior to delivering the oncolytic virus to the cancer cells.

[0186] In one example, such a composition comprises a pharmaceutically acceptable carrier and / or excipient.

[0187] The terms "carrier" and "excipient" refer to a composition of matter 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. A suitable carrier is, for example, stable and, for example, incapable of reacting with other ingredients in the carrier. In one example, a carrier causes no significant local or systemic adverse effects in the recipient at the dosages and concentrations used for therapy.

[0188] Suitable carriers for the present disclosure include those conventionally used, such as water, saline, aqueous dextrose, lactose, Ringer's solution, buffered solutions, hyaluronan, and glycols, which are exemplary liquid carriers, particularly for solutions (when isotonic). 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, glycerol, propylene glycol, water, ethanol, and the like.

[0189] In another example, the carrier is, for example, a media composition that the cells are grown or suspended in. Such a media composition does not induce any adverse effects in the subject to which it is administered.

[0190] 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.

[0191] In one example, the carrier or excipient provides buffering activity to maintain the cells and / or soluble factors at a suitable pH, thereby exerting biological activity; for example, the carrier or excipient is phosphate buffered saline (PBS). PBS represents 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 application directly to the bloodstream or for application directly to tissues or areas surrounding or adjacent to the tissue, e.g., by injection.

[0192] The cell compositions disclosed herein can be administered alone or as a mixture with other cells. Different types of cells can be mixed with the disclosed compositions immediately or shortly before administration, or can be co-cultured for a period of time before administration.

[0193] In one example, the composition comprises an effective or therapeutically effective amount of cells. For example, the composition comprises about 1 x 10 5 cells ~ approx. 1 x 10 9 cells, or approximately 1.25 x 10 3 cells ~ approx. 1.25 x 10 7 The exact amount of cells to be administered will depend on various factors, including the age, weight, and sex of the subject, as well as the extent and severity of the disorder being treated.

[0194] Exemplary dosages include at least about 1.2 x 10 8 ~Approx. 8×10 10 cells, e.g., about 1.3 x 10 8 ~Approx. 8×10 9 cells, approximately 1.4 x 10 8 ~Approx. 8×10 8 cells, approximately 1.5 x 10 8 ~Approx. 7.2×10 8 cells, approximately 1.6 x 10 8 ~Approx. 6.4×10 8 cells, approximately 1.7 x 108 ~Approx. 5.6×10 8 cells, approximately 1.8 x 10 8 ~Approx. 4.8×10 8 cells, approximately 1.9 x 10 8 ~Approx. 4.0×10 8 cells, approximately 2.0 x 10 8 ~Approx. 3.2×10 8 cells, approximately 2.1 x 10 8 ~Approx. 2.4×10 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 The cell may include a cell.

[0195] In other words, an exemplary dose is at least about 1.5 x 10 6 In one example, the dose is at least about 2.5 x 10 cells / kg (80 kg subject). 6 In another example, the dose can include about 1.5 x 10 cells / kg. 6 ~Approx. 1×10 9 cells / kg, approximately 1.6 x 10 6 ~Approx. 1×10 8 cells / kg, approximately 1.8 x 10 6 ~Approx. 1×10 7 cells / kg, approximately 1.9 x 10 6 ~Approx. 9×10 6 cells / kg, approximately 2.0 x 10 6 ~Approx. 8×10 6 cells / kg, approximately 2.1 x 10 6 ~Approx. 7×10 6 cells / kg, approximately 2.3 x 10 6 ~about 6×10 6 cells / kg, approximately 2.4 x 10 6 ~Approx. 5×10 6 cells / kg, approximately 2.5 x 10 6 ~Approx. 4×10 6 cells / kg, approximately 2.6 x 10 6 ~Approx. 3×10 6 The cells / kg may be included.

[0196] 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.

[0197] The compositions of the present disclosure can be cryopreserved. Cryopreservation of mesenchymal precursors or stem cells can be performed using slow cooling methods or "rapid" freezing protocols known in the art. Preferably, the cryopreservation method maintains a similar phenotype, cell surface markers, and proliferation rate of the cryopreserved cells compared to unfrozen cells.

