Patent Application
20200087398
The invention relates to T cell-targeted T cells, and methods employed in their production.
Cell therapy is therapy in which cellular material is introduced into a patient. Generally, intact, living cells are introduced to the patient. For example, T cells targeting disease-causing cells, such as cancer cells or immune cells, may be introduced to a patient. In this way, cancer cells may be destroyed or host immunity ablated.
Therapeutic T cells may comprise modifications associated with their therapeutic effect. For instance, a therapeutic cell may be modified to be targeted towards an antigen of interest, or to express a particular therapeutic molecule. T cells express heterodimeric antigen specific T cell receptors comprising disulphide bridged α and β chains or γ and δ chains in association with a multimeric CD3 complex. Exogenous molecules (e.g. an antigen receptor or therapeutic molecule) can be introduced to a therapeutic cell by transfecting or transducing the therapeutic cell with a nucleic acid sequence or construct encoding the molecule (i.e. a transgene). Such transfection and transduction is well known in the art.
In this way, therapeutic T cells may be engineered to direct their cytotoxic effects towards a particular antigen of interest. For example, T cells for killing tumour cells may be specific for a tumour antigen. T cell specificity may directed by endogenous αβ T cell receptors, or via introduced recombinant αβ receptors or by chimeric antigen receptors (CARs). The latter usually incorporates a single chain variable fragment (scfv) derived from the antigen binding regions of an antibody, linked to transmembrane and intracellular activation domains.
Recently, T cells transduced to express a CAR specific for CD19 (CAR19 T cells) have reached the clinic. These T cells have been shown to effectively target B cell malignancies expressing CD19. So far, though, it has not been possible to use CAR-expressing T cells therapeutically to effectively target T cell malignancies or unwanted T cells. One of the major problems in this respect is “T on T” cytotoxicity, where T cell-targeted therapeutic T cells target other such cells. Because T cell-targeted therapeutic T cells recognise and attack any cell expressing the T cell antigen for which they are specific, they recognise and attack desirable, therapeutic T cells as well as the malignant or unwanted T cells they are designed to ablate. In other words, T cell-targeted T cells have traditionally been subject to fratricide i.e. killing by other T cell-targeted therapeutic T cells. Thus, during manufacture and also presumably if T cell-targeted T cells were administered to a patient, the engineered cells attack each other. This depletes the population and its associated therapeutic effect.
Accordingly, there exists a need for T cell-targeted T cells that are not subject to fratricide.
The present invention aims to provide a T cell that expresses a CAR specific for a T cell marker, but which is not itself targeted by T cells specific for a T cell marker. Such a T cell-targeted T cell may be use therapeutically to deplete malignant or unwanted T cells, for instance to treat cancer or ablate host immunity. In addition, the T cell-targeted T cell has improved longevity, because it is not recognised and attacked by other T cell-targeted T cells. Thus, the T cell-targeted T cell of the invention has a reduced susceptibility to fratricide.
In more detail, the present inventors have found that a T cell-targeted T cell's susceptibility to fratricide can be reduced by disrupting the endogenous expression of one or more T cell-specific markers in the T cell. For instance, fratricide can be reduced by disrupting the endogenous expression of one or more T cell receptor (TCR)/CD3 complex components in the T cell. Similarly, fratricide can be reduced by disrupting the endogenous expression of CD5 in the T cell. Fratricide can be reduced by disrupting the endogenous expression of CD7 in the T cell. When the expression of a TCR complex component, CD5 or CD7 is disrupted in the T cell, the T cell becomes less visible to T cells expressing an antigen receptor (such as a CAR or a TCR) specific for a TCR/CD3 complex component, CD5 or CD7 respectively. Therefore, the T cell-targeted T cell is less able to be targeted by other T cell-specific T cells.
The present inventors have also developed a highly efficient method for generating such T cell-targeted T cells. The method of the invention provides a surprisingly high yield of T cell-targeted T cells that resist fratricide. The population of T cell-targeted T cells is surprisingly pure. When T cell-targeted T cells of the invention are produced according to the new method, any residual T cell-targeted T cells that remain visible to T cells expressing an antigen receptor specific for the T cell marker whose endogenous expression is disrupted in the T cell-targeted T cells (such as a TCR/CD3 complex component, CD5 or CD7) are attacked by the other T-cell targeted T cells in the population. Thus, T cell-targeted T cells of the invention whose endogenous TCR/CD3 complex, CD5 or CD7 expression is disrupted acquire a survival advantage, and these populations are enriched or ‘self-selected’ in culture as they lyse non-modified T cells. This means that further enrichment steps (such as antibody-mediated selection of transduced cells or depletion of residual TCR/CD3-, CD5- or CD7-expressing cells, by fluorescence activated cell sorting or magnetic activated cell sorting) are not required.
Accordingly, the invention provides
It is to be understood that different applications of the disclosed products and methods may be tailored to the specific needs in the art. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments of the invention only, and is not intended to be limiting.
In addition, as used in this specification and the appended claims, the singular forms “a”, “an”, and “the” include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to “a CAR” includes “CARs”, reference to “a T cell” includes two or more such T cells, reference to “a component” includes two or more such components, and the like.
All publications, patents and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety.
The invention provides a method for producing a T cell expressing one or more chimeric antigen receptors (CARs) specific for CD3, CD5 and/or CD7. The method comprises (i) disrupting endogenous expression of one or more of a T cell receptor (TCR)/CD3 complex component, CD5 and CD7 in a T cell, (ii) introducing a nucleic acid sequence encoding the CAR to said T cell of (i), and subsequently (iii) expressing the CAR of (ii) in said T cell of (i).
In the method, (i) may be carried out before (ii). Alternatively, (i) and (ii) may be carried out together. Step (iii) is carried out after (i) and after (ii).
The method may comprise, after (iii), formulating the T cell for administration to an individual. For example, pharmaceutically compatible diluents or excipients may be added to the T cell. For instance, isotonic phosphate buffered saline, EDTA, DMSO and/or human albumin serum may be added to the T cell. Preferably, isotonic phosphate buffered saline, EDTA, DMSO and human albumin serum are added to the T cell to provide a formulation comprising the T cell, isotonic phosphate buffered saline, EDTA, DMSO and human albumin serum. The formulation may comprise about 7.5% DMSO, such as about 3%, about 4%, about 5%, about 6%, about 8%, about 9% or about 10% DMSO. The formulation may comprise about 4% human albumin serum, such as about 1%, about 2%, about 3%, about 5%, about 6% or about 7% human albumin serum.
