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WO2018184074A1 - Procédé de traitement d'un effet secondaire d'une thérapie cellulaire par des lymphocytes t récepteurs d'antigènes chimériques (car) - Google Patents

Procédé de traitement d'un effet secondaire d'une thérapie cellulaire par des lymphocytes t récepteurs d'antigènes chimériques (car) Download PDF

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Publication number
WO2018184074A1
WO2018184074A1 PCT/AU2018/050321 AU2018050321W WO2018184074A1 WO 2018184074 A1 WO2018184074 A1 WO 2018184074A1 AU 2018050321 W AU2018050321 W AU 2018050321W WO 2018184074 A1 WO2018184074 A1 WO 2018184074A1
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Prior art keywords
cells
car
cell
msc
cell therapy
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PCT/AU2018/050321
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English (en)
Inventor
Kilian KELLY
Igor Slukvin
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Cynata Therapeutics Ltd
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Cynata Therapeutics Ltd
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Priority claimed from AU2017901273A external-priority patent/AU2017901273A0/en
Priority to JP2019554822A priority Critical patent/JP2020516605A/ja
Priority to KR1020197032910A priority patent/KR20190133254A/ko
Priority to RU2019133327A priority patent/RU2019133327A/ru
Priority to MX2019011847A priority patent/MX2019011847A/es
Priority to EP18781756.4A priority patent/EP3606540A1/fr
Application filed by Cynata Therapeutics Ltd filed Critical Cynata Therapeutics Ltd
Priority to BR112019020848-8A priority patent/BR112019020848A2/pt
Priority to CA3059249A priority patent/CA3059249A1/fr
Priority to CN201880031863.1A priority patent/CN110678191A/zh
Priority to SG11201909201S priority patent/SG11201909201SA/en
Priority to AU2018248071A priority patent/AU2018248071A1/en
Publication of WO2018184074A1 publication Critical patent/WO2018184074A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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Definitions

  • the invention relates to treating a side effect of chimeric antigen receptor (CAR) T cell therapy.
  • CAR chimeric antigen receptor
  • 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.
  • ACT adoptive cell transfer
  • 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 .
  • 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.
  • CAR T cell therapy treatments that have been subjected 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.
  • ALL acute lymphoblastic leukaemia
  • CLL chronic lymphocytic leukaemia
  • NHL non-Hodgkin lymphoma
  • DLBCL diffuse large B cell lymphoma
  • follicular lymphoma follicular lymphoma
  • CAR T cell therapy is not without side effects and significant risk.
  • side effects include cytokine release syndrome (CRS) that is related to macrophage activation 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
  • CRS cytokine release syndrome
  • MAS macrophage activation syndrome
  • GVHD graft-versus-host disease
  • TLS tumour lysis syndrome
  • neurotoxicity such as cerebral oedema
  • anaphylaxis caused by a subject's IgG response to CARs
  • CRS has been treated with standard supportive therapies, including steroids.
  • 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.
  • 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.
  • MSC mesenchymal stem cell
  • 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.
  • MSC mesenchymal stem cell
  • 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.
  • the MSC has a
  • the MSC expresses miR-145-5p, miR-181b-5p, and miR-214-3p, but not miR-127-3p and miR-299-5p.
  • treating comprises administering about lxlO 6 to about lxlO 7 MSCs to the subject.
  • 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.
  • the side effect or symptom is: cytokine release syndrome (CRS) , optionally release of interleukin-6 (IL-6) , interferon- ⁇ (IFN- ⁇ ) , 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.
  • CRS cytokine release syndrome
  • IL-6 interleukin-6
  • IFN- ⁇ interferon- ⁇
  • TNF tumour necrosis factor
  • MAS macrophage activation syndrome
  • TLS tumour lysis syndrome
  • neurotoxicity optionally cerebral oedema
  • cerebral oedema optionally cerebral oedema
  • 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) .
