Method for producing engineered cells expressing chimeric antigen receptors

Abstract

A process for producing a population of engineered cells, the process comprising: i) providing a starting population of cells; ii) activating the starting population of cells; iii) splitting the starting population of cells into a first cell population and a second cell population; iv) transducing the first cell population with a first vector encoding a first chimeric antigen receptor and transducing the second cell population with a second vector encoding a second chimeric antigen receptor; v) separately expanding the first cell population and the second cell population in a culture medium for a maximum of 6 days, or until a target dose is reached; and vi) combining the first cell population and the second cell population to form a final engineered cell population.

Description

[0001] METHOD

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to a process for producing engineered cells. The process comprises providing a starting population of cells; activating the cells; splitting the starting population of cells into a first cell population and a second cell population; transducing the first cell population with a first vector encoding a first chimeric antigen receptor and transducing the second cell population with a second vector encoding a second chimeric antigen receptor; separately expanding the first cell population and the second cell population in a culture medium for a maximum of 6 days, or until a target dose is reached; and combining the first cell population and the second cell population to form a final cell population. In a preferred embodiment, the first chimeric antigen receptor is an anti-CD19 chimeric antigen receptor and the second chimeric antigen receptor is an anti-CD22 chimeric antigen receptor.

[0004] BACKGROUND TO THE INVENTION

[0005] Adoptive cell therapy (ACT) is a personalised therapy that involves administration to the subject of immune cells with activity directed against a specific disease related antigen. One such approach is the administration of lymphocytes genetically engineered to express a chimeric antigen receptor (CAR). CARs are artificial receptors that can be constructed by linking the variable regions of the antibody heavy and light chains to intracellular signalling chains alone or in combination with other signalling moieties. CARs recognise antigens which are presented on the tumour cell surface, but do not need to be MHC-restricted. For example, CARs against the B cell antigen CD19 have been shown to mediate regression of an advanced B cell lymphoma. Transgenic T cell receptors (TCRs) are also being investigated for use in ACT.

[0006] A particular problem in the field of oncology is provided by the Goldie-Coldman hypothesis: which describes that the sole targeting of a single antigen may result in tumour escape by modulation of said antigen due to the high mutation rate inherent in most cancers. This modulation of antigen expression may reduce the efficacy of known immunotherapeutics, including those which target CD19.

[0007] Thus a problem with immunotherapeutics targeted against CD19 is that a B-cell malignancy may mutate and become CD 19-negative. This may result in relapse withCD19-negative cancers which are not responsive to CD19 targeted therapeutics. For example, in one paediatric study, Grupp et al. reported that half of all relapses following CD19-targeted chimeric antigen receptor therapy for B-acute Lymphoblastic leukaemia (B-ALL) were due to CD19-negative disease (56thAmerican Society of Hematology Annual Meeting and Exposition).

[0008] There is thus a need for immunotherapeutic agents which are capable of targeting more than one cell surface structure to reflect the complex pattern of marker expression that is associated with many cancers, including CD19-positive cancers.

[0009] DESCRIPTION OF THE FIGURES

[0010] Figure 1 - Modified GMP manufacturing methodology incorporating shortened manufacture to preserve TSCM and separate transduction to reduce CAR T cell AICD / exhaustion.

[0011] Figure 2 - Dual transduced CD19 / 22CAR T cells may be more susceptible to AICD.

[0012] A. Single cell CITESeq analysis showing upregulation of FasL in dual transduced CAR T cells. B. Progressive increase in early apoptosis (Annexin V +ve, 7-AAD -ve) in CD19 / 22 CAR dual positive population after repetitive stimulation in vitro. CAR T cells from 5 co-transduced products from CARPALL Cohort 3 were stimulated every 5 days at 1:1 ratio with CD19+CD22+ Raji cells in the absence of cytokines. Flow cytometric analysis of single and dual CAR+ cells was performed at the timepoints shown.

[0013] Figure 3- In vitro function of CD19+CD22 CAR T cells and comparison of functionality following manufacture with a 9-day or 5-day process. Functional assays were performed with PBMCs from leukapheresis or CD19 / 22 CAR T cells from three patients (CPL-27, CPL-29 and CPL-31) produced using either Cohort 3 (9-day process) or Cohort 4 (5-day process) methods. (A) Cytotoxicity assays were carried with cocultures of CD19 / 22 CAR T cells with SupT1 (CD19-), Raji (CD19+) or Nalm6 cells (CD19+) for ~18h (E:T ratio = 1:1). Flow cytometric analysis was used to determine the number of viable target cells. Percentage killing was determined relative to number of target cells in the co-cultures of leukapheresis+target cells. (B) 3H-thymidine incorporation assays were carried out with CAR T cells co-cultured with irradiated target cells at 1:1 E:T ratio for 72 hours. P / I=phorbol 12-myristate 13-acetate / ionomycin. Cells were pulsed with 3H-thymidine for the last 18h of co-culture. Cytometric bead assays were performed using supernatant taken at 48 h of co-culture (from the same wells asfor panel B) to determine levels of (C) IFN-y or (D) IL-2. All data are mean±s.d. for triplicate wells. Statistical analysis was carried out using Students t-test to compare Cohort 3 vs Cohort 4.

[0014] Figure 4 - In vivo functionality comparison of Cohort 3 (9-day process) and Cohort 4 (5-day process) CD19+CD22 CAR T cells. A. Xenograft experimental schematic. NSG mice were irradiated with 2.5Gy, followed by i.v. injection of 1x105 GFP+ / Luc+ NALM6 cells. PBS, 2x106 Cohort 3 (CPL-27), or 2x106 Cohort 4 (CPL-27) CD19+CD22 CAR T were delivered i.v. on the following day. Leukaemia progression was monitored by weekly bioluminescence imaging. On day 28, the three mice with the highest MS signal from each CAR-treated group were harvested to examine early CAR-T cells persistence. B. Bioluminescence images of GFP+ / Luc+ NALM6 xenograft mice. C.

[0015] Percent-age of GFP+ / Luc+ NALM6 cells of total mononuclear cells (MNCs) in blood, bone marrow, spleen, and liver in the sacrificed mice on day 28. One-way ANOVA test was performed. D. Percentage of CAR T cells (hCD45+hCD3+) cells of total MNCs in blood, bone marrow, spleen, and liver in the sacrificed mice on day 28. Mann-Whitney test was performed. E. Survival curves for xenograft mice up to 40 days post NALM6 injection. Logrank test was performed. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns, not significant.

[0016] SUMMARY OF ASPECTS OF THE INVENTION

[0017] The present inventors provide a process for producing a population of engineered cells which are capable of targeting multiple antigens.

[0018] The present inventors have shown that preparing engineered cells expressing multiple chimeric antigen receptors using a dual transduction approach leads to increased levels of activation induced cell death. To address this issue, the inventors have split the starting population of cells into a first and second population which can be separately transduced with one of two chimeric antigen receptors.

[0019] Another advantage of the present invention is that genetically modified cells produced by the method reach the desired dose level more quickly. This is particularly important for autologous applications, where critically ill patients may only be treatable if short vein-to-vein times can be achieved.In a first aspect the invention provides a process for producing a population of engineered cells, the process comprising:

[0020] i) providing a starting population of cells;

[0021] ii) activating the starting population of cells;

[0022] iii) splitting the starting population of cells into a first cell population and a second cell population;

[0023] iv) transducing the first cell population with a first vector encoding a first chimeric antigen receptor and transducing the second cell population with a second vector encoding a second chimeric antigen receptor;

[0024] v) separately expanding the first cell population and the second cell population in a culture medium for a maximum of 6 days, or until a target dose is reached;

[0025] vi) combining the first cell population and the second cell population to form a final engineered cell population.

[0026] In a preferred embodiment, the first chimeric antigen receptor is an anti-CD19 chimeric antigen receptor and the second chimeric antigen receptor is an anti-CD22 chimeric antigen receptor.

[0027] Preferably, the starting population of cells comprises T cells.

[0028] The process may further comprise a CD4 and CD8 selection step prior to step (iii).

[0029] The process may be carried out in the absence of exogenous cytokines.

[0030] The expansion in step (v) may be for a maximum of 6, 5, 4, or 3 days.

[0031] The ratio of the first cell population and the second cell population in the final cell population may be 1:1.

[0032] The cells may be activated in step (ii) using anti-CD3 and anti-CD28 beads.

[0033] The chimeric antigen receptors may comprise:

[0034] a. an antigen-binding domain;

[0035] b. a spacer domain;

[0036] c. a transmembrane domain;d. a 4-1 BB endodomain; and

[0037] e. a CD3zeta endodomain.

[0038] Preferably, where the first CAR is an anti-CD19 CAR and the second CAR is an anti-CD22 CAR, the anti-CD19 CAR comprises the amino acid sequence of SEQ ID NO: 1 and the anti-CD22 CAR comprises the amino acid sequence of SEQ ID NO: 3.