The method may comprise, after (iii), cyropreserving the T cell. Cryopreservation may take place after the T cell is formulated for administration to an individual as set out above. Cells may be cryopreserved using a controlled rate freezer stored in the vapour phase of liquid nitrogen until required. If the method comprises cryopreserving the T cell, the T cell may be thawed before use, e.g. before administration to an individual. For example, the cell may be thawed at the bedside. The cell may be thawed in a water bath. infused into a vein over a period of 5 minutes
The method may comprise, after (iii), (iv) administering the cell to an individual. Step (iv) may take place after (a) the T cell is formulated for administration to an individual as set out above and/or (b) the T cell is cryopreserved as set out above. If the T cell is cryopreserved, it may be thawed before administration to the individual in (iii), as set out above. The T cell may be administered by any route. Suitable routes include, but are not limited to, intravenous, intramuscular, intraperitoneal or other appropriate administration routes. When the T cell is administered intravenously, a population of such T cells may be administered over five minutes.
The individual into which the T cell is administered may have a disease, such as cancer, an autoimmune condition, an autoinflammatory condition, GVHD, or HIV. Exemplary cancers, autoimmune conditions and autoinflammatory conditions are set out below in relation to medical uses of the T cells produced by the method of the invention.
The T cell may be a CD8+ T cell, or cytotoxic T cell. The T cell may be a CD4+ T cell, or helper T cell (TH cell), such as a TH1, TH2, TH3, TH17, TH9, or TFH cells. The T cell may be a regulatory T cell (Treg). The T cell may be a naive, effector, memory, effector memory, central memory, memory stem T cell. The T cell may be a cord blood cell. The T cell may be a peripheral lymphocyte. The T cell may be expanded from PBMNCs. The T cell may be autologous with respect to an individual into which it is to be administered. The T cell may be allogeneic with respect to an individual into which it is to be administered. The T cell may be partially HLA-mismatched with respect to an individual into which it is to be administered.
Preferably, the T cell is generated from a cord blood T cell. The use of cord blood T cells is advantageous to the invention because they have a naive phenotype, an immense proliferative potential and potent in vivo activity in transplant recipients. Thus, the method of the invention may begin with a sample of cord blood. The sample of cord blood may be any type of sample. For instance, the sample of cord blood may be fresh cord blood or frozen cord blood. The sample of cord blood may have been derived from one individual. The sample of cord blood may have been derived from multiple individuals, i.e. a pooled cord blood sample.
Cord blood T cells may be obtained from the cord blood sample by separating cells that express CD62L from the sample. Any appropriate method may be used to separate cells that express CD62L from the sample. For instance, the cells that express CD62L may be separated from the sample based on their ability to bind an anti-CD62L antibody. Fluorescence activated cell sorting (FACS) or magnetic activated cell sorting (MACS) may be used to separate the cells that express CD62L from the sample. In MACS, magnetic beads are conjugated to the anti-CD62L antibody. Binding of the CD62L-expressing cells to the anti-CD62L antibody therefore tags the cells with magnetic beads. Magnetism can therefore be used to separate the tagged cells from the sample.
The separation step may be manually performed. Alternatively, the separation step may be performed in a system designed for the automated separation of cells. In one aspect, the system is configured for automated production of cord T cells. The system may be a CliniMacs system or a Miltenyi Prodigy system. Other automated cell separation systems are known in the art.
In some aspects, the T cell is stimulated before step (i) of the method of the invention. For instance, the T cell may be contacted with an anti-CD3 antibody and/or an anti-CD28 antibody. In this way, the T cell may be activated or expanded. The anti-CD3 antibody and/or the anti-CD28 antibody may be present on microbeads. Cytokines such as IL2, IL7, or IL15 may additionally be present alone or in combination.
In step (i) of the method, endogenous expression of one or more of a TCR/CD3 complex component, CD5 and CD7 in a T cell is disrupted. Any combination of a TCR/CD3 complex component, CD5 and CD7 may be disrupted. For instance, endogenous expression of (a) a T cell receptor (TCR)/CD3 complex component (b) CD5, (c) CD7, (d) a T cell receptor (TCR)/CD3 complex component and CD5, (e) a T cell receptor (TCR)/CD3 complex component and CD7, (f) CD5 and CD7 or (g) a T cell receptor (TCR)/CD3 complex component, CD5 and CD7 may be disrupted in step (i) of the method. Endogenous expression of CD52, MEW and/or a molecule involved in a checkpoint pathway may also be disrupted.
Disruption of the expression a TCR/CD3 complex component, CD5 or CD7 refers to changing the amount of expression of the TCR/CD3 complex component, CD5 or CD7 on the surface of the T-cell. Preferably, the expression of the TCR/CD3 complex component, CD5 or CD7 is reduced or completely eliminated. A T-cell having reduced expression of a TCR/CD3 complex component, CD5 or CD7 has a reduced amount of the TCR/CD3 complex component, CD5 or CD7 on its surface. A T-cell with completely eliminated expression of the TCR/CD3 complex component, CD5 or CD7 has none of the TCR/CD3 complex component, CD5 or CD7 on its surface.
Methods for determining surface expression of TCR/CD3 complex components, CD5 or CD7 are known in the art. For instance, T cells surface-stained (i.e. stained without a permeablisation step) with an antibody or other molecule that binds to the TCR or CD3 elements, CD5 or CD7 may be analysed by flow cytometry or fluorescence microscopy. Using flow cytometry, reduction in the mean fluorescence intensity (MFI) of TCR/CD3 complex component, CD5 or CD7 surface-staining in study T cells compared to control T cells indicates a reduction in surface expression of the TCR/CD3 complex component, CD5 or CD7 respectively. For instance, the MFI (and thus surface expression) may be reduced by up to 100%, such as up to 99%, up to 98%, up to 95%, up to 90%, up to 85%, up to 80%, up to 75%, up to 70%, up to 60%, up to 50%, up to 40%, or up to 25%, in study T cells compared to control T cells. The absence of expression of a TCR/CD3 complex component, CD5 or CD7 is indicated by the absence of surface-staining for the TCR/CD3 complex component, CD5 or CD7 respectively (i.e. by an MFI equivalent or similar to that of a negative control sample).
The TCR/CD3 complex, otherwise known as the T-cell receptor complex, is a multimeric complex on the T-cell surface whose activation leads to the activation of the T-cell. The complex comprises (i) TCR, (ii) CD3 T-cell activation receptor. As set out below, the TCR comprises alpha (α) and beta (β) chains. The CD3 T-cell receptor comprises a CD3-gamma (CD3γ) chain, a CD3-delta (CD3δ) chain, two CD3-epsilon (CD3ϵ) chains and two zeta-chain (ζ-chain) accessory molecules.