  • DLBCL diffuse large B cell lymphoma
  • NHL non-Hodgkin lymphoma
  • PMBCL primary mediastinal B cell lymphoma
  • CLL chronic lymphocytic leukaemia
  • TTL transformed follicular lymphoma
  • MM multiple myeloma
  • MCL mantle cell lymphoma
  • AML acute myeloid leukaemia
  • ALL acute lymph
  • the CAR is an anti-CD19 CAR.
  • 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 comprises a mesenchymal stem cell (MSC) , wherein the MSC is made by a method comprising:
  • M-CFM mesenchymal- colony forming medium
  • 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-.
  • a third aspect provides a container comprising the
  • 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.
  • Figure 10 is a set of graphs showing CD69 expression on human
  • Figure 11 is a set of graphs showing CD69 expression on human CD8+ T cells in spleen of mice of Example 18.
  • a MSC refers to one or more, for example, “a MSC,” is understood to represent one or more MSCs.
  • the terms “a” or “an”, “one or more,” and “at least one” may be used interchangeably herein.
  • 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.
  • 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
  • CAR T cells includes T cells or NK cells.
  • 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.
  • 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.
  • NILV non-integrating lentiviral
  • S/MAR scaffold/matrix attachment region
  • CARs may comprise four domains :
  • 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 0 ⁇ 3 ⁇ , 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.
  • NFAT nuclear factor of activated 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.
  • 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) ;
  • DLBCL diffuse large B cell lymphoma
  • NHL non-Hodgkin lymphoma
  • PMBCL primary mediastinal B cell lymphoma
  • CLL chronic lymphocytic leukaemia
  • TBL transformed follicular lymphoma
  • MM multiple myeloma
  • MCL mantle cell lymphoma
  • AML acute myeloid leukaemia
  • ALL lymphoblastic leukaemia
  • CAR T cells may be used to treat: B cell
  • malignancies paediatric or young adults with B cell malignancies; 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;
  • CD70 relapsed and refractory aggressive B cell NHL; relapsed/refractory CD30+ Hodgkin lymphoma or CD30+ NHL;
  • 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.
  • FAP -positive malignant pleural mesothelioma
  • CML chronic myelogenous leukaemia
  • chemotherapy refractory EGFR epidermatitis
  • adenocarcinoma or type 1 diabetes.
  • a dose of CAR T cells may be lxlO 5 cells/kg. In another embodiment, a dose of CAR T cells may be lxlO 10 cells/kg. In other embodiments, a dose of CAR T cells may be 5xl0 5 cells/kg, lxlO 6 cells/kg, 5xl0 6 cells/kg, lxlO 7 cells/kg, 5xl0 7 cells/kg, lxlO 8 cells/kg, 5xl0 8 cells/kg, lxlO 9 cells/kg, or 5xl0 9 cells/kg.
  • a dose of CAR T cells may be
  • a dose of CAR T cells may be lxlO 10 cells/m 2 .
  • a dose of CAR T cells may be 5xl0 5 cells/m 2 , lxlO 6 cells/m 2 , 5xl0 6 cells/m 2 , lxlO 7 cells/m 2 ,
  • a dose of CAR T cells may be lxlO 5 cells. In another embodiment, a dose of CAR T cells may be lxlO 10 cells. In other embodiments, a dose of CAR T cells may be 5xl0 5 cells, lxlO 6 cells, 5xl0 6 cells, lxlO 7 cells, 5xl0 7 cells, lxlO 8 cells, 5xl0 8 cells, lxlO 9 cells, or 5xl0 9 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.
  • Cytokine-release syndrome (CRS) is a serious side effect of
  • CRS 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- ⁇ (IF - ⁇ ) , tumour necrosis factor (TNF) , IL-2, IL-2-receptor a, IL-8, IL-10, and granulocytes
  • cytokines including interleukin-6 (IL-6) , interferon- ⁇ (IF - ⁇ ) , tumour necrosis factor (TNF) , IL-2, IL-2-receptor a, IL-8, IL-10, and granulocytes.
  • GMCSF macrophage colony-stimulating factor
  • 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.
