WO2021250420A1

WO2021250420A1 - Culture medium

Info

Publication number: WO2021250420A1
Application number: PCT/GB2021/051453
Authority: WO
Inventors: Ana RUBIO GÓMEZ; Monica GÓMEZ PALOU; Abigail CULSHAW
Original assignee: Autolus Ltd
Current assignee: Autolus Ltd
Priority date: 2020-06-12
Filing date: 2021-06-11
Publication date: 2021-12-16
Anticipated expiration: 2022-12-12
Also published as: EP4165170A1; GB202008957D0

Abstract

The present invention relates to a culture medium suitable for the production of T-cells comprising molecules that are able to uncouple the processes of proliferation and differentiation allowing for effective expansion of cells that maintain a naïve or 'early' memory phenotype.

Description

CULTURE MEDIUM

FIELD OF THE INVENTION

The present invention relates to a culture medium suitable for the production of T-cells comprising molecules that are able to uncouple the processes of proliferation and differentiation allowing for effective expansion of cells that maintain a naive or ‘early’ memory phenotype.

BACKGROUND TO THE INVENTION

Whilst many factors are likely to be involved in determining the effectiveness of CAR T cell therapies, the ability of the transferred cells to engraft and persist appears to play in important role. Cells displaying a naive or early memory phenotype have been demonstrated to persist for longer following transfer and subsequently mediate improved tumour control as compared to cells with an effector phenotype. This however presents a challenge during the manufacturing process as efficient viral transduction and expansion relies on T cell activation and division that will lead to differentiation. Accordingly, changes that can be implemented to the CAR T cell manufacturing method that uncouple the process of expansion from differentiation would be of great benefit.

Targeting the PBK/Akt pathway in order to modulate the memory phenotype has been investigated previously. In vivo studies have demonstrated that T cells generated in the presence of inhibitors of this pathway express higher percentages of naivety markers (CD45RA+CCR7+) and perform better following adoptive transfer. Antigen-specific T cells, including CAR-T cells, expanded in the presence of PBK/Akt inhibitors have been shown to possess enhanced control of tumour growth in various mouse models.

Therefore, there is a need in the art for an improved method of producing CAR T cells.

SUMMARY OF ASPECTS OF THE INVENTION The present inventors have found that class-IIA specific histone deacetylase inhibitors have an effect in maintaining a more undifferentiated phenotype in T-cells compared to T cells that are incubated in standard T cell culture medium. The compound 3-[5-(3-(3- Fluorophenyl)-3-oxopropen- l-yl)-l -methyl- lH-pyrrol-2-yl]-N-hydroxy-2-propenamide (MCI 568) has been found to be particularly effective. In addition, the combination of a class-IIA specific histone deacetylase inhibitor and an AKT inhibitor has been found to result in a synergistic effect. The small molecule AKT inhibitor VIII (l,3-Dihydro-l-(l-((4-(6- phenyl-lH-imidazo[4,5-g]quinoxalin-7-yl)phenyl)methyl)-4-piperidinyl)-2H- benzimidazol-2-one; AKTi VIII) has been found to be particularly effective in combination with MCI 568. Accordingly, these compounds have been found to be useful for generating central memory T cells or less differentiated T-cell progeny.

The Examples provided herein demonstrate that using these supplements in combination has a synergistic effect, enhancing the percentage of T stem cell-like memory cells to a greater extent than using each one alone.

Thus, in a first aspect, the present invention provides a culture medium suitable for culturing T-cells comprising a class-IIA specific histone deacetylase inhibitor. Preferably the class- IIA specific histone deacetylase inhibitor is 3-[5-(3-(3-Fluorophenyl)-3-oxopropen-l-yl)-l- methyl-lH-pyrrol-2-yl]-N-hydroxy-2-propenamide (MCI 568).

The concentration of class-IIA specific histone deacetylase inhibitor may be 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, 1, 1.05, 1.1, 1.2, 1.3, 1.4 or 1.5 mM. Preferably the concentration of the class-IIA specific histone deacetylase inhibitor is 0.75 or 1 pM

The culture medium may additionally comprise one or more of platelet lysate, an inhibitor of the PI3K/AKT/mTOR pathway and IL-21. In some cases, the culture medium does not contain IL-21.

The inhibitor of the PI3K/AKT/mTOR pathway may be an AKT inhibitor.

The AKT inhibitor may be AKT inhibitor VIII. In a preferred embodiment, the culture medium comprises MCI 568 and AKT inhibitor VIII.

The concentration of AKT inhibitor may be 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, or 1 mM. Preferably the concentration of the AKT inhibitor is 1 mM.

The platelet lysate may be a human platelet lysate.

The human platelet lysate may be a pathogen reduced human platelet lysate.

The concentration of platelet lysate may be 3%.

The concentration of IL-21 may be 10 ng/ml.

The culture medium according to the first aspect of the invention may contain no serum.

The culture medium according to the first aspect of the invention may further comprise interleukin 7 (IL-7) and interleukin 15 (IL-15).

The concentration of IL-7 may be 10 ng/ml and the concentration of IL-15 may be 10 ng/ml.

In a second aspect, the present invention provides a method of culturing a T-cell, comprising culturing the T-cell in the presence of a culture medium according to the first aspect of the invention.

In a third aspect, the present invention provides a method of activating a T-cell, comprising activating the T-cell in the presence of a culture medium according to the first aspect of the invention.

The method of activating a T-cell may further comprise a step of stimulating the T-cells with a mitogen.

The mitogen may be provided as a polymeric matrix or a virus-like particle. In a fourth aspect, the present invention provides a method of modifying a T-cell, comprising culturing the T-cell in the presence of a modifying agent and a culture medium according to the first aspect of the invention, wherein the modifying agent is selected from the group consisting of a viral vector, a transposon, a plasmid vector, an RNA, and a genome editing system.

The modifying agent may be a viral vector.

The viral vector may be a lentiviral vector or a retroviral vector.

The modifying agent may be a transposon.

The modifying agent may be a plasmid vector.

The modifying agent may be a genome editing system.

The genome editing system may be CRISPR/Cas9 system.

In a fifth aspect, the present invention provides a method of expanding a T-cell, comprising culturing the T-cell in the presence of a culture medium according to the first aspect of the invention.

In a sixth aspect, the present invention provides a method of producing an engineered T-cell, comprising the steps of:

(i) activating a T-cell according to the method of the third aspect of the invention; and

(ii) modifying the activated T-cell obtained in (i) according to the method of fourth aspect of the invention.

The method of the sixth aspect of the invention may further comprise a step of expanding the T-cell obtained in (ii) according to the fifth aspect of the invention.

The T-cell used in the methods of the second, third, fourth, fifth and sixth aspects of the invention may be an engineered T-cell. The engineered T-cell may be a CAR-T cell.

The T-cell may be obtained from a patient or an allogeneic donor.

The methods of the second, third, fourth, fifth and sixth aspects of the invention may be conducted in a closed culture system.

In a seventh aspect, the present invention provides a T-cell obtained by the methods of the second, third, fourth, fifth and sixth aspects of the invention.

In an eighth aspect, the present invention provides a use of the culture medium according to the first aspect of the invention for producing a population of T-cells.

The population of T-cells may produced according to the method of the second, third, fourth, fifth and sixth aspects of the invention.

The T-cells may be engineered T-cells.

In a ninth aspect, the present invention provides a kit comprising a class-IIA specific histone deacetylase inhibitor and an AKT inhibitor.

The class-IIA specific histone deacetylase inhibitor may be 3-[5-(3-(3-Fluorophenyl)-3- oxopropen-1 -yl)- 1 -methyl- lH-pyrrol-2-yl]-N-hydroxy-2-propenamide (MC 1568).

The AKT inhibitor may be AKT inhibitor VIII

The kit may further comprise IL-7 and IL-15.

DESCRIPTION OF THE FIGURES

Figure 1. T cell differentiation process and cell surface marker expression. Figure 2. Overview of chimeric antigen receptors and their endodomains. (a) Basic schema of a chimeric antigen receptor; (b) First generation receptors; (c) Second generation receptors; (d) Third generation receptors.

Figure 3. Flow diagram showing the manufacturing process of CAR-T cells drug product.

Figure 4. Memory phenotype results obtained from flow cytometry analysis. (A) Percent cell expression of CCR7 and CD45RA markers in which naive=CCR7+/CD45RA+, Tcm=CCR7+CD45RA-, Tem=CCR7-/CD45RA- and Teff=CCR7-/CD45RA+. (B) Percent cell expression of CD27 and CD62L markers. (C) Percent cell expression of TSCM markers (CCR7+, CD45RA+, CD27+. CD62L+, CD95+ and CD45RO+). Measurement of cell surface markers involved in memory phenotype were obtained from the viable CD3+CAR+ population of cells.

Figure 5. Memory phenotype results obtained from flow cytometry analysis. (A) Percent cell expression of CCR7 and CD45RA markers in which naive=CCR7+/CD45RA+, Tcm=CCR7+CD45RA-, Tem=CCR7-/CD45RA- and Teff=CCR7-/CD45RA+. (B) Percent cell expression of CD27 and CD62L markers. (C) Percent cell expression of TSCM markers (CCR7+, CD45RA+, CD27+. CD62L+, CD95+ and CD45RO+). Measurement of cell surface markers involved in memory phenotype were obtained from the viable CD3+CAR+ population of cells.

Figure 6. Memory phenotype results obtained from flow cytometry analysis of cells transduced with lentiviral vector. (A) Percent cell expression of CCR7 and CD45RA markers in which naiVe=CCR7+/CD45RA+, Tcm=CCR7+CD45RA-, Tem=CCR7-/CD45RA- and Teff=CCR7-/CD45RA+. (B) Percent cell expression of CD27 and CD62L markers. (C) Percent cell expression of TSCM markers (CCR7+, CD45RA+, CD27+. CD62L+, CD95+ and CD45RO+). Measurement of cell surface markers involved in memory phenotype were obtained from the viable CD3+CAR+ population of cells. DETAILED DESCRIPTION OF THE INVENTION

1. Culture medium

In a first aspect, the present invention provides a culture medium suitable for culturing T- cells, hereinafter “the culture medium of the invention” comprising a class-IIA specific histone deacetylase inhibitor.

The term “culture medium” or “cell culture medium” or “medium”, as used herein, refers to any medium capable of supporting the in vitro proliferation of mammalian cells, and T-cells in particular. Typically, this will comprise a buffering system to keep an isotonic solution at approximately pH 7.4 containing any combination of amino acids, such as glutamine, proteins, trace elements, vitamins, inorganic salts, and energy sources, such as glucose. Non limiting examples of media for culturing T cells include RPMI 1640 supplemented with FBS in research laboratories, whereas for the biomanufacturing of T cells for adoptive cell therapy, complete formulations such as X-VIVO 15 (Lonza, Inc), TexMACS (Miltenyi Biotec) and CTS OpTimizer (Thermofisher, Inc) supplemented with human serum are more common.

Additional supplements for successful cultivation can vary depending on the type of cell or subset of cell required. For T cells, interleukin-2 (IL-2) is a potent cytokine which modulates proliferation and differentiation into effector and memory T cells. Culture conditions may be further refined to polarise T cells to a specific phenotype during expansion. For example, IL-4, IL-7 and IL-15 have been reported to be essential for induction, survival or turnover of memory T cells, respectively.

The term “culture” or “culturing”, as used herein, refers to the process of growing cells in vitro or ex vivo. Culturing may refer to the ex vivo expansion of T cells.

T cells may be T cells or T lymphocytes which are a type of lymphocyte that play a central role in cell-mediated immunity. They can be distinguished from other lymphocytes, such as B cells and natural killer cells (NK cells), by the presence of a T-cell receptor (TCR) on the cell surface. There are various types of T cell, as summarised below. Helper T helper cells (TH cells) assist other white blood cells in immunologic processes, including maturation of B cells into plasma cells and memory B cells, and activation of cytotoxic T cells and macrophages. TH cells express CD4 on their surface. TH cells become activated when they are presented with peptide antigens by MHC class II molecules on the surface of antigen presenting cells (APCs). These cells can differentiate into one of several subtypes, including TH1, TH2, TH3, TH17, Th9, or TFH, which secrete different cytokines to facilitate different types of immune responses.

