CA3059249A1

CA3059249A1 - Method for treating a side effect of chimeric antigen receptor (car) t cell therapy

Info

Publication number: CA3059249A1
Application number: CA3059249A
Authority: CA
Inventors: Kilian KELLY; Igor Slukvin
Original assignee: Cynata Therapeutics Ltd
Current assignee: Cynata Therapeutics Ltd
Priority date: 2017-04-07
Filing date: 2018-04-09
Publication date: 2018-10-11
Also published as: RU2019133327A; SG11201909201SA; BR112019020848A2; KR20190133254A; AU2018248071A1; WO2018184074A1; MX2019011847A; EP3606540A1; JP2020516605A; CN110678191A

Abstract

The invention relates to a method for treating a side effect of chimeric antigen receptor (CAR) T cell therapy, the method comprising administering a mesenchymal stem cell (MSC) to a subject who has been or is being administered CAR T cell therapy.

Description

METHOD FOR TREATING A SIDE EFFECT OF CHIMERIC ANTIGEN RECEPTOR (CAR) T CELL THERAPY
FIELD
The invention relates to treating a side effect of chimeric antigen receptor (CAR) T cell therapy.
BACKGROUND
Immunotherapy is a biological therapy designed to improve a subject's native immune system to combat disease. Commonly, immunotherapy refers to cancer immunotherapy.
A developing area of cancer immunotherapy is adoptive cell transfer (ACT), in which T cells are isolated, engineered to recognise and attack cancer cells, then expanded and reintroduced to a subject with cancer. This may involve isolating and engineering a subject's own T cells for autologous ACT, although use of donor T
cell for allogeneic (sometimes called homologous) ACT is also being investigated.
For ACT, T cells or NK cells are engineered to express a receptor that recognises an antigen expressed on a cancer cell. The receptor may be a T cell receptor (TCR) or a chimeric antigen receptor (CAR). T cells expressing a CAR are referred to as CAR T
cells.
To date, ACT and CAR T cell therapy has been used primarily for blood cancers and have been restricted to small clinical trials, although there have been very limited trials with solid tumours.
Nevertheless, CAR T cell therapy has demonstrated impressive responses in subjects with advanced cancer.
Cancers that have been subjected to CAR T cell therapy, usually with CARs directed to CD19, a cell surface antigen present on B cells, include acute lymphoblastic leukaemia (ALL), chronic lymphocytic leukaemia (CLL), some types of non-Hodgkin lymphoma (NHL) including diffuse large B cell lymphoma (DLBCL) and follicular lymphoma, and multiple myeloma.
Despite its promise, CAR T cell therapy is not without side effects and significant risk. Observed side effects include cytokine release syndrome (CRS) that is related to macrophage activation

2 syndrome (MAS), on-target, off-cancer effects leading to outcomes similar to graft-versus-host disease (GVHD) and B cell aplasia, tumour lysis syndrome (TLS), neurotoxicity such as cerebral oedema, and anaphylaxis caused by a subject's IgG response to CARs comprising non-human antigens.
CRS has been treated with standard supportive therapies, including steroids. However, steroids may affect CAR T cells' activity or proliferation in the subject. Another therapy for CRS
has been administration of inhibitors of pro-inflammatory cytokines that are elevated in CRS. Tocilizumab, an anti-IL-6 receptor antibody, and etanercept, a TNF inhibitor, have been used to treat CRS.
B cell aplasia, resulting in reduced antibody production, has been treated with intravenous immunoglobulin to prevent infection.
TLS has been managed by standard supportive therapy, including hydration, diuresis, administration of allopurinol and recombinant urate oxidase, and haemodialysis as required.
Although these side effects have been managed with varying levels of success, they have not been entirely successful with adverse events occurring regularly, and even subject deaths occurring in a number of clinical trials.
Clearly an improved prophylactic and/or therapy for side effects of ACT, in particular CAR T cell therapy, is required.
It is to be understood that if any prior art publication is referred to herein, such reference does not constitute an admission that the publication forms a part of the common general knowledge in the art in Australia or any other country.
SUMMARY
A first aspect provides a method for treating a side effect of CAR T cell therapy, the method comprising administering a mesenchymal stem cell (MSC) to a subject who has been or is being administered CAR T cell therapy.
An alternative or additional embodiment of the first aspect provides use of a mesenchymal stem cell (MSC) in the manufacture of a medicament for treating a side effect of CAR T cell therapy in a subject who has been or is being administered CAR T cell therapy.

3 A further alternative or additional embodiment of the first aspect provides a mesenchymal stem cell (MSC) for use in treating a side effect of CAR T cell therapy in a subject who has been or is being administered CAR T cell therapy.
In one embodiment, the MSC has a CD73+CD105+CD9O+CD146+CD44+CD1O+CD31-CD45- phenotype.
In one embodiment, the MSC expresses miR-145-5p, miR-181b-5p, and miR-214-3p, but not miR-127-3p and miR-299-5p.
In one embodiment, treating comprises administering about 1x106 to about 1x107 MSCs to the subject.
In one embodiment, treating comprises administering the MSC(s) before, during or after receipt of the CAR T cell therapy by the subject. In one embodiment, treating comprises administering the MSC(s) after receipt of the CAR T cell therapy by the subject. In one embodiment, treating comprises administering the MSC(s) within 24 hours to 72 hours after receipt of the CAR T cell therapy by the subject.
In one embodiment, the side effect or symptom is: cytokine release syndrome (CRS), optionally release of interleukin-6 (IL-6), interferon-y (IFN-y), tumour necrosis factor (TNF), IL-2, IL-2-receptor a, IL-8, IL-10, or granulocyte macrophage colony-stimulating factor (GMCSF); macrophage activation syndrome (MAS); an on-target, off-cancer effect, optionally B cell aplasia; tumour lysis syndrome (TLS); neurotoxicity, optionally cerebral oedema; or anaphylaxis.
In one embodiment, the CAR T cell therapy is for treating:
diffuse large B cell lymphoma (DLBCL); non-Hodgkin lymphoma (NHL);
primary mediastinal B cell lymphoma (PMBCL); chronic lymphocytic leukaemia (CLL); transformed follicular lymphoma (TFL); multiple myeloma (MM); mantle cell lymphoma (MCL); acute myeloid leukaemia (AML); or acute lymphoblastic leukaemia (ALL).
In one embodiment, the CAR is an anti-CD19 CAR.
In one embodiment, the subject is mammalian, optionally human.
A second aspect provides a therapeutic composition for treating, ameliorating, or reducing a side effect of CAR T cell therapy in a mammalian subject, wherein said therapeutic composition

4 comprises a mesenchymal stem cell (MSC), wherein the MSC is made by a method comprising:
(a) culturing a primitive mesoderm cell in a mesenchymal-colony forming medium (M-CFM) comprising LiC1 and FGF2, but excluding PDGF, under normoxic conditions for sufficient time for a mesenchymal colony to form; and (b) culturing the mesenchymal colony of (a) adherently to produce the MSC, wherein the MSC of (b) expresses miR-145-5p, miR-181b-5p, and miR-214-3p, but not miR-127-3p and miR-299-5p, and/or has phenotype CD73+CD105+CD9O+CD146+CD44+CD1O+CD31-CD45-.
A third aspect provides a container comprising the therapeutic composition of the second aspect.
BRIEF DESCRIPTION OF THE FIGURES
Figure 1 is a nucleic acid sequence identified as SEQ ID
NO: 7 encoding a co-stimulatory signaling element from human CD28 including transmembrane and extracellular portions.
Figure 2 is a nucleic acid sequence identified as SEQ ID
NO: 8 encoding the cytoplasmic domain of human CD3 chain.
Figure 3 is a schematic representation of the experimental design of Example 18.
Figure 4 is a graph showing the rectal temperature of control and test mice of Example 18.
Figure 5 is a graph showing clinical score of control and test mice of Example 18. 0 = Normal activity; 1 = Normal activity, piloerection, tiptoe gait; 2 = Hunched, reduced activity but still mobile; 3 = Hypomotile, but mobile when prompted; 4 = Moribund, euthanized.
Figure 6 is a set of graphs showing percent mouse CD45+
cells, percent human CD45+ cells, CD4+ cells as a percent of human CD45+ cells, and CD8+ cells as a percent of human CD45+ cells in peripheral blood of mice of Example 18.
Figure 7 is a set of graphs showing CD69 expression on human CD4+ T cells in peripheral blood of mice of Example 18.
Figure 8 is a set of graphs showing CD69 expression on human CD8+ T cells in peripheral blood of mice of Example 18.

Figure 9 is a set of graphs showing percent mouse CD45+
cells, percent human CD45+ cells, CD4+ cells as a percent of human CD45+ cells, and CD8+ cells as a percent of human CD45+ cells in spleen of mice of Example 18.

5 Figure 10 is a set of graphs showing CD69 expression on human CD4+ T cells in spleen of mice of Example 18.
Figure 11 is a set of graphs showing CD69 expression on human CD8+ T cells in spleen of mice of Example 18.
DETAILED DESCRIPTION
Unless defined otherwise in this specification, technical and scientific terms used herein have the same meaning as commonly understood by the person skilled in the art to which this invention belongs and by reference to published texts.
It is to be noted that the term "a" or "an" refers to one or more, for example, "a MSC," is understood to represent one or more MSCs. As such, the terms "a" or "an", "one or more," and "at least one" may be used interchangeably herein.
In the claims which follow and in the description of the invention, except where the context requires otherwise due to express language or necessary implication, the word "comprise" or variations such as "comprises" or "comprising" is used in an inclusive sense, i.e. to specify the presence of the stated features, but not to preclude the presence or addition of further features in various embodiments of the invention.
The term "about" as used herein contemplates a range of values for a given number of 25% the magnitude of that number. In other embodiments, the term "about" contemplates a range of values for a given number of 20%, 15%, 10%, or 5% the magnitude of that number. For example, in one embodiment, "about 3 grams" indicates a value of 2.7 grams to 3.3 grams (i.e. 3 grams 10%), and the like.
Similarly, the timing or duration of events may be varied by at least 25%. For example, while a particular event may be disclosed in one embodiment as lasting one day, the event may last for more or less than one day. For example, "one day" may include a period of about 18 hours to about 30 hours. In other embodiments, periods of time may vary by 20%, 15%, 10%, or 5% of that period of time.