[0039] The vector may be a gamma retroviral vector or a lentiviral vector.

[0040] The culture medium may contain human serum.

[0041] The culture medium may be exchanged during manufacture. Culture medium may be exchanged 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 times during expansion.

[0042] The process may additionally comprise a step of harvesting the cells, which may be conducted by any suitable method. The process may further comprise a washing and / or formulation step. Additionally, or alternatively, the process may further comprise a cryopreservation step.

[0043] The starting population of cells may be from any suitable source. The cells may be from a leukapheresis product (leukapheresate) from a patient or a donor. The cells may be derived from peripheral blood mononuclear cells (PBMCs).

[0044] The retroviral vector may be a gamma retroviral vector or a lentiviral vector. Preferably the retroviral vector is a lentiviral vector.

[0045] In a preferred embodiment, the first CAR is a CAR targeting CD 19. A particularly preferred CAR is described in WO2016 / 139487.

[0046] In a second aspect, the present invention relates to a population of engineered cells obtainable by the process of the first aspect.

[0047] In a third aspect, the present invention provides a pharmaceutical composition which comprises a population of engineered cells according to the second aspect.

[0048] The pharmaceutical composition according to the third aspect may be for use in treating and / or preventing a disease.In a fourth aspect, the present invention provides a method for treating and / or preventing a disease, which comprises the step of administering a pharmaceutical composition according to the third aspect to a subject in need thereof.

[0049] Also provided herein is the use of a pharmaceutical composition according to the third aspect in the manufacture of a medicament for the treatment and / or prevention of a disease.

[0050] The disease may be cancer. The disease may be a haematological malignancy. The disease may be a leukaemia or lymphoma. The disease may be a cytokine-driven disease. The disease may be an autoimmune disease.

[0051] The engineered cells according to the present invention may have increased expression of CCR7, CD45RA, CD27 and / or CD62L compared with engineered cells prepared by other methods.

[0052] Suitably, the engineered cells according to the present invention may be more naive than engineered cells prepared by other methods.

[0053] Suitably, the engineered cells according to the present invention may be less exhausted compared with engineered cells prepared by other methods.

[0054] The engineered cells in accordance with the present invention may have decreased expression of one or more exhaustion markers compared with engineered cells prepared by other methods.

[0055] The one or more exhaustion markers may be selected from the group consisting of: PD1, Lag3 and Tim3.

[0056] DETAILED DESCRIPTION

[0057] The present invention provides a method of preparing a population of engineered cells which are capable of targeting multiple antigens.

[0058] Chimeric antigen receptors have shown great promise in the treatment of haematological malignancies. Currently, most approved CAR-T therapies target theCD19 protein, which is expressed on B cells. Relapse due to antigen escape has been observed in several studies and there are ongoing efforts to address this issue. The targeting of a second antigen is one option for avoiding relapse due to antigen escape and several secondary targets are currently being investigated.

[0059] One option for a second target is CD22, the expression of which is, like CD19, restricted to the B-cell lineage. Several different strategies to co-target CD19 and CD22 are possible. One approach is to generate a tandem CAR (TanCAR) whereby both scFvs are co-expressed on the same CAR. An alternative strategy is to co-express two CARs on the same T-cell thus avoiding the need to link two scFvs. This can be achieved using a bicistronic construct. Alternatively, T-cells can be co-transduced with two vectors. Finally, CD19 and CD22CAR T cells can be generated separately and either pooled or infused sequentially.

[0060] Although dual-targeting shows promise, these products can suffer from poor persistence in some patients, leading to antigen-positive relapse. The optimum approach to dual targeting therefore still needs to be identified.

[0061] The mechanisms of early loss of CAR T cell persistence are poorly understood but likely to be multifactorial including the phenotype and intrinsic fitness of CAR T cells, activation induced cell death / exhaustion and immune mediated rejection. It is possible that higher CAR T expression and increased signalling in dual transduced CAR T cells may result in activation induced cell death (AICD) or exhaustion. Single cell CITESeq analysis of the CAR T cell products from 3 patients shows massive upregulation of FasL on dual transduced CAR T cells compared to single CD19 or CD22CAR T cells (Figure 2A). When we repetitively stimulated our CAR T cell products from 5 donors in vitro with CD19+CD22+ RAJI cells, we observed a progressive increase in early apoptotic (Annexin V+7-AAD-) cells specifically in the dual CAR transduced T cells in all donors consistent with AICD (Figure 2). There was no clear evidence of preferential increase in the exhaustion markers PD-1, LAG-3 and TIM-3 in dual CAR transduced T cells. These data imply that dual transduced CAR T cells may be more susceptible to activation induced cell death after repetitive antigenic exposure and suggests that coinfusion of separately transduced CAR T cells may be preferable to co-transduction.

[0062] Additionally, it has previously been shown that Stem Cell Memory T cells (TSCM) appear to be critical for long term CAR T cell persistence. We have shown that a shortened manufacturing methodology (5 days rather than 9) results in a CAR T cell productenriched for TSCM with equivalent transduction efficiency. Based on these findings, we have developed a GMP compliant manufacturing methodology, incorporating a shortened 5 day manufacture to enhance persistence and separate transduction with the 2 lentiviral vectors in order to reduce CAR T cell exhaustion / AICD (Figure 1).

[0063] CHIMERIC ANTIGEN RECEPTOR (CAR)

[0064] A classical chimeric antigen receptor (CAR) is a chimeric type I trans-membrane protein which connects an extracellular antigen-recognizing domain (binder) to an intracellular signalling domain (endodomain). The binder is typically a single-chain variable fragment (scFv) derived from a monoclonal antibody (mAb), but it can be based on other formats which comprise an antibody-like antigen binding site. A spacer domain is usually necessary to isolate the binder from the membrane and to allow it a suitable orientation. A common spacer domain used is the Fc of lgG1. More compact spacers can suffice e.g., the stalk from CD8a and even just the lgG1 hinge alone, depending on the antigen. A trans-membrane domain anchors the protein in the cell membrane and connects the spacer to the endodomain.

[0065] Early CAR designs had endodomains derived from the intracellular parts of either the y chain of the FCER1 or CD3 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 CD3 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 41 BB 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.

[0066] CAR-encoding nucleic acids may be transferred to T cells using, for example, retroviral vectors. Lentiviral vectors may be employed. In this way, a large number of antigenspecific cells can be generated for adoptive cell transfer. When a CAR binds the targetantigen, this results in the transmission of an activating signal to the T-cell it isexpressed on. Thus, the CAR directs the specificity and cytotoxicity of the T cell towards tumour cells expressing the targeted antigen.

[0067] CARs typically therefore comprise: (i) an antigen-binding domain; (ii) a spacer; (iii) a transmembrane domain; and (iii) an intracellular domain which comprises or associates with a signalling domain.

[0068] ANTIGEN BINDING DOMAIN

[0069] The antigen binding domain is the portion of the CAR which recognizes antigen.

[0070] Numerous antigen-binding domains are known in the art, including those based on the antigen binding site of an antibody, antibody mimetics, and T-cell receptors. For example, the antigen-binding domain may comprise: a single-chain variable fragment (scFv) derived from a monoclonal antibody; a natural ligand of the target antigen; a peptide with sufficient affinity for the target; a single domain antibody; an artificial single binder such as a Darpin (designed ankyrin repeat protein); or a single-chain derived from a T-cell receptor.

[0071] The antigen binding domain may comprise a domain which is not based on the antigen binding site of an antibody. For example the antigen binding domain may comprise a domain based on a protein / peptide which is a soluble ligand for a tumour cell surface receptor (e.g. a soluble peptide such as a cytokine or a chemokine); or an extracellular domain of a membrane anchored ligand or a receptor for which the binding pair counterpart is expressed on the tumour cell.

[0072] The antigen binding domain may be based on a natural ligand of the antigen.

[0073] The antigen binding domain may comprise an affinity peptide from a combinatorial library or a de novo designed affinity protein / peptide.

[0074] CD19

[0075] The human CD19 antigen is a 95 kd transmembrane glycoprotein belonging to the immunoglobulin superfamily. CD19 is classified as a type I transmembrane protein, with a single transmembrane domain, a cytoplasmic C-terminus, and extracellular N-terminus.CD19 is a biomarker for normal and neoplastic B cells, as well as follicular dendritic cells. In fact, it is present on B cells from earliest recognizable B-lineage cells during development to B-cell blasts but is lost on maturation to plasma cells. It primarily acts as a B cell co-receptor in conjunction with CD21 and CD81. Upon activation, the cytoplasmic tail of CD19 becomes phosphorylated, which leads to binding by Src-family kinases and recruitment of PI-3 kinase. CD19 is expressed very early in B-cell differentiation and is only lost at terminal B-cell differentiation into plasma cells. Consequently, CD19 is expressed on all B-cell malignancies apart from multiple myeloma.