Cytolytic T cells (TC cells, or CTLs) destroy virally infected cells and tumour cells, and are also implicated in transplant rejection. CTLs express the CD8 at their surface. These cells recognize their targets by binding to antigen associated with MHC class I, which is present on the surface of all nucleated cells. Through IL-10, adenosine and other molecules secreted by regulatory T cells, the CD8+ cells can be inactivated to an anergic state, which prevent autoimmune diseases such as experimental autoimmune encephalomyelitis.

Memory T cells are a subset of antigen-specific T cells that persist long-term after an infection has resolved. They quickly expand to large numbers of effector T cells upon re exposure to their cognate antigen, thus providing the immune system with “memory” against past infections. Memory T cells comprise three subtypes: central memory T cells (TCM cells) and two types of effector memory T cells (TEM cells and TEMRA cells). Memory cells may be either CD4+ or CD8+. Memory T cells typically express the cell surface protein CD45RO.

Regulatory T cells (Treg cells), formerly known as suppressor T cells, are crucial for the maintenance of immunological tolerance. Their major role is to shut down T cell-mediated immunity toward the end of an immune reaction and to suppress auto-reactive T cells that escaped the process of negative selection in the thymus.

Two major classes of CD4+ Treg cells have been described: naturally occurring Treg cells and adaptive Treg cells. Naturally occurring Treg cells (also known as CD4+CD25+FoxP3+ Treg cells) arise in the thymus and have been linked to interactions between developing T cells with both myeloid (CD1 lc+) and plasmacytoid (CD123+) dendritic cells that have been activated with TSLP. Naturally occurring Treg cells can be distinguished from other T cells by the presence of an intracellular molecule called FoxP3. Mutations of the FOXP3 gene can prevent regulatory T cell development, causing the fatal autoimmune disease IPEX.

Adaptive Treg cells (also known as Trl cells or Th3 cells) may originate during a normal immune response.

The culture medium of the present invention a class-IIA specific histone deacetylase inhibitor. Preferably the class-IIA specific histone deacetylase inhibitor is 3-[5-(3-(3- Fluorophenyl)-3-oxopropen- l-yl)-l -methyl- lH-pyrrol-2-yl]-N-hydroxy-2-propenamide (MCI 568)..

In another embodiment, the culture medium of the invention additionally comprises an AKT inhibitor. Preferably the AKT inhibitor is AKT inhibitor VIII (l,3-Dihydro-l-(l-((4-(6- phenyl-lH-imidazo[4,5-g]quinoxalin-7-yl)phenyl)methyl)-4-piperidinyl)-2H- benzimidazol-2-one).

The term “culture medium supplement” or “medium supplement” “culture medium additive” or “medium additive”, as used herein, refers to additional compounds that are supplemented to a culture medium with the purpose of improving certain of the medium properties or to adapt the medium for the culture of a given cell type.

1.1. Inhibitors of histone deacetylase

Histone deacetylases (HDACs) are a class of enzymes that remove acetyl groups from an e- N-acetyl lysine amino acid on a protein. They typically act on histones, but they are also known to act on other proteins.

HDACs, are classified in four classes depending on sequence homology to the yeast original enzymes and domain organization. Class I includes HDACl, 2, 3, and 8. Class IIA includes HDAC4, 5, 7, and 9. Class IIB includes HDAC6 and HDAC10. Class III includes sirtuins in mammals. Class IV includes HDAC11.

Recent studies have suggested epigenetic modifications as new a therapeutic option to maintain naivety in CAR-T cells. Bae et al. used ACY241, a class IIB histone deacetylase (HDAC) inhibitor, to increase the percentage of TCM cells and enhance its anti-tumour activities against multiple myeloma and solid tumours (Bae et al. Leukemia 32, 1932-1947 (2018)).

An inhibitor may inhibit all HDACs (a broad spectrum or pan-inhibitor) or a class of HDACs (a selective or specific inhibitor).

The term “inhibitor of class IIA histone deacetylase” or “antagonist of class IIA histone deacetylase” or “class IIA specific histone deacetylase inhibitor”, as used herein, refers to an agent, such as a molecule or drug, inhibits, decreases, or reduces one or more activities of a Class IIA histone deacetylase.

In some cases the inhibitor may inhibit a single HDAC. Accordingly, an inhibitor of class IIA histone deacetylase may inhibit all class IIA histone deacetylases, or it may inhibit a single class IIA histone deacetylase.

In some cases, the inhibitor of class IIA histone deacetylase may have activity against other histone deacetylases. A selective inhibitor of class IIA histone deacetylase can be understood to refer to an agent that exhibits an IC50 concentration with respect to a class IIA histone deacetylase that is at least 10-fold, at least 20-fold, at least 30-fold, at least 50-fold, at least 100-fold, at least 1000-fold, or more, lower than the inhibitor’s IC50 with respect to other histone deacetylases.

Inhibition may also be irreversible or reversible. The class IIA HD AC inhibitor may inhibit class IIA HD AC with an IC50 (concentration that inhibits 50% of the activity) of about 200 nM or less, preferably about 100 nm or less, even more preferably about 60 nM or less, about 25 nM, about 10 nM, about 5 nM, about 1 nM.

The “half maximal inhibitory concentration” or “IC50”, as used herein, is a measure of the potency of a substance in inhibiting a specific biological or biochemical function. IC50 is a quantitative measure that indicates how much of a particular inhibitory substance (e.g. drug) is needed to inhibit, in vitro , a given biological process or biological component by 50%. The biological component could be an enzyme, cell, cell receptor or microorganism. IC50 values are typically expressed as molar concentration.

The IC50 of a drug can be determined by constructing a dose-response curve and examining the effect of different concentrations of the drug on inhibiting the biological process or biological component, such as HDAC. IC50 values can be calculated for a given antagonist by determining the concentration needed to inhibit half of the maximum biological response of the agonist. IC50 values can be used to compare the potency of two antagonists.

IC50 values are very dependent on conditions under which they are measured. In general, the higher the concentration of inhibitor, the more agonist activity will be lowered. IC50 value increases as agonist concentration increases. Furthermore, depending on the type of inhibition other factors may influence IC50 value; for ATP dependent enzymes IC50 value has an interdependency with concentration of ATP, especially so if inhibition is all of it competitive.

Non-limiting examples of class IIA HDAC inhibitors that can be used in the context of the present invention include, without limitation, the class IIA HDAC inhibitors shown in Table 1

Table 1. List of class IIA HD AC inhibitors.

In a preferred embodiment, the class IIA HD AC inhibitor is MCI 568.

The concentration of the class IIA HD AC inhibitor in the culture medium may be between 1 nM and 500 mM, or between 10 nM and 100 pM, or between 100 nM and 50 pM, or between 500 nM and 10 pM, or between 750 nM and 5 pM, or about 1 pM. In an embodiment, the concentration of the class IIA HD AC inhibitor is 1 pM.

1.2. Inhibitor of the PI3K/AKT/mTOR pathway

The term “inhibitor of the PI3K/AKT/mTOR pathway” or “antagonist of the PBK/AKT/mTOR pathway”, as used herein, refers to an agent, such as a molecule or drug, inhibits, decreases, or reduces one or more activities of a molecule in a PBK/AKT/mTOR pathway including, without limitation, a PI3K, an AKT or an mTOR (or mTORCl, mTORC2 complex). The PI3K-Akt Pathway is an intracellular signal transduction pathway which serves as a conduit to integrate growth factor signalling with cellular proliferation, differentiation, metabolism, and survival. This is mediated through serine and/or threonine phosphorylation of a range of downstream substrates. Key proteins involved are phosphatidylinositol 3-kinase (PI3K) and AKT/Protein Kinase B. A schematic representation of the PI3K/AKT/mTOR pathway is shown in Figure 1.

The inhibitors may target one or more activities in the pathway or a single activity.

The inhibitor may be a dual molecule inhibitor.

The inhibitor may inhibit a class of molecules have the same or substantially similar activities (a pan-inhibitor) or may specifically inhibit a molecule’s activity (a selective or specific inhibitor). Inhibition may also be irreversible or reversible.

A. AKT inhibitor The term “AKT inhibitor” or “AKTi”, as used herein, refers to a nucleic acid, peptide, compound, or small organic molecule that inhibits at least one activity of an AKT protein. The term “serine/threonine kinase AKT” or “AKT”, as used herein, refers to a serine/threonine-specific protein kinase with three isoforms (AKTI, AKT2 and AKT3) and which is also known as protein kinase B (PKB). AKT plays a critical role in regulating diverse cellular functions (Fig. 1) including cell size/growth, proliferation, survival, glucose metabolism, genome stability, transcription and protein synthesis, and neovascularization.

The term "AKT inhibitor", as used herein, refers to a nucleic acid, peptide, compound, or small organic molecule that inhibits at least one activity of AKT. AKT inhibitors can be grouped into several classes, including lipid-based inhibitors (e.g., inhibitors that target the pleckstrin homology domain of AKT which prevents AKT from localising to plasma membranes), ATP-competitive inhibitors, and allosteric inhibitors. The AKT inhibitor may act by binding to the AKT catalytic site. The AKT inhibitor may act by inhibiting phosphorylation of downstream AKT targets such as mTOR. The AKT inhibitor may be inhibited by inhibiting the input signals to activate AKT by inhibiting, for example, DNA- PK activation of AKT, PDK-1 activation of AKT, and/or mTORC2 activation of AKT.

AKT inhibitors may target all three AKT isoforms, AKTI, AKT2, AKT3 or may be isoform selective and target only one or two of the AKT isoforms. In an embodiment, the AKT inhibitor may be selective for AKTI and AKT2. In another embodiment, the AKT inhibitor may be selective for AKTI.

The AKT inhibitor may target AKT as well as additional proteins in the PI3K-AKT-mTOR pathway. An AKT inhibitor that only targets AKT can be referred to as a selective AKT inhibitor. A selective AKT inhibitor can be understood to refer to an agent that exhibits an IC50 concentration with respect to AKT that is at least 10-fold, at least 20-fold, at least 30- fold, at least 50-fold, at least 100-fold, at least 1000-fold, or more, lower than the inhibitor’s IC50 with respect to other proteins in the pathway.

The AKT inhibitor may inhibit AKT with an IC50 (concentration that inhibits 50% of the activity) of about 200 nM or less, preferably about 100 nm or less, even more preferably about 60 nM or less, about 25 nM, about 10 nM, about 5 nM, about 1 nM. The “half maximal inhibitory concentration” or “IC50”, as used herein, is a measure of the potency of a substance in inhibiting a specific biological or biochemical function. IC50 is a quantitative measure that indicates how much of a particular inhibitory substance (e.g. drug) is needed to inhibit, in vitro , a given biological process or biological component by 50%. The biological component could be an enzyme, cell, cell receptor or microorganism. IC50 values are typically expressed as molar concentration.

The IC50 of a drug can be determined by constructing a dose-response curve and examining the effect of different concentrations of the drug on inhibiting the biological process or biological component, such as AKT. IC50 values can be calculated for a given antagonist by determining the concentration needed to inhibit half of the maximum biological response of the agonist. IC50 values can be used to compare the potency of two antagonists.

Non-limiting examples of AKT inhibitors that can be used in the context of the present invention include, without limitation, the AKT inhibitors shown in Table 2.

Table 2. List of AKT inhibitors.

Additional examples that may be used in the present invention include AKT inhibitor VIII, AKT inhibitor IV, AKT inhibitor III (SH-6), Palomid 529, AKT inhibitor X (10-DEBC hydrochloride), Fisetin, AKT inhibitor XI (FPA 124), AKT inhibitor II (SH-5), Tetrahydro Curcumin, Miltefosine, perifosine (KRX-0401), CH5132799, Akt Inhibitor IX (API-59CJ- OMe), BML-257, API-1, AKT inhibitor XII, AKT inhibitor XIII, VQD-002, XL418, CCT128930, PX316, and Akt 1/2 inhibitor (l,3-Dihydro-l-(l-((4-(6-phenyl-lH-imidazo[4,5- g]quinoxalin-7-yl)phenyl)methyl) -4-piperidinyl)-2H-benzimidazol-2-one trifluoroacetate salt hydrate).