6 As used herein, "adoptive cell transfer" or "ACT" refers to a process in which T cells or NK cells are isolated, engineered to recognise a specific antigen, then expanded and reintroduced to a subject. T cells or NK cells used for ACT may be "autologous", derived from the subject to be treated, or "allogeneic" (sometimes called "homologous"), derived from a donor subject with an immunogenic profile similar enough not to be rejected by the subject receiving ACT.
As used herein, cells to be transferred in ACT are "chimeric antigen receptor T cells", "chimeric antigen receptor NK cells", or "CAR T cells". As used herein, "CAR T cells" includes T cells or NK
cells. As used herein, "CAR T cells" includes cells engineered to express a CAR or a T cell receptor (TCR). CAR T cells can be T
helper CD4+ and/or T effector CD8+ cells, optionally in defined proportions. CAR T cells may comprise total CD3+ cells.
Producing CAR-T cells CAR T cells may be produced by using genome-integrating vectors such as viral vectors, including retrovirus, lentivirus or transposon, or non-genome-integrating (episomal) DNA/RNA vectors, such as plasmids or mRNA. Genome-integrating vectors are stable, but may negatively affect endogenous gene expression in the recipient T
or NK cell. Episomal vectors are unlikely to negatively affect endogenous gene expression in the recipient T or NK cell, but are unstable with CAR expression often lost within about 1 week. Another episomal approach relies on a non-integrating lentiviral (NILV) vector comprising a scaffold/matrix attachment region (S/MAR) element. This approach has the benefit of stable maintenance/ long term expression in the CAR T cell, without genome integration.
Production of CARs and CAR T cells is known in the art and is described in: (i) numerous patent publications, including US 7,446,190, US 7,741,465, and US 9,181,527; and (ii) journal publications, including Kalos et al. Sci Transl Med. 2011, 3(95):95ra73, Milone et al. Mol Ther. 2009, 17(8):1453-64, and Maude et al. N Engl J Med. 2014, 371(16):1507-17; and (iii) Examples 1 to 4 of this disclosure. Each of the examples of (i) and (ii) is incorporated in full by this cross-reference.
Without wishing to be bound to theory, CARs may comprise four

7 domains:
(1) single chain antibody variable fragment (scFv) extracellular binding domain that targets specific cell surface antigen(s) (2) hinge domain (3) transmembrane domain (4) cytoplasmic signalling domain that triggers T cell proliferation, survival and cytokine production Again, without wishing to be bound to theory, CARs may fall into one of a number of categories: first generation (earliest), second generation, third generation, or fourth generation (latest).
First generation CARS possess a single signalling domain derived from CD3 zeta chain (CD3). Second generation CARS possess a signalling domain and a co-stimulatory domain, for example derived from CD3 and CD28 or 4-1BB (CD137), respectively. Third generation CARs possess a signalling domain, for example derived from CD3, and multiple co-stimulatory domains, for example derived from and CD28 and 4-1BB (CD137). Fourth generation CARs combine second generation CARs with additional sequences, e.g. nuclear factor of activated T
cell (NFAT), to induce expression of a cassette encoding a cytokine, e.g. IL-12, or co-stimulatory ligand, thereby enhancing cell killing capacity of the CAR T cell.
The relevant antigen to which a CAR is directed may be alpha V beta 6 integrin, CAIX, CD19, CD20, CD22, CD30, CD33, CD138, CD171, CEA, EGFR, ErbB (HER), ErbB2 (HER2), FAP, folate receptor-alpha, GD2, Glypican 3, IL-13, kappa light chain, Lewis Y, mesothelin, MUC16, NKG2D, PSMA, RORI (ROR alpha), or VEGFR, for example.
Indications CAR T cells may be used to treat: diffuse large B cell lymphoma (DLBCL); non-Hodgkin lymphoma (NHL); primary mediastinal B cell lymphoma (PMBCL); chronic lymphocytic leukaemia (CLL);
transformed follicular lymphoma (TFL); multiple myeloma (MM); mantle cell lymphoma (MCL); acute myeloid leukaemia (AML); acute lymphoblastic leukaemia (ALL). This list is not intended to be exhaustive.
Further, CAR T cells may be used to treat: B cell malignancies; paediatric or young adults with B cell malignancies;

8 B cell leukaemia; CD19+ lymphoma; CD19+ B cell lymphoma; CD19 positive malignant B cell derived leukaemia or lymphoma; relapsed or refractory CD19+ lymphoma; refractory/ relapsed B cell hematologic malignancies; recurrent or persistent B cell malignancies after allogeneic stem cell transplantation; chemotherapy resistant or refractory ALL; paediatric or young adult patients with relapsed B
cell ALL; glioblastoma; glioblastoma multiforme; recurrent glioblastoma multiforme; CD7 positive leukaemia or lymphoma; CD30 positive lymphomas; EGFR VIII+ glioblastoma; advanced liver malignancy; hepatocellular carcinoma; neuroblastoma; refractory and/or recurrent neuroblastoma; relapsed or refractory neuroblastoma in children; GD2+ solid tumours; paediatric solid tumours; MUC1 positive relapsed or refractory solid tumour; breast cancer;
advanced sarcoma; carcinoembryonic antigen (CEA) positive cancer;
CD70 expressing cancers; relapsed and refractory aggressive B cell NHL; relapsed/refractory CD30+ Hodgkin lymphoma or CD30+ NHL;
relapsed or refractory CD19+ CLL or small lymphocytic lymphoma (SLL); malignant glioma; relapsed/refractory CD33+ AML;
nasopharyngeal carcinoma; mesothelin positive advanced malignancies;
recurrent or metastatic malignant tumours; non-resectable pancreatic cancer; metastatic pancreatic cancer; advanced pancreatic carcinoma;
advanced ROR1+ malignancies; recurrent or refractory DLBCL; CD19 positive systemic lupus erythematosus (SLE); refractory or metastatic GD2-positive sarcoma; myeloma; myelodysplastic syndrome;
stomach cancer; recurrent or refractory acute non T lymphocyte leukaemia; T cell malignancies expressing CD5 antigen; recurrent or refractory lung squamous cell carcinoma; advanced hepatocellular carcinoma; GPC3-positive advanced hepatocellular carcinoma (HCC);
advanced MG7 positive liver metastases; persistent/recurrent blastic plasmacytoid dendritic cell neoplasm; head and neck cancer; HER2 positive malignancy; chemotherapy refractory human epidermal growth factor receptor-2 (HER-2) positive advanced solid tumours;
chemotherapy resistant or refractory CD20+ lymphoma; (FAP)-positive malignant pleural mesothelioma; chronic myelogenous leukaemia (CML);
chemotherapy refractory EGFR (epidermal growth factor receptor) positive advanced solid tumours; adenocarcinoma; or type 1 diabetes.
In one embodiment, a dose of CAR T cells may be

9 1x105 cells/kg. In another embodiment, a dose of CAR T cells may be 1x101 cells/kg. In other embodiments, a dose of CAR T cells may be 5x105 cells/kg, 1x106 cells/kg, 5x106 cells/kg, 1x107 cells/kg, 5x107 cells/kg, 1x108 cells/kg, 5x108 cells/kg, 1x109 cells/kg, or 5x109 cells/kg.
In one embodiment, a dose of CAR T cells may be 1x105 cells/m2. In another embodiment, a dose of CAR T cells may be 1x101 cells/m2. In other embodiments, a dose of CAR T cells may be 5x105 cells/m2, 1x106 cells/m2, 5x106 cells/m2, 1x107 cells/m2, 5x107 cells/m2, 1x108 cells/m2, 5x108 cells/m2, 1x109 cells/m2, or 5x109 cells/m2.
In one embodiment, a dose of CAR T cells may be 1x105 cells.
In another embodiment, a dose of CAR T cells may be 1x101 cells. In other embodiments, a dose of CAR T cells may be 5x105 cells, 1x106 cells, 5x106 cells, 1x107 cells, 5x107 cells, 1x108 cells, 5x108 cells, 1x109 cells, or 5x109 cells.
CAR T cells may be administered in a single dose, a split dose, or in multiple doses.
The dose of CAR T cells will be related to the target antigen and target cells expressing the target antigen, and therefore may vary by indication to be treated.
The dose of CAR T cells will be determined according to the principles for CAR T cell therapy by administering clinicians.
Side effects and symptoms Cytokine-release syndrome (CRS) is a serious side effect of CAR T cell therapy. CRS is thought to result from proliferating T
cells that release large quantities of cytokines, including interleukin-6 (IL-6), interferon-y (IFN- y), tumour necrosis factor (TNF), IL-2, IL-2-receptor a, IL-8, IL-10, and granulocyte macrophage colony-stimulating factor (GMCSF).
Symptoms of CRS include: high fever, malaise, fatigue, myalgia, nausea, anorexia, tachycardia/ hypotension, capillary leak, cardiac dysfunction, renal impairment, hepatic failure, and disseminated intravascular coagulation.
Thus, subjects with CRS may experience fever, cardiovascular symptoms including tachycardia, hypotension, arrhythmias, decreased cardiac ejection fraction, pulmonary symptoms including oedema, hypoxia, dyspnoea, and pneumonitis, acute renal injury usually caused by reduced renal perfusion, hepatic and gastrointestinal symptoms including elevated serum transaminases and bilirubin, diarrhoea, colitis, nausea, and abdominal pain, hematologic symptoms 5 including cytopenias such as grade 3-4 anaemia, thrombocytopenia, leukopenia, neutropenia, and lymphopenia, derangements of coagulation including prolongation of the prothrombin time and activated partial thromboplastin time (PTT), D-dimer elevation, low fibrinogen, disseminated intravascular coagulation, macrophage