TCRs allow for the antigen-specific activation of T-cells. Every T-cell expresses clonal TCRs which recognize specific peptide/MHC complex during physical contact between T-cell and antigen-presenting cell-APC (via MHC class II) or any other cell type (via MHC class I). The TCR is a disulfide-linked membrane-anchored heterodimeric protein normally consisting of the highly variable alpha (α) and beta (β) chains. Each chain of the TCR comprises two extracellular domains: a variable (V) region and a constant (C) region, both of immunoglobulin superfamily (IgSF) domain forming antiparallel β-sheets. The constant region is proximal to the cell membrane, followed by a transmembrane region and a short cytoplasmic tail, while the variable region binds to the peptide/MHC complex. The variable domain of the TCR α-chain and the TCR β-chain each have three hypervariable or complementarity determining regions (CDRs), that contribute to the TCR's specificity for a particular peptide/MHC complex. The variable region of the β-chain also has an additional area of hypervariability (HV4) that does not normally contact antigen. Subsets of T cells may express γδ TCR/CD3 complexes rather than αβ TCR and these cells can operate without MHC restriction
CD3 is required for the antigen-specific activation of T-cells. In particular, CD3 links antigen recognition by the TCR with intracellular signalling events downstream of the TCR CD3 zeta-chain. CD3 is a protein complex comprising six distinct chains. In mammals, CD3 comprises a CD3-gamma (CD3γ) chain, a CD3-delta (CD3δ) chain, two CD3-epsilon (CD3ϵ) chains and two zeta-chain (ζ-chain) accessory molecules. The CD3-gamma, CD3-delta and CD3-epsilon chains are highly related cell-surface proteins of the immunoglobulin superfamily comprising a single extracellular immunoglobulin domain. The transmembrane region of the CD3 chains is negatively charged, allowing the chains to associated with TCR chains, which are positively charged. The zeta-chain (also known as T-cell surface glycoprotein CD3 zeta-chain or CD247) plays an important role in coupling antigen recognition to several intracellular signal-transduction pathways. Low expression of the zeta-chain results in an impaired immune response.
Accordingly, TCR/CD3 normally comprises several components: TCR α-chain, TCR β-chain, CD3-gamma chain, CD3-delta chain, two CD3-epsilon chains, and two CD3-zeta-chains. In the method of the invention, endogenous expression of one or more of these components is disrupted. For example, endogenous expression of two or more, three or more, four or more, five or more, or six or more of these components may be disrupted. When endogenous expression of two or more of (i) TCR α-chain, (ii) TCR β-chain, (iii) CD3-gamma chain, (iv) CD3-delta chain, (v) CD3-epsilon chain, and (v) zeta-chain are disrupted, these TCR complex components may be disrupted in any combination, i.e. (i); (ii); (iii); (iv); (v); (i) and (ii); (i) and (iii); (i) and (iv); (i) and (v); (ii) and (iii); (ii and (iv); (ii) and (v); (iii) and (iv); (iii) and (v); (iv) and (v); (i), (ii) and (iii); (i), (ii) and (iv); (i), (ii) and (v); (i), (iii) and (iv); (i), (iii) and (v); (i), (iv) and (v); (ii), (iii) and (iv); (ii), (iii) and (v); (ii), (iv) and (v); (iii), (iv) and (v); (i), (ii), (iii) and (iv); (i), (ii), (iii) and (v); (i), (ii), (iv) and (v); (i), (iii), (iv) and (v); (ii), (iii), (iv) and (v); or (i), (ii), (iii), (iv) and (v).
Endogenous expression of CD52 may be disrupted. CD52 is a glycoprotein present on the surface of mature lymphocytes, but not on the stem cells from which these lymphocytes were derived.
Endogenous expression of MEW may be disrupted. MEW class 1 may be disrupted by targeting the transporter associated with antigen processing (TAP1 or TAP2) locus, whichever method of disruption is used. MEW class 1 may be disrupted by Beta-2 microglobulin (B2M) locus, whichever method of disruption is used (see gene editing techniques below). MHC molecules may also be disrupted by targeting transcription factors controlling MHC expression such as CIITA, RFX5, RFXAP or RFXANK.
Disrupting expression of MHC may reduce or eliminate the effects of using HLA-mismatched (or partially mismatched) allogeneic T cells in the method. Specifically, if allogeneic cells are used, allogeneic 3CAR T cells will be generated. In this case, consideration must be given to the consequences of potential HLA-mismatch between the donor and recipient. For all types of therapeutic cells, minimising human leukocyte antigen (HLA)-mismatch reduces rejection of the therapeutic cells by the patient, improving their longevity and therapeutic potential. Furthermore, it is particularly important to reduce HLA-mismatch for therapeutic immune cells or hematopoietic stem cells, due to their ability to cause graft versus host disease (GVHD) when transplanted to an HLA-mismatched patient. GVHD arises when the native T-cell receptor (TCR) of T cells in or arising from the donated tissue (the “graft”) recognise antigens in the recipient (the “host”) as foreign. Thus, transplanted T cells attack host cells and tissues, causing damage to the host organs.
Endogenous expression of a molecule involved in a checkpoint pathway, such as PD-1, may be disrupted. Disruption of checkpoint inhibitor pathways unleashes an immune system attack on cancer cell, such as malignant T cells.
A gene editing technique may be used to disrupt endogenous expression of a TCR complex component, CDS, CD7, CD52, MHC and/or a molecule involved in a checkpoint pathway. Gene editing is a type of genetic engineering in which DNA is inserted, deleted or replaced in the genome of a living organism using engineered nucleases, or “molecular scissors.” These nucleases create site-specific double-strand breaks (DSBs) at desired locations in the genome. The induced double-strand breaks are repaired through nonhomologous end-joining (NHEJ) or homologous recombination (HR), resulting in targeted mutations (“edits”).
Four families of engineered nucleases may be used for gene editing: (a) meganucleases, (b) zinc finger nucleases (ZFNs), (c) transcription activator-like effector-based nucleases (TALENs), (d) mega-TALENs and (e) the CRISPR-Cas system. Techniques using each of these engineered nucleases are well known in the art. All of these genome editing methods can disrupt a gene, entirely knocking out all of its output. One or more of these techniques may be used alone or in any combination to disrupt endogenous expression of a TCR complex component in the method of the invention, i.e. (a); (b); (c); (d); (e); (a) and (b); (a) and (c); (a) and (d); (a) and (e); (b) and (c); (b) and (d); (b) and (e); (c) and (d); (c) and (e); (d) and (e); (a), (b) and (c); (a), (b) and (d); (a), (b) and (e); (a), (c) and (d); (a), (c) and (e); (a), (d) and (e); (b), (c) and (d); (b), (c) and (e); (b), (d) and (e); (c), (d) and (e); (a), (b), (c) and (d); (a), (b), (c) and (e); (a), (b), (d) and (e); (a), (c), (d) and (e); (b), (c), (d) and (e); and (a), (b), (c), (d) and (e).