In a preferred embodiment, the AKT inhibitor is AKT inhibitor VIII (AKTiVIII).

The concentration of the AKT inhibitor in the culture medium may be between 1 nM and 500 mM, or between 10 nM and 100 pM, or between 100 nM and 50 pM, or between 500 nM and 10 pM, or between 750 nM and 5 pM, or about 1 pM. In an embodiment, the concentration of the AKT inhibitor is 1 pM. B. PI3K inhibitor

The term “PI3K inhibitor” or “PI3Ki”, as used herein, refers to an agent, such as a nucleic acid, peptide, compound, or small organic molecule, that binds to and inhibits at least one activity of PI3K. The PI3K proteins can be divided into three classes, class 1 PI3Ks, class 2 PI3Ks, and class 3 PI3Ks. Class 1 PI3Ks exist as heterodimers consisting of one of four pi 10 catalytic subunits (pi 10a, pi 10b, pi 105, and pi 10g) and one of two families of regulatory subunits. A PI3K inhibitor of the present invention preferably targets the class 1 PI3K inhibitors. A PI3K inhibitor may display selectivity for one or more isoforms of the class 1 PI3K inhibitors (i.e., selectivity for pi 10a, pi 10b, pi 105, and pi 10g or one or more of pi 10a, pi 10b, pi 105, and pi 10g). In another aspect, a PI3K inhibitor will not display isoform selectivity and be considered a “pan-PI3K inhibitor”. A PI3K inhibitor may compete for binding with ATP to the PI3K catalytic domain.

A PI3K inhibitor may be able to, for example, target PI3K as well as additional proteins in the PI3K/AKT/mTOR pathway. For example, PI3K inhibitor that targets both mTOR and PI3K can be referred to as either a mTOR inhibitor or a PI3K inhibitor. A PI3K inhibitor that only targets PI3K can be referred to as a selective PI3K inhibitor. A selective PI3K inhibitor can be understood to refer to an agent that exhibits a 50% inhibitory concentration with respect to PI3K that is at least 10-fold, at least 20-fold, at least 30-fold, at least 50-fold, at least 100-fold, at least 1000- fold, or more, lower than the inhibitor's IC50 with respect to mTOR and/or other proteins in the pathway.

Exemplary PI3K inhibitors inhibit PI3K with an IC50 (concentration that inhibits 50% of the activity) of about 200 nM or less, preferably about 100 nm or less, even more preferably about 60 nM or less, about 25 nM, about 10 nM, about 5 nM, about 1 nM, 100 uM, 50 mM, 25 mM, 10 pM, 1 pM, or less. The PI3K inhibitor may inhibit PI3K with an IC50 from about 2 nM to about 100 nm, more preferably from about 2 nM to about 50 nM, even more preferably from about 2 nM to about 15 nM.

Non-limiting examples of PI3K inhibitors that can be used in the context of the present invention include, without limitation, the PI3K inhibitors shown in Table 3. Table 3. List of PI3K inhibitors.

Illustrative examples of PI3K inhibitors suitable for use in the T cell manufacturing methods contemplated herein include, but are not limited to, Exelixis (pan-PBK inhibitor), and PX- 866 (class 1 PI3K inhibitor; pi 10a, pi 10b, and pi 10g isoforms). Other illustrative examples of selective PI3K inhibitors include, but are not limited to AS25242, and IPI-145. Further illustrative examples of pan-PI3K inhibitors include, but are not limited to BEZ235, and GSK1059615.

The concentration of the PI3K inhibitor in the culture medium may be between 1 nM and 500 mM, or between 10 nM and 100 mM, or between 100 nM and 50 pM, or between 500 nM and 10 pM, or between 750 nM and 5 pM, or about 1 pM.

C. mTOR inhibitor

The term “mTOR inhibitor”, as used herein, refers to a nucleic acid, peptide, compound, or small organic molecule that inhibits at least one activity of an mTOR protein, such as, for example, the serine/threonine protein kinase activity on at least one of its substrates (e.g., p70S6 kinase 1, 4E-BP1, A T/PKB and eEF2). mTOR inhibitors are able to bind directly to and inhibit mTORCl, mTORC2 or both mTORCl and mTORC2.

Inhibition of mTORCl and/or mTORC2 activity can be determined by a reduction in signal transduction of the PI3K/Akt/mTOR pathway. A wide variety of readouts can be utilized to establish a reduction of the output of such signaling pathway. Some non-limiting exemplary readouts include (1) a decrease in phosphorylation of Akt at residues, including but not limited to 5473 and T308; (2) a decrease in activation of Akt as evidenced, for example, by a reduction of phosphorylation of Akt substrates including but not limited to Fox01/O3a T24/32, GSIOa/b; S21/9, and TSC2 T1462; (3) a decrease in phosphorylation of signaling molecules downstream of mTOR, including but not limited to ribosomal S6 S240/244, 70S6K T389, and 4EBP1 T37/46; and (4) inhibition of proliferation of cancerous cells.

The mTOR inhibitor may be an active site inhibitor. These is an mTOR inhibitor that binds to the ATP binding site (also referred to as ATP binding pocket) of mTOR and inhibits the catalytic activity of both mTORC 1 and mTORC2. One class of active site inhibitors suitable for use in the methods contemplated herein are dual specificity inhibitors that target and directly inhibit both PI3K and mTOR. Dual specificity inhibitors bind to both the ATP binding site of mTOR and PI3K. Illustrative examples of such inhibitors include, but are not limited to, imidazoquinazolines, wortmannin, LY294002, PI-103, SF1126 (Semafore), BGT226, XL765, and NVP-BEZ235.

Another class of mTOR active site inhibitors suitable for use in the methods contemplated herein selectively inhibit mTORC 1 and mTORC2 activity relative to one or more type I phophatidylinositol 3 -kinases, e.g., PI3 kinase a, b, g, or d. These active site inhibitors bind to the active site of mTOR but not PI3K. Illustrative examples of such inhibitors include, but are not limited to, pyrazolopyrimidines, PP242 (2-(4-Amino-l-isopropyl-lH-pyrazolo[3,4- d]pyrimidin-3-yl)-lH-indol-5-ol), PP30, Ku-0063794, WAY-600 (Wyeth), WAY-687 (Wyeth), WAY-354 (Wyeth), and AZD8055 (Liu et ah, 2009, Nature Review 8:627-44).

A selective mTOR inhibitor may refer to an agent having an IC50 with respect to mTORC 1 and/or mTORC2, that is at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold, at least 1000-fold, or more, lower than the inhibitor’s IC50 with respect to one, two, three, or more Class I PI3Ks or to all of the Class I PI3Ks.

Another class of mTOR inhibitors for use in the present invention are referred to herein as rapalogs. The term “rapalogs”, as used herein, refers to compounds that specifically bind to the mTOR FRB domain (FKBP rapamycin binding domain), are structurally related to rapamycin, and retain the mTOR inhibiting properties. The term rapalogs excludes rapamycin. Rapalogs include esters, ethers, oximes, hydrazones, and hydroxylamines of rapamycin, as well as compounds in which functional groups on the rapamycin core structure have been modified, for example, by reduction or oxidation. Pharmaceutically acceptable salts of such compounds are also considered to be rapamycin derivatives. Illustrative examples of rapalogs suitable for use in the methods contemplated herein include, without limitation, temsirolimus (CC1779), everolimus (RAD001), deforolimus (AP23573), AZD8055 (AstraZeneca), and OSI-027 (OSI).

The mTOR inhibitor may be rapamycin (sirolimus).

Exemplary mTOR inhibitors for use in the present invention inhibit either mTORC 1 , mTORC2 or both mTORC 1 and mTORC2 with an IC50 of about 200 nM or less, preferably about 100 nm or less, even more preferably about 60 nM or less, about 25 nM, about 10 nM, about 5 nM, about 1 nM, 100 uM, 50 mM, 25 mM, 10 mM, 1 mM, or less. In one aspect, a mTOR inhibitor for use in the present invention inhibits either mTORCl, mTORC2 or both mTORC 1 and mTORC2 with an IC50 from about 2 nM to about 100 nm, more preferably from about 2 nM to about 50 nM, even more preferably from about 2 nM to about 15 nM.

Exemplary mTOR inhibitors inhibit either PI3K and mTORCl or mTORC2 or both mTORCl and mTORC2 and PI3K with an IC50 of about 200 nM or less, preferably about 100 nm or less, even more preferably about 60 nM or less, about 25 nM, about 10 nM, about 5 nM, about 1 nM, 100 uM, 50 mM, 25 mM, 10 mM, 1 mM, or less. In one aspect, a mTOR inhibitor for use in the present invention inhibits PI3K and mTORCl or mTORC2 or both mTORCl and mTORC2 and PI3K with an IC50 from about 2 nM to about 100 nm, more preferably from about 2 nM to about 50 nM, even more preferably from about 2 nM to about 15 nM.

Further illustrative examples of mTOR inhibitors include, but are not limited to AZD8055, INK128, rapamycin, PF-04691502, and everolimus.

The concentration of the mTOR I3K inhibitor in the culture medium may be between 1 nM and 500 mM, or between 10 nM and 100 mM, or between 100 nM and 50 mM, or between 500 nM and 10 mM, or between 750 nM and 5 mM, or about 1 mM .

1.3. Platelet lysate

The use of foetal bovine serum (FBS) as a culture supplement carries a risk of pathogen transmission as well as xeno-immunization against bovine antigens. Human AB serum, another cell culture option for T cells, has supply limitations and therefore may not be sufficient to meet the expected demand for immunotherapies. Human platelet lysate (hPL) obtained from transfusable donor platelets is widely recognized as a valuable alternative to both FBS and human AB serum for production of clinical cellular therapies. A higher proportion of naive and central memory T cell phenotypes, which may be associated with improved long-term tumour killing, has been reported using hPL as a substitute of FBS and human AB serum (Dann et ak, A new platelet lysate alternative to serum for ex vivo transduction and expansion of human T cells. Poster presented at the American Society of Gene & Cell Therapy 2018 Annual Meeting). The term “platelet lysate” or “PL”, as used herein, refers to the content rich in growth factors that is released from platelets by various methods, such as freeze/thaw cycles that result in platelet lysis. In an embodiment, the platelet lysate is human platelet lysate (hPL). hPL may be generated from huffy coat, platelet rich (platelet-rich plasma (PRP)), or platelet concentrates derived from whole blood or apheresis plasma. The platelets undergo lysis, usually through a freeze/thaw process. hPL may contain an anticoagulant, such as heparin, to prevent coagulation. Alternatively, hPL may go through further manufacturing steps to inhibit the clotting factors. hPL is available commercially through a number of manufacturers, which include AventaCell BioMedical, Mill Creek Life Sciences, Compass Biomedical, Inc., Cook Regentec, Macopharma SA, iBiologics, PL BioScience GmbH, Life Science Productions Ltd (UK) and Trinova Biochem GmbH under the product lines UltraGRO, PLTMax, PLUS, Stemulate, Human Platelet Lysate, XcytePlus, PLSOLUTION, PLMATRIX and CRUX RUFA Media Supplements. Some companies provide different grades of platelet lysate including GMP versions and clinical grade for use in human clinical trials. hPL may comprise different growth factors, including fibroblast growth factor (FGF), endothelial growth factor (EGF), platelet derived growth factor AB (PDGF-AB), tissue growth factor beta (TGF-b), and platelet derived growth factor BB (PDGF-BB). hPL may have a reduced content of pathogens, including enveloped and non-enveloped viruses. There are many routing methods to decrease or complete deplete the pathogen content in hPL, such as electron-beam irradiation. This is available commercially from a number of manufacturers, such as nLiven PR™ (Cook Regentec).

The concentration of hPL in the culture medium may be between 1 and 20%, or between 2 and 10%, or between 3 and 5%, or about 10%. In an embodiment, the concentration of hPL is 3%.