10 activation syndrome (MAS), haemorrhage, B-cell aplasia, and hypogammaglobulinemia, infectious diseases including bacteremia, Salmonella, urinary tract infections, viral infections such as influenza, respiratory syncytial virus, and herpes zoster virus, musculoskeletal symptoms including elevated creatine kinase, myalgias and weakness, neurological symptoms including delirium, confusion, and seizure. Steroids and anti-cytokine therapies have been used to treat CRS, for example etanercept, and anti-TNF
molecule, and tocilizumab, an anti-IL-6 receptor antibody.
MAS overlaps clinically with CRS with subjects potentially experiencing hepatosplenomegaly, lymphadenopathy, pancytopenia, liver dysfunction, disseminated intravascular coagulation, hypofibrinogenemia, hyperferritinemia, and hypertriglyceridemia.
Like CRS, subjects with MAS exhibit elevated levels of cytokines, including IFN-y and GMCSF.
On-target, off-cancer effects may lead to outcomes similar to graft-versus-host disease (GVHD) and B cell aplasia, which is caused when the target cancer antigen is expressed endogenously on other healthy/ normal cells types.
For instance, B cell aplasia occurs because anti-CD19 CARs also target normal B cells that express CD19. The consequence of B
cell aplasia is a reduced capacity to fight infection because of hypoimmunoglobulinemia. Intravenous immunoglobulin replacement therapy is used to prevent infection.
Another side effect of CAR T cell therapy is tumour lysis syndrome (TLS), which occurs when the contents of cells are released as a result of therapy causing cell death, most often with lymphoma and leukaemia. TLS is characterised by blood ion and metabolite

11 imbalance, and symptoms include nausea, vomiting, acute uric acid nephropathy, acute kidney failure, seizures, cardiac arrhythmias, and death.
Neurotoxicity may result from CAR T cell therapy and symptoms may include cerebral oedema, delirium, hallucinations, dysphasia, akinetic mutism, headache, confusion, alterations in wakefulness, ataxia, apraxia, facial nerve palsy, tremor, dysmetria, and seizure.
Anaphylaxis can arise from non-host proteins, such as murine-derived proteins forming part of the CAR.
Management of side-effects In general, side-effects of CAR T cell therapy are managed with standard supportive therapy for any presenting symptoms.
However, given the myriad side effects and symptoms that may occur as a result of CAR T cell therapy, multiple supportive therapies may be required simultaneously.
Mesenchymal stem cells Accordingly, the invention provides an improved therapy for reducing the number, severity and duration of side effects caused by CAR T therapy, by administration of CAR T cells and mesenchymal stem cells (MSCs). MSCs exert their effect through their immunomodulatory properties, so for many side effects and symptoms, MSCs are able to act directly at the immunogenic cause of the side effect or symptom.
MSCs secrete bioactive molecules such as cytokines, chemokines and growth factors and have the ability to modulate the immune system. MSCs have been shown to facilitate regeneration and effects on the immune system without relying upon engraftment. In other words, the MSCs themselves do not necessarily become incorporated into the host - rather, they exert their effects and are then eliminated within a short period of time. However, MSCs may be engrafted.
As used herein, "mesenchymal stem cell" or "MSC" refers to a particular type of stem cell that may be isolated from a wide range of tissues, including bone marrow, adipose tissue (fat), placenta and umbilical cord blood. Alternatively, MSCs may be produced from pluripotent stem cells (PSCs). MSCs are also known as "mesenchymal stromal cells".
For the avoidance of doubt, as used herein a "side effect"

12 includes a "symptom" and both terms refer to an undesired or adverse effect of CAR T cell therapy, determined either qualitatively, i.e. undesired in any form, or quantitatively, undesired above or below a specific threshold. Such a symptom may also be referred to as an "adverse symptom" to distinguish an effect from a necessary or desired effect of CAR T cell therapy. A side effect or symptom of CAR T cell therapy may also be referred to as an "adverse event", an "immune-mediated adverse event", or an "immune-related adverse event".
Production of MSCs from PSCs is described in international patent application no. PCT/AU2017/050228 filed 14 March 2017, which is incorporated in full by this cross-reference, and is described in Examples 5 and 6.
MSCs have been shown to exert immunomodulatory activities against T cells, B cells, dendritic cells, macrophages, and natural killer cells. While not wishing to be bound by theory, the underlying mechanisms may comprise immunomodulatory mediators, for example nitric oxide, indoleamine 2,3, dioxygenase, prostaglandin E2, tumour necrosis factor-inducible gene 6 protein, CCL-2, and programmed death ligand 1. These mediators are expressed at a low level until stimulated, for example by an inflammatory cytokine, such as IFNy, TNFa, and IL-17.
MSCs may be administered systemically or peripherally, for example by routes including intravenous (IV), intra-arterial, intramuscular, intraperitoneal, intracerobrospinal, intracranial, subcutaneous (SC), intra-articular, intrasynovial, intrathecal, intracoronary, transendocardial, surgical implantation, topical and inhalation (e.g. intrapulmonary). MSCs may be administered in combination with a scaffold of biocompatible material.
In one embodiment, MSCs are pre-treated prior to administration. Pre-treatment may be with a growth factor or by gene editing, for example, where a growth factor may prime the MSC and gene editing may confer a new therapeutic property on the MSC.
As used herein, "piuripotent stem cell" or "PSC' refers to a cell that has the ability to reproduce itself indefinitely, and to differentiate into any other cell type. There are two main types of

13 PSC: embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs).
As used herein, "embryonic stem cell" or "ESC" refers to a cell isolated from a five to seven day-old embryo donated with consent by subjects who have completed in vitro fertilisation therapy, and have surplus embryos. The use of ESCs has been hindered to some extent by ethical concerns about the extraction of cells from human embryos.
Suitable human PSCs include H1 and H9 human embryonic stem cells.
As used herein, "induced pluripotent stem cell" or "iPSC"
refers to an ESC-like cell derived from adult cells. iPSCs have very similar characteristics to ESCs, but avoid the ethical concerns associated with ESCs, since iPSCs are not derived from embryos.
Instead, iPSCs are typically derived from fully differentiated adult cells that have been "reprogrammed" back into a pluripotent state.
Suitable human iPSCs include, but are not limited to, iPSC
19-9-7T, MIRJT6i-mND1-4 and MIRJT7i-mND2-0 derived from fibroblasts and iPSC BM119-9 derived from bone marrow mononuclear cells. Other suitable iPSCs may be obtained from Cellular Dynamics International (CDI) of Madison, WI, USA.
In one embodiment, MSCs used according to the invention are formed from primitive mesodermal cells. The primitive mesoderm cells may have mesenchymoangioblast (MCA) potential. The primitive mesoderm cells may have a -1"11ilin-KDR+APLNR-PDGFRalpha' phenotype. In one embodiment, MSCs used according to the invention are formed from gmulin--KDR-APLNR+PDGFRalpha+ primitive mesoderm cells with MCA
potential.
As used herein, "EmrIlin-KDREAPLNR+PDGFRalphaf primitive mesoderm cell with MCA potential" refers to a cell expressing typical primitive streak and lateral plate/ extraembryonic mesoderm genes.
These cells have potential to form MCA and hemangioblast colonies in serum-free medium in response to fibroblast growth factor 2 (FGF2).
When cultured according to example 6, these cells become MSCs.
The term Ewilin denotes lack of expression of CD31, VE-cadherin endothelial markers, CD73 and CD105 mesenchymal/
endothelial markers, and CD43 and CD45 hematopoietic markers.

14 In one embodiment, MSCs used according to the invention exhibit a CD73 CD105 CD90 CD146 CD44 CD10 CD31-CD45- phenotype.
In one embodiment, MSCs used according to the invention express each of the microRNAs miR-145-5p, miR-181b-5p, and miR-214-3p, but not miR-127-3p and miR-299-5p.
MSCs possess "immunomodulatory activities", which may be assessed in vitro as the capacity of a MSC to suppress proliferation of T helper (CD4) lymphocytes. Immunomodulatory activities may be quantified in vitro relative to a reference, for example as determined using an ImmunoPotency Assay.
A suitable ImmunoPotency Assay uses an irradiated test MSC
(e.g. iPSC-MSC produced according to the method disclosed herein) and an irradiated reference sample MSC, which are plated separately at various concentrations with carboxyfluorescein succinimidyl ester-labelled leukocytes purified from healthy donor peripheral blood. T helper (CD4) lymphocytes that represent a subset of the reference sample are stimulated by adding CD3 and CD28 antibodies.
CD4 labelled T cells are enumerated using flow cytometry to assess T
cell proliferation. IC50 values are reported as a function of the reference sample. A higher IC50 value indicates a greater magnitude of suppression of proliferation of T helper (CD4) lymphocytes and thus is indicative of superior T-cell immunomodulatory properties.
MSC samples are irradiated prior to use in this assay to eliminate the confounding factor of their proliferative potential.
It will be appreciated by the person skilled in the art that the exact manner of administering to a subject a therapeutically effective amount of MSCs for treating a side effect or symptom of CAR T cell therapy in a subject will be at the discretion of the medical practitioner. The mode of administration, including dose, combination with other agents, timing and frequency of administration, and the like, may be affected by the subject's condition and history.
The MSC may be administered as a therapeutic composition. As used herein, the term "therapeutic composition" refers to a composition comprising an MSC or population of MSCs as described herein that has been formulated for administration to a subject.