[0198] The cryopreserved composition may comprise a cryopreservation solution, the pH of which is typically 6.5 to 8, preferably 7.4.

[0199] Cryopreservation solutions may include, for example, a sterile, non-pyrogenic, isotonic solution such as Plasmalyte A™. 100 mL of Plasmalyte A™ contains 526 mg of sodium chloride, USP (NaCl), 502 mg of sodium gluconate (C6H11NaO7), 368 mg of sodium acetate trihydrate, USP (C2H3NaO2·3H2O), 37 mg of potassium chloride, USP (KCl), and 30 mg of magnesium chloride, USP (MgCl2·6H2O). No antimicrobial agents are included. The pH is adjusted with sodium hydroxide. The pH is 7.4 (6.5-8.0).

[0200] The cryopreservation solution may include Profreeze™. The cryopreservation solution may additionally or alternatively include culture medium, for example, αMEM.

[0201] 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, provide high survival rates after thawing, and allow transplantation without washing. However, the most commonly used cryoprotectant, DMSO, exhibits some degree of cytotoxicity. Hydroxyethyl starch (HES) can be used as an alternative or in combination with DMSO to reduce the cytotoxicity of cryopreservation solutions.

[0202] The cryopreservation solution may include one or more of DMSO, hydroxyethyl starch, human serum components, and other protein bulking agents. In one example, the cryopreservation solution includes about 5% human serum albumin (HSA) and about 10% DMSO. The cryopreservation solution may further include one or more of methylcellulose, polyvinylpyrrolidone (PVP), and trehalose.

[0203] In one embodiment, cells are suspended in 42.5% Profreeze™ / 50% αMEM / 7.5% DMSO and cooled in a controlled-rate freezer.

[0204] The cryopreserved composition can be thawed and administered directly to a subject, or can be added to another solution containing, for example, hyaluronic acid. Alternatively, the cryopreserved composition can be thawed and the mesenchymal precursors or stem cells resuspended in an alternative carrier prior to administration.

[0205] In one example, the cell compositions described herein can be administered as a single dose. In another example, the cell compositions are administered in multiple doses, such as at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten doses.

[0206] In one example, mesenchymal precursors or stem cells can be cultured and expanded before administration.Various methods for mesenchymal precursor or stem cell culture are known in the art.In one example, mesenchymal precursors or stem cells are cultured and expanded in serum-free medium before administration.For example, mesenchymal precursors 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.

[0207] The mesenchymal precursors or stem cells can be administered systemically, for example, by intravenous, intraarterial, or intraperitoneal administration. The mesenchymal precursors or stem cells can also be administered intranasally, intramuscularly, or intracardially. In one example, the mesenchymal precursors or stem cells are administered directly to the subject's tumor. [Example]

[0208] Example 1 - Evaluation of viral delivery systems for mesenchymal precursor cells The efficacy of three different viral delivery systems was evaluated in human mesenchymal precursor cells (MPCs). Two batches of MPCs were procured from frozen stocks and were cultured at 5,000, 10,000, and 15,000 cells / cm. 2 Cells were seeded directly into 96-well plates at 100°C. Cells were allowed to adhere overnight at 37°C with 5% CO2 before adding viral particles. Three viral delivery systems were tested: lentivirus, adenovirus, and rAAV, each encoding GFP under the control of a CMV promoter.

[0209] Each viral delivery system was added to each cell density at an MOI of 3. a. Lentiviral particles were added at MOIs of 10, 50, and 100. b. Adenovirus particles were added at MOIs of 50, 100, and 200. c. Both rAAV serotypes were tested at MOIs of 1,000, 10,000, and 100,000.