1.4. Interleukin 21 The term “interleukin 21” or “interleukin-21” or “IL21” or “IL-21”, as used herein, refers to a cytokine that has potent immunoregulatory effects on cells of the immune system, including natural killer (NK) cells and B cells and T cells. It has been reported that IL-21 modulates the differentiation of various CD4 and CD8 T cell subsets. Two isoforms have been described for human IL-21, i.e. isoform 1, which is the canonical sequence and has an amino acid sequence depicted under Uniprot Accession No. Q9HBE4-1 (version 3, 10^th April 2019), and isoform 2, which is also known as IL-21iso and has an amino acid sequence depicted under Uniprot Accession No. Q9HBE4-2 on 27^th May 2019.

The IL-21 may be isoform 1.

The IL-21 may be an IL-21 polypeptide, a human IL-21 polypeptide, an active fragment thereof, or a fusion protein comprising an IL-21 polypeptide, such as a fusion protein comprising IL21 and one or more of IL-7 and IL-15 as described in WO 2019/046313.

The concentration of the IL-21 in the culture medium may be between 1 ng/ml and 50 ng/ml, or between 2 ng/ml and 25 ng/ml, or between 5 ng/ml and 15 ng/ml, or about 10 ng/ml. In an embodiment, the concentration of the IL-21 is 10 ng/ml.

1.5. Further culture medium supplements

The culture medium of the invention may be supplemented with further culture medium supplements, such as other interleukins or growth factors, serum, a buffering system, amino acids, carbohydrates, lipids, inorganic salts, trace elements and/or vitamins.

In an embodiment, the culture medium of the invention comprises interleukin 7 (IL-7) and/or interleukin 15 (IL-15). In another embodiment, the culture medium of the invention comprises IL-7 and IL-15. IL-7 and IL-15 are enriched in the lymph node and support the survival of memory T cells.

The term “interleukin 7” or “interleukin-7” or “IL7” or “IL-7”, as used herein, refers to a cytokine that stimulates the differentiation of multipotent (pluripotent) hematopoietic stem cells into lymphoid progenitor cells. It also stimulates proliferation of all cells in the lymphoid lineage (B cells, T cells and NK cells). Three isoforms of human IL-7 have been described, i.e. isoform 1, which is the canonical sequence and has an amino acid sequence depicted under Uniprot Accession No. P13232-1 (version 1, 1^st January 1990), and isoforms 2 and 3, which have an amino acid sequence depicted under Uniprot Accession No. P13232- 2 and PI 3232-3, respectively, on 27^th May 2019.

The IL-7 may be isoform 1.

The concentration of the IL-7 in the culture medium may be between 1 ng/ml and 50 ng/ml, or between 2 ng/ml and 25 ng/ml, or between 5 ng/ml and 15 ng/ml, or about 10 ng/ml. In an embodiment, the concentration of the IL-7 is 10 ng/ml.

The term “interleukin 15” or “interleukin- 15” or “IL15” or “IL-15”, as used herein, refers to a cytokine that stimulates the proliferation of T-lymphocytes. Stimulation by IL-15 requires interaction of IL-15 with components of the IL-2 receptor, including IL-2RB and probably IL-2RG but not IL-2RA. Two isoforms of human IL-15 have been described, i.e. isoform IL15-S48AA, which is the canonical sequence and has an amino acid sequence depicted under Uniprot Accession No. P10933-1 (version 1, 1^st February 1995), and isoforms IL- 15- S21 AA, which have an amino acid sequence depicted under Uniprot Accession No. P 10933- 2 on 27^th May 2019.

The IL-15 may be isoform IL15-S48AA.

The concentration of the IL-15 in the culture medium may be between 1 ng/ml and 50 ng/ml, or between 2 ng/ml and 25 ng/ml, or between 5 ng/ml and 15 ng/ml, or about 10 ng/ml. In an embodiment, the concentration of the IL-15 is 10 ng/ml.

The culture medium of the invention may contain a source of serum, such as foetal bovine serum (FBS) or human serum (HS), such as human AB serum. HS is preferred over FBS, since FBS is of animal origin. However, HS is expensive and there is variability in quality, availability and efficacy. The concentration of serum in the culture medium may be 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, or more.

There are risks associated with the use of serum. For example, the use of FBS as a culture supplement carries a risk of pathogen transmission as well as xeno-immunisation against bovine antigens whilst the use of human serum has supply limitations. Alternatives for FBS and HS include serum replacements such as PhysiologixTM XF SR (Nucleus Biologies) that utilize cGMP grade, xeno-free growth factor and cytokine mixtures to deliver optimal performance while maintaining favourable T cell phenotypes and enhancing transduction efficiency.

In an embodiment, the culture medium of the invention contains no serum. Serum-free medium may contain components derived from serum or plasma, i.e. animal-derived components such as bovine serum albumin (BSA).

Cells have a narrow physiologically acceptable pH range that they require their culture environment to fall within, usually between 7.0-7.4 but that can vary by cell type. T cells can be particularly sensitive to pH and it has been shown that neutral (7.0) and acidic pH can severely inhibit activation. Buffer systems are used in media formulations to monitor and maintain pH levels. The culture medium of the invention may contain a buffering system. Non-limiting examples of buffering systems that may be used in the culture medium of the invention include sodium bicarbonate (NaHCCh) buffer and HEPES (4-(2-hy droxy ethyl)- 1- piperazineethanesulfonic acid) buffer.

Sodium bicarbonate, so-called “natural” buffering system, is a non-toxic buffer that approximates physiological conditions which is commonly included in stem cell medium to stabilise changes in pH within a CO2 incubator.

HEPES is an organic zwitterion that can modulate pH independent of CO2 levels (useful in hypoxic culture conditions). It can be used in combination with sodium bicarbonate to increase buffering capacity. A common method to monitor pH in cell culture media is through the inclusion of phenol red. Its colour changes depending on the pH, where media below pH 6.8 (acidic) appearing yellow and above pH 8.2 (basic) appearing fuchsia. Activated T cells shift their metabolism to aerobic glycolysis which culminates in lactic acid production. At physiologic pH lactic acid dissociates into its corresponding [H⁺] and lactate anion which are exported. This results in extracellular acidification in metabolically active T cells. Other visual pH indicators may be used.

The culture medium of the invention may contain an amino acid.

Amino acids are the building blocks of proteins and facilitate the storage and transfer of nitrogen to the cells in culture. Cells can produce non-essential amino acids (NEAA) but may not produce enough to replenish those depleted during rapid growth. Adding supplements of NEAA to media can both stimulate growth and prolong the viability of the cells in culture. Essential amino acids, however, cannot be synthesised so they must be added to culture media for cells to proliferate.

L-glutamine is an essential amino acid and is a major fuel for many cells including lymphocytes ex vivo. The concentration of extracellular glutamine appears to regulate T cell proliferation, IL-2 production and IL-2 receptor expression with the ideal concentration range being 0.6-2.0mM for lymphocytes.

The culture medium of the invention may contain a carbohydrate. The main source of energy for cells is derived from carbohydrates in the form of sugars. Glucose and galactose are the most common additives; however, maltose or fructose are also used. The culture medium of the invention may contain glucose.

The culture medium of the invention may contain a lipid, such as a fatty acid. Fatty acids serve as fuel for cells but are also precursors to produce cholesterol and membrane phospholipids. The culture medium of the invention may contain an inorganic salt. Inorganic salts, such as calcium, magnesium and potassium are important for regulating the osmotic balance. They also release ions which regulate membrane potential and serve as cofactors for enzymes.

The culture medium of the invention may contain a trace element. Trace elements may include zinc, copper, selenium, tricarboxylic acid intermediates, or any combination thereof.

The culture medium of the invention may contain a vitamin. Vitamins are precursors for numerous co-factors and many are necessary for cell growth and proliferation. The culture medium of the invention may contain riboflavin, thiamine and/or biotin.

The culture medium of the invention may contain B-Mercaptoethanol (BME, 2-ME). This chemical acts as a reducing agent to maintain the intracellular redox environment. Particularly for T cells grown in serum-free conditions, the addition of 2-ME was found to promote T cell proliferation in vitro.

2. Methods of producing an engineered T-cell

Without wishing to be bound by any theory, class IIA HDAC inhibitors generally impact cell properties relating to T-cell differentiation. As a result, the present inventors have discovered that a T-cell incubated in the presence of these compounds is more undifferentiated, or to maintain “sternness” and decreased effector functions, than a T-cell that is incubated in standard culture medium. Accordingly, these compounds have been found to be useful for generating central memory T cells or less differentiated T-cell progeny.

2.1. Method of culturing a T-cell

In a second aspect, the present invention relates to a method of culturing a T-cell, hereinafter “the method of culturing a T-cell of the invention” comprising culturing the T-cell in the presence of a culture medium according to the first aspect of the invention. The terms “T-cell” and “culture medium of the invention” have been described in detail in the context of the first aspect of the invention and their features and embodiments apply equally to the second aspect of the invention.

The T-cell may be an engineered T-cell.

The term “engineered T-cell”, as used herein, refers to a T cell which has been genetically engineered to express artificial receptors, such as a chimeric antigen receptor (CAR) or an engineered T cell receptor (TCR). CARs and TCRs confer to T cells the ability to recognise, for example, given tumour-associated antigens and kill tumour cells, respectively, via HLA- dependent (TCR) and HLA-independent (CAR) mechanisms. These modified cells are genetically engineered ex vivo , then culture-expanded and re-infused back to the patient in a process called adoptive cell transfer. The method of the invention is particularly suited for expanding engineered T-cell s in adoptive cell transfer procedures.

The term “chimeric antigen receptor” or “CAR” or “chimeric T cell receptor” or “artificial T cell receptor” or “chimeric immunoreceptor”. as used herein, refers to a chimeric type I trans-membrane protein which connects an extracellular antigen-recognising domain (binder) to an intracellular signalling domain (endodomain). The binder is typically a single chain variable fragment (scFv) derived from a monoclonal antibody (mAh), but it can be based on other formats which comprise an antigen binding site. A spacer domain is usually necessary to separate the binder from the membrane and to allow it a suitable orientation. A common spacer domain used is the Fc of IgGl. More compact spacers can suffice e.g. the stalk from CD8a and even just the IgGl hinge alone, depending on the antigen. A trans membrane domain anchors the protein in the cell membrane and connects the spacer to the endodomain.

Early CAR designs had endodomains derived from the intracellular parts of either the g chain of the FceRl or Oϋ3z. Consequently, these first-generation receptors transmitted immunological signal 1, which was sufficient to trigger T-cell killing of cognate target cells but failed to fully activate the T-cell to proliferate and survive. To overcome this limitation, compound endodomains have been constructed: fusion of the intracellular part of a T-cell co-stimulatory molecule to that of CD3z results in second generation receptors which can transmit an activating and co-stimulatory signal simultaneously after antigen recognition. The co-stimulatory domain most commonly used is that of CD28. This supplies the most potent co-stimulatory signal - namely immunological signal 2, which triggers T-cell proliferation. Some receptors have also been described which include TNF receptor family endodomains, such as the closely related 0X40 and 4- IBB which transmit survival signals. Even more potent third generation CARs have now been described which have endodomains capable of transmitting activation, proliferation and survival signals.

When the CAR binds the target-antigen, this results in the transmission of an activating signal to the T-cell it is expressed on. Thus, the CAR directs the specificity and cytotoxicity of the T cell towards tumour cells expressing the targeted antigen.

Therefore, CARs typically comprise (Figure 2): i) an antigen-binding domain; ii) a spacer; iii) a transmembrane domain; and iv) an intracellular domain which comprises or associates with a signalling domain.

A CAR may have the general structure:

Antigen-binding domain - spacer domain - transmembrane domain - intracellular signalling domain (endodomain).

The CAR may bind specifically to CD 19, CD22, CD20, TRBCl, TRBC2, GD2, BCMA, TACI, PSMA.

The engineered T cell may express CARs with a single specificity. Alternatively, the engineered T cell may express two or more CARs having different specificities, such as CAR-T cells expressing the so-called “logic gates”.

The CAR may be used in a combination with one or more other activatory or inhibitory CARs. For example, it may be used in combination with one or more other CARs in a "logic- gate", a CAR combination which, when expressed by a cell, such as a T cell, is capable of detecting a particular pattern of expression of at least two target antigens. If the at least two target antigens are arbitrarily denoted as antigen A and antigen B, the three possible options are as follows:

“OR GATE” - T cell triggers when either antigen A or antigen B is present on the target cell;

“AND GATE” - T cell triggers only when both antigens A and B are present on the target cell;

“AND NOT GATE” - T cell triggers if antigen A is present alone on the target cell, but not if both antigens A and B are present on the target cell.