  • 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 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 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
  • hypertriglyceridemia hypertriglyceridemia
  • subjects with MAS exhibit elevated levels of cytokines, including IF - ⁇ and GMCSF.
  • GVHD graft-versus-host disease
  • B cell aplasia which is caused when the target cancer antigen is expressed endogenously on other healthy/ normal cells types.
  • 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.
  • TLS tumour lysis syndrome
  • 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.
  • 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
  • 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
  • MSCs may be engrafted.
  • MSC meenchymal stem cell
  • MSCs may be produced from pluripotent stem cells (PSCs) .
  • PSCs pluripotent stem cells
  • MSCs are also known as “mesenchymal stromal cells”.
  • a side effect 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.
  • 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” .
  • 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
  • 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 I FNY, TNFct, 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) .
  • IV intravenous
  • SC subcutaneous
  • MSCs may be administered in combination with a scaffold of biocompatible material.
  • MSCs are pre-treated prior to
  • 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.
  • pluripotent stem cell refers to a cell that has the ability to reproduce itself indefinitely, and to differentiate into any other cell type.
  • PSC embryonic stem cells
  • iPSCs induced pluripotent stem cells
  • 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 HI and H9 human embryonic stem cells.
  • iPSC induced pluripotent stem cell
  • 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-mNDl-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.
  • MSCs used according to the invention are formed from primitive mesodermal cells .
  • the primitive mesoderm cells may have mesenchymoangioblast (MCA) potential.
  • MCA mesenchymoangioblast
  • the primitive mesoderm cells may have a EMH lin " KDR + APLNR * PDGFRalpha + phenotype.
  • MSCs used according to the invention are formed from EMH lin " KDR * APLNR + PDGFRalpha + primitive mesoderm cells with MCA potential .
  • ⁇ linKDR + APLNR + PDGFRalpha 1 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.
  • FGF2 fibroblast growth factor 2
  • MSCs used according to the invention exhibit a CD73 + CD105 + CD90 + CD146 + CD44 + CD10 + CD31-CD45- phenotype.
  • 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.
  • administration may be affected by the subject's condition and history.
  • the MSC may be administered as a therapeutic composition.
  • 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.
  • the therapeutic composition is sterile.
  • the therapeutic composition is pyrogen-free.
  • the therapeutic composition is provided in a container, preferably a sterile container, preferably a pyrogen- free container.
  • the container is a syringe, for example suitable for bolus administration.
  • 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.
  • the therapeutically effective amount of the MSC to be administered will be governed by such considerations .
  • Doses of MSCs may range from about 10 3 cells/m 2 to about 10 10 cells/m 2 , for example about 10 6 cells/m 2 to about 2xl0 8 cells/m 2 , or about 10 3 cells/m 2 , about 5xl0 3 cells/m 2 , about 10 4 cells/m 2 , about 5xl0 4 cells/m 2 , about 10 5 cells/m 2 , about 5xl0 5 cells/m 2 , about 10 6 cells/m 2 , about 5xl0 6 cells/m 2 , about 10 7 cells/m 2 , about 5xl0 7 cells/m 2 , about 10 8 cells/m 2 , about 5xl0 8 cells/m 2 , about
  • Doses of MSCs may range from about 10 3 cells/kg to about
  • 10 10 cells/kg for example about 10 6 cells/kg to about 2xl0 8 cells/kg, or about 10 3 cells/kg, about 5xl0 3 cells/kg, about
  • 5xl0 8 cells/kg about 10 9 cells/kg, about 5xl0 9 cells/kg, about 10 10 cells/kg, or about 5xl0 10 cells/kg.
  • Doses of MSCs may range from about 10 3 cells to about
  • 10 10 cells for example about 10 6 cells to about 2xl0 8 cells, or about 10 3 cells, about 5xl0 3 cells, about 10 4 cells, about
  • 5xl0 4 cells about 10 5 cells, about 5xl0 5 cells, about 10 6 cells, about 5xl0 6 cells, about 10 7 cells, about 5xl0 7 cells, about 10 8 cells, about 5xl0 8 cells, about 10 9 cells, about 5xl0 9 cells, about 10 10 cells, or about 5xl0 10 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.