[0210] The viral particles were incubated with the cells overnight at 37°C in 5% CO2. The following day, the lentiviral and adenoviral particles were removed and replaced with fresh medium. The rAAV particles remained on the cells throughout the assay. GFP fluorescence and cell confluence were determined using an Incucyte ZOOM™ cell imager (Essen) at 24, 48, and 72 hours post-infection. A contrast-based algorithm was used to determine cell confluence and GFP-expressing cells. For each well, GFP / phase confluence was calculated by dividing the GFP confluence by the phase confluence.

[0211] Lentiviral delivery at an MOI of 100 was the most efficient, with nearly all cells expressing GFP 72 hours after infection (Figures 1 and 2). The delivery efficiency of each batch of MPCs was roughly equivalent. The percentage of cells expressing GFP after adenovirus or rAAV infection was much lower than with lentivirus, with only a handful of cells expressing GFP using these methods (Figures 3-5).

[0212] Example 2 - HSV-P10 Loading of Mesenchymal Stem Cells (MSCs) The oncolytic herpes simplex virus (HSV-P10) expressing PTENα was generated using a modified PTENα gene sequence, whereby the PTENα CUG start codon was mutated to AUG to enhance translation of the full-length N-terminal extension protein, and the internal canonical PTEN AUG start codon was mutated to AUA to abolish 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 locus. Figure 6 shows the constructs engineered within the ICP6 locus in the control (HSVQ) and HSV-P10 viruses used in this study.

[0213] 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 by detecting intracellular GFP over time (Figures 7A and 2E). GFP was monitored over time using a Cytation 5 Cell Imaging Multi-Mode Reader in conjunction with a BioSpa8 automated incubator (Biotek Instruments, Inc.). The number of GFP objects was quantified and graphed as an average of four wells per treatment group ± SEM. The intracellular replication rate correlated with the MOI of HSVQ or HSV-P10 used to infect mesenchymal stem cells.

[0214] To determine the kinetics of HSV-P10 and HSVQ viral replication in mesenchymal stem cells, a comparison of HSV-P10 and HSVQ-loaded mesenchymal stem cells was performed (Figure 7A). 6 Mesenchymal stem cells (MSCs) were plated in 6-well plates and cultured for 24 hours. The plated MSCs 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, followed by further culture for 24 hours. The HSVQ- or HSV-P10-loaded MSCs and conditioned medium were collected and titrated on Vero cells.

[0215] HSV-P10 appeared to have superior viral replication kinetics compared to HSVQ (Figure 7A). However, the viral load of HSV-P10-loaded mesenchymal stem cells was comparable to that of HSVQ-loaded mesenchymal stem cells (Figure 7B). Viral replication of HSV-P10 and HSVQ was observed in loaded mesenchymal stem cells even after five in vitro passages.

[0216] To determine the effect of viral loading on mesenchymal stem cell viability, cytoplasmic activity (Aqua Live / Dead dye) and GFP expression were determined in loaded mesenchymal stem cells, which were assessed by flow cytometry, quantified, and represented as histograms (Figure 8). The data demonstrate that HSV-10 and HSVQ loaded mesenchymal stem cells were viable 24 hours after infection (Figure 8A). Flow cytometry quadrants are shown in Figure 8B.

[0217] Example 3 - 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 HSV-P10-loaded mesenchymal stem cells. Western blot analysis revealed increased AKT in HSVQ-loaded mesenchymal stem cells, whereas HSV-P10-loaded mesenchymal stem cells expressing PTENα had reduced phosphorylated AKT compared to control virus-loaded mesenchymal stem cells (Figure 9A). PTENα was detected in the conditioned medium of HSV-P10-loaded mesenchymal stem cells, suggesting the secretion of PTENα by HSV-P10-loaded mesenchymal stem cells (Figure 9B).

[0218] Example 4 - 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, Boyden chamber assays were performed and migration was performed by monitoring viral GFP over time using a Cytation 5 Cell Imaging Multi-Mode Reader in conjunction with a BioSpa8 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 with HSVQ-loaded mesenchymal stem cells (Figure 10).