Logic gates have been described in, for example, patent applications WO2015/075468, WO20 15/075469 and WO2015/075470.

The engineered T-cell may express a CAR which binds specifically to CD 19 and a CAR which binds specifically to CD22.

The term “engineered T-cell receptor” or “engineered TCR” or “modified-T cell receptor” or “modified TCR”. as used herein, refers to a wild type TCR or recombinant TCR that expresses a pair of a and b chains which has been selected for its specific binding to a tumour or virus-derived ELLA peptide complex, or a pair of a and b chains which has been engineered to enhance affinity to that ELLA peptide complex. T-cell receptors consist of two associated protein chains: the a and b chains, which are associated with d, e, g, and signalling z chains. Each of the a and b chains has two regions: a variable region and a constant region. The constant region sits next to the T-cell membrane and the variable region of the two chains binds to the target peptides. The variable region of each TCR chain has three hyper-variable complementarity determining regions, or CDRs, which may be modified to enhance the affinity of the TCR to its cognate ELLA peptide complex.

The engineered TCR may bind specifically to CD 19, CD22, CD20, TRBCl, TRBC2, GD2, BCMA, TACI, PSMA.

In an embodiment, the engineered T-cell is a CAR-T cell. In another embodiment, the engineered T-cell expresses an engineered TCR. The T-cell may be isolated from the subject or from other sources. The T cell may be isolated from a subject’s own peripheral blood (1^st party), or in the setting of a haematopoietic stem cell transplant from donor peripheral blood (2^nd party), or peripheral blood from an unconnected donor (3^rd party). Thus, the T-cell is obtained from a patient or an allogeneic donor.

Thus, the T-cell may be obtained from a patient, i.e. autologous, or from an allogeneic donor.

“Autologous”, as used herein, refers to cells from the same subject. “Allogeneic”, as used herein, refers to cells of the same species that differ genetically to the cell in comparison.

The T cell may be an ex vivo cell from a subject. The T cell may be from a peripheral blood mononuclear cell (PBMC) sample.

The isolation of the sample containing a T-cell may be obtained from the donor subject by any suitable method used in the art. For example, the population of T cells may be obtained by any suitable extracorporeal method, venipuncture, or other blood collection method by which a sample of blood and/or lymphocytes is obtained. In one embodiment, the sample containing a T-cell is obtained by apheresis, such as leukapheresis.

Alternatively, T cells may be derived from ex vivo differentiation of inducible progenitor cells to T cells. Alternatively, an immortalised T cell line which retains its lytic function and could act as a therapeutic may be used.

The method of the invention may further comprise a step of isolating the T cells from a T cell-containing sample from a subject or from other sources as listed above, prior to the modification of the cell. This may be attained by enriching the sample containing a T-cell for T-cells. Enrichment of T-cells may be accomplished by any suitable separation method including, but not limited to, the use of a separation medium (e.g., Ficoll-Paque™, RosetteSep™ HLA Total Lymphocyte enrichment cocktail, Lymphocyte Separation Medium (LSA) (MP Biomedical Cat. No. 0850494X), or the like), cell size, shape or density separation by filtration or elutriation, immunomagnetic separation (e.g., magnetic-activated cell sorting system, MACS), fluorescent separation (e.g., fluorescence activated cell sorting system, FACS), or bead-based column separation using CD3/CD28 specific reagents.

The isolated T cell may be in the culture medium of the invention.

The T cell or the sample containing isolated T cells may be cryopreserved until needed.

The method of culturing a T-cell of the invention is performed at conditions that are suitable for culturing or growing T-cells and are well known in the art. Typically, the T-cell is incubated in the culture medium of the invention at a predetermined temperature, for a predetermined amount of time, and/or in the presence of a predetermined level of CO2.

The predetermined temperature for culturing T cells may be about 34 °C, about 35 °C, about 36 °C, about 37 °C, about 38 °C, or about 39 °C. In certain embodiments, the temperature may be about 34-39 °C. In certain embodiments, the temperature may be from about 35-37 °C. In certain embodiments, the preferred temperature may be from about 36-38 °C. In certain embodiments, the temperature may be about 36-37 °C or more preferably about 37 °C.

Culturing T cells may be for a predetermined time. In certain embodiments, the time for culturing T cells may be about may be 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, or more than 21 days.

Culturing T cells may be in the presence of a predetermined level of CO2. In certain embodiments, the level of CO2 may be about 1.0-10% CO2. In certain embodiments, the level of CO2 may be about 1.0%, about 2.0%, about 3.0%, about 4.0%, about 5.0%, about 6.0%, about 7.0%, about 8.0%, about 9.0%, or about 10.0% CO2. In certain embodiments, the level of CO2 may be about 3-7% CO2. In certain embodiments, the level of CO2 may be about 4-6% CO2. In certain embodiments, the level of CO2 may be about 4.5-5.5% CO2. In certain embodiments, the level of CO2 may be about 5% CO2. In one embodiment, culturing T cells may comprise incubating the cells at a temperature of about 37 °C, for an amount of time of about 21 days, and in the presence of a level of CO2 of about 5% CO2.

The method of culturing a T-cell of the invention may be conducted in a closed culture system or bioreactor. The closed culture system may be a closed bag culture system, using any suitable cell culture bags, such as Mitenyi Biotec MACS® GMP Cell Differentiation Bags and Origen Biomedical PermaLife™ Cell Culture bags. Other closed culture systems that are available commercially include CliniMACS Prodigy (Miltenyi Biotec), G- REX500MCS vessel (Wilson Wolf Manufacturing), Xuri™ Cell Expansion System W25 (GE Healthcare).

2.2. Method of activating a T-cell

In a third aspect, the present invention relates to a method of activating a T-cell, hereinafter “the method of activating a T-cell of the invention” comprising activating the T-cell in the presence of a culture medium according to the first aspect of the invention.

The T-cell may be an engineered T-cell.

In an embodiment, the engineered T-cell is a CAR-T cell. In another embodiment, the engineered T-cell expresses an engineered TCR.

The T-cell may be obtained from a patient or an allogeneic donor.

The terms “T-cell”, “culture medium of the invention”, “engineered T-cell”, “CAR-T cell”, and “engineered TCR” have been described in detail in the context of the method of culturing a T-cell of the invention and their features and embodiments apply equally to the method of activating a T-cell of the invention..

A T cell may be activated prior to being transduced or transfected with nucleic acid encoding the molecules providing the CAR or the engineered TCR. The method of activating a T-cell of the invention comprises activating the T-cell in the presence of the culture medium of the invention.

The activation of the T-cell may be achieved by stimulating the cells with one or more mitogens.

The term “mitogen” or “T cell mitogen” or “stimulating agent” or “T cell stimulating agent”, as used herein, refers to a molecule which is capable of binding to the TCR or CD3 chains of the T-cell and trigger intracellular signalling, resulting in T-cell activation and proliferation. Non-limiting examples of mitogens include lectins, such as phytohemagglutinin (PHA), wheat germ agglutinin (WGA), concanavalin A (Con A), and pokeweed mitogen (PWM), as well as an agonist for CD2, CD3, CD28, CD134 or CD137, such as specific monoclonal antibodies or functional fragments thereof, and a T cell cytokine (e.g., any isolated, wildtype, or recombinant cytokines such as interleukin 1 (IL-1), interleukin 2, (IL-2), interleukin 4 (IL-4), interleukin 5 (IL-5), IL-7, IL-15, tumour necrosis factor a (TNFa)). Non-limiting examples of mitogen antibodies include any soluble or immobilised anti-CD2, anti-CD3 and/or anti-CD28 antibody or functional fragment thereof, such as clone OKT3 (anti-CD3), clone 145-2C11 (anti-CD3), clone UCHT1 (anti-CD3), clone L293 (anti-CD28), and clone 15E8 (anti-CD28).

The T cell mitogen may include an anti-CD3 antibody at a concentration of 20 ng/mL-100 ng/mL. In certain embodiments, the concentration of anti-CD3 antibody may be about 20 ng/mL, about 30 ng/mL, about 40 ng/mL, about 50 ng/mL, about 60 ng/mL, about 70 ng/mL, about 80 ng/mL, about 90 ng/mL, or about 100 ng/mL. In certain embodiments, the concentration of anti-CD3 antibody may be about 50 ng/mL.

In an embodiment, the T cell is activated and/or expanded by treatment with an anti-CD3 monoclonal antibody, such as OKT3.

The mitogen may be provided as a polymeric matrix, a viral vector or a virus-like particle. An example of a polymeric matrix is TransAct™ (Miltenyi Biotec), which is a colloidal polymeric nanomatrix covalently attached to humanised recombinant agonists against human CD3 and CD28. The viral vector may be a retroviral or lentiviral vector having a viral envelope which comprises a mitogenic T-cell activating transmembrane protein, such as proteins which bind CD3, CD28, CD134 or CD137, and/or a cytokine-based T-cell activating transmembrane protein.

The activation of the T-cells may comprise stimulating the T-cells with one or more T-cell mitogens at a predetermined temperature, for a predetermined amount of time, and/or in the presence of a predetermined level of CO2.

The predetermined temperature for T cell activation may be about 34 °C, about 35 °C, about 36 °C, about 37 °C, about 38 °C, or about 39 °C. In certain embodiments, the temperature for T cell activation may be about 34-39 °C. In certain embodiments, the temperature for T cell activation may be from about 35-37 °C. In certain embodiments, the preferred temperature for T cell activation may be from about 36-38 °C. In certain embodiments, the temperature for T cell activation may be about 36-37 °C or more preferably about 37 °C.

The step of T cell activation may comprise stimulating T cells with one or more T-cell mitogens for a predetermined time. In certain embodiments, the time for T cell activation may be about 24-72 hours. In certain embodiments, the time for T cell activation may be about 24-36 hours, about 30-42 hours, about 36-48 hours, about 40-52 hours, about 42-54 hours, about 44-56 hours, about 46-58 hours, about 48-60 hours, about 54-66 hours, or about 60-72 hours. In certain embodiments, the time for T cell activation may be about 48 hours or at least about 48 hours.

In certain embodiments, the T cell activation may comprise stimulating T cells with one or more T-cell mitogens in the presence of a predetermined level of CO2. In certain embodiments, the level of CO2 may be about 1.0-10% CO2. In certain embodiments, the level of CO2 may be about 1.0%, about 2.0%, about 3.0%, about 4.0%, about 5.0%, about 6.0%, about 7.0%, about 8.0%, about 9.0%, or about 10.0% CO2. In certain embodiments, the level of CO2 may be about 3-7% CO2. In certain embodiments, the level of CO2 may be about 4-6% CO2. In certain embodiments, the level of CO2 may be about 4.5-5.5% CO2. In certain embodiments, the level of CO2 may be about 5% CO2. In one embodiment, the T cell activation may comprise stimulating T cells with one or more T-cell mitogens at a temperature of about 36-38 °C, for an amount of time of about 44-52 hours, and in the presence of a level of CO2 of about 4.5-5.5% CO2.