[0076] A preferred CD19 binding domain is the CAT19 binding domain described in WO2016139487, the contents of which are hereby incorporated by reference. The CAR used in the present invention may preferably be the CAR described in WO2016139487. Preferably, the amino acid sequence of the anti-CD19 CAR is the sequence given in SEQ ID NO: 1. The nucleotide sequence for this CAR is provided in SEQ ID NO: 2.

[0077] CD22

[0078] The human CD22 antigen is a molecule belonging to the SIGLEC family of lectins. It is found on the surface of mature B cells and on some immature B cells. Generally speaking, CD22 is a regulatory molecule that prevents the overactivation of the immune system and the development of autoimmune diseases.

[0079] CD22 is a sugar binding transmembrane protein, which specifically binds sialic acid with an immunoglobulin (Ig) domain located at its N-terminus. The presence of Ig domains makes CD22 a member of the immunoglobulin superfamily. CD22 functions as an inhibitory receptor for B cell receptor (BCR) signaling.

[0080] CD22 is a molecule of the IgSF which may exist in two isoforms, one with seven domains and an intra-cytoplasmic tail comprising of three ITIMs (immune receptor tyrosine-based inhibitory motifs) and an ITAM; and a splicing variant which instead comprises of five extracellular domains and an intra-cytoplasmic tail carrying one ITIM. CD22 is thought to be an inhibitory receptor involved in the control of B-cell responses to antigen. Like CD19, CD22 is widely considered to be a pan-B antigen, although expression on some non-lymphoid tissue has been described. Targeting of CD22 with therapeutic monoclonal antibodies and immunoconjugates has entered clinical testing.Examples of anti-CD22 CARs are described by Haso et al. (Blood; 2013; 121(7)). Specifically, anti-CD22 CARs with antigen-binding domains derived from m971, HA22 and BL22 scFvs are described.

[0081] A preferred CD22 binding domain is the 9A8 binding domain described in WO2019220109, the contents of which are hereby incorporated by reference. The CAR used in the present invention may preferably be the CAR described in WO2019220109. Preferably, the amino acid sequence of the anti-CD22 CAR is the sequence given in SEQ ID NO: 3. The nucleotide sequence for this CAR is provided in SEQ ID NO: 4.

[0082] SPACER DOMAIN

[0083] The CAR may comprise a spacer sequence to connect the antigen-binding domain with the transmembrane domain and spatially separate the antigen-binding domain from the endodomain. A flexible spacer allows the antigen-binding domain to orient in different directions to facilitate binding.

[0084] The spacer sequence may, for example, comprise an lgG1 Fc region, an lgG1 hinge or a human CD8 stalk or the mouse CD8 stalk. The spacer may alternatively comprise an alternative linker sequence which has similar length and / or domain spacing properties as an lgG1 Fc region, an lgG1 hinge or a CD8 stalk. A human I gG 1 spacer may be altered to remove Fc binding motifs.

[0085] TRANSMEMBRANE DOMAIN

[0086] The transmembrane domain is the sequence of the CAR that spans the membrane.

[0087] A transmembrane domain may be any protein structure which is thermodynamically stable in a membrane. This is typically an alpha helix comprising of several hydrophobic residues. The transmembrane domain of any transmembrane protein can be used to supply the transmembrane portion of the invention.

[0088] The presence and span of a transmembrane domain of a protein can be predicted by those skilled in the art using bioinformatics tools such as the TMHMM algorithm (http: / / www.cbs. dtu.dk / services / TMHMM-2.0 / ). Further, given that the transmembrane domain of a protein is a relatively simple structure, i.e., a polypeptide sequencepredicted to form a hydrophobic alpha helix of sufficient length to span the membrane, an artificially designed TM domain may also be used (for example as described in US 7052906 B1 which is incorporated herein by reference).

[0089] The transmembrane domain may be derived from CD28, which gives good receptor stability.

[0090] ACTIVATING ENDODOMAIN

[0091] The endodomain is the signal-transmission portion of the CAR. It may be part of or associate with the intracellular domain of the CAR. After antigen recognition, receptors cluster, native CD45 and CD148 are excluded from the synapse and a signal is transmitted to the cell. The most commonly used endodomain component is that of CD3-zeta which contains 3 ITAMs. This transmits an activation signal to the T cell after antigen is bound. CD3-zeta may not provide a fully competent activation signal and additional co-stimulatory signalling may be needed. For example, chimeric CD28 and 0X40 can be used with CD3-Zeta to transmit a proliferative I survival signal, or all three can be used together.

[0092] Where a CAR comprises an activating endodomain, it may comprise the CD3-Zeta endodomain alone, the CD3-Zeta endodomain with that of either CD28 or 0X40 or the CD28 endodomain and 0X40 and CD3-Zeta endodomain.

[0093] Any endodomain which contains an ITAM motif can act as an activation endodomain.

[0094] SIGNAL PEPTIDE

[0095] The CARs of the cell of the present invention may comprise a signal peptide so that when the CAR is expressed inside a cell, such as a T-cell, the nascent protein is directed to the endoplasmic reticulum and subsequently to the cell surface, where it is expressed.

[0096] The core of the signal peptide may contain a long stretch of hydrophobic amino acids that has a tendency to form a single alpha-helix. The signal peptide may begin with a short positively charged stretch of amino acids, which helps to enforce proper topology of the polypeptide during translocation. At the end of the signal peptide there is typically a stretch of amino acids that is recognized and cleaved by signal peptidase. Signalpeptidase may cleave either during or after completion of translocation to generate a free signal peptide and a mature protein. The free signal peptides are then digested by specific proteases.

[0097] The signal peptide may be at the amino terminus of the molecule.

[0098] The signal peptide may comprise the SEQ ID No. 5, 6 or 7 or a variant thereof having 5, 4, 3, 2 or 1 amino acid mutations (insertions, substitutions or additions) provided that the signal peptide still functions to cause cell surface expression of the CAR.

[0099] SEQ ID No. 5: MGTSLLCWMALCLLGADHADG

[0100] The signal peptide of SEQ ID No. 5 is compact and highly efficient. It is predicted to give about 95% cleavage after the terminal glycine, giving efficient removal by signal peptidase.

[0101] SEQ ID No. 6: MSLPVTALLLPLALLLHAARP

[0102] The signal peptide of SEQ ID No. 6 is derived from lgG1.

[0103] SEQ ID No. 7: MAVPTQVLGLLLLWLTDARC

[0104] The signal peptide of SEQ ID No. 7 is derived from CD8.

[0105] The signal peptide for the anti-CD19 CAR may have a different sequence from the signal peptide of the anti-CD22 CAR.

[0106] CELL POPULATION

[0107] The present invention also relates to a population of engineered cells, obtainable (or obtained) by the process of the invention.

[0108] A “starting population of cells” as used herein refers to a sample of cells which will be used to produce engineered cells which comprise a CAR.

[0109] The starting population of cells may be obtained from any source of blood cells or peripheral blood mononuclear cells (PBMCs). The source cells may be provided freshor may be cryopreserved prior to use. The starting population of cells may be used without any further manipulation or may be used after an isolation or enrichment step. Methods for isolating or enriching white blood cells are known in the art. For example, white blood cells or PBMCs may be obtained from whole blood by various methods e.g., density gradient separation, such as using Ficoll-Paque density gradient media; by magnetic bead separation, such as MACS Milteyni Biotec CD3, CD4 or CD8 beads; by elutriation or any other method. Separation or isolation of cells may be automated or may be performed manually.

[0110] The starting population of cells may be from blood e.g., from a peripheral blood sample or from a biopsy. The starting population of cells may be peripheral blood mononuclear cells. The starting population of cells may be a leukapheresate.

[0111] Suitably, the starting population of cells may be obtained from the subject (1stparty). Suitably, the starting population of cells may be obtained from a donor (2ndparty). Suitably, the starting population of cells may be obtained from a donor who is an unconnected donor (3rdparty).

[0112] Alternatively, the cells may be derived from ex vivo differentiation of inducible progenitor cells or embryonic progenitor cells to, for example, T cells. Alternatively, an immortalized cell line which retains its lytic function and could act as a therapeutic may be used.

[0113] Suitably, the starting population may be whole blood obtained from the subject. Suitably, the starting population may be PBMCs obtained from the subject. Suitably, the starting population may be a leukapheresate obtained from the subject.

[0114] Suitably, the starting population may be whole blood obtained from a donor. Suitably, the starting population may be PBMCs obtained from a donor. Suitably, the starting population may be a leukapheresate obtained from a donor.

[0115] “Transduced or transfected cells” as used herein refers to the cell population which has undergone the transduction or transfection process. This population of cells may contain a mixture of cells which have been successfully genetically modified and those which have not.A “genetically modified cell” or “engineered cell” as used herein means a cell which has been modified to comprise or express a CAR. Methods for engineering cells are known in the art and include but are not limited to genetic modification of cells e.g., by transduction such as gammaretroviral or lentiviral transduction, transfection (such as transient transfection - DNA or RNA based) including lipofection, polyethylene glycol, calcium phosphate and electroporation. Any suitable method may be used to introduce a nucleic acid sequence into a cell which encodes a CAR.