Preferably, the therapeutic composition is sterile. In one embodiment, the therapeutic composition is pyrogen-free.
In one embodiment, the therapeutic composition is provided in a container, preferably a sterile container, preferably a pyrogen-5 .. free container. In one embodiment, the container is a syringe, for example suitable for bolus administration. In another embodiment, the container is an infusion bag suitable for infusion.
The MSC will be formulated, dosed, and administered in a fashion consistent with good medical practice. Factors for 10 consideration in this context include the particular type of disorder being treated and anticipated side effects or symptoms, the particular subject being treated, the clinical condition of the subject, the site of administration, the method of administration, the scheduling of administration, and other factors known to medical

15 practitioners. The therapeutically effective amount of the MSC to be administered will be governed by such considerations.
Doses of MSCs may range from about 103 cells/m2 to about 1010 cells/m2, for example about 106 cells/m2 to about 2x108 cells/m2, or about 103 cells/m2, about 5x103 cells/m2, about 104 cells/m2, about .. 5x104 cells/m2, about 105 cells/m2, about 5x105 cells/m2, about 106 cells/m2, about 5x106 cells/m2, about 105 cells/m2, about 5x105 cells/m2, about 108 cells/m2, about 5x108 cells/m2, about 109 cells/m2, about 5x109 cells/m2, about 1010 cells/m2, or about 5x101 cells/m2.
Doses of MSCs may range from about 103 cells/kg to about 101 cells/kg, for example about 106 cells/kg to about 2x108 cells/kg, or about 103 cells/kg, about 5x103 cells/kg, about 104 cells/kg, about 5x104 cells/kg, about 105 cells/kg, about 5x105 cells/kg, about 106 cells/kg, about 5x106 cells/kg, about .. 105 cells/kg, about 5x107 cells/kg, about 108 cells/kg, about 5x108 cells/kg, about 109 cells/kg, about 5x109 cells/kg, about 1010 cells/kg, or about 5x101 cells/kg.
Doses of MSCs may range from about 103 cells to about 1010 cells, for example about 106 cells to about 2x108 cells, or about 103 cells, about 5x103 cells, about 104 cells, about 5x104 cells, about 105 cells, about 5x105 cells, about 106 cells, about 5x106 cells, about 102 cells, about 5x102 cells, about

16 108 cells, about 5x108 cells, about 109 cells, about 5x109 cells, about 1010 cells, or about 5x101 cells.
The MSCs may be administered in a single dose, a split dose, or in multiple doses.
The MSCs may be administered to a subject by any suitable method including intravenous (IV), intra-arterial, intramuscular, intraperitoneal, intracerobrospinal, intracranial, subcutaneous (SC), intra-articular, intrasynovial, intrathecal, intracoronary, transendocardial, surgical implantation, topical and inhalation (e.g. intrapulmonary) routes. Most preferably, the MSCs are administered IV.
The MSCs may be administered to the subject before, during or after receipt of the CAR T cell therapy by the subject. In one embodiment, MSCs are administered during inflammation. Accordingly, in one embodiment, MSCs are administered after administration of CAR
T cells, optionally after inflammation has commenced and/or pro-inflammatory cytokine release has commenced or increased relative to a control, for example relative to pre-administration of the CAR T
cells. Without wishing to be bound by theory, it is thought that most benefit will be gained by administering the MSCs after administering CAR T cell therapy. This is because CAR T cell therapy is intrinsically an immune/ inflammatory response, whereas MSCs exert immunomodulatory and anti-inflammatory effects. Thus, administering MSCs before, during or too early after administering CAR T cell therapy may dampen the effect of the CAR T cell therapy.
Despite this theory, the invention is not restricted to such.
However, this apparent paradox is understood and accepted by those skilled in the art. For example, a primary treatment for CRS
caused by CAR T cell therapy is administration of steroids, which are profoundly immunosuppressive. Advantages of MSCs, for example, may include local immunomodulation versus systemic immunosuppression by steroids, lack of persistence in the body, providing a further line of defence in subjects who fail to respond to steroids or other immunosuppressive therapies, reduced toxicity and increased .. specificity versus steroids, and self-regulation by MSCs versus steroids. Reduced toxicity, increased specificity, and self-regulation are related, and by self-regulation, it is meant that

17 MSCs are thought to have a capacity to reduce their immunomodulatory activity as the immune response of the side effect or symptom of CAR
T cell therapy dissipates, whereas steroids for example must be withdrawn by the physician, with an ensuing period of half-lives before the steroid concentration drops below the therapeutic concentration.
Accordingly, in one embodiment, the MSCs may be administered to the subject receiving CAR T cell therapy about 1 week after administering the CAR T cells. In another embodiment, the MSCs may be administered to the subject receiving CAR T cell therapy about 5 min after administering the CAR T cells. In another embodiment, the MSCs may be administered to the subject receiving CAR T cell therapy about 6 days, about 5 days, about 4 days, about 72 hours, about 48 hours, about 36 hours, about 24 hours, about 16 hours, about 12 hours, about 8 hours, about 4 hours, about 2 hours, about 60 min, about 45 min, about 30 min, about 15 min, or about 5 min after administering the CAR T cells. In one embodiment, the MSCs may be administered to the subject receiving CAR T cell therapy within about 24 hours to about 72 hours after administering the CAR T
cells.
In one embodiment, the MSCs may be administered to the subject receiving CAR T cell therapy about 1 week before administering the CAR T cells. In another embodiment, the MSCs may be administered to the subject receiving CAR T cell therapy about 5 min before administering the CAR T cells. In another embodiment, the MSCs may be administered to the subject receiving CAR T cell therapy about 6 days, about 5 days, about 4 days, about 72 hours, about 48 hours, about 36 hours, about 24 hours, about 16 hours, about 12 hours, about 8 hours, about 4 hours, about 2 hours, about 60 min, about 45 min, about 30 min, or about 15 min before administering the CAR T cells.
In another embodiment, the MSCs may be administered to the subject receiving CAR T cell therapy at about the same time as or during administering the CAR T cells.
The term "therapeutically effective amount" refers to an amount of MSCs effective to treat a side effect or symptom of CAR T
cell therapy in a subject.

18 The terms "treat", "treating" or "treatment" refer to both therapeutic treatment and prophylactic or preventative measures, wherein the aim is to prevent, reduce, or ameliorate a side effect or symptom of CAR T cell therapy in a subject or slow down (lessen) progression of a side effect or symptom of CAR T cell therapy in a subject. Subjects in need of treatment include those already with the side effect or symptom of CAR T cell therapy as well as those in which the side effect or symptom of CAR T cell therapy is to be prevented or ameliorated.
The terms "preventing", "prevention", "preventative" or "prophylactic" refers to keeping from occurring, or to hinder, defend from, or protect from the occurrence of a side effect or symptom of CAR T cell therapy. A subject in need of prevention may be prone to develop the side effect or symptom of CAR T cell therapy.
The term "ameliorate" or "amelioration" refers to a decrease, reduction or elimination of a side effect or symptom of CAR T cell therapy.
A side effect or symptom of CAR T cell therapy may be quantified as a binary event, i.e. presence or absence, 0 or 1.
Alternatively, a side effect or symptom of CAR T cell therapy may be quantified on a semi-quantitative scale, for example 0 to 5, where 0 represents absence, 1 to 4 represent identifiable increases in severity, and 5 represents maximum severity. Clinical trials often use a 1 to 5 scale where: 1 represents a mild adverse event (side effect); 2 represents a moderate adverse event (side effect);
3 represents a severe adverse event (side effect); 4 represents a life-threatening or disabling adverse event (side effect); and 5 represents death related to adverse event (side effect). Other semi-quantitative scales will be readily apparent to the person skilled in the art. In another embodiment, a side effect or symptom of CAR T
cell therapy may be quantified on a quantitative scale, for instance: mass per volume such as mass of cytokine per volume of tissue fluid; temperature; duration; rate; enzyme activity; oxygen saturation; and so on.
The person skilled in the art will readily understand how to assess and quantify any side effect or symptom of CAR T cell

19 therapy, and be able to do so without difficulty or undue burden.
For example, the person skilled in the art will be able to measure:
a cytokine concentration in plasma or serum; temperature (fever);
heart rate (tachycardia); blood pressure (hypotension); cardiac dysfunction; renal impairment; serum or plasma enzyme concentrations (hepatic function); and so on.
Any quantification of a side effect or symptom of CAR T cell therapy may be compared to a control, for example a healthy control subject receiving neither CAR T cell therapy nor MSCs, an affected control subject treated with CAR T cell therapy, but not treated with MSCs, or a population.
Treating a side effect or symptom of CAR T cell therapy by administering a MSC may be about a 1% decrease, about a 2% decrease, about a 3% decrease, about a 4% decrease, about a 5% decrease, about a 6% decrease, about a 7% decrease, about an 8% decrease, about a 9%
decrease, about a 10% decrease, about a 20% decrease, about a 30%
decrease, about a 40% decrease, about a 50% decrease, about a 60%
decrease, about a 70% decrease, about an 80% decrease, about a 90%
decrease, about a 100%, or greater decrease in the side effect or symptom of CAR T cell therapy. Alternatively, treating a side effect or symptom of CAR T cell therapy may be about a 2-fold, about a 3-fold, about a 4-fold, about a 5-fold, about a 6-fold, about a 7-fold, about an 8-fold, about a 9-fold, about a 10-fold, or more decrease in the side effect or symptom of CAR T cell therapy.
As used herein, the term "subject" may refer to a mammal.
The mammal may be a primate, particularly a human, or may be a domestic, zoo, or companion animal. Although it is particularly contemplated that the method disclosed herein is suitable for medical treatment of humans, it is also applicable to veterinary treatment, including treatment of domestic animals such as horses, cattle and sheep, companion animals such as dogs and cats, or zoo animals such as felids, canids, bovids and ungulates.
The following examples assist in describing the invention, which is not to be limited to such examples.