In certain embodiments, the method is used for activating a population of T cells. The population of T cells that is used may be at a predetermined concentration of T cells. The concentration of T cells may be about 0.1-10.0 x 10⁶ cells/mL. In certain embodiments, the concentration of lymphocytes may be about 0.1-1.0 x 10⁶ cells/mL, 1.0-2.0 x 10⁶ cells/mL, about 1.0-3.0 x 10⁶ cells/mL, about 1.0-.0 x 10⁶ cells/mL, about 1.0-5.0 x 10⁶ cells/mL, about 1.0-6.0 x 10⁶ cells/mL, about 1.0-7.0 x 10⁶ cells/mL, about 1.0-8.0 x 10⁶ cells/mL, 1.0-9.0 x 10⁶ cells/mL, or about 1.0-10.0 x 10⁶ cells/mL. In certain embodiments, the concentration of T cells may be about 1.0-3.0 x 10⁶ cells/mL. In certain embodiments, the concentration of T cells may be about 1.0-1.2 x 10⁶ cells/mL, about 1.0-1.4 x 10⁶ cells/mL, about 1.0-1.6 x 10⁶ cells/mL, about 1.0-1.8 x 10⁶ cells/mL, or about 1.0-2.0 x 10⁶ cells/mL. In certain embodiments, the concentration of T cells may be at least about 0.1 x 10⁶ cells/mL, at least about 1.0 x 10⁶ cells/mL, at least about 1.1 x 10⁶ cells/mL, at least about 1.2 x 10⁶ cells/mL, at least about 1.3 x 10⁶ cells/mL, at least about 1.4 x 10⁶ cells/mL, at least about 1.5 x 10⁶ cells/mL, at least about 1.6 x 10⁶ cells/mL, at least about 1.7 x 10⁶ cells/mL, at least about 1.8 x 10⁶ cells/mL, at least about 1.9 x 10⁶ cells/mL, at least about 2.0 x 10⁶ cells/mL, at least about 4.0 x 10⁶ cells/mL, at least about 6.0 x 10⁶ cells/mL, at least about 8.0 x 10⁶ cells/mL, or at least about 10.0 x 10⁶ cells/mL.

The method of activating a T-cell of the invention may be conducted in a closed culture system or bioreactor. The closed culture system may be a closed bag culture system, using any suitable cell culture bags, such as Mitenyi Biotec MACS® GMP Cell Differentiation Bags and Origen Biomedical PermaLife™ Cell Culture bags. Other closed culture systems that are available commercially include CliniMACS Prodigy (Miltenyi Biotec), G- REX500MCS vessel (Wilson Wolf Manufacturing), Xuri™ Cell Expansion System W25 (GE Healthcare).

2.3. Method of modifying a T-cell In a third aspect, the present invention relates to a method of transducing a T-cell, hereinafter “the method of modifying a T-cell of the invention” comprising a step of culturing the T- cell in the presence of a modifying agent and a culture medium according to the first aspect of the invention, wherein the modifying agent is selected from the group consisting of a viral vector, a transposon, a plasmid vector, an RNA, and a genome editing system.

The T-cell may be an engineered T-cell.

The T-cell may be obtained from a patient or an allogeneic donor.

The terms “T-cell”, “culture medium of the invention”, “engineered T-cell”, “CAR-T cell”, and “engineered TCR” have been described in detail in the context of the method of culturing a T-cell of the invention and their features and embodiments apply equally to the method modifying a T-cell of the invention.

The term “modifying”, as used herein, refers to the process of engineering genetically the T- cells by introducing a nucleic acid (DNA or RNA) encoding a protein of interest into the cell. The protein of interest may be a CAR or an engineered TCR. The cells resulting from this modification are termed “modified T-cells” or “engineered T-cells”. There are many means of introducing a nucleic acid into a cell, including transduction with a viral vector, transfection with a nucleic acid DNA or RNA, transformation with a plasmid vector. Accordingly, the method of modifying a T-cell of the invention may comprise a step of transducing the T-cell with a viral vector, or a step of transfecting the T-cell with a nucleic acid DNA or RNA, or a step of transforming a plasmid vector. All these methods and steps are routine in the art.

Several recombinant viruses have been used as viral vectors to deliver genetic material to a cell. Viral vectors that may be used in accordance with the transduction step may be any ecotropic or amphotropic viral vector including, but not limited to, retroviral vectors, lentiviral vectors, adenoviral vectors, and adeno-associated viral (AAV) vectors. In one embodiment, the viral vector is a retroviral vector. In another embodiment, the viral vector is a lentiviral vector.

A transposon-based vector or synthetic mRNA may be used for introducing the genetic material into the T cell by transfection.

Alternatively, the term “modifying” may also be used to refer to the process of editing the genome of a T cell. This may be useful in the production of a universal allogeneic T-cell to serve as an “off-the-shelf’ ready -to-use therapeutic agent. Non-limiting examples of genome editing technologies that are suitable for this invention include, without limitation, the clustered regularly interspaced short palindromic repeats/CRISPR associated nuclease9 (CRISPR/Cas9) technology, zinc finger nuclease (ZFN), and transcription activator-like effector nuclease (TALEN), all of which are routine in the art.

A “modification agent”, as used herein, refers to an entity or group of entities which are suitable for genetically modifying a T-cell. The modification agent depends upon the method of modifying the T-cell that is used, and includes, without limitation, a viral vector, a transposon, a plasmid vector, and a genome editing system.

The method of modifying a T-cell of the invention is performed at conditions that are suitable for the modifying agent to be functional, which are well known in the art. Typically, the T- cell is incubated in the culture medium of the invention in the presence of the modifying agent at a predetermined temperature, for a predetermined amount of time, and/or in the presence of a predetermined level of CO2.

The predetermined temperature for modifying T cells may be about 34 °C, about 35 °C, about 36 °C, about 37 °C, about 38 °C, or about 39 °C. In certain embodiments, the temperature may be about 34-39 °C. In certain embodiments, the temperature may be from about 35-37 °C. In certain embodiments, the preferred temperature may be from about 36- 38 °C. In certain embodiments, the temperature may be about 36-37 °C or more preferably about 37 °C. The step of culturing the T-cell in the presence of a modifying agent and the culture medium of the invention may occur at a predetermined time after the T-cell has been obtained or, where applicable, defrosted, which is considered t=0. The T-cell modification may occur at day 0, or 1 day, or 2 days, or 3 days, or 4 days, or 5 days, or 6 days, or 7 days, or 8 days, or 9 days, or 10 days, or 11 days, or 12 days, or 13 days, or 14 days, or 15 days, or 16 days, or 17 days, or 18 days, or 19 days, or 20 days, or 21 days, or longer after the T-cell has been obtained or, where applicable, defrosted. In an embodiment, the T-cell modification occurs between 0 and 2 days after the T-cell has been obtained or, where applicable, defrosted. In another embodiment, the T-cell modification occurs at day 0. In another embodiment, the T- cell modification occurs at day 1. In another embodiment, the T-cell modification occurs at day 2.

The step of culturing the T-cell in the presence of a modifying agent and the culture medium of the invention, as described herein, may be performed for a predetermined time. In certain embodiments, the time for modifying the T-cell may be about 5-36 hours. In certain embodiments, the time for modifying the T-cell may be about 5-12 hours, about 12-16 hours, about 12-20 hours, about 12-24 hours, about 12-28 hours, or about 12-32 hours. In certain embodiments, the time for modifying the T-cell may be about 20 hours or at least about 20 hours. In certain embodiments, the time for modifying the T-cell may be about 16-24 hours. In certain embodiments, the time for modifying the T-cell may be at least about 14 hours, at least about 16 hours, at least about 18 hours, at least about 20 hours, at least about 22 hours, at least about 24 hours, or at least about 26 hours.

Modifying T cells may be in the presence of a predetermined level of CO2. In certain embodiments, the level of CO2 may be about 1.0-10% CO2. In certain embodiments, the level of CO2 may be about 1.0%, about 2.0%, about 3.0%, about 4.0%, about 5.0%, about 6.0%, about 7.0%, about 8.0%, about 9.0%, or about 10.0% CO2. In certain embodiments, the level of CO2 may be about 3-7% CO2. In certain embodiments, the level of CO2 may be about 4-6% CO2. In certain embodiments, the level of CO2 may be about 4.5-5.5% CO2. In certain embodiments, the level of CO2 may be about 5% CO2.

In one embodiment, modifying T cells in the presence of a modifying agent and the culture medium of the invention may comprise incubating the cells at a temperature of about 37 °C, for an amount of time of about 16 hours (overnight), and in the presence of a level of CO2 of about 5% CO2.

The method of modifying a T-cell of the invention may be conducted in a closed culture system or bioreactor. The closed culture system may be a closed bag culture system, using any suitable cell culture bags, such as Mitenyi Biotec MACS® GMP Cell Differentiation Bags and Origen Biomedical PermaLife™ Cell Culture bags. Other closed culture systems that are available commercially include CliniMACS Prodigy (Miltenyi Biotec), G- REX500MCS vessel (Wilson Wolf Manufacturing), Xuri™ Cell Expansion System W25 (GE Healthcare).

2.4. Method of expanding a T-cell

In another aspect, the present invention relates to a method of expanding a T-cell, hereinafter “the method of expanding a T-cell of the invention” comprising culturing the T-cell in the presence of a culture medium according to the invention.

The T-cell may be an engineered T-cell.

The T-cell may be obtained from a patient or an allogeneic donor.

The terms “T-cell”, “culture medium of the invention”, “engineered T-cell”, “CAR-T cell”, and “engineered TCR” have been described in detail in the context of the method of culturing a T-cell of the invention and their features and embodiments apply equally to the method expanding a T-cell of the invention.

The method of expanding a T-cell of the invention is particularly useful for expanding the modified or engineered T-cell to obtain a sufficient number of cells, especially for adoptive T-cell therapy indications. The method of expanding a T-cell of the invention comprises a step of culturing the T-cell in the presence of a culture medium according to the first aspect of the invention. Conditions suitable for expanding T-cells are well known in the art and are similar to those for growing or culturing T-cells. Typically, the T-cell is incubated in the culture medium of the invention at a predetermined temperature, for a predetermined amount of time, and/or in the presence of a predetermined level of CO2.

The predetermined time for expansion may be any suitable time which allows for the production of (i) a sufficient number of cells in the population of T cells or engineered T cells for at least one dose for administering to a patient, (ii) a population of of T cells or engineered T cells with a favourable proportion of undifferentiated or less differentiated T- cells compared to a typical longer process, or (iii) both (i) and (ii). This time will depend on the cell surface receptor expressed by the T cells, the modifying agent used (e.g. vector or nucleic acid), the method of modifying the T cells (e.g. transduction or transfection), the dose that is needed to have a therapeutic effect, and other variables. Thus, the predetermined time for expansion may be 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, or more than 21 days. In some aspects, the predetermined time for expansion is shorter than expansion methods known in the art. For example, the predetermined time for expansion may be shorter by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, or may be shorter by more than 75%.

In one embodiment, the time for expansion is about 3 days. In another embodiment, the time for expansion is about 7 days. In another embodiment, the time for expansion is about 10 days. In another embodiment, the time for expansion is about 14 days. In another embodiment, the time for expansion is about 15 days. In another embodiment, the time for expansion is about 21 days.

Alternatively, the length of time for expanding the T-cells may be determined by the total number of T-cells to be produced, e.g. the total number of cells needed for adoptive T-cell therapy. Thus, the T-cells may be incubated for a length of time that is necessary to obtain 10⁶ cells, 5 x 10⁶ cells, 10⁷ cells, 5 x 10⁷ cells, 10⁸ cells, 5 x 10⁸ cells, 10⁹ cells, 5 x 10⁹ cells, or more.

The step of expanding the T cell or engineered T cell is performed by incubating the engineered T cell in the culture medium of the invention at a predetermined temperature and in the presence of a predetermined level of CO2. The particular embodiments relating to the temperature and level of CO2 have been described previously in the context of the conditions suitable for culturing or growing T-cells and apply equally to the expansion of T cells.

The predetermined temperature for expanding T cells may be about 34 °C, about 35 °C, about 36 °C, about 37 °C, about 38 °C, or about 39 °C. In certain embodiments, the temperature may be about 34-39 °C. In certain embodiments, the temperature may be from about 35-37 °C. In certain embodiments, the preferred temperature may be from about 36- 38 °C. In certain embodiments, the temperature may be about 36-37 °C or more preferably about 37 °C.

Expanding T cells may be in the presence of a predetermined level of CO2. In certain embodiments, the level of CO2 may be about 1.0-10% CO2. In certain embodiments, the level of CO2 may be about 1.0%, about 2.0%, about 3.0%, about 4.0%, about 5.0%, about 6.0%, about 7.0%, about 8.0%, about 9.0%, or about 10.0% CO2. In certain embodiments, the level of CO2 may be about 3-7% CO2. In certain embodiments, the level of CO2 may be about 4-6% CO2. In certain embodiments, the level of CO2 may be about 4.5-5.5% CO2. In certain embodiments, the level of CO2 may be about 5% CO2.

In one embodiment, expanding T cells may comprise incubating the cells at a temperature of about 37 °C, for an amount of time of about 10 days, and in the presence of a level of CO2 of about 5% CO2.