[0116] Suitably, a genetically modified cell is a cell whose genome has been modified e.g., by transduction or by transfection. Suitably, a genetically modified cell is a cell whose genome has been modified by gammaretroviral transduction. Suitably, a genetically modified cell is a cell whose genome has been modified by lentiviral transduction.

[0117] As used herein, the term “introduced” refers to methods for inserting foreign DNA or RNA into a cell. As used herein the term introduced includes both transduction and transfection methods. Transfection is the process of introducing nucleic acids into a cell by non-viral methods. Transduction is the process of introducing foreign DNA or RNA into a cell via a viral vector.

[0118] Genetically modified cells according to the invention may be generated by introducing DNA or RNA coding for the CAR by one of many means including transduction with a viral vector, transfection with DNA or RNA.

[0119] Cells may be activated and / or expanded prior to the introduction of a nucleic acid sequence encoding a CAR, for example by treatment with an anti-CD3 monoclonal antibody or both anti-CD3 and anti-CD28 monoclonal antibodies.

[0120] Suitably, the genetically modified cell may be autologous.

[0121] Suitably, the cell may be allogeneic.

[0122] In one embodiment, the genetically modified cell may be a PBMC.

[0123] Suitably, the genetically modified cell may be a B cell. Suitably the genetically modified cell may be an NK cell. Suitably the genetically modified cell may be a T cell.

[0124] The genetically modified cell may be a cytolytic immune cell.“Cytolytic immune cell” as used herein is a cell which directly kills other cells. Cytolytic cells may kill cancerous cells; virally infected cells or other damaged cells. Cytolytic immune cells include T cells and Natural killer (NK) cells.

[0125] Cytolytic immune cells can be T cells or T lymphocytes which are a type of lymphocyte that play a central role in cell-mediated immunity. T cells can be distinguished from other lymphocytes, such as B cells and NK cells, by the presence of a TCR on their cell surface. There are various types of T cell, as summarised below.

[0126] Helper T 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, TH 17, Th9, orTFH, which secrete different cytokines to facilitate different types of immune responses.

[0127] 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. CTLs may be known as CD8+ T cells. 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.

[0128] 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.

[0129] 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.

[0130] Two major classes of CD4+ Treg cells have been described — naturally occurring Treg cells and adaptive or induced Treg cells.

[0131] 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 (CD11c+) and plasmacytoid (CD123+) dendritic cells that have been activated with TSLP. Naturally occurring T reg 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.

[0132] As used herein, the term “natural T reg” means a thymus-derived Treg. Natural T regs are CD4+CD25+FOXP3+ Helios+ Neuropilin 1+. Compared with iTregs, nTregs have increased expression of PD-1 (programmed cell death-1, pdcdl), neuropilin 1 (Nrp1), Helios (Ikzf2), and CD73. nTregs may be distinguished from iTregs on the basis of the expression of Helios protein or Neuropilin 1 (Nrp1) individually.

[0133] Adaptive Treg cells (also known as Tr1 cells orTh3 cells) may originate during a normal immune response.

[0134] Peripherally generated Tregs may be referred to as induced Treg (iTreg) cells.

[0135] As used herein, the term “induced regulatory T cell” (iTreg) means a CD4+ CD25+ FOXP3+ Helios- Neuropilin 1- T cell which develops from mature CD4+ conventional T cells outside of the thymus. For example, iTregs can be induced in vitro from CD4+ CD25-FOXP3- cells in the presence of IL-2 and TGF-p.

[0136] Suitably the cell may be a T cell. Suitably the cell may be a helper T cell. Suitably the cell may be a cytolytic T cell. Suitably the cell may be a memory T cell. Suitably the cell may be a regulatory T cell (Treg). Suitably the cell may be a naturally occurring Treg or an adaptive Treg.

[0137] Natural Killer Cells (or NK cells) are a type of cytolytic cell which form part of the innate immune system. NK cells provide rapid responses to innate signals from virally infected cells in an MHC independent manner.NK cells (belonging to the group of innate lymphoid cells) are defined as large granular lymphocytes (LGL) and constitute the third kind of cells differentiated from the common lymphoid progenitor generating B and T lymphocytes. NK cells are known to differentiate and mature in the bone marrow, lymph node, spleen, tonsils, and thymus where they then enter into the circulation.

[0138] Suitably the cell may be a natural killer cell.

[0139] Suitably, the cell may be a stem cell.

[0140] In one embodiment, the cell may be a progenitor cell.

[0141] As used herein, the term “stem cell” means an undifferentiated cell which is capable of indefinitely giving rise to more stem cells of the same type, and from which other, specialised cells may arise by differentiation. Stem cells are multipotent. Stem cells may be for example, embryonic stem cells or adult stem cells.

[0142] As used herein, the term “progenitor cell” means a cell which is able to differentiate to form one or more types of cells but has limited self-renewal in vitro.

[0143] Suitably, the cell may be any cell capable of differentiating into a cytolytic immune cell.

[0144] Suitably, the cell may be capable of being differentiated into a T cell or NK cell.

[0145] Suitably, the cell may be an embryonic stem cell (ESC). Suitably, the cell may be a haematopoietic stem cell or haematopoietic progenitor cell. Suitably, the cell may be an induced pluripotent stem cell (iPSC). Suitably, the cell may be obtained from umbilical cord blood. Suitably, the cell may be obtained from adult peripheral blood.

[0146] In some aspects, hematopoietic stem and progenitor cell (HSPCs) may be obtained from umbilical cord blood. Cord blood can be harvested according to techniques known in the art (e.g., U.S. Pat. Nos. 7,147,626 and 7,131,958 which are incorporated herein by reference).

[0147] In one aspect, HSPCs may be obtained from pluripotent stem cell sources, e.g., induced pluripotent stem cells (iPSCs) and embryonic stem cells (ESCs).As used herein, the term “hematopoietic stem and progenitor cell” or “HSPC” refers to a cell which expresses the antigenic marker CD34 (CD34+) and populations of such cells. In particular embodiments, the term “HSPC” refers to a cell identified by the presence of the antigenic marker CD34 (CD34+) and the absence of lineage (lin) markers. The population of cells comprising CD34+ and / or Lin(-) cells includes haematopoietic stem cells and hematopoietic progenitor cells.

[0148] HSPCs can be obtained or isolated from bone marrow of adults, which includes femurs, hip, ribs, sternum, and other bones. Bone marrow aspirates containing HSPCs can be obtained or isolated directly from the hip using a needle and syringe. Other sources of HSPCs include umbilical cord blood, placental blood, mobilized peripheral blood, Wharton's jelly, placenta, fetal blood, fetal liver, or fetal spleen. In particular embodiments, harvesting a sufficient quantity of HSPCs for use in therapeutic applications may require mobilizing the stem and progenitor cells in the subject.

[0149] As used herein, the term “induced pluripotent stem cell” or “iPSC” refers to a non-pluripotent cell that has been reprogrammed to a pluripotent state. Once the cells of a subject have been reprogrammed to a pluripotent state, the cells can then be programmed to a desired cell type, such as a hematopoietic stem or progenitor cell (HSC and HPC respectively).

[0150] As used herein, the term “reprogramming” refers to a method of increasing the potency of a cell to a less differentiated state.

[0151] As used herein, the term “programming” refers to a method of decreasing the potency of a cell or differentiating the cell to a more differentiated state.

[0152] The cells of the invention may be any of the cell types mentioned above.

[0153] A “population of genetically modified cells” as used herein means one or more genetically modified cells according to the present invention.

[0154] Suitably a population of genetically modified cells as used herein may mean two or more (or a plurality of) genetically modified cells according to the present invention.Optionally, the method may additionally comprise isolating a cell containing sample from a subject. This cell containing sample may be used as the staring population of cells.

[0155] Optionally, the cells for use in the present invention may be activated and / or expanded prior to the introduction of a nucleic acid sequence which encodes a CAR.

[0156] Any method known in the art for activating and / or expanding cells may be used in the method of the invention. For example, cells for use in the present invention e.g., T cells may be activated and / or expanded by treatment with an anti-CD3 monoclonal antibody or both anti-CD3 and anti-CD28 monoclonal antibodies.

[0157] As used herein “activated” means that a cell has been stimulated, causing the cell to proliferate, differentiate or initiate an effector function.

[0158] Methods for measuring cell activation are known in the art and include, for example, measuring the expression of activation markers by flow cytometry, such as the expression of CD69, CD25, CD71, CD38 or HLA-DR or measuring intracellular cytokines.

[0159] As used herein “expanded” means that a cell or population of cells has been induced to proliferate.

[0160] The expansion of a population of cells may be measured for example by counting the number of cells present in a population. The phenotype of the cells may be determined by methods known in the art such as flow cytometry.