When the gene editing technique is used to disrupt expression of a TCR complex component (and, optionally, CD5 and/or CD7), the gene editing technique may target one or more of (i) the TRAC locus; (ii) a locus controlling TCR beta expression; (iii) a locus controlling TCR gamma expression; (iv) a locus controlling TCR delta expression (v) a locus controlling expression of a CD3 chain. For instance, the gene editing technique may target (i); (ii); (iii); (iv); (v); (i) and (ii); (i) and (iii); (i and (iv); (i) and (v); (ii) and (iii); (ii and (iv); (ii) and (v); (iii) and (iv); (iii) and (v); (iv) and (v); (i), (ii) and (iii); (i), (ii) and (iv); (i), (ii) and (v); (i), (iii) and (iv); (i), (iii) and (v); (i), (iv) and (v); (ii), (iii) and (iv); (ii), (iii) and (v); (ii), (iv) and (v); (iii), (iv) and (v); (i), (ii), (iii) and (iv); (i), (ii), (iii) and (v); (i), (ii), (iv) and (v); (i), (iii), (iv) and (v); (ii), (iii), (iv) and (v); or (i), (ii), (iii), (iv) and (v). CD3 chains are described above. The CD3 chain may be CD3γ, CD3δ, CD3ϵ or CD3ζ.
As set out in Example 2 and shown in
The present inventors have surprisingly found that by disrupting expression of a TCR/CD3 complex component before a 3CAR is introduced to the T cells is expressed, suitable yields of engineered, CD3-specific T cells can be generated. Accordingly, in the present invention, endogenous expression of a TCR complex component in a T cell is disrupted. Subsequently, a 3CAR is expressed. The 3CAR is encoded by a nucleic acid sequence which is introduced to the T cell at the same time as the endogenous expression of a TCR/CD3 complex component is disrupted, or after the endogenous expression of a TCR/CD3 complex component is disrupted. The same approach can be used to generate CD5-specific T cells lacking endogenous CD5 expression, or CD7-specific T cells lacking endogenous CD7 expression. The CD3-specific T cells may additional have disrupted expression of endogenous CD5 and/or CD7. The CD5-specific T cells may additional have disrupted expression of endogenous CD3 and/or CD7. The CD7-specific T cells may additional have disrupted expression of endogenous CD3 and/or CD5.
Thus, one or more nucleic acid sequences encoding one or more of a CD3-specific CAR, a CD5-specific CAR or a CD7-specific CAR may be introduced to the T cell at the same time as, or after, steps are taken to disrupt endogenous expression of a TCR/CD3 complex component, CD5 or CD7 respectively. In either case, the CAR protein is expressed after endogenous expression of the TCR/CD3 complex component, CD5 or CD7 is disrupted.
When (i) disruption of endogenous expression of a TCR/CD3 complex component, CD5 or CD7 is carried out before (ii) introduction of a nucleic acid sequence encoding a CD3-specific, CD5-specific or CD7-specific CAR, the nucleic acid sequence encoding the CAR may be introduced to the T cell 2 to 48 hours after endogenous expression of a TCR complex component, CD5 or CD7 is disrupted. The nucleic acid sequence encoding the CAR may be introduced to the T cell 12 hours after steps are taken to disrupt endogenous expression of a TCR complex component, CD5 or CD7. The nucleic acid sequence encoding the CAR may be introduced to the T cell 26 hours after steps are taken to disrupt endogenous expression of a TCR complex component, CD5 or CD7. The nucleic acid sequence encoding the CAR may be introduced to the T cell 36 hours after steps are taken to disrupt endogenous expression of a TCR complex component, CD5 or CD7. The nucleic acid sequence encoding the CAR may be introduced to the T cell 48 hours after steps are taken to disrupt endogenous expression of a TCR complex component, CD5 or CD7. The nucleic acid sequence encoding the CAR may be introduced to the T cell 2 to 48, 2 to 46, 2 to 44, 2 to 42, 2 to 40, 2 to 38, 2 to 36, 2 to 34, 2 to 32, 2 to 30, 2 to 38, 2 to 26, 2 to 24, 2 to 22, 2 to 20, 2 to 18, 2 to 16, 2 to 14, 2 to 12, 2 to 10, 2 to 8, 2 to 6, 2 to 4, 4 to 48, 4 to 46,4 to 44,4 to 42,4 to 40,4 to 38,4 to 36,4 to 34, 4 to 32, 4 to 30, 4 to 38, 4 to 26, 4 to 24, 4 to 22, 4 to 20, 4 to 18, 4 to 16, 4 to 14, 4 to 12, 4 to 10, 4 to 8, 4 to 6, 6 to 48, 6 to 46, 6 to 44, 6 to 42, 6 to 40, 6 to 38, 6 to 36, 6 to 34, 6 to 32, 6 to 30, 6 to 38, 6 to 26, 6 to 24, 6 to 22, 6 to 20, 6 to 18, 6 to 16, 6 to 14, 6 to 12, 6 to 10, 6 to 8, 8 to 48, 8 to 46, 8 to 44, 8 to 42, 8 to 40, 8 to 38, 8 to 36, 8 to 34, 8 to 32, 8 to 30, 8 to 38, 8 to 26, 8 to 24, 8 to 22, 8 to 20, 8 to 18, 8 to 16, 8 to 14, 8 to 12, 8 to 10, 10 to 48, 10 to 46, 10 to 44, 10 to 42, 10 to 40, 10 to 38, 10 to 36, 10 to 34, 10 to 32, 10 to 30, 10 to 38, 10 to 26, 10 to 24, 10 to 22, 10 to 20, 10 to 18, 10 to 16, 10 to 14, 10 to 12, 12 to 48, 12 to 46, 12 to 44, 12 to 42, 12 to 40, 12 to 38, 12 to 36, 12 to 34, 12 to 32, 12 to 30, 12 to 38, 12 to 26, 12 to 24, 12 to 22, 12 to 20, 12 to 18, 12 to 16, 12 to 14, 14 to 48, 14 to 46, 14 to 44, 14 to 42, 14 to 40, 14 to 38, 14 to 36, 14 to 34, 14 to 32, 14 to 30, 14 to 38, 14 to 26, 14 to 24, 14 to 22, 14 to 20, 14 to 18, 14 to 16, 