[0219] Example 5 - Effect of HSV-P10 loaded mesenchymal stem cells on primary human glioma cells HSVQ and HSV-P10-loaded mesenchymal stem cells were cocultured with RPFs expressing GMB12 primary human glioma cells (Figure 11A). The functionality of PTENα expressed by HSV-P10-loaded mesenchymal stem cells on 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 11B).

[0220] Example 6 - Effect of HSV-P10 loaded mesenchymal stem cells on breast cancer cells Co-culture of HSV-P10-loaded mesenchymal stem cells with DB7 mouse breast cancer cells resulted in the transfer of HSV-P10 to the cancer cells, leading to the induction of cell death in the cancer cells as determined by cytoplasmic activity (Aqua Live / Dead dye) and GFP expression. After co-culture with HSV-Q-loaded mesenchymal stem cells, an increase in the total number of dead DB7 mouse breast cancer cells was observed compared to unloaded mesenchymal stem cells (control) (Figure 12). After co-culture with HSV-P10-loaded mesenchymal stem cells, a further increase in the total number of dead DB7 mouse breast cancer cells was observed compared to unloaded mesenchymal stem cells (control) and HSV-Q-loaded cells (Figure 12).

[0221] Example 7 - Effect of oncolytic HSV on MSCs and MPCs Mesenchymal stem cells (MSCs) and mesenchymal precursor cells (MPCs) were loaded with oncolytic herpes simplex virus (HSV) at increasing multiplicities of infection (MOI) of 0.1 to 5. Infection was determined by detection of fluorescence in cells over time.

[0222] Viral replication was determined by harvesting virus from cells at 24, 48, and 72 hours postinfection and titrating by plaque assay on Vero cells. Surprisingly, increased HSV replication was observed in MPCs compared to MSCs at all time points and at both MOIs tested (Figure 13A, MOI 0.1; Figure 13B, MOI 1).

[0223] HSV cytotoxicity in MSCs and MPCs was determined by MTT assay 72 hours after infection. Again, surprisingly, increased cell viability was observed in MPCs compared to MSCs, especially when the MOI was increased above 0.1 (Figure 14).

[0224] These findings support the general concept of using MPCs as carriers of oncolytic viral payloads and in applications such as cancer therapy.

[0225] Example 8 - Oncolytic viruses in MPCs and MSCs method Several cancer cell lines were infected with respiratory syncytial virus (RSV), including the lung cancer cell lines A549 (passage 15), H1299 (passage 13), H1650 (passage 8), and LLC (passage 12), the sarcoma cell lines U2-OS (passage 9) and SK-ES1 (passage 9), and the breast cancer cell lines MCF-7 (passage 13) and 4T1 (passage 9). The cancer cell lines were plated in 96-well plates and infected with RSV at multiplicities of infection (MOIs) of 1, 5, and 10 using Opti-Mem medium for 90 minutes. After 90 minutes, the medium was replaced with complete medium for each cell line. Cell viability assays were performed at 48 and 72 hours postinfection using the Cell Titer Glo assay.

[0226] Human mesenchymal precursor cells (MPCs) and mesenchymal stem cells (MSCs) were also infected with RSV at MOIs of 1, 5, and 10. Cell viability was also assessed at 48 and 72 hours post-infection.

[0227] At 72 hours post-infection, supernatants from infected MSCs and MPCs were collected from wells at various MOIs (1, 5, and 10) and used to infect cancer cell lines. After overnight infection with the supernatant from each MOI, the infected supernatant was replaced and complete medium was added. Cell viability was measured 72 hours later. The titers of the supernatants from infected MPCs and MSCs were determined via plaque assay using Vero cells. References to an MOI of 0 in the results represent supernatants from mock-infected, i.e., uninfected, control wells.

[0228] result Infection of various cancer cell lines with RSV oncolytic virus induced significant cancer cell death, with higher cell death generally observed after 72 hours post-infection and at higher MOIs.