[0161] In one aspect, the method according to the present invention produces a population of engineered cells (e.g., genetically modified cells) which comprise a chimeric antigen receptor or a transgenic T-cell receptor.

[0162] Suitably a genetically modified cell or population of genetically modified cells according to the present invention may be made by the method according to the invention.

[0163] As used herein “differentiated” refers to the stage of development of a particular cell within the linear progression of differentiation of that cell type. For example, CD4+ and CD8+ T cells can be categorized into distinct memory subsets based on theirdifferentiation states. CD4+ and CD8+ T cells follow a progressive pathway of differentiation from naive T cells into central memory and effector memory cell populations. The differentiation state of CD8+ T cells is inversely related to their capacity to proliferate and persist.

[0164] Preclinical studies suggest that improved antitumor responses are achieved when genetically modified T cells are in the early stages of differentiation (such as naive or central memory cells). Central memory cells have improved in vivo persistence compared with effector memory cells.

[0165] In one aspect, the population of genetically modified cells according to the invention or obtainable by a method according to the invention are more naive than genetically modified cells which were not prepared according to a method of the invention.

[0166] As used herein, “naive” means a cell which is not fully differentiated. A naive T cell may not have encountered antigen.

[0167] Naive T cells may be characterised by the surface expression of L selection (CD62L), the absence of activation markers CD25, CD71, CD44 or CD69 and the absence of memory CD45RO isoform e.g., naive T cells may be CD62LHiCD25LoCD44LoCD69Lo. Naive T cells also express functional IL-7 receptors, consisting of subunits IL-7 receptor-a, CD127, and common-y chain, CD132.

[0168] In one aspect, a naive cell subset may be defined as CCR7+ / CD45RA+ cells. Suitably, a naive cell subset may be further defined as CCR7+ / CD45RA+ / CD62L+ / CD27+ cells.

[0169] Suitably, the genetically modified cells according to the present invention or obtainable (e.g., obtained) by a method according to the present invention may have increased expression of CD27 and / or CD62L compared with genetically modified cells which were not prepared according to a method of the present invention.

[0170] Without wishing to be bound by theory, a more naive or immature genetically modified cell population is advantageous for use in therapy because naive cells exhibit enhanced persistence in vivo and enhanced cytolytic activity when compared to cells with a more differentiated phenotype.Suitably, the method according to the present invention may produce more naive or central memory cells than prior methods. Suitably, at least 75% of the genetically modified cells may be naive or central memory cells. Suitably, at least 80% of the genetically modified cells may be naive or central memory cells. Suitably, at least 85% of the genetically modified cells may be naive or central memory cells.

[0171] Suitably, the method according to the present invention may produce fewer effector and effector memory cells than prior methods. Suitably, fewer than 25% of the genetically modified cells may be effector or effector memory cells. Suitably, fewer than 20% of the genetically modified cells may be effector or effector memory cells. Suitably, fewer than 15% of the genetically modified cells may be effector or effector memory cells.

[0172] In one aspect, the population of genetically modified cells according to the present invention or obtainable (or obtained) by a method according to the present invention are less exhausted compared with genetically modified cells which were not prepared according to a method of the invention.

[0173] As used herein “exhaustion” or “exhausted” means that the cell exhibits decreased effector functions and / or altered phenotype. Immune cell exhaustion describes the status of dysfunction of immune cells, usually under the setting of tumours or chronic infection. Exhaustion may be accompanied by phenotypic changes, epigenetic modifications, and alterations in transcriptional profiles.

[0174] Effector functions may include the production of effector cytokines and direct cytotoxic activity.

[0175] Suitably the population of genetically modified cells according to the present invention or obtainable (or obtained) by a method according to the present invention may have decreased expression of one or more exhaustion markers compared with genetically modified cells which were not prepared according to a method of the present invention.

[0176] Suitably, one or more exhaustion markers may be two exhaustion markers. Suitably, one or more exhaustion markers may be three exhaustion markers. Suitably, one or more exhaustion markers may be four exhaustion markers. Suitably, one or more exhaustion markers may be five exhaustion markers. Suitably, one or more exhaustionmarkers may be six exhaustion markers. Suitably, one or more exhaustion markers may be seven exhaustion markers.

[0177] For example, in the context of NK cells, effector functions may include production of interferon gamma (IFN-y). Other effector functions of NK cells include direct cytotoxic activity, such as activity dependent on perforin and granzyme, or induction of target cell apoptosis by tumour necrosis factor alpha (TNF-a), Fas ligand (FasL) and TNF-related apoptosis-inducing ligand (TRAIL).

[0178] Suitably, exhausted NK cells may produce decreased amounts of effector cytokines e.g., IFN-y, compared with non-exhausted NK cells. Suitably, exhausted NK cells may have decreased cytolytic activity and may, for example, produce decreased amounts of CD107a and / or granzyme B and / or perforin compared with non-exhausted NK cells.

[0179] Suitably the one or more exhaustion markers may be selected from the group consisting of: IFN-y, TNF-a, FasL, TRAIL, CD107a, granzyme B and perforin. Suitably the one or more exhaustion markers may be selected from the group consisting of: IFN-y, TNF-a, FasL, TRAIL, CD107a, granzyme B and perforin wherein the genetically modified cell is an NK cell.

[0180] Suitably, the one or more exhaustion markers may comprise decreased IFN-y production. Suitably, the one or more exhaustion markers may comprise decreased TNF-a production. Suitably, the one or more exhaustion markers may comprise decreased expression of FASL. Suitably, the one or more exhaustion markers may comprise decreased expression of TRAIL. Suitably, the one or more exhaustion markers may comprise decreased expression of CD 107a. Suitably, the one or more exhaustion markers may comprise decreased production of granzyme B. Suitably, the one or more exhaustion markers may comprise decreased production of perforin.

[0181] For example, in the context of T cells, exhaustion may be defined by poor effector function, sustained expression of inhibitory receptors and / or a transcriptional state distinct from that of functional effector or memory T cells. For example, exhausted T cells may express high levels of PD1, Tim3, Lag3, CD43 (1B11), TIGIT, CD69 and inhibitory receptors but low levels of CD62L and CD127 and decreased interleukin-2 (IL-2), TNF-a and IFN-y production.Suitably, the one or more exhaustion markers may comprise increased (e.g., high) expression of PD1. Suitably, the one or more exhaustion markers may comprise increased (e.g., high) expression of Tim3. Suitably, the one or more exhaustion markers may comprise increased (e.g., high) expression of Lag3 orTIGIT. Suitably, the one or more exhaustion markers may comprise increased (e.g., high) expression of CD43 (1B11). Suitably, the one or more exhaustion markers may comprise increased (e.g., high) expression of CD69. Suitably, the one or more exhaustion markers may comprise increased (e.g., high) expression of inhibitory receptors. Suitably, the one or more exhaustion markers may comprise decreased (e.g., low) expression of CD62L. Suitably, the one or more exhaustion markers may comprise decreased (e.g., low) expression of CD127. Suitably, the one or more exhaustion markers may comprise decreased (e.g., low) IL-2 production upon target encounter. Suitably, the one or more exhaustion markers may comprise decreased (e.g., low) TNF-a production upon target encounter. Suitably, the one or more exhaustion markers may comprise decreased (e.g., low) IFN-y production upon target encounter.

[0182] Suitably, the one or more exhaustion markers may be selected from the group consisting of: PD1, Lag3, Tim3 and TIGIT. Suitably, the one or more exhaustion markers may comprise PD1. Suitably, the one or more exhaustion markers may comprise Lag3. Suitably, the one or more exhaustion markers may comprise Tim3. Suitably, the one or more exhaustion markers may be selected from the group consisting of: PD1, Lag3 and Tim3 wherein the genetically modified cell is a T cell.

[0183] In one aspect, more than 10%, more than 15%, more than 20%, more than 25%, more than 30%, or more than 35% of the population of genetically modified cells according to the present invention or obtainable (or obtained) by a method according to the present invention may be naive (CCCR7+ / CD45RA+) and CD62L+ / CD27+. Suitably more than 10% of the genetically modified cells according to the invention may be naive (CCCR7+ / CD45RA+) and CD62L+ / CD27+. Suitably more than 15% of the genetically modified cells according to the invention may be naive (CCCR7+ / CD45RA+) and CD62L+ / CD27+. Suitably more than 20% of the genetically modified cells according to the invention may be naive (CCCR7+ / CD45RA+) and CD62L+ / CD27+. Suitably more than 30% of the genetically modified cells according to the invention may be naive (CCCR7+ / CD45RA+) and CD62L+ / CD27+. Suitably more than 40% of the genetically modified cells according to the invention may be naive (CCCR7+ / CD45RA+) and CD62L+ / CD27+.Suitably, the proportion of naive cells may be measured in the CD8+ T cell subset.