EXAMPLES
Example 1. CARs and retroviral vectors All CARs in these examples contain a scEv derived from the J591 hybridoma as described (Gong, M. C. et al. Neoplasia 1, 123-127 5 (1999)) specific for prostate specific membrane antigen (PSMA). To facilitate detection of transduced cells, all constructs contained the encephalomyocarditis virus internal ribosome entry site (EMCV-IRES) and the eGFP gene inserted in the SFG vector. In Pzl, the J591 scEv (P) is coupled through human CD8a hinge and transmembrane 10 sequences to the intracellular domain of human TCR (z) (Gong et al.). P28 comprises a fusion of the J591 scFli (P) to human CD28 (28) as described (Krause, A. et al. J. Exp. Med. 188, 619-626 (1998) and Krause, A. et al. Mbl. Ther. 1, S260, 713 (2000)). To construct P28z, nucleotides 336-660 of 0028 were amplified using primers 15 5'-GGCGGCCGCAATTGAAGTTA1GTATC-3' (SEQ ID NO: 1) and 5'-TGCGCTCCTGCTGAACTTCACTCTGGAGCGATAGGCTGCTAAGTCGCG-3 SEQ ID NO: 2).
The intracellular domain of TCR7, was amplified using primers 5'-AGAGTGAAGTTCAGCAGGAGCGCA-3' (SEQ ID NO: 3) and 5'-CTCGAGTGGCTGTTAGCCAGA-3' (SEQ ID NO: 4). The products were fused

20 in a separate PCR reaction driven by primers of SEQ ID NOs: 1 and 4, A-tailed with Taq polymerase, and subcloned as a NotI/XhoI ligament into SFG-Pzl. To generate Pz28, the intracellular domain of CD28 was amplified using 5'-GCACTTCACATGCAGGCTCTGCCACCTCGCAGGAGTAAGAGGAGCAGG
CTCCTGCAC-3' (SEQ ID NO: 5) and 5'-CGCTCGAGTCAGGAGCGATAGGCTGCGAAGTC
GCGT-3' (SEQ ID NO: 6) (two silent mutations introduced to interrupt cytosine repeats are underlined). The resultant PCR product represents a fusion of the distal nine codons of TCR (minus stop codon) to the intracellular domain of CD28 and contains a convenient 5' NspI site. This fragment was subcloned, digested with NspI/XhoI, and ligated into SFG-Pzl. SFG-c-fms encodes the human macrophage colony-stimulating factor receptor. This resulted in a series of receptors that comprise a PSMA-specific scEv fragment coupled to signaling elements derived from TCR and/or CD28. Pzl and P28 are designed to respectively deliver signals in a PSMA-dependent manner.
In P28z, the intracellular portion of TCR has been joined to the C
terminus of P28, while in Pz28, the CD28 signaling domain was added at the C terminus of Pzl. All chimeric complementary DNAs (cDNAs)

21 were cloned in bicistronic onco-retroviral vectors upstream of enhanced green fluorescent protein.
Example 2. Culture and retroviral transduction of primary human T cells Peripheral blood mononuclear cells from healthy donors were established in RPMI+10% (vol/vol) human serum, activated with phytohemagglutinin (2 pg/ml) for two days, and transferred to non-tissue culture plates (FALCON, Becton Dickinson, Franklin Lakes, N.J.) precoated with retronectin (15 pg/ml; Takara Biomedicals, Shiga, Japan). Gibbon ape leukemia virus envelope-pseudotyped retroviral particles comprising the CAR constructs were generated as described(Gallardo, H. F. et al. Blood 90, 952-957 (1997) and Riviere, I. et al. Mol. Biotechnoi. 15, 133-142 (2000)).
Three days after transduction of mitogen-activated PBLs, gene transfer efficiency ranged from 20% to 70%. CD4+ and CD8 T-cells subsets were transduced at similar efficiencies. Expression of chain containing fusion receptors was also analyzed by Western blotting, confirming homodimer formation and little, if any, heterodimerization with endogenous CD8 or CD28. Both P28z and Pz28 receptors, but not P28, mediated specific lysis of fibroblasts expressing human PSMA. When T-cells were stimulated on NIH3T3 cells expressing PSMA, Pzl, P28z, and Pz28 elicited IL-2 secretion in the presence of the PSMA and B7.1. In the absence of the co-stimulatory ligand, IL-2 production was only observed in cultures of P28z-transduced T-cells. When stimulation was provided by NIH3T3 cells expressing PSMA alone, T-cells expressing Pzl underwent limited expansion. Pz28-transduced cells also grew poorly. By contrast, P28z-transduced T-cells consistently proliferated and after 7 days of co-culture onto a PSMA+ fibroblast monolayer, T-cells expressing the P28z retained the ability to specifically lyse PSMA+ targets.
Example 3. CD19-specific scEv To construct a CD19-specific scFv, the heavy (VH) and light (VL) chain variable regions were cloned from hybridoma cell line SJ25C1 using degenerate primers a described (Orlandi et. al. Proc Natl Acad Sci USA 86, 3833-3837 (1989)). These coding regions were fused with a DNA fragment encoding a (Gly3Ser)4 spacer region. A
costimulatory signaling element from human CD28 was ligated,

22 including transmembrane and extracellular portions (SEQ ID NO: 7) to the 3' end of the resulting scFv and the cytoplasmic domain of the human- (SEQ ID NO: 8) to the 3' end of the CD28 portion to form fusion gene 19-28z.
The 19-28z fusion was tested for its ability to reduce tumour growth and enhance survival in mice injected with NALM6 T cells.
NAIM6 cells express CD19, MHC I, and MHC II, but not B7.1 or B7.2.
Most (-80%) untreated SCID-Beige mice develop hind-limb paralysis 4-5 weeks after tumour cell injection, remaining mice develop weight loss and/or other CNS symptoms (i.e. vestibular symptoms). When the 19-28z fusion was present, T cell stimulation was enhanced nearly ten-fold, and survival of some of the mice was greatly extended as compared to mice treated with Pz1 (a PSMA specific construct) or 19z1, a CD19-specific construct lacking the costimulatory signaling element.
Example 4. CD19 specific CAR
CAR comprising a CD19 binding element, 4-1BB (CD137) as the costimulatory region and the intracellular domain of the CD3 chain in that order will be prepared using the methodology of Example 1.
The 4-1BB will be amplified using the following primers GCGGCCGCA-CCATCTCCAGCCGAC SEQ ID NO: 9) and CTTCACTCT-CAGTTCACATCCTTC SEQ ID
NO: 10) to generate a 4-1BB amplicon with CD19 scFv and zeta tails with restriction cleavage sites to facilitate ligation to the CD19 scFv and zeta chain portions. The hyphen in the sequence indicates the transition from the 4-113B sequence to the tail. The same primer can be used for other binding elements such as PSMA which end in the same sequence.
A CAR comprising a CD19 binding element, ICOS as the costimulatory region and the intracellular domain of the CD3 chain in that order will be prepared using the methodology of Example 1.
The ICOS will be amplified using the following primers GCGGCCGCA-CTATCAATTTTTGATCCT SEQ ID NO: 11) and CTTCACTCT-TAGGGTCACATCTGTGAG
SEQ ID NO: 12) to generate a ICOS amplicon with CD19 scFv and zeta tails with restriction cleavage sites to facilitate ligation to the CD19 scFv and zeta chain portions. The hyphen in the sequence indicates the transition from the ICOS sequence to the tail. The

23 same primer can be used for other binding elements such as PSMA
which end in the same sequence.
A CAR comprising a CD19 binding element, DAP-10 as the costimulatory region and the intracellular domain of the CD3 chain in that order will be prepared using the methodology of Example 1.
The DAP-10 is amplified using the following primers GCGGCCGCA-CAGACGACCCCAGGA (SEQ ID NO: 13) and CTTCACTCT-GCCCCTGCCTGGCATG (SEQ
ID NO: 14) to generate a DAP-10 amplicon with CD19 scEv and zeta tails with restriction cleavage sites to facilitate ligation to the CD19 scEv and zeta chain portions. The hyphen in the sequence indicates the transition from the DAP-10 sequence to the tail. The same primer can be used for other binding elements such as PSMA
which end in the same sequence.
Example 5. Reagents for MSC production Table 1. Reagents Description Vendor / Cat # or Ref #
DMEM/F12 Base Medium Invitrogen / A1516901 E8 supplement Invitrogen / A1517101 vitronectin Life Technologies / A14700 collagen IV Sigma / C5533 H-1152 ROCK Inhibitor EMD Millipore / 555550 Y27632 dihydrochloride ROCK
Tocris / 1254 Inhibitor Waisman Biomanufacturing / WC-human endothelial-SFM Life Technologies / 11111-044 stemline II hematopoietic stem cell Sigma / S0192 expansion medium GLUTAMAX Invitrogen / 35050-061 insulin Sigma / 19278 lithium chloride (LiC1) Sigma / L4408 collagen I solution Sigma / C2249 fibronectin Life Technologies / 33016-015 DMEM/F12 Invitrogen / 11330032 recombinant human BMP4 Peprotech / 120-05E1 activin A Peprotech / 120-14E
Iscove's modified Dulbecco's medium Invitrogen / 12200036 (IMDM) Ham's F12 nutrient mix Invitrogen / 21700075 sodium bicarbonate Sigma / S5761 L-ascorbic acid 2-phosphate Mg2 Sigma / A8960