16 to 48, 16 to 46, 16 to 44, 16 to 42, 16 to 40, 16 to 38, 16 to 36, 16 to 34, 16 to 32, 16 to 30, 16 to 38, 16 to 26, 16 to 24, 16 to 22, 16 to 20, 16 to 18, 18 to 48, 18 to 46, 18 to 44, 18 to 42, 18 to 40, 18 to 38, 18 to 36, 18 to 34, 18 to 32, 18 to 30, 18 to 28, 18 to 26, 18 to 24, 18 to 22, 18 to 20, 20 to 48, 20 to 46, 20 to 44, 20 to 42, 20 to 40, 20 to 38, 20 to 36, 20 to 34, 20 to 32, 20 to 30, 20 to 28, 20 to 26, 20 to 24, 20 to 22, 22 to 48, 22 to 46, 22 to 44, 22 to 42, 22 to 40, 22 to 38, 22 to 36, 22 to 34, 22 to 32, 22 to 30, 22 to 38, 22 to 26, 22 to 24, 24 to 48, 24 to 46, 24 to 44, 24 to 42, 24 to 40, 24 to 28, 24 to 36, 24 to 34, 24 to 32, 24 to 30, 24 to 38, 24 to 26, 26 to 48, 26 to 46, 26 to 44, 26 to 42, 26 to 40, 26 to 38, 26 to 36, 26 to 34, 26 to 32, 26 to 30, 26 to 28, 28 to 48, 28 to 46, 28 to 44, 28 to 42, 28 to 40, 28 to 38, 28 to 36, 28 to 34, 28 to 32, 28 to 30, 30 to 48, 30 to 46, 30 to 44, 30 to 42, 30 to 40, 30 to 38, 30 to 36, 30 to 34, 30 to 32, 32 to 48, 32 to 46, 32 to 44, 32 to 42, 32 to 40, 32 to 38, 32 to 36, 32 to 34, 34 to 48, 34 to 46, 34 to 44, 34 to 42, 34 to 40, 34 to 38, 34 to 36, 36 to 48, 36 to 46, 36 to 44, 36 to 42, 36 to 40, 36 to 38, 38 to 48, 38 to 46, 38 to 44, 38 to 42, 38 to 40, 40 to 48, 40 to 46, 40 to 44, 40 to 42, 42 to 48, 42 to 46, 42 to 44, 44 to 48, 44 to 46, or 46 to 48 hours after endogenous expression of a TCR complex component, CD5 or CD7 is disrupted. Preferably, the nucleic acid encoding the CAR is introduced to the T cell 10 to 40, such as 15 to 35 or 20 to 30, hours after endogenous expression of a TCR complex component, CD5 or CD7 is disrupted
In other words, step (i) of the method of the invention may be carried out 2 to 48, 2 to 46, 2 to 44, 2 to 42, 2 to 40, 2 to 38, 2 to 36, 2 to 34, 2 to 32, 2 to 30, 2 to 38, 2 to 26, 2 to 24, 2 to 22, 2 to 20, 2 to 18, 2 to 16, 2 to 14, 2 to 12, 2 to 10, 2 to 8, 2 to 6, 2 to 4, 4 to 48, 4 to 46, 4 to 44, 4 to 42, 4 to 40, 4 to 38, 4 to 36, 4 to 34, 4 to 32, 4 to 30, 4 to 38, 4 to 26, 4 to 24, 4 to 22, 4 to 20, 4 to 18, 4 to 16, 4 to 14, 4 to 12, 4 to 10, 4 to 8, 4 to 6, 6 to 48, 6 to 46, 6 to 44, 6 to 42, 6 to 40, 6 to 38, 6 to 36, 6 to 34, 6 to 32, 6 to 30, 6 to 38, 6 to 26, 6 to 24, 6 to 22, 6 to 20, 6 to 18, 6 to 16, 6 to 14, 6 to 12, 6 to 10, 6 to 8, 8 to 48, 8 to 46, 8 to 44, 8 to 42, 8 to 40, 8 to 38, 8 to 36, 8 to 34, 8 to 32, 8 to 30, 8 to 38, 8 to 26, 8 to 24, 8 to 22, 8 to 20, 8 to 18, 8 to 16, 8 to 14, 8 to 12, 8 to 10, 10 to 48, 10 to 46, 10 to 44, 10 to 42, 10 to 40, 10 to 38, 10 to 36, 10 to 34, 10 to 32, 10 to 30, 10 to 38, 10 to 26, 10 to 24, 10 to 22, 10 to 20, 10 to 18, 10 to 16, 10 to 14, 10 to 12, 12 to 48, 12 to 46, 12 to 44, 12 to 42, 12 to 40, 12 to 38, 12 to 36, 12 to 34, 12 to 32, 12 to 30, 12 to 38, 12 to 26, 12 to 24, 12 to 22, 12 to 20, 12 to 18, 12 to 16, 12 to 14, 14 to 48, 14 to 46, 14 to 44, 14 to 42, 14 to 40, 14 to 38, 14 to 36, 14 to 34, 14 to 32, 14 to 30, 14 to 38, 14 to 26, 14 to 24, 14 to 22, 14 to 20, 14 to 18, 14 to 16, 16 to 48, 16 to 46, 16 to 44, 16 to 42, 16 to 40, 16 to 38, 16 to 36, 16 to 34, 16 to 32, 16 to 30, 16 to 38, 16 to 26, 16 to 24, 16 to 22, 16 to 20, 16 to 18, 18 to 48, 18 to 46, 18 to 44, 18 to 42, 18 to 40, 18 to 38, 18 to 36, 18 to 34, 18 to 32, 18 to 30, 18 to 28, 18 to 26, 18 to 24, 18 to 22, 18 to 20, 20 to 48, 20 to 46, 20 to 44, 20 to 42, 20 to 40, 20 to 38, 20 to 36, 20 to 34, 20 to 32, 20 to 30, 20 to 28, 20 to 26, 20 to 24, 20 to 22, 22 to 48, 22 to 46, 22 to 44, 22 to 42, 22 to 40, 22 to 38, 22 to 36, 22 to 34, 22 to 32, 22 to 30, 22 to 38, 22 to 26, 22 to 24, 24 to 48, 24 to 46, 24 to 44, 24 to 42, 24 to 40, 24 to 28, 24 to 36, 24 to 34, 24 to 32, 24 to 30, 24 to 38, 24 to 26, 26 to 48, 26 to 46, 26 to 44, 26 to 42, 26 to 40, 26 to 38, 26 to 36, 26 to 34, 26 to 32, 26 to 30, 26 to 28, 28 to 48, 28 to 46, 28 to 44, 28 to 42, 28 to 40, 28 to 38, 28 to 36, 28 to 34, 28 to 32, 28 to 30, 30 to 48, 30 to 46, 30 to 44, 30 to 42, 30 to 40, 30 to 38, 30 to 36, 30 to 34, 30 to 32, 32 to 48, 32 to 46, 32 to 44, 32 to 42, 32 to 40, 32 to 38, 32 to 36, 32 to 34, 34 to 48, 34 to 46, 34 to 44, 34 to 42, 34 to 40, 34 to 38, 34 to 36, 36 to 48, 36 to 46, 36 to 44, 36 to 42, 36 to 40, 36 to 38, 38 to 48, 38 to 46, 38 to 44, 38 to 42, 38 to 40, 40 to 48, 40 to 46, 40 to 44, 40 to 42, 42 to 48, 42 to 46, 42 to 44, 44 to 48, 44 to 46, or 46 to 48 hours before step (ii) of the method of the invention. Preferably, step (i) is carried out 10 to 40, such as 15 to 35 or 20 to 30, hours before step (ii). This particular window between step (i) and step (ii) improves the efficiency of generating T-cell targeted T cells, as shown in the Examples. This is surprising, given that Osborn et al. (2016) Molecular Therapy, Volume 25, No. 3, pages 570 to 581) demonstrated that disrupting the expression of the T cell receptor before introducing a CAR is detrimental to T cell viability. Increasing the window between step (i) and step (ii) to e.g. 20 to 30 hours would not, therefore, have been predicted to improve the efficiency of generation.