[0184] In one aspect, fewer than 30%, fewer than 25%, fewer than 20%, fewer than 15%, fewer than 10%, fewer than 5% of the population of genetically modified cells according to the present invention or obtainable (or obtained) by a method according to the present invention may express multiple exhaustion markers. Suitably fewer than 30% of the genetically modified cells according to the invention may exhibit multiple exhaustion markers. Suitably fewer than 25% of the genetically modified cells according to the invention may exhibit multiple exhaustion markers. Suitably fewer than 20% of the genetically modified cells according to the invention may exhibit multiple exhaustion markers. Suitably fewer than 15% of the genetically modified cells according to the invention may exhibit multiple exhaustion markers.

[0185] Suitably the multiple exhaustion markers may be selected from increased expression (e.g., high levels) of PD1, Tim3, Lag3, CD43 (1B11), CD69 and inhibitory receptors and decreased expression of (e.g., low levels) of CD62L and CD127 and decreased (e.g., low) interleukin-2 (IL-2), TNF-a and IFN-y production. Suitably the multiple exhaustion markers may be selected from increased expression of (e.g., high levels) of Lag3, PD1 and Tim3.

[0186] In one aspect, the population of genetically modified cells according to the present invention or obtainable (or obtained) by a method according to the present invention have increased levels of CAR transduction efficiency compared with genetically modified cells which were not prepared according to a method of the present.

[0187] Suitably, the level of transduction efficiency in the population of genetically modified cells according to the present invention or obtainable (or obtained) by a method according to the present invention may be at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% higher than transduction efficiency of genetically modified cells which were not prepared according to a method of the present.

[0188] Suitably, the level of transduction efficiency in the population of genetically modified cells according to the present invention or obtainable (or obtained) by a method according to the present invention may be at least 50% higher than transduction efficiency of genetically modified cells which were not prepared according to a method of the present invention. Suitably, the level of transduction efficiency in the population of genetically modified cells according to the present invention or obtainable (orobtained) by a method according to the present invention may be at least 60% higher than transduction efficiency of genetically modified cells which were not prepared according to a method of the present invention. Suitably, the level of transduction efficiency in the population of genetically modified cells according to the present invention or obtainable (or obtained) by a method according to the present invention may be at least 70% higher than transduction efficiency of genetically modified cells which were not prepared according to a method of the present invention. Suitably, the level of transduction efficiency in the population of genetically modified cells according to the present invention or obtainable (or obtained) by a method according to the present invention may be at least 80% higher than transduction efficiency of genetically modified cells which were not prepared according to a method of the present invention. Suitably, the level of transduction efficiency in the population of genetically modified cells according to the present invention or obtainable (or obtained) by a method according to the present invention may be at least 90% higher than transduction efficiency of genetically modified cells which were not prepared according to a method of the present invention.

[0189] In one aspect, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50% or at least 60% of the population of the transduced or transfected cells comprise a CAR. Suitably at least 40% of the population of transduced or transfected cells comprises a CAR. Suitably at least 50% of the population of the transduced or transfected cells comprise a CAR. Suitably at least 60% of the population of transduced or transfected cells comprises a CAR.

[0190] METHOD

[0191] There is provided a method of preparing a population of genetically modified cells which comprise a chimeric antigen receptor (CAR) or a transgenic T-cell receptor (TCR).

[0192] Methods of preparing a population of genetically modified cells for cellular therapy are known generally in the art. Methods of preparing genetically modified cells for cellular therapy may include some or all of the following steps:

[0193] The starting material may initially be frozen e.g., the starting population of cells may be frozen once it is obtained from the donor e.g., source of cells. If frozen material is used then thawing and an optional rest period may occur before proceeding to the next step. Alternatively, fresh starting material may be used. The starting material may undergo initial purification / enrichment for white cells (e.g., Ficoll gradient) or for T cells.The starting material may then be activated e.g., the T cells may be activated. This may be performed by any methods known in the art e.g., using soluble CD3 / CD28 antibodies, or CD3 / CD28 beads (e.g., Dynabeads), or CD3 / 28 nanomatrix (e.g TransAct). As is understood in the art, the length of the activation step may be varied e.g., from under an hour to beyond 72 hours before proceeding to the next step.

[0194] The activated cells may then be transduced with the viral vector (e.g., gammaretroviral or lentiviral). This may be done in the presence of a transduction enhancer (e.g., retronectin, or polybrene), or by spinoculation or by simple incubation. Non-viral vectors may also be used for the genetic modification step (e.g., using RNA electroporation or transposition using DNA).

[0195] The cells may then undergo an expansion step that may last from hours to several days, depending on the final dose of cells required. Generally, the more cells required, the longer the expansion step.

[0196] The cells at the end of the manufacturing process may be used fresh, or may be frozen before use.

[0197] The overall process may therefore take from 2 to 18 days. Typically, the overall process takes 2-6 days.

[0198] During the process the cells may be cultured in cell growth medium that may contain additional supplements. Such supplements may be human serum, fetal bovine serum, and human serum albumin.

[0199] “MOI7”multiplicity of infection” as used herein indicates the number of infectious vector particles per cell used in transduction. For example, a MOI of 1 means the addition of 104infectious vector particles to 104cells. The number of infectious particles is obtained by titration of the viral vector on a permissive cell line or primary cells.

[0200] As is known in the art, the use of transduction enhancers may alter the MOI required for transduction e.g., may lower the MOI required for transduction. Transduction enhancers are known in the art, such as VectoFusin or RetroNectin.

[0201] Suitably, the MOI may be chosen to achieve about 10-95% transduction. Suitably, the MOI may be chosen to achieve about 15-90% transduction. Suitably, the MOI may be chosen to achieve about 20-85% transduction.

[0202] Suitably, the MOI may be about 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.PHARMACEUTICAL COMPOSITION

[0203] The present invention also relates to a pharmaceutical composition comprising a genetically modified cell of the invention or a population of genetically modified cells according to the invention.

[0204] In one aspect, there is provided a pharmaceutical composition which comprises a population of genetically modified cells according to the present invention or obtainable by a method according to the present invention.

[0205] Suitably, the pharmaceutical composition may comprise cryopreserved genetically modified cells according to the present invention or obtainable by a method according to the present invention.

[0206] The pharmaceutical composition may additionally comprise a pharmaceutically acceptable carrier, diluent, or excipient. The pharmaceutical composition may optionally comprise one or more further pharmaceutically active polypeptides and / or compounds. Such a formulation may, for example, be in a form suitable for intravenous infusion.

[0207] In one aspect, the present invention provides a method of preparing a pharmaceutical composition comprising:

[0208] i) providing a starting population of cells;

[0209] ii) activating the starting population of cells;

[0210] iii) splitting the starting population of cells into a first cell population and a second cell population;

[0211] iv) transducing the first cell population with a first vector encoding a first chimeric antigen receptor and transducing the second cell population with a second vector encoding a second chimeric antigen receptor;

[0212] v) separately expanding the first cell population and the second cell population in a culture medium for a maximum of 6 days, or until a target dose is reached;

[0213] vi) combining the first cell population and the second cell population to form a final engineered cell population.In a preferred embodiment, the first chimeric antigen receptor is an anti-CD19 chimeric antigen receptor and the second chimeric antigen receptor is an anti-CD22 chimeric antigen receptor.

[0214] The cells may be activated and / or expanded by any method known in the art, for example by treatment with an anti-CD3 monoclonal antibody or both anti-CD3 and anti-CD28 monoclonal antibodies.

[0215] Suitably, the starting population of cells may previously have been frozen. If frozen cells are used, then the cells may be thawed and optionally, may be allowed to recover in culture before being processed.

[0216] The method may additionally comprise a step of enriching the starting population for white blood cells. Any methods for isolating or enriching white blood cells known in the art may be used. For example, white blood cells or PBMCs may be obtained from whole blood by various methods e.g., density gradient separation, such as using Ficoll-Paque density gradient media; by magnetic bead separation, such as MACS Milteyni Biotec CD3, CD4 or CD8 beads; by elutriation or any other method. Separation or isolation of cells may be automated or may be performed manually.

[0217] Suitably, the CAR may be introduced into the cell by transduction or transfection. Suitably, the CAR may be introduced into the cell by transduction and the multiplicity of infection may be chosen to be sufficient to transduce cells.

[0218] Successful transduction or transfection of a cell with a nucleic acid encoding a CAR may be identified by methods known in the art, for example by flow cytometry.

[0219] In one aspect, the population of genetically modified cells according to the present invention is the active ingredient of the pharmaceutical composition.