24 Description Vendor / Cat # or Ref #
1-thioglycerol Sigma / M6145 sodium selenite Sigma / S5261 non-essential amino acids HyClone / 5H30853.01 chemically defined lipid Invitrogen / 11905031 concentrate embryo transfer grade water Sigma / W1503 polyvinyl alcohol (PVA) MP Bio / 151-941-83 holo-transferrin Sigma / T0665 ES-CULT M3120 Stem Cell Technologies / 03120 STEMSPAN serum-free expansion Stem Cell Technologies / 09650 medium (SFEM) L-ascorbic acid Sigma / A4544 Platelet-derived growth factor Peprotech / 110-14B
subunit B homodimer (PDGF-BB) The reagents listed in Table 1 are known to the person skilled in the art and have accepted compositions, for example IMDM
and Ham's F12. GLUTAMAX comprises L-alanyl-L-glutamine dipeptide, usually supplied at 200 mM in 0.85% NaCl. GLUTAMAX releases L-glutamine upon cleavage of the dipeptide bond by the cells being cultured. Chemically defined lipid concentrate comprises arachidonic acid 2 mg/L, cholesterol 220 mg/L, DL-alpha-tocopherol acetate 70 mg/L, linoleic acid 10 mg/L, linolenic acid 10 mg/L, myristic acid 10 mg/L, oleic acid 10 mg/L, palmitic acid 10 mg/L, palmitoleic acid 10 mg/L, pluronic F-68 90 g/L, stearic acid 10 mg/L, TWEEN 80 2.2 g/L, and ethyl alcohol. H-1152 and Y27632 are highly potent, cell-permeable, selective ROCK (Rho-associated coiled coil forming protein serine/threonine kinase) inhibitors.
Table 2. IF6S medium (10X concentration) 10X IF6S Quantity Final Conc.
IMDM 1 package, 5X
powder for 1 L
Ham's F12 nutrient mix 1 package, 5X
powder for 1 L
sodium bicarbonate 4.2 g 21 mg/mL
L-ascorbic acid 2-phosphate Mg2 128 mg 640 pg/mL
1-thioglycerol 80 pL 4.6 rnM
sodium selenite (0.7 mg/mL solution) 24 pL 84 ng/mL
GLUTAMAX 20 mL 10X
non-essential amino acids 20 mL 10X
chemically defined lipid concentrate 4 mL 10X
embryo transfer grade water To 200 mL NA

Table 3. IF9S medium (1X concentration; based on IF6S) IF9S Quantity Final Concentration IF6S 5 mL lx polyvinyl alcohol (PVA; 20 mg/mL 25 mL 10 mg/mL
solution) holo-transferrin (10.6 mg/mL 50 pL 10.6 pg/mL
solution) insulin 100 pL 20 pg/mL
embryo transfer grade water To 50 mL NA
Table 4. Differentiation medium (1X concentration; based on 5 IF9S) Differentiation Medium Quantity Final Concentration IF9S 36 mL lx FGF2 1.8 pg 50 ng/mL
LiC1 (2M solution) 36 pL 2mM
BMP4 (100 pg/mL solution) 18 pL 50 ng/mL
Activin A (10 mg/mL solution) 5.4 pL 1.5 ng/mL
Table 5. Mesenchymal colony forming medium (1X concentration) Mesenchymal colony forming medium Quantity Final (M-CFM) Concentration ES-CULT M3120 40 mL 40%
STEMSPAN SFEM 30 mL 30%
human endothelial-SFM 30 mL 30%
GLUTAMAX 1 mL lx L-ascorbic acid (250 mM solution) 100 pL 250 pM
LiC1 (2M solution) 50 pL 1 mM
1-thioglycerol (100 mM solution) 100 pL 100 pM
FGF2 600 ng 20 ng/mL
Table 6. Mesenchymal serum-free expansion medium (1X
10 concentration) Mesenchymal serum-free expansion Quantity Final medium (M-SFEM) Concentration human endothelial-SFM 5 L 50%
STEMLINE II HSFM 5 L 50%
GLUTAMAX 100 mL lx 1-thioglycerol 87 pL 100 pM
FGF2 100 pg 10 ng/mL
Example 6. Protocol for differentiating human PSC into MSC
1. Thawed iPSCs in E8 Complete Medium (DMEM/F12 Base Medium + E8 Supplement) + 1 pM H1152 on Vitronectin coated (0.5 pg/ckg) 15 plastic ware. Incubated plated iPSCs at 37 C, 5% CO2, 20% 02 (normoxic).

2. Expanded iPSCs three passages in E8 Complete Medium (without ROCK inhibitor) on Vitronectin coated (0.5 pg/ckg) plastic ware and incubated at 37 C, 5% CO2, 20% 02 (normoxic) prior to initiating differentiation process.
3. Harvested and seeded iPSCs as single cells/small colonies at 5x103 cells/ckg on Collagen IV coated (0.5 pg/ckg) plastic ware in E8 Complete Medium + 10 pM Y27632 and incubated at 37 C, 5% CO2, 20% 02 (normoxic) for 24 h.
4. Replaced E8 Complete Medium + 10 pM Y27632 with Differentiation Medium and incubated at 37 C, 5% CO2, 5% 02 (hypoxic) for 48 h to produce primitive mesoderm cells.
5. Harvested colony forming primitive mesoderm cells from Differentiation Medium adherent culture as a single cell suspension, transferred to M-CFM suspension culture and incubated at 37 C, 5% CO2, 20% 02 (normoxic) for 12 days, until mesenchymal colonies formed.
6. Harvested and seeded mesenchymal colonies on Fibronectin/Collagen I coated (0.67 pg/ckg Fibronectin, 1.2 pg /ckg Collagen I) plastic ware in M-SFEM and incubated at 37 C, 5% CO2, 20% 02 (normoxic) for 3 days to produce MSCs (Passage 0).
7. Harvested colonies and seeded as single cells (Passage 1) at 1.3x104 cells/ckg on Fibronectin/Collagen 1 coated plastic ware in M-SFEM and incubated at 37 C, 5% CO2, 20% 02 (normoxic) for 3 days.
8. Harvested and seeded as single cells (Passage 2) at 1.3x104 cells/ckg on Fibronectin/Collagen 1 coated plastic ware in M-SFEM and incubated at 37 C, 5% CO2, 20% 02 (normoxic) for 3 days.
9. Harvested and seeded as single cells (Passage 3) at 1.3x104 cells/ckg on Fibronectin/Collagen 1 coated plastic ware in M-SFEM and incubated at 37 C, 5% CO2, 20% 02 (normoxic) for 3 days.
10. Harvested and seeded as single cells (Passage 4) at 1.3x104 cells/ckg on Fibronectin/Collagen 1 coated plastic ware in M-SFEM and incubated at 37 C, 5% CO2, 20% 02 (normoxic) for 3 days.

11. Harvested and seeded as single cells (Passage 5) at 1.3x104 cells/ckg on Fibronectin/Collagen 1 coated plastic ware in M-SFEM and incubated at 37 C, 5% CO2, 20% 02 (normoxic) for 3 days.
12. Harvested as single cells and froze final product.
Two experiments (TC-A-96 and DAD-V-90) were executed to investigate the impact of supplementing M-CFM with PDGF-BB
(10 ng/mL) and/or LiC1 (1 mM) on T cell suppression of iPSC-MSCs. T
cell suppression was evaluated generated using Waisman Biomanufacturing's ImmunoPotency Assay (IPA).
As outlined in Table 7, the following combinations of platelet-derived growth factor (PDGF) and LiC1 were evaluated:
PDGF+/LiC1+, PDGF-/LiC1-, PDGF+/LiC1- and PDGF-/LiC1+. Note that two different Dneg1 seed densities (5x103 cells/ckg and 1x104 cells/ckg) and two different concentrations of activin A (AA) in the Differentiation Medium (1X AA = 15ng/mL and 0.1X AA =
1.5ng/mL) were compared in the TC-A-96 experiment. A single Dneg1 seed density (5x10e3 cells/ckg) and activin A concentration (1.5 ng/mL) were used in the DAD-V-90 experiment. Also note that a single leukopak (LPK7) was used in the first IPA (IPA 1) and two leukopaks (LPK7 and LPK8) were used in the second IPA (IPA 2).
This assay is designed to assess the degree to which each MSC
line can suppress the proliferation of T helper (CD4) lymphocytes.
Cryopreserved MSCs were tested using cryopreserved leukocytes purified from the peripheral blood of healthy individuals (peripheral blood mononucleocyte cells (PBMC) derived from Leucopaks (LPK)). As such, LPK cell population variation is expected from donor to donor. Each MSC test sample was tested against the PMBC
from two different individuals for clinical grade material with the option to limit testing to a single PMBC sample for research grade material. The assay for each MSC test sample was run in conjunction with a reference standard MSC line to ensure assay integrity/
reproducibility and to normalize test samples. The assay is described in Bloom et al. Cytotherapy, 2015, 17(2):140-51.
In brief, test MSCs were exposed to 21 Gy of gamma irradiation. In a 48-well tissue culture plate 4x10e5, 2x10e5, 4x10e4, and 2x10e4 irradiated MSCs were plated into individual wells.

PMBC were separately labelled with carboxyfluorescein succinimidyl ester. Labelled PMBC cells are plated at 4x105 cells per well containing the MSCs above. This results in titrated PBMC:MSC ratios of 1:1, 1:0.5, 1:0.1, and 1:0.05. An additional well was plated with stimulated PBMCs alone, another with MSCs alone, and another 1:0.05 ratio without stimulation, all which serve as controls.
Subsequently, T cell-stimulatory monoclonal antibodies, anti-human CD3-epilson and anti-human CD28 (R&D Systems, Inc., Minneapolis, MN), were added to each well.
On day four of culture, cells were harvested from individual wells. Cells from each well were incubated with allophycocyanin-labelled anti-human CD4. CD4+ cells were then analysed for proliferation via carboxyfluorescein intensity using a flow cytometer. The MSC alone control served to gate out MSCs from co-culture wells. The PBMC alone control served as the positive control for maximum T cell proliferation against which the degree of MSC
mediated suppression is measured. The non-stimulated 1:0.05 ratio well was used to generate a negative control gate against which proliferation was measured.
From test sample ratios a best fit curve was used to generate IC50 values. The IC50 values were normalized to the reference standard (IC50 Ref Std/IC50 Test Sample). This normalized IC50 yields larger values for more potent (more suppressive) samples and smaller values for less potent samples.
Results IC50 data presented in Table 7 show that M-CFM supplemented with LiC1, but excluding PDGF (i.e. PDGF-/LiCl+) was optimal for differentiating iPSCs to produce iPSC-MSCs that are immunomodulatory. Furthermore, a lower concentration of activin A
also improved the immunosuppression of iPSC-MSCs.