The method may be used to product a T cell expressing one or more CARs specific for CD3, CD5 and/or CD7. Thus, step (ii) of the method may comprise the introduction of one or more of a nucleic acid sequence encoding a CD3-specific CAR, a nucleic acid sequence encoding a CD5-specific CAR, and a nucleic acid sequence encoding a CD7-specific CAR. For example, step (ii) may comprise introducing (t) a nucleic acid sequence encoding a CD3-specific CAR, (u) a nucleic acid sequence encoding a CD5-specific CAR, (v) a nucleic acid sequence encoding a CD7-specific CAR, (w) a nucleic acid sequence encoding a CD3-specific CAR and a nucleic acid sequence encoding a CD5-specific CAR, (x) a nucleic acid sequence encoding a CD3-specific CAR and a nucleic acid sequence encoding a CD7-specific CAR, (y) a nucleic acid sequence encoding a CD5-specific CAR and a nucleic acid sequence encoding a CD7-specific CAR or (z) a nucleic acid sequence encoding a CD3-specific CAR, a nucleic acid sequence encoding a CD5-specific CAR and a nucleic acid sequence encoding a CD7-specific CAR to said T cell of (i). In any of these cases, step (iii) comprises expressing the CAR or CARs of (ii) in the T cell of (i). When two or more CAR-encoding nucleic acid sequences are introduced in step (ii), In this case, each CAR-encoding nucleic acid sequence may be introduced using a separate vector. Alternatively, each CAR-encoding nucleic acid sequence may be introduced using the same vector. When two CAR-encoding nucleic acid sequences are introduced in step (ii), the CAR-encoding nucleic acid sequences may be encoded by a nucleic acid sequence encoding a bispecific CAR.
When the nucleic acid sequence encoding the CAR is introduced to the T cell after disruption of endogenous expression of a TCR/CD3 complex component, CD5 or CD7, the CAR protein may be expressed immediately after the nucleic acid sequence encoding the CAR is introduced to the T cell. Alternatively, there may be a delay in expression of the CAR protein following introduction of the nucleic acid sequence encoding the CAR to the T cell. The delay may be around 24 hours in duration. The delay may be 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 in duration. The delay may be 12 to 36 hours in duration, such as 12 to 34, 12 to 32, 12 to 30, 12 to 28, 12 to 26, 12 to 24, 12 to 22, 12 to 20, 12 to 18, 12 to 16, 12 to 14, 14 to 36, 14 to 34, 14 to 32, 14 to 30, 14 to 28, 14 to 26, 14 to 24, 14 to 22, 14 to 20, 14 to 18, 14 to 16, 16 to 36, 16 to 34, 16 to 32, 16 to 30, 16 to 28, 16 to 26, 16 to 24, 16 to 22, 16 to 20, 16 to 18, 18 to 36, 18 to 34, 18 to 32, 18 to 30, 18 to 28, 18 to 26, 18 to 24, 18 to 22, 18 to 20, 20 to 36, 20 to 34, 20 to 32, 20 to 30, 20 to 28, 20 to 26, 20 to 24, 20 to 22, 22 to 36, 22 to 34, 22 to 32, 22 to 30, 22 to 28, 22 to 26, 22 to 24, 24 to 36, 24 to 34, 24 to 32, 24 to 30, 24 to 28, 24 to 26, 26 to 36, 26 to 34, 26 to 32, 26 to 30, 26 to 28, 28 to 36, 28 to 34, 28 to 32, 28 to 30, 30 to 36, 30 to 34, 30 to 32, 32 to 36, 32 to 34 or 34 to 36 hours. The delay may occur because the vector comprising the nucleic acid sequence encoding the CAR has to enter cells, reverse transcribe, translocate to nucleus, integrate and then express mRNA and then protein. A further period of delay may occur when TALENs are used as the gene editing technique and the cells are cultured at 30° C. to 32° C. for a period of time. The duration of the further period of delay will depend on the length of culture at this temperature. For instance, the cells may be cultured for up to 12 hours, 24 hours, 36 hours, 48 hours or 72 hours, in which case the duration of the further delay up to12 hours, 24 hours, 36 hours, 48 hours or 72 hours respectively.
Expression of the CAR protein may be apparent from the presence of CAR protein on the surface of the T cell. Methods for identifying the presence of CAR protein on the surface of the T cell are well known in the art. For instance, flow cytometry may be used. Expression of the CAR protein may be apparent from its functional effects on the T cell in which it is expressed. In particular, the T cell is antigen-specific for CD3 when the 3CAR protein is expressed. The T cell is antigen-specific for CD5 when the 5CAR protein is expressed. The T cell is antigen-specific for CD7 when the 7CAR protein is expressed. Methods for determining the CD3-specificify, CD5-specificify or CD7-specificify of the T cell are known in the art. For instance, a CD3-specific T cell may be capable of targeting or killing a CD3-expressing cell (such as a non-engineered T cell) in an in vitro assay.
The CAR may be expressed by the T cell about one, two, three, four, five, six or seven days after disruption of endogenous expression of a TCR complex component For example, the CAR may be expressed by the T cell about 0 hours, about 6 hours, about 12 hours, about 24 hours, about 36 hours or about 48 hours after disruption of endogenous expression of a TCR/CD3 complex component, CD5 or CD7. The CAR may be expressed by the T cell about 6 to 24 hours after disruption of endogenous expression of a TCR complex component, CD5 or CD7. For instance, the CAR may be expressed by the T cell 6 to 8, 6 to 10, 6 to 12, 6 to 14, 6 to 16, 6 to 18, 6 to 20, 6 to 22, 6 to 24, 8 to 10, 8 to 12, 8 to 14, 8 to 16, 8 to 18, 8 to 20, 8 to 22, 8 to 24, 10 to 12, 10 to 14, 10 to 16, 10 to 18, 10 to 20, 10 to 22, 10 to 24, 12 to 14, 12 to 16, 12 to 18, 12 to 20, 12 to 22, 12 to 24, 14 to 16, 14 to 18, 14 to 20, 14 to 22, 14 to 24, 16 to 18, 16 to 20, 16 to 22, 16 to 24, 18 to 20, 18 to 22, 18 to 24, 20 to 22, 20 to 24 or 22 to 24 hours after disruption of endogenous expression of a TCR complex component, CD5 or CD7. The CAR may be expressed by the T cell 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, or 47 hours after disruption of endogenous expression of a TCR complex component, CD5 or CD7.