[0220] As will be understood by those skilled in the art, a pharmaceutical composition may additionally comprise impurities. In the context of the present invention, an impurity may be a cell which expresses the target antigen. Suitably, in the context of the present invention, an impurity may be a cell which expresses the target antigen and which expresses the CAR. In the context of the present invention, an impurity may be a cell which does not express the CAR.Suitably, a pharmaceutical composition according to the present invention comprises a population of cells wherein less than 10% of the cells are impurities. Suitably, a pharmaceutical composition according to the present invention comprises a population of cells wherein less than 5% of the cells are impurities. Suitably, a pharmaceutical composition according to the present invention comprises a population of cells wherein less than 3% of the cells are impurities. Suitably, a pharmaceutical composition according to the present invention comprises a population of cells wherein less than 2% of the cells are impurities. Suitably, a pharmaceutical composition according to the present invention comprises a population of cells wherein less than 1 % of the cells are impurities.

[0221] METHOD OF TREATMENT

[0222] The genetically modified cells of the present invention may be capable of killing target cells, such as cancer cells, virally infected cells, or other damaged cells.

[0223] The genetically modified cells of the present invention may be used in therapy. The genetically modified cells of the present invention may be used for the treatment and / or prevention of disease. Suitably, a pharmaceutical composition comprising genetically modified cells according to the present invention may be used in therapy. Suitably, a pharmaceutical composition comprising genetically modified cells according to the present invention may be used for the treatment and / or prevention of disease.

[0224] It will be understood that the target antigen of the CAR will be chosen based on the required therapy. For example, if the CAR is for treating cancer, the target antigen of the CAR may be an antigen associated with cancer.

[0225] The genetically modified cells of the present invention may be used for the treatment of an infection, such as a viral infection.

[0226] The genetically modified cells of the invention may also be used for the control of pathogenic immune responses, for example in autoimmune diseases, allergies, and graft-vs-host rejection. Autoimmune conditions include but are not limited to celiac disease, post-infectious IBS, diabetes mellitus type 1, Henoch-Schbnlein purpura (HSP) sarcoidosis, systemic lupus erythematosus (SLE), Sjogren syndrome, eosinophilic granulomatosis with polyangiitis, Hashimoto's thyroiditis, Graves' disease, idiopathic thrombocytopenic purpura, Addison's disease, rheumatoid arthritis (RA),ankylosing spondylitis, polymyositis (PM), dermatomyositis (DM), and multiple sclerosis (MS)

[0227] The present invention provides a method for treating and / or preventing a disease which comprises the step of administering a genetically modified cell of the present invention to a subject.

[0228] The present invention provides a method for treating and / or preventing a disease which comprises the step of administering a pharmaceutical composition of the present invention to a subject.

[0229] The present invention also provides a genetically modified cell of the present invention for use in treating and / or preventing a disease.

[0230] The present invention also provides a pharmaceutical composition of the present invention for use in treating and / or preventing a disease.

[0231] The invention also relates to the use of a genetically modified cell according to the present invention in the manufacture of a medicament for treating and / or preventing a disease.

[0232] Suitably, the present methods of treatment may relate to the administration of a pharmaceutical composition of the present invention to a subject.

[0233] Suitably, the present invention provides a method of treatment comprising:

[0234] a) providing a starting population comprising at least 400 million cells; b) activating the cells;

[0235] c) transducing the starting population with a retroviral vector carrying a transgene of interest;

[0236] d) expanding the transduced cells in a culture medium for a maximum of 6 days, or until a target dose is reached; and

[0237] e) administering the cells from (d) to a subject.

[0238] Suitably, the method may additionally comprise a cell expansion step before administration to the patient e.g., the cells may be cultured before administration to the patient.The genetically modified cells or pharmaceutical composition of the present invention may be used for the treatment and / or prevention of a cancerous disease, such as a haematological malignancy, bladder cancer, breast cancer, colon cancer, endometrial cancer, kidney cancer (renal cell), lung cancer, melanoma, pancreatic cancer, prostate cancer and thyroid cancer, cancers of the oral cavity and pharynx which includes cancer of the tongue, mouth and pharynx; cancers of the digestive system which includes oesophageal, gastric and colorectal cancers; cancers of the liver and biliary tree which includes hepatocellular carcinomas and cholangiocarcinomas; cancers of the respiratory system which includes bronchogenic cancers and cancers of the larynx; cancers of bone and joints which includes osteosarcoma; cancers of the skin which includes melanoma; breast cancer; cancers of the genital tract which include uterine, ovarian and cervical cancer in women, prostate and testicular cancer in men; cancers of the renal tract which include renal cell carcinoma and transitional cell carcinomas of the utterers or bladder; brain cancers including gliomas, glioblastoma multiforme and medullobastomas; cancers of the endocrine system including thyroid cancer, adrenal carcinoma and cancers associated with multiple endocrine neoplasm syndromes; and cancers of other and unspecified sites including neuroblastoma.

[0239] Suitably, the genetically modified cells or pharmaceutical composition of the present invention may be used for the treatment and / or prevention of a haematological malignancy.

[0240] As used herein, “haematological malignancy” refers to a cancer which affects the blood and lymph system and includes leukaemia, lymphoma, myeloma, and related blood disorders.

[0241] Suitably, the genetically modified cells or pharmaceutical composition of the present invention may be used for the treatment and / or prevention of a haematological malignancy.

[0242] Suitably, the genetically modified cells or pharmaceutical composition of the present invention may be used in the treatment and / or prevention of leukaemias both acute and chronic, myeloid or lymphoid including: acute lymphoblastic leukaemia (ALL), acute myeloid leukaemia (AML), acute premyelocytic leukaemia (APL), and B- or T-cell acute lympoblastic leukaemia (B-ALL or T-ALL respectively), chronic lymphocytic leukaemia (CLL), chronic myeloid leukaemia (CML), chronic myelomonocytic leukaemia (CMML), hairy cell leukaemia (HCL) and large granular lymphocytic leukaemia (LGLL);lymphomas including Hodgkin's lymphoma and non-Hodgkin lymphoma (NHL), both Low-grade NHL and High-grade NHL; myeloma (Multiple Myeloma (MM)), including: smouldering or asymptomatic myeloma and symptomatic myeloma and other conditions related to blood cancer such as monoclonal gammopathy of undetermined significance (MGUS), myelodysplastic syndromes (MDS), solitary plasmacytoma, and myeloproliferative neoplasms (MPN), including essential thrombocythaemia (ET), myelofibrosis (MF), and polycythaemia vera (PV).

[0243] Suitably, the genetically modified cells or pharmaceutical composition of the present invention may be used in the treatment and / or prevention of a T cell lymphoma. Suitably, the genetically modified cells or pharmaceutical composition of the present invention may be used in the treatment and / or prevention of a T cell leukaemia.

[0244] A method for treating a T-cell lymphoma and / or leukaemia relates to the therapeutic use of an genetically modified cell of the invention. The genetically modified cell may be administered to a subject having an existing disease of T-cell lymphoma and / or leukaemia in order to lessen, reduce or improve at least one symptom associated with the disease and / or to slow down, reduce or block the progression of the disease.

[0245] The following is an illustrative, non-exhaustive list of diseases which may be treated by the method of the present invention.

[0246] Suitably, the T-cell lymphoma or leukaemia may be peripheral T-cell lymphoma, not otherwise specified (PTCL-NOS). Suitably, the T-cell lymphoma or leukaemia may be angio-immunoblastic T-cell lymphoma (AITL). Suitably, the T-cell lymphoma or leukaemia may be anaplastic large cell lymphoma (ALCL). Suitably, the T-cell lymphoma or leukaemia may be enteropathy-associated T-cell lymphoma (EATL). Suitably, the T-cell lymphoma or leukaemia may be hepatosplenic T-cell lymphoma (HSTL). Suitably, the T-cell lymphoma or leukaemia may be extranodal NK / T-cell lymphoma nasal type. Suitably, the T-cell lymphoma or leukaemia may be cutaneous T-cell lymphoma (CTCL). Suitably, the T-cell lymphoma or leukaemia may be primary cutaneous (ALCL). Suitably, the T-cell lymphoma or leukaemia may be T cell prolymphocytic leukaemia. Suitably, the T-cell lymphoma or leukaemia may be T-cell acute lymphoblastic leukaemia.Treatment with the genetically modified cells of the present invention or pharmaceutical composition according to the present invention may help prevent the escape or release of tumour cells which often occurs with standard approaches.

[0247] The term “treat / treatment / treating” refers to administering a genetically modified cell, population of genetically modified cells, or pharmaceutical composition according to the present invention to a subject having an existing disease or condition in order to lessen, reduce or improve at least one symptom associated with the disease and / or to slow down, reduce or block the progression of the disease.

[0248] Reference to “prevention” / ”preventing” (or prophylaxis) as used herein refers to delaying or preventing the onset of the symptoms of the disease. Prevention may be absolute (such that no disease occurs) or may be effective only in some individuals or for a limited amount of time.

[0249] In a preferred embodiment of the present invention, the subject of any of the methods described herein is a mammal, preferably a cat, dog, horse, donkey, sheep, pig, goat, cow, mouse, rat, rabbit, or guinea pig. Preferably the subject is a human.