[0229] Lung cancer cell lines: - A549 cells: At 72 hours, significant cell death was observed at all MOIs. At an RSV MOI of 1, there was approximately 40% cell death. At MOIs of 5 and 10, there was 50% and 60% cell death, respectively (Figure 15). -H1299 cells: At 72 hours, significant cell death was observed at all MOIs of 5 and 10. At an RSV MOI of 10, there was approximately 40% cell death (Figure 16). -H1650 cells: Nearly 40% cell death was observed at an RSV MOI of 5 and 65% cell death at an MOI of 10 at both 48 and 72 hours post-infection (Figure 17). - LLC cells: At 48 hours, 35% cell death was observed at an RSV MOI of 10. At 72 hours, approximately 25% cell death was observed at an MOI of 1, 65% at an MOI of 5, and 75% at an MOI of 10 (Figure 18).

[0230] Sarcoma cell lines: -U2-OS cells: At 48 hours, significant cell death was observed at an MOI of 10. At 72 hours, significant cell death was observed at all MOIs, with nearly 60% cell death observed at an MOI of 10 (Figure 19). -SK-ES1 cells: At 48 hours post-infection, significant cell death was observed with RSV MOIs of 5 and 10. At 72 hours, nearly 90% cell death was observed with MOIs of 5 and 10 (Figure 20).

[0231] Breast cancer cell lines: -4T1 cells: At 72 hours, significant cell death was observed at MOIs of 5 and 10. 25% cell death was observed 72 hours after infection with RSV MOI 10 (Figure 21).

[0232] These data demonstrate that the oncolytic virus RSV is capable of infecting and killing multiple cancer cell lines of diverse lineages.

[0233] Stem cells: - MPC and MSC cells: At 72 hours after RSV infection, 40% cell death was observed at an MOI of 5 and 50% at an MOI of 10 (Figures 22 and 23). Similar results were observed for MSCs at 72 hours (Figures 24 and 25).

[0234] The data show that RSV oncolytic virus infects both MPCs and MSCs, and both MPCs and MSCs are viable at least 72 hours after infection. Consistent with the HSV infection results discussed in Example 7 above, 48 hours after RSV infection, more MPCs were viable than MSCs, especially at MOIs of 5 and 10, suggesting that MPCs are more resistant to viral infection, especially at 48 hours.

[0235] Both RSV-infected MPCs and MSCs produced new RSV present in the supernatant of cultured cells, which could infect cancer cell lines (Figures 26-31). Surprisingly, however, the data showed that MPCs shed more virus into the surrounding environment than MSCs, resulting in greater infection of cancer cells. This finding was particularly striking considering the increased number of cancer cells infected with MPC-derived supernatant compared with MSC-derived supernatant (see, in particular, the results at an MOI of 5 for A549, H1299, and H1650 lung cancer cells, U2-OS sarcoma cells, and 4T1 breast cancer cells shown in Figures 26-28, 30, and 31). Thus, we observed that media (v / v) from MPCs infected with oncolytic viruses resulted in greater infectivity of cancer cells than MSCs infected with oncolytic viruses.

[0236] These results further support the above findings and the general concept of using MPC as a carrier for oncolytic viruses. Thus, the inventors' findings represent an important advance in the art, particularly in view of the potential application of these findings to deliver oncolytic viruses to cancer cells.

[0237] Example 9 - Anticancer therapy The mesenchymal precursor cells contain an oncolytic virus, such as RSV or adenovirus, before being administered to a subject diagnosed with cancer. Approximately 200 million loaded mesenchymal precursor cells are administered to the subject.

[0238] Treated subjects are evaluated for safety and efficacy of the therapy over a period of approximately 2-6 weeks, and if necessary, additional doses of loaded mesenchymal precursor cells are administered.

[0239] Example 10 - Pancreatic Cancer Therapy Before being administered to a subject diagnosed with pancreatic cancer, mesenchymal precursor cells containing a conditionally replicating oncolytic adenovirus (CRAd) are loaded with approximately 10-50 infectious units (iu) per MPC by adding the oncolytic CRAd to the mesenchymal precursor cell culture medium. Approximately 200 million loaded mesenchymal precursor cells are administered to the subject.