The T cell may express 3CAR, 5CAR or 7CAR on its surface and/or be antigen specific for CD3, CD5 or CD7 respectively about one, two, three, four, five, six or seven days after disruption of endogenous expression of a TCR complex component, CD5 or CD7 respectively. For example, the T cell may express 3CAR on its surface and/or be antigen specific for CD3 about 0 hours, about 6 hours, about 12 hours, about 24 hours, about 36 hours or about 48 hours after disruption of endogenous expression of a TCR/CD3 complex component. The T cell may express 3CAR on its surface and/or be antigen specific for CD3 about 6 to 24 hours after disruption of endogenous expression of a TCR complex component. For instance, the T cell may express 3CAR on its surface and/or be antigen specific for CD36 to 8, 6 to 10, 6 to 12, 6 to 14, 6 to 16, 6 to 18, 6 to 20, 6 to 22, 6 to 24, 8 to 10, 8 to 12, 8 to 14, 8 to 16, 8 to 18, 8 to 20, 8 to 22, 8 to 24, 10 to 12, 10 to 14, 10 to 16, 10 to 18, 10 to 20, 10 to 22, 10 to 24, 12 to 14, 12 to 16, 12 to 18, 12 to 20, 12 to 22, 12 to 24, 14 to 16, 14 to 18, 14 to 20, 14 to 22, 14 to 24, 16 to 18, 16 to 20, 16 to 22, 16 to 24, 18 to 20, 18 to 22, 18 to 24, 20 to 22, 20 to 24 or 22 to 24 hours after disruption of endogenous expression of a TCR complex component. The T cell may express 3CAR on its surface and/or be antigen specific for CD3 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, or 47 hours after disruption of endogenous expression of a TCR complex component. The same time periods may apply to the expression of 5CAR and antigen specificity for CD5 after disruption of endogenous CD5 expression. The same time periods may apply to the expression of 7CAR and antigen specificity for CD7 after disruption of endogenous CD7 expression.
The T cell may be cultured or incubated between disruption endogenous expression of a TCR complex component and introduction and/or expression of a nucleic acid sequence encoding a CD3-specific, CD5-specific, or CD7-specific CAR. For instance, the T cell may be cultured at 30° C. to 37° C., such as at 31° C., 32° C., 33° C., 34° C., 35° C. or 36° C. Standard culture conditions are well known in the art.
The CD3-specific CAR (3CAR) may comprise a scFV from a CD3-specific antibody. The CD3-specific antibody may be a monoclonal antibody. The CD3-specific antibody may be a human antibody. The CD3-specific antibody may be a humanised antibody. The CD3-specific antibody may be a non-human antibody, such a, canine, equine, bovine, ovine, porcine, murine, feline, leporine, cavine or camelid antibody. If the CD3-specific antibody is a non-human antibody, it may comprise one or more human constant domains. For instance, the CD3-specific CAR may comprise a scFV from any of the following CD3-antibodies, which are well known in the art: OKT3, RIV-9, UCHT1, SK7, MEM57, MEM92, or HIT3A. The CD3-specific CAR may comprise a scFV from the OKT3 antibody. The CD3-specific CAR may comprise a scFV from an antibody that competes for CD3 binding with one or more of OKT3, RIV-9, UCHT1, SK7, MEM57, MEM92, or HIT3A. The CD3-specific CAR may comprise a scFV from an antibody that competes for CD3 binding with the OKT3 antibody.
The CD5-specific CAR (5CAR) may comprise a scFV from a CD5-specific antibody. The CD5-specific antibody may be a monoclonal antibody. The CD5-specific antibody may be a human antibody. The CD5-specific antibody may be a humanised antibody. The CD5-specific antibody may be a non-human antibody, such a, canine, equine, bovine, ovine, porcine, murine, feline, leporine, cavine or camelid antibody. If the CD5-specific antibody is a non-human antibody, it may comprise one or more human constant domains.
The CD7-specific CAR (7CAR) may comprise a scFV from a CD7-specific antibody. The CD7-specific antibody may be a monoclonal antibody. The CD7-specific antibody may be a human antibody. The CD7-specific antibody may be a humanised antibody. The CD7-specific antibody may be a non-human antibody, such as a canine, equine, bovine, ovine, porcine, murine, feline, leporine, cavine or camelid antibody. If the CD7-specific antibody is a non-human antibody, it may comprise one or more human constant domains.
The CAR may comprise a cytoplasmic domain comprising an activation domain. The activation domain serves to activate the T cell following engagement of the extracellular domain (e.g. scFv). For instance, the cytoplasmic domain may comprise one or more of a 41BB activation domain, a CD3ζ activation domain and a CD3e activation domain. Preferably, the cytoplasmic domain comprises a 41BB activation domain and/or a CD3ζ activation domain.
The CAR may comprise a transmembrane domain. The transmembrane domain serves to transmit activation signals to the cytoplasmic signal transduction domains following ligand binding of the extracellular domains (e.g. scFv). The CAR may comprise a spacer. The spacer connects the transmembrane domain to the extracellular domain. The spacer may confer steric effects that influence the strength of activation, cytotoxicity and signaling from the target cell and its surface receptors.
The nucleic acid sequence encoding the CAR may be introduced to the T cell using any method known in the art. In particular, the T cell may be transfected or transduced with the nucleic acid sequence. The CAR may be introduced to the T cell using a vector.
The term “transduction” may be used to describe virus mediated nucleic acid transfer. A viral vector may be used to transduce the cell with the one or more constructs. Conventional viral based expression systems could include retroviral, alpha-retroviral, lentivirus, adenoviral, adeno-associated (AAV) and herpes simplex virus (HSV) vectors for gene transfer. Non-viral transduction vectors include transposon based systems including PiggyBac and Sleeping Beauty systems. Methods for producing and purifying such vectors are know in the art. The vector is preferably a vector of the invention. Cord blood T cells may be transduced using any method known in the art. Transduction may be in vitro or ex vivo.
The term “transfection” may be used to describe non-virus-mediated nucleic acid transfer. The cord blood T cells may be transfected using any method known in the art. Transfection may be in vitro or ex vivo. Any vector capable of transfecting the cord blood T cells may be used, such as conventional plasmid DNA or RNA transfection. A human artificial chromosome and/or naked RNA may be used to transfect the cell with the nucleic acid sequence or nucleic acid construct. Human artificial chromosomes are described in e.g. Kazuki et al., Mol. Ther. 19 (9): 1591-1601 (2011), and Kouprina et al., Expert Opinion on Drug Delivery 11 (4): 517-535 (2014). Alternative non-viral delivery systems include DNA plasmids, naked nucleic acid, and nucleic acid complexed with a delivery vehicle, such as a liposome. Methods of non-viral delivery of nucleic acids include lipofection, microinjection, biolistics, virosomes, liposomes, immunoliposomes, polycation or lipid:nucleic acid conjugates, naked DNA, artificial virions, and agent-enhanced uptake of DNA. Lipofection is described in e.g., U.S. Pat. Nos. 5,049,386, 4,946,787; and 4,897,355) and lipofection reagents are sold commercially (e.g., Transfectam™ and Lipofectin™). Cationic and neutral lipids that are suitable for efficient receptor-recognition lipofection of polynucleotides include those of Felgner, WO 91/17424; WO 91/16024. The preparation of lipid:nucleic acid complexes, including targeted liposomes such as immunolipid complexes, is well known to one of skill in the art (see, e.g., Crystal, Science 270:404-410 (1995); Blaese et al., Cancer Gene Ther. 2:291-297 (1995); Behr et al., Bioconjugate Chem. 5:382-389 (1994); Remy et al., Bioconjugate Chem. 5:647-654 (1994); Gao et al., Gene Therapy 2:710-722 (1995); Ahmad et al., Cancer Res. 52:4817-4820 (1992); U.S. Pat. Nos. 4,186,183, 4,217,344, 4,235,871, 4,261,975, 4,485,054, 4,501,728, 4,774,085, 4,837,028, and 4,946,787).