Table 7. ImmunoPotency Assay Seed (LPK7) (LPK8) Sample PDGF LiC1 Activin A Density (D2) Not not 0.1X 5x103 TC-A-96-B3 +
Applicable suppressive (1.5ng/mL) cells/cm2 Not 5x103 0.17 TC-A-96-B1 + 1X (15ng/mL) Applicable cells/cm2 Not 0.1X 5x103 0.17 DAD-V-90-4 +
Applicable (1.5ng/mL) cells/cm2 Not 0.1X 1x104 0.19 TC-A-96-D3 +
Applicable (1.5ng/mL) cells/cm2 Not 0.1X 5x103 0.36 DAD-V-90-2 +
Applicable (1.5ng/mL) cells/cm2 Not 0.1X 5x103 0.57 DAD-V-90-1 -Applicable (1.5ng/mL) cells/cm2 0.39 0.54 TC-A-96-B2 - 1X (15ng/mL) 5x10 cells/cm2 0.37 0.58 TC-A-96-D2 - 1X (15ng/mL) lx10 cells/cm2 0.69 0.93 DAD-V-90-3 - 0.1X 5x103 (1.5ng/mL) cells/cm2 MSCs produced according to this example exhibit a CD73+CD105+CD9O+CD146+CD44+CD1O+CD31-CD45- phenotype.
Example 7. MSC microRNA analysis The MSC produced according to Example 6 underwent analysis against a microRNA (miRNA) microarray comprising 1194 miRNAs and a proprietary miRNA panel consisting of miR-127-3p, miR-145-5p, miR-181b-5p, miR-214-3p, miR-299-5p, validated against 71 MSC samples and 94 non-MSC samples.
The MSC produced according to Example 6 expressed each of miR-145-5p, miR-181b-5p, and miR-214-3p, but not miR-127-3p and miR-299-5p.
Example 8. Alternative immunopotency assay 1 Immunopotency of MSCs will be evaluated as follows: human PBMCs from various donors are pooled (to minimise inter-individual variability in immune response) in phosphate-buffered saline and stained with carboxyfluorescein succinimidyl ester (CFSE, 2 TIM) for 15 minutes at 37 C in the dark, at a cell density of 2 x 107 PBMCs/mL. The reaction will be stopped by adding an equal amount of RPMI-1640 medium supplemented with 10 % human blood group AB serum.
3 x 105 CFSE labelled PBMCs resuspended in RPMI-1640 medium supplemented with 10 % pooled human platelet lysate, 2 IU/mL
preservative-free heparin (Biochrom), 2 mM L-glutamine, 10 mM (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid ) (HEPES; Gibco), 100 IU/mL penicillin (Sigma) and 100 pg/mL streptomycin (Sigma) will be then plated per well in triplicate in 96-well flat-bottomed plates (Corning). T-cell proliferation will be determined using a Gallios 5 10-color flow cytometer and the Kaluza G1.0 software (both Coulter).
Viable 7-aminoactinomycin-D-excluding (7-AAD-; BD Pharmingen) CD3-APC+ (eBioscience) T cells will be analysed after 4 to 7 days.
Proliferation kinetics and population distribution will be analysed using Modfit 4.1 software (Verity).
10 Example 9. Alternative immunopotency assay 2 Immunopotency of MSCs will be evaluated as follows: T helper (CD4) lymphocytes will be stained with CellTrace violet (CTV;
Invitrogen) according to the manufacturer's instructions and then stimulated with anti-CD3/CD28-coated beads (Dynabeads, Invitrogen) 15 at a T-cell/bead ratio of 5:1 in 96-well U-bottomed plates.
Responder CD4 T cells will be then incubated with irradiated (at 100 Gy) Karpas 299 cells (K299 cells; Sigma) as a reference standard, or MSCs. The co-cultured cells will be incubated at 37 C in 5% CO2 in RPMI-1640 medium for 72 h. The cells will be then washed with 20 AnnexinV binding buffer (BD Biosciences) and stained with Annexin V-fluorescein isothiocyanate or APC (BD Biosciences) for 15 min in the dark at room temperature. After this incubation, the cells will be stained with propidium iodide (PI) (Molecular Probes) and then data immediately acquired on a LSRII Fortessa (BD Biosciences). Collected

25 data will be analysed with the use of FlowJo software (version 8.8.6; Tree Star). The viability is measured by the population of Annexin V-negative and PI-negative T cells. This proportion of viable cells will be analysed for CTV dim (% proliferation).
Suppression of T-cell proliferation will be calculated by means of 30 the equation: % Suppression = 100 - (a/b * 100), where a is the percentage proliferation in the presence of suppressor cells and b is the percentage proliferation in the absence of suppressor cells.
Example 10. Reduced occurrence of side effects of CAR T cell therapy for ALL
Subjects with refractory ALL will be divided into two groups and each group infused IV with autologous CAR T cells specific for CD19. The CAR T cells will have been transduced with a lentiviral vector encoding the CD19 CAR. Subjects will be infused with doses of 0.76x106 to 20.6x106 CD19 CAR T cells per kilogram body weight.
Patients will be monitored for response, toxicity and side effects, and expansion and persistence of circulating CD19 CAR T cells.
Within 3 hours after infusion with CD19 CAR T cells, one group of subjects will be infused IV with 1x106 to 1x107 MSCs.
Complete remission of ALL is expected in around 90% of subjects. CD19 CAR T cells are expected to proliferate and be detectable in blood, bone marrow, and cerebrospinal fluid of responding subjects. Sustained remission of ALL is expected with an anticipated 6-month event-free survival rate of around 67% and an anticipated overall survival rate of about 80%.
All subjects who are not infused with MSCs are expected to develop CRS. About 25% of subjects in this group are anticipated to develop severe CRS, which will be treated with tocilizumab.
All subjects who are infused with MSCs are expected to exhibit reduced CRS, presenting as reduced severity of symptoms compared with subjects not treated with MSCs. Subjects in this group are also expected to exhibit fewer and less severe side effects other than CRS.
Example 11. Treatment of side effects of CAR T cell therapy for ALL
Subjects with refractory ALL will be divided into two groups and each group infused IV with autologous CAR T cells specific for CD19. The CAR T cells will be transduced with a lentiviral vector encoding the CD19 CAR. Subjects will be infused with doses of 0.76x106 to 20.6x106 CD19 CAR T cells per kilogram body weight.
Patients will be monitored for response, toxicity and side effects, and expansion and persistence of circulating CD19 CAR T cells.
Twenty-four to 72 hours after infusion with CD19 CAR T cells, one group of subjects will be infused IV with 1x106 to 1x107 MSCs.
Complete remission of ALL is expected in around 90% of subjects. CD19 CAR T cells are expected to proliferate and be detectable in blood, bone marrow, and cerebrospinal fluid of responding subjects. Sustained remission of ALL is expected with an anticipated 6-month event-free survival rate of around 67% and an anticipated overall survival rate of about 80%.

All subjects who are not infused with MSCs are expected to develop CRS. About 25%, of subjects in this group are anticipated to develop severe CRS, which will be treated with tocilizumab.
All subjects who are infused with MSCs are expected to exhibit reduced CRS, presenting as reduced severity of symptoms compared with subjects not treated with MSCs. Subjects in this group are also expected to exhibit fewer and less severe side effects other than CRS.
Example 12. Reduced occurrence of side effects of CAR T cell therapy for CLL
Subjects with refractory CLL will be treated according to Example 10. Similar, but not identical, improvements in CLL subjects treated with MSCs over those treated without MSCs are expected compared with improvements in ALL subjects treated with MSCs over those treated without MSCs.
Example 13. Treatment of side effects of CAR T cell therapy for CLL
Subjects with refractory CLL will be treated according to Example 11. Similar, but not identical, improvements in CLL subjects treated with MSCs over those treated without MSCs are expected compared with improvements in ALL subjects treated with MSCs over those treated without MSCs.
Example 14. Reduced occurrence of side effects of CAR T cell therapy for NHL
Subjects with NHL will be treated according to Example 10.
Similar, but not identical, improvements in NHL subjects treated with MSCs over those treated without MSCs are expected compared with improvements in ALL subjects treated with MSCs over those treated without MSCs.
Example 15. Treatment of side effects of CAR T cell therapy for NHL
Subjects with NHL will be treated according to Example 11.
Similar, but not identical, improvements in NHL subjects treated with MSCs over those treated without MSCs are expected compared with improvements in ALL subjects treated with MSCs over those treated without MSCs.