The method of the invention may be conducted in a closed culture system or bioreactor. The closed culture system may be a closed bag culture system, using any suitable cell culture bags, such as Mitenyi Biotec MACS® GMP Cell Differentiation Bags and Origen Biomedical PermaLife™ Cell Culture bags. Other closed culture systems that are available commercially include CliniMACS Prodigy (Miltenyi Biotec), G-REX500MCS vessel (Wilson Wolf Manufacturing), Xuri™ Cell Expansion System W25 (GE Healthcare).

2.5. Method of producing a T-cell

In another aspect, the present invention relates to a method of producing an engineered T- cell, hereinafter “the method of producing a T-cell of the invention” comprising the steps of:

(i) activating a T-cell according to the method of activating a T-cell of the invention; and

(ii) modifying the activated T-cell obtained in (i) according to the method of modifying a T-cell of the invention.

The method of producing a T-cell of the invention may further comprise a step of expanding the T-cell obtained in step (ii) according to the method of expanding a T-cell of the invention.

The T-cell may be an engineered T-cell.

The T-cell may be obtained from a patient or an allogeneic donor.

The terms “T-cell”, “culture medium of the invention”, “engineered T-cell”, “CAR-T cell”, “engineered TCR”, “method of activating a T-cell of the invention”, “method of modifying a T-cell of the invention”, and “method of expanding a T-cell of the invention” have been described in detail in the context of previous aspects of the invention and their features and embodiments apply equally to the method producing a T-cell of the invention.

2.6. T-cell obtainable or obtained by the methods of the invention The T-cell, population of T-cells, engineered T cell or population of engineered T cells obtainable or obtained by any of the methods of the invention, as described herein, constitute additional aspects contemplated by the present invention.

The T-cell, population of T-cells, engineered T cell or population of engineered T cells produced by any of the methods of the invention may optionally be cryopreserved so that the cells may be used at a later date. This may be done by any routine method that is suitable with adoptive T cell therapy.

The T-cell, population of T-cells, engineered T cell or population of engineered T cells obtained using the method of the invention may be characterised in that it comprises a higher proportion of undifferentiated T cells compared with the proportion of effector T cells (effector memory T cells and effector T cells). This may be particularly advantageous in the context of adoptive T cell therapies since undifferentiated T cell phenotypes have been associated with improved long-term tumour killing (Hinrichs et al., 2009. Adoptively transferred effector cells derived from naive rather than central memory CD8+ T cells mediate superior antitumor immunity. Proc Natl Acad Sci USA 106:17469-74). For example, it may contain a higher proportion of naive T cells, or a higher proportion of central memory T cells, or a higher proportion of the combination of naive and central memory T cells. The population of engineered T cells obtained using the method of the invention may be characterised in that it comprises a higher proportion of memory stem T cells.

The phenotype of T cells may be evaluated by any routine method, such as FACS, using the relevant T cell markers of differentiation (Fig. 16).

3. Use

The present invention also contemplates the use of the culture medium of the invention for culturing, activating, modifying, expanding, or producing a T-cell following the methods of the invention. Therefore, in a third aspect, the present invention provides the use of the culture medium according to the first aspect of the invention for culturing, activating, modifying, expanding, or producing a T-cell, hereinafter “the use of the invention”. The T-cell may be an engineered T-cell.

The T-cell may be obtained from a patient or an allogeneic donor.

The population of T-cells is produced according to any of the methods of the invention.

4. Kit

The particular components of the culture medium of the invention may be provided in a format that is suitable for preparing the culture medium of the invention at a time when is needed.

Thus, in another aspect, the present invention also provides kit comprising a class-IIA specific histone deacetylase inhibitor and an ART inhibitor.

The AKT inhibitor may be AKT inhibitor VIII

The kit may further comprise IL-7 and IL-15. The terms “culture medium supplements”, “class-IIA specific histone deacetylase inhibitor”, and “AKT inhibitor” have been described in detail in the context of the first aspect of the invention and their features and embodiments apply equally to this aspect of the invention.

The kit of the invention may contain further culture medium supplements, such as other interleukins or growth factors, serum, a buffering system, amino acids, carbohydrates, lipids, inorganic salts, trace elements and/or vitamins. These have been described in detail in the context of the first aspect of the invention and their features and embodiments apply equally to this aspect of the invention.

It will be understood that the kit of the invention may be suitable for producing a population of T-cells.

The invention will now be further described by way of Examples, which are meant to serve to assist one of ordinary skill in the art in carrying out the invention and are not intended in any way to limit the scope of the invention.

EXAMPLES

In order to halt cellular differentiation and increase the percentage of naive or early memory cells in the CAR-T cell product we have tested inhibitors of AKT and HDAC signalling. There exist many molecules that can target AKT as well as others that act as HDAC inhibitors. The AKT gold standard inhibitory molecule used for induction of an early memory phenotype is AKT inhibitor VIII (Akt 1/2). The HDAC inhibitors tested in the following experiments are class II HDAC inhibitors that were chosen after performing a screening of a library of inhibitors involved in epigenetic modifications.

Flow cytometry was used to identify the percentage of cells in early memory phenotype by measuring cellular expression of naive markers. Cell surface markers used in our experiments to identify cells in a naive or early memory phenotype are shown in figure 1.

Materials and methods Materials

PBMCs were obtained from healthy donors by leukapheresis. CAR products were AUT02 (APRIL-CAR) and AUT03 (anti-CD 19 CAR and anti-CD22 CAR). Human AB serum was from Life Science Production (LSP). Human platelet lysate (hPL) was nLiven PR, from Cook Regentec.

Cell Count and Cell Viability

Cell counting was performed by acridine orange/4', 6-diamidino-2-phenylindole (DAPI) staining using an automated cell counter. For determination of the viability and cell number, two independent samples from the cell suspension were used for counting procedures. The counts were performed at several time points during the manufacturing process (Fig. 3).

Cell Phenotype/Purity

Cells were analysed by flow cytometry to determine the purity and immunophenotype of the leukapheresis and drug product. Five different flow cytometry assays will be used at different time points. The flow cytometry assays and the parameters used for the comparability assessment are described as follows:

1. Flow cytometry release assay

This assay was performed in fresh samples during manufacturing. Briefly, cells were incubated in darkness with an antibody cocktail comprising anti-CD3, anti-CD4, anti-CD8, anti-CD 19, anti-CD34, and anti-CD45. Anti-CD34 (RQR8) clone Hu37 (CD34-id-Hu37) was used to detect CAR-T cells in AUT02 product, and anti-CD19 clone HD37 (CD19-id- HD37) was used to detect CAR-T cells in AUT03. Following incubation, cells were spun and resuspended in PBS with 7-AAD to be analysed by FACS.

The quality attributes assessed with this assay were:

The percentage of T-cell expressing CAR; and

The percentage of CD45+CD3+ CAR+ cells on the DS on the last day of manufacture. 2 Memory flow cytometry assay

This assay was performed on leukapheresis and drug product. Briefly, cells were incubated in darkness with antibodies conjugated with different fluorochromes specific for the following markers: CD3, CD4, CD8, CD95, CD45RA, CD62L, CXCR3, CCR7/CD197, CD28, and CD27. Anti-CD34 (RQR8) clone Hu37 (CD34-id-Hu37) was used to detect CAR-T cells in AUT02 product, and anti-CD19 clone HD37 (CD19-id-HD37) was used to detect CAR-T cells in AUT03. Following incubation, cells were spun and resuspended in PBS with 7-AAD to be analysed by FACS.

The quality attributes assessed with this assay are the differentiation potential (memory), measured as the percentage change of CD45RA+CCR7+ T-cells and CD45RA+/CCR7+/CD27+/CD62L/CD45RO+/CD95+ between the starting leukapheresis material and the drug product.

3 Exhaustion flow cytometry assay

This assay was performed on leukapheresis and drug product. Briefly, cells were incubated in darkness with an antibody cocktail comprising anti-CD3, anti-CD8, anti-Tim, anti-LAG3, and anti-PDl antibodies conjugated with different fluorochromes. Anti-CD34 (RQR8) clone Hu37 (CD34-id-Hu37) was used to detect CAR-T cells in AUT02 product, and anti-CD19 clone HD37 (CD19-id-HD37) was used to detect CAR-T cells in AUT03. Following incubation, cells were spun and resuspended in PBS with 7-AAD to be analysed by FACS.

The quality attributes assessed with this assay are Cell Exhaustion, measured as the percentage of CD8+CAR+ triple positive (Lag3/TIM3/PD1) cells in drug product.

4 Cell-ID flow cytometry assay

This assay will be performed on leukapheresis and drug product. Briefly, cells were incubated in darkness with an antibody cocktail comprising anti-CD3-FITC, and anti-CD4, anti-CD8, anti-CD235a, anti-CD45, anti-CD 19, anti-CD56, anti-CD 14, anti-CD34, and anti- CD19/22 (Clone Hu37) antibodies conjugated with different fluorochromes. Following incubation, cells were spun and resuspended in PBS with 7-AAD to be analysed by FACS. The quality attribute assessed with this assay is the cell composition of the starting material and purity of drug product.

5 Activation flow cytometry assay

This assay was performed on activated PBMCs on Day 2 of the process. Briefly, cells were incubated in darkness with a primary antibody master mix comprising anti-CD3-FITC, anti- CD4-PE, anti -CD 8 APC-H7, and anti-CD45, anti-CD71, anti-CD25, and anti-CD69 conjugated with different fluorochromes. Following incubation, cells were spun and resuspended in PBS with 7-AAD to be analysed by FACS.

The quality attribute assessed with this assay is the percentage of T cells expressing markers of activation.

CAR T Cell Functional Testing

1. Cell Killing Assay

This assay was performed on thawed drug product vials to assess the efficacy of the product. Briefly, CD19/22 CAR T cells were incubated with target MM1S (AUT02) or Raji cells (AUT03) at an effectontarget (E:T) ratio 1 :4, in TexMACS medium supplemented with 3% HS, and incubated at 37°C, 5% CO2 for a predetermined time (e.g. 48 hours). The concentration of effector cells (transduced cells/ml) is calculated as:

No. of transduced cells/ml = % viable cells x % transduction x cell number/ml Then, cells were incubated in darkness with a primary antibody master mix comprising:

- for AUTO 2: anti-CD3-FITC antibody and CD34-id-Hu37 (RQR8), or

- for AUT03: anti-CD3-FITC antibody, anti-CD2-PE, and CD19-id AF647. Following incubation, cells were spun and resuspended in PBS with 7-AAD to be analysed by FACS.

The quality attribute assessed with this assay is the cytotoxicity potential of the drug product.

2. Cell activation assay The release of IFNy, TNFa, IL-2 and Granzyme B into the cell culture supernatants was determined using the SimplePlex Protein Assay (Ella) from Biotechne, following the manufacturer’s instructions. The analysis was performed using the SimplePlex Runner software.

Vector copy number (VCN)

Vector copy number analysis is performed via a quantitative real-time PCR (qPCR) assay. DNA is extracted and copies of the viral vector are detected using primers and probe targeting the packaging signal, Psi, and quantified against a plasmid standard. Simultaneously, primers and probe are used to detect copies of the albumin gene alb , which is quantified against the same plasmid standard. The number of vector copies is normalised to the number of cells in the sample.

Example 1: Standard production of CAR-T cells

The standard procedure for producing CAR-T cells is outlined in Figure 3. Briefly, PBMCs from healthy donors are incubated overnight in TexMACS medium supplemented with 3% human AB serum (HABS) in cell differentiation bags in an incubator (37°C, 5% CO2). Cells are activated using TransAct reagent (as per manufacturer’s instructions) in TexMACS media supplemented with 3% HABS, 10 ng/mL IL7 and 10 ng/mL IL15. After two days, a retroviral vector encoding the CAR was used to transduce the T cells in the presence of RetroNectin or Vectofusin-1. Cells, vector and transduction reagent are incubated overnight in TexMACS medium supplemented with 3% HABS and 10 ng/mL each IL7 and IL15 at 37°C, 5% CO2. Then, the remaining untransduced vector was washed and cells were resuspended in fresh TexMACS medium supplemented with 3% HABS and 10 ng/mL each IL7 and IL15 for expansion by incubating for up to 7 days in at 37°C, 5% CO2. The production process was carried out on shake flasks or using the CliniMACS Prodigy apparatus (Miltenyi Biotec).