[0240] Treated subjects are evaluated for safety and efficacy of the therapy over a period of approximately 2-6 weeks, and if necessary, additional doses of loaded mesenchymal precursor cells are administered.

[0241] It will be understood by those skilled in the art that numerous variations and / or modifications may be made to the present disclosure as illustrated in the specific embodiments without departing from the spirit or scope of the disclosure as broadly described. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.

[0242] All publications discussed above are incorporated herein in their entirety.

[0243] This application claims priority from 63 / 063,657, filed August 10, 2020, the disclosures of which are incorporated herein in their entireties.

[0244] 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 an admission that any or all of such matters formed part of the prior art or were common general knowledge in the art relevant to the present disclosure as they existed prior to the priority date of each claim of this application.

[0245] 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 composition for the treatment of cancer comprising a population of human mesenchymal precursor cells (MPCs) containing an oncolytic herpes simplex virus (HSV).

2. 2. The composition of claim 1, wherein the MPCs express one or more of the markers selected from the group consisting of α1, α2, α3, α4, and α5, αv, β1, and β3.

3. The composition of claim 1 , wherein the oncolytic HSV comprises a capsid protein that binds to a tumor-specific promoter and / or a tumor-specific cell surface molecule.

4. The composition of claim 3, 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.

5. 4. The composition of claim 3, wherein the tumor-specific cell surface molecule is selected from the group consisting of integrin, EGF receptor family member, proteoglycan, disialoganglioside, B7-H3, cancer antigen 125 (CA-125), epithelial cell adhesion molecule (EpCAM), vascular endothelial growth factor receptor 1, vascular endothelial growth factor receptor 2, carcinoembryonic antigen (CEA), tumor-associated glycoprotein, cluster of differentiation 19 (CD19), CD20, CD22, CD30, CD33, CD40, CD44, CD52, CD74, CD152, mucin 1 (MUC1), tumor necrosis factor receptor, insulin-like growth factor receptor, folate receptor a, transmembrane glycoprotein NMB, C-C chemokine receptor, prostate-specific membrane antigen (PSMA), recepteur d'originé Nantais (RON) receptor, and cytotoxic T-lymphocyte antigen 4.

6. 6. The composition of any one of claims 1 to 5, wherein the MPCs are culture-expanded from a population of STRO-1 + MPCs prior to introduction of the oncolytic HSV to generate a population of MPCs containing the oncolytic HSV.

7. The MPCs are substantially STRO-1 ｂｒｉ The composition according to any one of claims 1 to 5, wherein

8. The MPCs have a concentration of at least 0.1 μg / 10 ６ The composition of any one of claims 1 to 5, wherein the composition expresses angiopoietin-1 (Ang1) in an amount of 1 to 5 cells.

9. The MPCs are about 0.05 μg / 10 ６ The composition of any one of claims 1 to 5, wherein the composition expresses vascular endothelial growth factor (VEGF) in an amount of less than one cell.

10. 6. The method or composition of any one of claims 1 to 5, wherein the MPCs express Ang1:VEGF in a ratio of at least about 2:

1.

11. The composition of any one of claims 1 to 5, wherein the MLPSCs are derived from induced pluripotent stem (iPS) cells.

12. 6. The composition of any one of claims 1 to 5, wherein the MPCs form gap junctions with the cancer cells, whereby the oncolytic virus is delivered to the cancer cells by crossing the gap junctions.

13. The composition according to any one of claims 1 to 5, wherein the cancer is lung cancer, pancreatic cancer, colon cancer, liver cancer, cervical cancer, prostate cancer, osteosarcoma, breast cancer, or melanoma.

14. The composition of claim 13 , wherein the cancer is syncytial carcinoma.

15. 6. The composition of any one of claims 1 to 5, wherein the oncolytic HSV is modified to insert a nucleotide sequence that is complementary to an oligonucleotide expressed by the MPCs and not expressed by the cancer cells.

16. The composition of claim 15 , wherein the oligonucleotide is a miRNA.

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