[0250] ADMINISTRATION

[0251] The administration of the pharmaceutical composition can be accomplished using any of a variety of routes that make the genetically modified cells comprised in the pharmaceutical composition bioavailable to the subject. For example, the composition can be administered by oral and parenteral routes, intraperitoneally, intravenously, subcutaneously, transcutaneously, intramuscularly, via local delivery for example by catheter or stent.

[0252] Suitably, the genetically modified cell according to the invention or the pharmaceutical composition according to the invention is administered intravenously.

[0253] Those skilled in the art will appreciate, for example, that route of delivery (e.g., oral vs intravenous vs subcutaneous, etc.) may impact dose amount and / or required dose amount may impact route of delivery. For example, where particularly high concentrations of an agent within a particular site or location are of interest, focused delivery may be desired and / or useful. Other factors to be considered when optimizing routes and / or dosing schedule for a given therapeutic regimen may include, forexample, the disease being treated (e.g., type or stage, etc.), the clinical condition of a subject (e.g., age, overall health, etc.), the presence or absence of combination therapy, and other factors known to medical practitioners.

[0254] The dosage is such that it is sufficient to stabilise or improve symptoms of the disease.

[0255] Typically, a physician will determine the actual dosage which will be most suitable for an individual subject and it will vary with the age, weight, and response of the particular patient. The dosage is such that it is sufficient to reduce or deplete the number of cells expressing the target antigen.

[0256] USE

[0257] The present invention also provides a pharmaceutical composition or population of genetically modified cells according to the invention for use in treating disease. The pharmaceutical composition or population of genetically modified cells may be any as defined above.

[0258] The present invention also relates to the use of a population of genetically modified cells of the present invention as defined above in the manufacture of a medicament for the treatment of a disease.

[0259] 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.

[0260] EXAMPLES

[0261] Example 1 - Activation Induced Cell Death in Dual Transduced Cell Populations

[0262] The mechanisms of early loss of CAR T cell persistence are poorly understood but likely to be multifactorial including the phenotype and intrinsic fitness of CAR T cells, activation induced cell death / exhaustion and immune mediated rejection.It is possible that higher CAR T expression and increased signalling in dual transduced CAR T cells may result in activation induced cell death (AICD) or exhaustion. Single cell CITESeq analysis of the CAR T cell products from 3 patients shows massive upregulation of FasL on dual transduced CAR T cells compared to single CD19 or CD22CAR T cells (Figure 3A). When we repetitively stimulated our CAR T cell products from 5 donors in vitro with CD19+CD22+ RAJI cells, we observed a progressive increase in early apoptotic (Annexin V+7-AAD-) cells specifically in the dual CAR transduced T cells in all donors consistent with AICD (Figure 3). There was no clear evidence of preferential increase in the exhaustion markers PD-1, LAG-3 and TIM-3 in dual CAR transduced T cells. These data imply that dual transduced CAR T cells may be more susceptible to activation induced cell death after repetitive antigenic exposure and suggests that co-infusion of separately transduced CAR T cells may be preferable to co-transduction.

[0263] Example 2 - Comparison of the in vitro function of CD19 / 22 CAR T cells manufactured with a 9-day or 5-day process

[0264] Functional assays were performed with PBMCs from leukapheresis or CD19 / 22 CAR T cells produced using either 9-day process (Cohort 3) or 5-day process (Cohort 4) methods.51Cr release cytotoxicity assays were performed using51Cr-labelled SupT1 (CD19-) or Nalm6 (CD19+) target cells with different EffectorTarget (E / T) ratios for 4 hours. Maximum killing was determined by lysis of51Cr-labelled target cells with 5% Triton X-100. The results are shown in Figure 3A, which demonstrates that cells manufactured using a 5-day process (Cohort 4) showed improved target cell lysis when compared to cells manufactured using a 9-day process (Cohort 3).

[0265] In addition,3H-thymidine incorporation assays were carried out with CAR T cells cocultured with irradiated target cells at 1:1 E / T ratio for 72 hours. P / I=phorbol 12-myristate 13-acetate / ionomycin. As shown in Figure 3B, cells manufactured using a 5-day process (Cohort 4) showed improved proliferation when compared to cells manufactured using a 9-day process (Cohort 3).

[0266] CD19 / 22 CAR T cells were co-cultured with SupT1, Nalm6 and Raji (CD19+) for ~18h before flow cytometric analysis to determine number of viable target cells. Percentage killing was determined relative to number of target cells in co-cultures of leukapheresis+target cells. As shown in Figure 3C, cells manufactured using a 5-day process (Cohort 4) showed improved cytotoxicity when compared to cells manufactured using a 9-day process (Cohort 3).Cytometric bead assays were performed using supernatant taken at ~18h co-culture from the same wells as for panel C to determine levels of IFN-y (Figure 3D), IL-2 (Figure 3E) and TNF (Figure 3F). Cells manufactured using a 5-day process (Cohort 4) showed improved cytokine secretion when compared to cells manufactured using a 9-day process (Cohort 3).

[0267] Example 3- In vivo functionality of CD19+CD22CAR T cells

[0268] The activity of CD19+CD22 CAR T cells generated by the two manufacturing methods was com-pared in vivo using an NSG-NALM6 xenogeneic mouse of ALL (Figure 4A). GFP+ / Luciferase+ NALM6 cells were injected into NSG mice one day before treatment with PBS or CD19+CD22 CAR T cells from patient CPL-27. PBS treated mice rapidly developed leukaemia and were all culled by day 22 (Figure 4B and 4E) with GFP+ / Luc+ NALM6 cells detected in the blood, bone marrow, spleen and liver (Figure 4C). Leukaemia development was transiently delayed in mice treated with CD19 / 22 CAR T cells generated using the cohort 3 methodology, which correlated with prolonged survival compared to PBS treated mice (Figure 4B and 4E). Most animals in this cohort showed increasing leukaemic burden by day 28, correlating with detection of GFP+ / Luc+ NALM6 cells in the blood and liver with no / low levels of CAR T cells in the tissues analysed and 40% of mice were alive at day 40. In contrast, mice treated with CD19+CD22 CAR T cells generated from the same donor using the methodology to be used in cohort 4 showed better control of leukaemia burden, with low bioluminescence signals throughout, which correlated with undtectable levels of GFP+ leukaemic cells and better persistence of of CAR T cells in the tissues analysed, in a sample of animals analysed on day 28 (Figure 4B-4E). All Cohort 4 CD19+CD22 CAR T cell treated mice were alive at day 40. These data show that Cohort 4 CD19+CD22 CAR T cells function better than Cohort 3 CD19+CD22 CAR T cells and appear to have better persistence in vivo.

[0269] This application claims the benefit of United Kingdom application No. 2501676.7 filed on 5 February 2025. This application is incorporated herein by reference in its entirety.

[0270] All publications mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described methods and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that theinvention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in molecular biology or related fields are intended to be within the scope of the following claims.

Claims

CLAIMS1. A process for producing a population of engineered cells, the process comprising:i) providing a starting population of cells;ii) activating the starting population of cells;iii) splitting the starting population of cells into a first cell population and a second cell population;iv) transducing the first cell population with a first vector encoding a first chimeric antigen receptor and transducing the second cell population with a second vector encoding a second chimeric antigen receptor;v) separately expanding the first cell population and the second cell population in a culture medium for a maximum of 6 days, or until a target dose is reached;vi) combining the first cell population and the second cell population to form a final engineered cell population.

2. The process of claim 1, the first chimeric antigen receptor is an anti-CD19 chimeric antigen receptor and the second chimeric antigen receptor is an anti- CD22 chimeric antigen receptor.

3. The process of claim 1, wherein the starting population of cells comprises T cells.

4. The process of claim 3, wherein the process further comprises a CD4 and CD8 selection step prior to step (iii).

5. The process of claim 1, wherein the process is carried out in the absence of exogenous cytokines.

6. The process of claim 1, wherein the ratio of the first cell population and the second cell population in the final cell population is 1:1.

7. The process of claim 1, wherein the cells are activated in step (ii) using anti- CD3 and anti-CD28 beads.

8. The process of claim 1, wherein the chimeric antigen receptors comprise:a. an antigen-binding domain;b. a spacer domain;c. a transmembrane domain;d. a 4-1 BB endodomain; ande. a CD3zeta endodomain.

9. The process of claim 2, wherein the anti-CD19 CAR comprises the amino acid sequence of SEQ ID NO: 1 and the anti-CD22 CAR comprises the amino acid sequence of SEQ ID NO: 3.

10. The process of claim 1, wherein the vector is a gamma retroviral vector or a lentiviral vector.

11. A population of engineered cells obtainable by the process of claim 1.

12. A pharmaceutical composition comprising a population of engineered cells according to claim 11.

13. The pharmaceutical composition according to claim 12, for use in therapy.

14. The pharmaceutical composition of claim 12, for use in the treatment of disease.

15. The pharmaceutical composition of claim 14, wherein the disease is cancer.

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