  • IV intravenous
  • intra-arterial intramuscular
  • intraperitoneal intracerobrospinal
  • intracranial subcutaneous
  • SC subcutaneous
  • intra-articular intrasynovial
  • intrathecal intracoronary
  • transendocardial surgical implantation
  • topical and inhalation e.g. intrapulmonary
  • the MSCs may be administered to the subject before, during or after receipt of the CAR T cell therapy by the subject.
  • MSCs are administered during inflammation.
  • MSCs are administered after administration of CAR T cells, optionally after inflammation has commenced and/or proinflammatory cytokine release has commenced or increased relative to a control, for example relative to pre-administration of the CAR T cells .
  • CAR T cell therapy is intrinsically an immune/ inflammatory response, whereas MSCs exert immunomodulatory and anti-inflammatory effects.
  • administering MSCs before, during or too early after administering CAR T cell therapy may dampen the effect of the CAR T cell therapy.
  • the invention is not restricted to such.
  • a primary treatment for CRS caused by CAR T cell therapy is administration of steroids, which are profoundly immunosuppressive.
  • Advantages of MSCs 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.
  • 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 .
  • 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.
  • the MSCs may be administered to the subject receiving CAR T cell therapy about 1 week before
  • the MSCs may be administered to the subject receiving CAR T cell therapy about 5 min before administering the CAR T cells.
  • 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
  • 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 .
  • terapéuticaally effective amount refers to an amount of MSCs effective to treat a side effect or symptom of CAR T cell therapy in a subject.
  • 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.
  • 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.
  • 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.
  • 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) ;
  • 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.
  • CAR T cell therapy any side effect or symptom of CAR T cell therapy, and be able to do so without difficulty or undue burden.
  • 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.
  • 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.
  • 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.
  • a primate particularly a human
  • a domestic, zoo, or companion animal may be a domestic, zoo, or companion animal.
  • 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.
  • All CARs in these examples contain a scFv derived from the J591 hybridoma as described (Gong, M. C. et al. Neoplasia 1, 123-127 (1999)) specific for prostate specific membrane antigen (PSMA) .
  • PSMA prostate specific membrane antigen
  • all constructs contained the encephalomyocarditis virus internal ribosome entry site (EMCV- IRES) and the eGFP gene inserted in the SFG vector.
  • EMCV- IRES encephalomyocarditis virus internal ribosome entry site
  • the J591 scFv (P) is coupled through human CD8a hinge and transmembrane sequences to the intracellular domain of human TCR ⁇ (z) (Gong et al.) .
  • P28 comprises a fusion of the J591 scFv (P) to human CD28 (28) as described (Krause, A. et al. J. Exp. Med. 188, 619-626 (1998) and Krause, A. et al. Mol. Ther. 1, S260, 713 (2000)).
  • nucleotides 336-660 of CD28 were amplified using primers 5 '-GGCGGCCGCAATTGAAGTTATGTATC-3 ' (SEQ ID NO: 1) and
  • 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' Nspl site.
  • SFG-c-fms encodes the human macrophage colony-stimulating factor receptor. This resulted in a series of receptors that comprise a PSMA-specific scFv 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) were cloned in bicistronic onco-retroviral vectors upstream of enhanced green fluorescent protein.
  • Peripheral blood mononuclear cells from healthy donors were established in RPMI+10% (vol/vol) human serum, activated with phytohemagglutinin (2 ⁇ g/ml) for two days, and transferred to non- tissue culture plates (FALCON, Becton Dickinson, Franklin Lakes, N.J.) precoated with retronectin (15 ⁇ g/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. Biotechnol. 15, 133-142 (2000)).
  • VH heavy chain variable region
  • VL light chain variable region
  • a costimulatory signaling element from human CD28 was ligated, 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.
  • NALM6 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).