Example 16. Reduced occurrence of side effects of CAR T cell therapy for sarcoma or GD2-positive solid tumour Subjects with recurrent/ refractory GD2-positive sarcoma will be divided into two groups. Following collection of T cells, subjects will receive cyclophosphamide 1800mg/m2/d as a lymphodepleting regimen. The CAR T cells will be transduced with a lentiviral vector encoding the GD2 CAR. Each group of subjects will then be infused IV with 1x105 to 1x107 CAR T cells/kg. Subjects will be monitored for response and side effects, and expansion and persistence of circulating GD2 CAR T cells.
Three days prior to CAR T cell infusion, subjects will receive cyclophosphamide, 1800 mg/m2 per day IV over 2 hours daily x2, and Mesna, 1800 mg/m2 per day by continuous IV infusion daily x2. On the day of CAR T cell infusion, 30-60 minutes prior, subjects will be administered diphenhydramine 1 mg/kg/d (maximum 50 mg) IV or p.o., acetaminophen 15 mg/kg/dose (maximum 650 mg) p.o. GD2-CAR T
cells will be infused over 15-30 minutes.
Within 3 hours after infusion with GD2 CAR T cells, one group of subjects will be infused IV with 1x106 to 1x107 MSCs.
All subjects who are not infused with MSCs are expected to develop at least one symptom of CRS or side effect other than CRS.
All subjects who are infused with MSCs are expected to exhibit reduced severity and/or duration of at least one symptom of CRS or side effect other than CRS.
Example 17. Treatment of side effects of CAR T cell therapy for sarcoma or GD2-positive solid tumour Subjects with recurrent/ refractory GD2-positive sarcoma will be divided into two groups. Following collection of T cells, subjects will receive cyclophosphamide 1800mg/m2/d as a lymphodepleting regimen. The CAR T cells will be transduced with a lentiviral vector encoding the GD2 CAR. Each group of subjects will then be infused IV with 1x105 to 1x107 CAR T cells/kg. Subjects will be monitored for response and side effects, and expansion and persistence of circulating GD2 CAR T cells.
Three days prior to CAR T cell infusion, subjects will receive cyclophosphamide, 1800 mg/m2 per day IV over 2 hours daily x2, and Mesna, 1800 mg/m2 per day by continuous IV infusion daily x2. On the day of CAR T cell infusion, 30-60 minutes prior, subjects will be administered diphenhydramine 1 mg/kg/d (maximum 50 mg) IV or p.o., acetaminophen 15 mg/kg/dose (maximum 650 mg) p.o. GD2-CAR T
cells will be infused over 15-30 minutes.
Twenty-four to 72 hours after infusion with GD2 CAR T cells, one group of subjects will be infused IV with 1x106 to 1x107 MSCs.
All subjects who are not infused with MSCs are expected to develop at least one symptom of CRS or side effect other than CRS.
All subjects who are infused with MSCs are expected to exhibit reduced severity and/or duration of at least one symptom of CRS or side effect other than CRS.
Example 18. Prevention or Treatment of Cytokine Release Syndrome This example uses NOD.Cg-Prkdc scid ISzJ (NSG) mice that are severely immunodeficient, which allows these mice to be humanized by engraftment and differentiation of peripheral blood mononuclear cells (PBMC) resulting in high percentages of human CD4+
and CD8+ T cells in the peripheral blood and the spleens of the mice. The OKT3 antibody binds to the human T cells and causes a strong induction of human cytokines, thereby modelling CRS in humans.
On day zero, 8- to 12-weeks-old female NOD.Cg-Prkdcscic' 112,gtmiwi1 /SzJ (NSG) mice were injected intravenously through the tail vein with 20x106 human PBMC(huPBMC). A schematic representation of the experimental design is provided in Figure 3.
Frozen human PBMC were purchased from StemCell Technologies and NSG mice were purchased from The Jackson Laboratory.
Frozen huPBMC samples were stored and thawed following the manufacturer's instructions. Briefly, the vial of frozen cells was thawed in a 37 C water bath, the outside of the vial was cleaned with 70% ethanol, the cells were transferred to a 15mL conical tube containing 10 mL of RPMI 10% FBS pre-warmed at 37 C, centrifuged at 1 500 rpm for 10 min, washed once with 10 mL of PBS and suspended in 1 mL of PBS for cell count by Trypan Blue dye exclusion. 20x106 huPBMC aliquots in 150 pl of PBS were prepared and kept on ice while preparing the mice for injection.
Mice were placed in a cage and warmed for 2 to 3 minutes with a lamp to induce dilatation of the tail vein. Next, mice were placed in a mouse restrainer, the tails were cleaned with 70% ethanol, and mice were injected through the tail vein with 20x106 huPBMC
administered with a 1 ml syringe, 27G needle. After the injection, 5 light pressure was applied to the site of the injection to prevent bleeding. Mice were monitored daily for signs of disease until the day of euthanasia.
10-12 days after huPBMC engraftment, mice were assigned to either a control (n=2) or test (n=5) cohort. Control cohorts 10 received muromonab-CD3 (OKT3) antibody at a dose of 10 pg, via intraperitoneal injection. OKT3 antibody is an anti-CD3 antibody used as an immunosuppressant agent to treat acute rejection after organ transplant. OKT3 antibody may be purchased from commercial suppliers such as abcam (catalog no. ab86883) or ThermoFisher 15 Scientific (catalog no. 14-0037-82) or other sources such as Walter and Eliza Hall Institute's Antibody Facility. The control and test cohort received OKT3 antibody via intraperitoneal injection 12 hours after huPBMC administration. The test cohort received 2x106 MSCs by tail vein injection 5 hours before OKT3 administration (i.e. 7 hours 20 after huPBMC administration). In other examples, MSCs will be administered at the same time as OKT3 administration or 1 h, 3 h, 5 h or 24 h after OKT3 administration (Figure 3).
Temperatures of mice were acquired 0, 1, 3, 5, and 24 hours after administration of OKT3 antibody. Temperatures were taken using 25 a non-contact, infrared thermometer that has been calibrated against a standard rectal thermometer to adjust for differences between rectal and surface/skin temperatures.
Five hours after administration of OKT3 antibody, peripheral blood samples were obtained via cheek vein puncture using a sterile 30 4 mm Goldenrod Animal Lancet or by withdrawing blood from the tail vein.
Mice were sacrificed 5 or 24 hours after OKT3 administration, depending on clinical score and body temperature. Peripheral blood samples were obtained immediately upon euthanasia via cardiac 35 puncture, then spleens were harvested. Percent human CD45, CD4 and CD8 T cells found in circulation and in spleens was determined by standard flow cytometric staining and analysis. CD69 expression on circulating and splenic CD4 and CD8 T cells was assessed by surface staining and flow cytometric analysis.
Plasma samples collected at 5 and 24 hours after OKT3 administration will be evaluated for expression of IL-113, IL-2, IL-6, IFNy, TNF, IL-10, and optionally IL-4 and IL-5.
In this model of CRS, test mice exhibited a higher rectal temperature compared with control mice (Figure 4). Also, test mice exhibited reduced clinical scores compared with test mice (Figure 5) in this model of CRS.
No difference was observed between control and test mice in the percentage of CD45+ cells in peripheral blood (Figure 6) or spleen (Figure 9).
However, CD69 expression was reduced in test mice compared with control mice in human CD4+ cells in both peripheral blood (Figure 7) and spleen (Figure 10) and in human CD8+ cells in both peripheral blood (Figure 8) and spleen (Figure 11).
In view of reduced CD69 expression in human CD4+ cells and human CD8+ cells of test mice relative to control mice, expression of one or more of IL-113, IL-2, IL-6, IFNy, TNF, IL-10, IL-4 and IL-5 is expected to be reduced in test mice relative to control mice.

Claims

37

1. A method for treating a side effect of CAR T cell therapy, the method comprising administering a mesenchymal stem cell (MSC) to a subject who has been or is being administered CAR T cell therapy.

2. Use of a mesenchymal stem cell (MSC) in the manufacture of a medicament for treating a side effect of CAR T cell therapy in a subject who has been or is being administered CAR T cell therapy.

3. The method of claim 1 or use of claim 2, wherein the MSC has a CD73+CD105+CD9O+CD146+CD44+CD10+CD31-CD45- phenotype.

4. The method or use of any one of claims 1 to 3, wherein the MSC
expresses miR-145-5p, miR-181b-5p, and miR-214-3p, but not miR-127-3p and miR-299-5p.

5. The method or use of any one of claims 1 to 4, wherein treating comprises administering about 1×10 6 to about 1×10 7 MSCs to the subject.

6. The method or use of any one pf claims 1 to 5, wherein treating comprises administering the MSC(s) before, during or after receipt of the CAR T cell therapy by the subject.

7. The method or use of any one of claims 1 to 6, wherein treating comprises administering the MSC(s) after receipt of the CAR
T cell therapy by the subject.

8. The method or use of claim 7, wherein treating comprises administering the MSC(s) within 24 hours to 72 hours after receipt of the CAR T cell therapy by the subject.

9. The method or use of any one of claims 1 to 8, wherein the side effect or symptom is: cytokine release syndrome (CRS), optionally release of interleukin-6 (IL-6), interferon-.gamma. (IFN-.gamma.), tumour necrosis factor (TNF), IL-2, IL-2-receptor .alpha. IL-8, IL-10, or granulocyte macrophage colony-stimulating factor (GMCSF); macrophage activation syndrome (MAS); an on-target, off-cancer effect, optionally B cell aplasia; tumour lysis syndrome (TLS);
neurotoxicity, optionally cerebral oedema; or anaphylaxis.

10. The method or use of any one of claims 1 to 9, wherein the CAR
T cell therapy is for treating: diffuse large B cell lymphoma (DLBCL); non-Hodgkin lymphoma (NHL); primary mediastinal B cell lymphoma (PMBCL); chronic lymphocytic leukaemia (CLL); transformed follicular lymphoma (TFL); multiple myeloma (MM); mantle cell lymphoma (MCL); acute myeloid leukaemia (AML); or acute lymphoblastic leukaemia (ALL).

11. The method or use of any one of claims 1 to 10, wherein the CAR is an anti-CD19 CAR.

12. The method or use of any one of claims 1 to 11, wherein the subject is mammalian, optionally human.
13. A therapeutic composition for treating, ameliorating, or reducing a side effect of CAR T cell therapy in a mammalian subject, wherein said therapeutic composition comprises a mesenchymal stem cell (MSC), wherein the MSC is made by a method comprising:
(a) culturing a primitive mesoderm cell in a mesenchymal-colony forming medium (M-CFM) comprising LiCl and FGF2, but excluding PDGF, under normoxic conditions for sufficient time for a mesenchymal colony to form; and (b) culturing the mesenchymal colony of (a) adherently to produce the MSC, wherein the MSC of (b) expresses miR-145-5p, miR-181b-5p, and miR-214-3p, but not miR-127-3p and miR-299-5p, and/or has phenotype CD73+CD105+CD90+CD146+CD44+CD10+CD31-CD45-.
14. A container comprising the therapeutic composition of

claim 13.