Example 2: Comparison of AKT and HDAC inhibitors The conditions chosen in the first experiment were the following: untreated, AKTiVIII (ImM), TMP269 (1.25 nM), MC1568 (ImM) and Bufexamac (10 mM). The experiment was run at a small scale (96 well plate) in three healthy donors, following the process steps for the AUTO 3 program. In summary, cells were thawed at day -1 and activated with TransAct + IL-7/15 on day 0. Transduction was performed on day 2 and there was a wash on day 3. Compounds were added to the media at each step of the process except in day -1 and phenotypic analysis was performed on day 6.

Results show a significant decrease in the naive phenotype in cells treated with MCI 568 versus cells not treated with compounds. In this case, we are observing a decrease in CCR7+ and CD45RA+ marker expression. However, cells treated with TMP269 and Bufexamac (a class IIB HD AC inhibitor) didn’t show any significant difference versus the untreated group (Figure 4A).

As mentioned in figure 1, there are additional markers of naivety that can be measured such as CD27 and CD62L. Cells expressing CD27 and CD62L simultaneously are also in early phases of differentiation. Cells treated with MCI 568 and Bufexamac show a significant increase in the percentage of double positive cells compared to the untreated group. Moreover, a significant increase of this population was observed when comparing both MCI 568 and Bufexamac with AktiVIII (Figure 4B).

Finally, we looked at stem cell memory-like markers which are a combination of CCR7, CD45RA, CD27 and CD62L positive populations plus CD95+ and CD45RO+ expression. Results correlated with what we had observed so far, we observed a significantly higher percentage of cells expressing stem cell memory-like markers in cells treated with MCI 568 and Bufexamac versus the untreated group and the Bufexamac group was also significantly increased versus AKTiVIII (Figure 4C).

Additional parameters were analysed for each compound (%recovery, viability, TE, exhaustion phenotype, cytotoxicity and cytokine release) and after scoring the results from each compound we decided to move forward with compound MC1568. Example 3: Testing MC1568 as media supplement

Next, a medium scale experiment was performed in order to assess reproducibility of effects by MCI 568. In addition, we tested MCI 568 in combination with AKTiVTTT to observe if both compounds would have an additive effect increasing the percentage of naive or early memory cells in the CAR-T cell product.

The conditions chosen for the next experiment were the following: untreated, AktiVIII alone (ImM), MC1568 alone (ImM) and AKTiVIII+MC1568. We ran the experiment at a medium scale (24 well plate) in six healthy donors, and we followed the process steps for the AUTO 3 program. In summary, cells were thawed at day -1 and activated with TransAct + IL-7/15 on day 0. Transduction was performed on day 2 and there was a wash on day 3 and media addition at day 6. Compounds were added to media at each step of the process except in day -1 and phenotypic analysis was performed on day 7.

Results show a significant increase in the naive phenotype in cells treated with AKTiVIII+ MCI 568 versus cells not treated with compounds. In this case, we are observing an increase in CCR7+ and CD45RA+ marker expression. What is most interesting is that the increase in the naive phenotype is also significant when we compare the group of cells treated with the combination versus cells treated with AKTiVIII alone. These results not only demonstrate the ability of the combination to improve upon the memory phenotype, but they show that this combination leads to better results than treating the cells with AKTiVIII alone (Figure 5A).

Results show a significant increase in the percentage of double positive cells in the combination group compared to the untreated group. These results corroborate what we had initially observed with CCR7 and CD45RA expression, which is that the combination of AKTiVIII with MCI 568 induces a more naive phenotype in the CAR-T product. Additionally, we observed a significant increase in the MCI 568 group versus untreated which is due to a higher expression of CD27 in that group (Figure 5B).

Finally, we looked at stem cell memory-like markers which are a combination of CCR7, CD45RA, CD27 and CD62L positive populations plus CD95+ and CD45RO+ expression. Results correlated with what we had observed so far, we observed a significantly higher percentage of cells expressing stem cell memory-like markers versus the untreated group and the AKTiVIII+ MCI 568 group was also significantly increased versus AKTiVIII alone (Figure 5C).

Aside from the memory phenotype, we studied additional features of cells treated in this experiment. We looked at effects on exhaustion as well as cell functionality by measuring %killing and cytokine production and release. Co-culture of CAR-T cells with target cells was set up on day 7 and samples for cytotoxicity and cytokine release were obtained for analysis after 48 hours (day 9). Table 4 presents the different analyses performed at each day

Table 4. Table presenting analyses performed during the matrix experiment with information on the day of completion

In summary, viability, recovery and transduction efficiency were not affected by the combination of compounds. The analysis on exhaustion and cytotoxicity revealed some values that were affected by the combination of compounds. We observed increased expression of LAG3+ and PD1+ in the combination group while TIM3+ was significantly reduced. In addition, we observed a significant decrease in cytotoxicity at 48hr after culture compared to the untreated group. To improve the exhaustion phenotype and cytotoxicity results, we tested a matrix of concentrations and evaluated the optimal dose to use for each compound in the combination (we used Design of Experiments (DoE) to analyse this data). With DoE software we were able to identify the optimal concentration for each compound in order to minimize exhaustion and maintain cytotoxicity levels. Concentrations chosen were AKTiVIII at ImM and MC1568 at 0.75mM.

Example 4: Confirmation of Effect in Cells Transduced with a Lentiviral Vector A medium scale experiment was performed to assess the reproducibility of effects of AKTiVIII and MCI 568 when transducing cells using a lentiviral platform. AKTiVIII was tested alone and in combination with MCI 568 with the goal to reproduce the increased percentage of naive or early memory cells in the CAR-T product observed previously when transducing cells retrovirally.

The conditions chosen for the next experiment were the following: untreated, AktiVIII alone (ImM) and AKTiVIII+MC1568 (ImM for each compound). We ran the experiment in three donors at a medium scale. Transduction was carried out in T10 flasks and cells were transferred into larger flasks for expansion. In summary, cells were thawed at day -1 and activated with TransAct + IL-7/15 on day 0. Transduction was performed on day 1. Culture media volume was doubled at day 3 and a wash and refresh of media was performed on day 4. Compounds were added to media at each step of the process except in day -1 and phenotypic analysis was performed on day 7.

Results show a significant increase in the naive phenotype in cells treated with AKTiVIII+ MCI 568 versus cells not treated with compounds. In this case, we are observing an increase in CCR7+ and CD45RA+ marker expression. The increase in the naive phenotype is also significant when we compare the group of cells treated with the combination versus cells treated with AKTiVIII alone as we had observed previously using AUT03. These results repeatedly show that this combination leads to better results than treating the cells with AKTiVTTT alone (Figure 6A).

Results show a significant increase in the percentage of double positive cells (CD27+CD62L+) in the combination group compared to the untreated group. These results support the data observed with CCR7 and CD45RA expression, which is that the combination of AKTiVTTT with MCI 568 induces a more naive phenotype in the CAR-T product. (Figure 6B).

Finally, we looked at stem cell memory-like markers which are a combination of CCR7, CD45RA, CD27 and CD62L positive populations plus CD95+ and CD45RO+ expression. Results correlated with what we had observed so far, we observed a significantly higher percentage of cells expressing stem cell memory -like markers in the AKTiVTTT+ MCI 568 group versus the untreated group and there was also a significantly increase on this group versus AKTiVIII alone (Figure 6C).

Additional features of cells treated in this experiment were measured. We looked at effects on exhaustion as well as cell functionality by measuring %killing and cytokine production and release. Co-culture of CAR-T cells with target cells was set up on day 7 and samples for cytotoxicity and cytokine release were obtained for analysis after 48 hours (day 9). In summary, viability, transduction efficiency, cytotoxicity, cytokine release and exhaustion were not affected by the combination of compounds.

Claims

1. A culture medium suitable for culturing T-cells comprising a class-IIA specific histone deacetylase inhibitor.

2. The culture medium according to claim 1, wherein the class-IIA specific histone deacetylase inhibitor is 3-[5-(3-(3-Fluorophenyl)-3-oxopropen-l-yl)-l-methyl-lH-pyrrol- 2-yl]-N-hydroxy-2-propenamide (MC 1568).

3. The culture medium according to any of claims 1 or 2, wherein the concentration of class-IIA specific histone deacetylase inhibitor is 0.75 or 1 mM

4. The culture medium according to any of claims 1 to 3, wherein the culture medium culture additionally comprises an AKT inhibitor.

5. The culture medium according to claim 4, wherein the AKT inhibitor is AKT inhibitor VIII (1,3 -Dihydro- 1 -( 1 -((4-(6-phenyl- 1 H-imidazo[4, 5 -g] quinoxalin-7 - yl)phenyl)methyl)-4-piperidinyl)-2H-benzimidazol-2-one).

6. The culture medium according to any of claims 4 or 5, wherein the concentration of AKT inhibitor is 1 mM.

7. The culture medium according to any of claims 1 to 6, which contains no serum.

8. The culture medium according to any of claims 1 to 7, further comprising interleukin 7 (IL-7) and interleukin 15 (IL-15).

9. The culture medium according to claim 8, wherein the concentration of IL-7 is 10 ng/ml and the concentration of IL-15 is 10 ng/ml.

10. A method of culturing a T-cell, comprising culturing the T-cell in the presence of a culture medium according to any of claims 1 to 9.

11. A method of activating a T-cell, comprising culturing the T-cell in the presence of a culture medium according to any of claims 1 to 9.

12. The method according to claim 11, comprising a step of stimulating the T-cells with a mitogen.

13. The method according to claim 12, wherein the mitogen is provided as a polymeric matrix or a virus-like particle.

14. A method of modifying a T-cell, comprising culturing the T-cell in the presence of a modifying agent and a culture medium according to any of claims 1 to 9, wherein the modifying agent is selected from the group consisting of a viral vector, a transposon, a plasmid vector, an RNA, and a genome editing system.

15. The method of claim 14, wherein the modifying agent is a viral vector.

16. The method according to claim 14, wherein the viral vector is a lentiviral vector or a retroviral vector.

17. The method according to claim 14, wherein the modifying agent is a transposon.

18. The method according to claim 14, wherein the modifying agent is a plasmid vector.

19. The method according to claim 14, wherein the modifying agent is a genome editing system.

20. The method according to claim 19, wherein the genome editing system is CRISPR/Cas9 system.

21. A method of expanding a T-cell, comprising culturing the T-cell in the presence of a culture medium according to any of claims 1 to 9.

22. A method of producing an engineered T-cell, comprising the steps of:

(i) activating a T-cell according to the method of any of claims 11 to 13; and

(ii) modifying the activated T-cell obtained in (i) according to the method of claim 14 to 20.

23. The method of claim 22, further comprising a step of expanding the T-cell obtained in (ii) according to the method of claim 21.

24. The method according to any of claims 10 to 23, wherein the T-cell is an engineered T-cell.

25. The method according to claim 24, wherein the engineered T-cell is a CAR-T cell.

26. The method according to any of claims 10 to 25, wherein the T-cell is obtained from a patient or an allogeneic donor.

27. The method according to any of claims 10 to 26, wherein the method is conducted in a closed culture system.

28. T-cell obtained by the method according to any of claims 10 to 27.

29. Use of the culture medium according to any of claims 1 to 9 for producing a population of T-cells.

30. Use according to claim 29, wherein the population of T-cells is produced according to the method of any of claims 10 to 27.

31. Use according to claim 30, wherein the T-cells are engineered T-cells.

32. Kit comprising a class-IIA specific histone deacetylase inhibitor and an AKT inhibitor.

33. Kit according to claim 32, wherein the a class-IIA specific histone deacetylase inhibitor i s 3 - [5 -(3 -(3 -Fluorophenyl)-3 -oxopropen- 1 -yl)- 1 -methyl- 1 H-pyrrol-2-yl] -N- hydroxy-2-propenamide (MCI 568).

34. Kit according to claim 32, wherein the AKT inhibitor is AKT inhibitor VIII.

35. Kit according to any of claims 32 to 34, further comprising IL-7 and IL-15.