  • T cell stimulation was enhanced nearly ten-fold, and survival of some of the mice was greatly extended as compared to mice treated with Pzl (a PSMA specific construct) or 19zl, a CD19-specific construct lacking the costimulatory signaling element.
  • CAR comprising a CD19 binding element, 4-1BB (CD137) as the costimulatory region and the intracellular domain of the 0 ⁇ 3 ⁇ 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 4-1BB 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 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 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 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.
  • GLUTAMAX comprises L-alanyl-L-glutamine dipeptide, usually supplied at 200 mM in 0.85% NaCl. GLUTAMAX releases
  • 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.
  • Differentiation Medium adherent culture as a single cell suspension, transferred to M-CFM suspension culture and incubated at 37 e C, 5% CO2, 20% O2 (normoxic) for 12 days, until mesenchymal colonies formed.
  • Fibronectin/Collagen I coated (0.67 ⁇ g/ckg Fibronectin, 1.2 ⁇ /ckg Collagen I) plastic ware in M-SFEM and incubated at 37 e C, 5% C0 2 , 20% 0 2 (normoxic) for 3 days to produce MSCs (Passage 0) .
  • T cell suppression was evaluated generated using Waisman
  • PDGF platelet-derived growth factor
  • 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
  • PBMC peripheral blood mononucleocyte cells
  • LPK Leucopaks
  • test MSCs were exposed to 21 Gy of gamma
  • irradiation In a 48-well tissue culture plate 4xl0e 5 , 2xl0e 5 , 4xl0e 4 , and 2xl0e 4 irradiated MSCs were plated into individual wells. PMBC were separately labelled with carboxyfluorescein succinimidyl ester. Labelled PMBC cells are plated at 4xl0 5 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.
  • T cell-stimulatory monoclonal antibodies anti-human CD3-epilson and anti-human CD28 (R&D Systems, Inc., Minneapolis, MN) , were added to each well.
  • 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.
  • 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.
  • IC50 data presented in Table 7 show that M-CFM supplemented with LiCl, but excluding PDGF (i.e. PDGF-/LiCl+) was optimal for differentiating iPSCs to produce iPSC-MSCs that are
  • 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.
  • miRNA microRNA
  • 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.
  • 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 uM) for 15 minutes at 37 e C in the dark, at a cell density of 2 x 10 7 PBMCs/mL. The reaction will be stopped by adding an equal amount of RPMI-1640 medium supplemented with 10 % human blood group AB serum.
  • CFSE carboxyfluorescein succinimidyl ester
  • T-cell proliferation will be determined using a Gallios 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.
  • T helper (CD4 + ) lymphocytes will be stained with CellTrace violet (CTV;
  • 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 e C in 5% CO 2 in RPMI-1640 medium for 72 h. The cells will be then washed with
  • 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 data will be analysed with the use of FlowJo software (version
  • 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 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.
  • 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 0.76*10 6 to 20.6*10 6 CD19 CAR T cells per kilogram body weight.
  • 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%.
  • 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 0.76*10 6 to 20.6*10 6 CD19 CAR T cells per kilogram body weight.
  • 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.
  • 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 .
  • 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 11 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/m 2 /d as a
  • 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 lxlO 5 to lxlO 7 CAR T cells/kg. Subjects will be monitored for response and side effects, and expansion and persistence of circulating GD2 CAR T cells.
  • CAR T cell infusion Three days prior to CAR T cell infusion, subjects will receive cyclophosphamide, 1800 mg/m 2 per day IV over 2 hours daily x2, and Mesna, 1800 mg/m 2 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.
  • 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/m 2 /d as a
  • 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 lxlO 5 to lxlO 7 CAR T cells/kg. Subjects will be monitored for response and side effects, and expansion and persistence of circulating GD2 CAR T cells.
  • CAR T cell infusion Three days prior to CAR T cell infusion, subjects will receive cyclophosphamide, 1800 mg/m 2 per day IV over 2 hours daily x2, and Mesna, 1800 mg/m 2 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.
  • 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.
  • This example uses NOD. Cg-Prkdc acid I12rg tml ⁇ 1 /SzJ (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.
  • PBMC peripheral blood mononuclear cells
  • the OKT3 antibody binds to the human T cells and causes a strong induction of human cytokines, thereby modelling CRS in humans .
  • mice I12rg tml *i 1 /SzJ (NSG) mice were injected intravenously through the tail vein with 20xl0 6 human PBMC (huPBMC) .
  • huPBMC human PBMC
  • 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 e 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 e 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. 20xl0 6 huPBMC aliquots in 150 ⁇ 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 20xl0 6 huPBMC administered with a 1 ml syringe, 27G needle. After the injection, 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.
  • Control cohorts received muromonab-CD3 (OKT3) antibody at a dose of 10 ⁇ , 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 abeam (catalog no. ab86883) or ThermoFisher 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 2xl0 6 MSCs by tail vein injection 5 hours before OKT3 administration (i.e. 7 hours after huPBMC administration) .
  • 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 a non-contact, infrared thermometer that has been calibrated against a standard rectal thermometer to adjust for differences between rectal and surface/skin temperatures.
  • peripheral blood samples were obtained via cheek vein puncture using a sterile 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 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- ⁇ , IL-2, IL-6, IFNY, TNF, IL-10, and optionally IL-4 and IL-5.
  • 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.
  • 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) .

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Abstract

L'invention concerne un procédé de traitement d'un effet secondaire d'une thérapie cellulaire par des lymphocytes T récepteurs d'antigènes chimériques (CAR), le procédé comprenant l'administration d'une cellule souche mésenchymateuse (CSM) à un sujet qui a été ou étant administré une thérapie cellulaire par des lymphocytes T CAR.
PCT/AU2018/050321 2017-04-07 2018-04-09 Procédé de traitement d'un effet secondaire d'une thérapie cellulaire par des lymphocytes t récepteurs d'antigènes chimériques (car) Ceased WO2018184074A1 (fr)

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AU2018248071A AU2018248071A1 (en) 2017-04-07 2018-04-09 Method for treating a side effect of chimeric antigen receptor (CAR) T cell therapy
CA3059249A CA3059249A1 (fr) 2017-04-07 2018-04-09 Procede de traitement d'un effet secondaire d'une therapie cellulaire par des lymphocytes t recepteurs d'antigenes chimeriques (car)
RU2019133327A RU2019133327A (ru) 2017-04-07 2018-04-09 Способ лечения побочного эффекта терапии Т-клетками с химерными антигенными рецепторами (CAR)
MX2019011847A MX2019011847A (es) 2017-04-07 2018-04-09 Metodo para tratar un efecto secundario de la terapia de celulas t con receptor de antigeno quimerico (car).
EP18781756.4A EP3606540A1 (fr) 2017-04-07 2018-04-09 Procédé de traitement d'un effet secondaire d'une thérapie cellulaire par des lymphocytes t récepteurs d'antigènes chimériques (car)
JP2019554822A JP2020516605A (ja) 2017-04-07 2018-04-09 キメラ抗原受容体（ｃａｒ）ｔ細胞療法の副作用を治療する方法
BR112019020848-8A BR112019020848A2 (pt) 2017-04-07 2018-04-09 Método para tratar um efeito colateral da terapia com célula t receptor de antígeno quimérico (car)
KR1020197032910A KR20190133254A (ko) 2017-04-07 2018-04-09 키메라 항원 수용체 (car) t 세포 요법의 부작용을 치료하는 방법
CN201880031863.1A CN110678191A (zh) 2017-04-07 2018-04-09 治疗嵌合抗原受体(car)t细胞疗法的副作用的方法
SG11201909201S SG11201909201SA (en) 2017-04-07 2018-04-09 Method for treating a side effect of chimeric antigen receptor (car) t cell therapy

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US20230400451A1 (en) * 2022-06-10 2023-12-14 Chun-Hsuan Ho Method for detecting immune efficacy and method of treating a cancer
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