Nanoparticle delivery systems may be used to transfect the T cell with the nucleic acid sequence. Such delivery systems include, but are not limited to, lipid-based systems, liposomes, micelles, microvesicles and exosomes. With regard to nanoparticles that can deliver RNA, see, e.g., Alabi et al., Proc Natl Acad Sci USA. 2013 Aug. 6; 110 (32):12881-6; Zhang et al., Adv Mater. 2013 Sep. 6; 25 (33):4641-5; Jiang et al., Nano Lett. 2013 Mar. 13; 13 (3):1059-64; Karagiannis et al., ACS Nano. 2012 Oct. 23; 6 (10):8484-7; Whitehead et al., ACS Nano. 2012 Aug. 28; 6 (8):6922-9 and Lee et al., Nat Nanotechnol. 2012 Jun. 3; 7 (6):389-93. Lipid Nanoparticles, Spherical Nucleic Acid (SNA™) constructs, nanoplexes and other nanoparticles (particularly gold nanoparticles) are also contemplated as a means for delivery of a construct or vector in accordance with the invention.
Uptake of nucleic acid constructs may be enhanced by several known transfection techniques, for example those including the use of transfection agents. Examples of these agents includes cationic agents, for example, calcium phosphate and DEAE-Dextran and lipofectants, for example, lipofectAmine, fugene and transfectam.
The T cell may be transfected or transduced under suitable conditions. Suitable conditions are known in the art. For instance, the T cell may be transfected or transduced following activation with combinations of antibodies such as anti-CD3 and anti-CD28 which may be conjugated to beads or polymers and used with or without cytokines such as IL2, IL7, and IL15, used alone or in combination. The T cell and agent or vector may, for example, be contacted for between five minutes and ten days, preferably from an hour to five days, more preferably from five hours to two days and even more preferably from twelve hours to one day after activation.
The nucleic acid sequence transduced or transfected into the T cell gives rise to expression of 3CAR, 5CAR or 7CAR in the T cells. Methods for determining expression of CARs are known in the art. For instance, flow cytometry for FAB surface staining may be used to detect CAR expression.
If the nucleic acid sequence is transduced into the T cell, the vector used for transduction may comprise a further nucleic acid sequence encoding another molecule useful to the generation of 3CAR, 5CAR or 7CAR cells, such as a suicide gene. Suicide genes are discussed in more detail below.
The method of the invention may comprise (ii) introducing a nucleic acid sequence encoding a suicide gene to the T cell.
Suicide genes are well known in the art. The suicide gene may be herpes simplex virus thymidine kinase, or an inducible caspase. The suicide gene may encode an epitope recognised by a monoclonal antibody specific for HerB2, EGFR or CD20.
The suicide gene provides an “off switch” that allows the 3CAR T cells produced by the method to be eliminated from an individual into whom they are administered. This provides a safety mechanism to stop the effects of the 3CAR, 5CAR or 7CAR T cells when their advantageous (therapeutic) effects are outweighed by their deleterious effects.
The nucleic acid sequence encoding the suicide gene may be introduced to the T cell together with the nucleic acid sequence encoding the 3CAR, 5CAR or 7CAR or at the same time as the nucleic acid sequence encoding the 3CAR, 5CAR or 7CAR. The nucleic acid sequence encoding the suicide gene may be introduced to the cell using any method known in the art. The nucleic acid sequence encoding the suicide gene may be transfected or transduced into the T cell. Transfection and transduction are known in the art and are set out in detail above. The nucleic acid sequence encoding the suicide gene may be introduced to the cell using a vector comprising the nucleic acid sequence encoding the suicide gene. Suitable vectors are described above. The vector may also comprise a nucleic acid sequence encoding a 3CAR, 5CAR or 7CAR.
Accordingly, the nucleic acid sequence encoding the 3CAR, 5CAR or 7CAR and the nucleic acid sequence encoding the suicide gene may be introduced to the T cell using the same vector. The nucleic sequence encoding the suicide gene may be linked to the nucleic acid sequence encoding the CD3-specific CAR, CD5-specific CAR or CD7-specific CAR by an IRES element or a nucleic acid sequence encoding a self-cleaving peptide. The nucleic sequence encoding the suicide gene may linked to the nucleic acid sequence encoding the CD3-specific CAR, CD5-specific CAR or CD7-specific CAR such that the suicide gene and the CAR are expressed as an in-frame fusion construct.
As set out above, step (i) of the method of the invention may comprise disrupting endogenous expression of (a) a T cell receptor (TCR)/CD3 complex component (b) CD5, (c) CD7, (d) a T cell receptor (TCR)/CD3 complex component and CD5, (e) a T cell receptor (TCR)/CD3 complex component and CD7, (f) CD5 and CD7 or (g) a T cell receptor (TCR)/CD3 complex component, CD5 and CD7. Step (ii) of the method may comprise introducing (t) a nucleic acid sequence encoding a CD3-specific CAR, (u) a nucleic acid sequence encoding a CD5-specific CAR, (v) a nucleic acid sequence encoding a CD7-specific CAR, (w) a nucleic acid sequence encoding a CD3-specific CAR and a nucleic acid sequence encoding a CD5-specific CAR, (x) a nucleic acid sequence encoding a CD3-specific CAR and a nucleic acid sequence encoding a CD7-specific CAR, (y) a nucleic acid sequence encoding a CD5-specific CAR and a nucleic acid sequence encoding a CD7-specific CAR or (z) a nucleic acid sequence encoding a CD3-specific CAR, a nucleic acid sequence encoding a CD5-specific CAR and a nucleic acid sequence encoding a CD7-specific CAR to said T cell of (i).
Step (i) of the method may comprise disrupting endogenous expression of the target corresponding to the specificity of a CAR that is to be introduced in step (ii). The endogenous expression of other targets may also be disrupted. For example, if a CD3-specific CAR is to be introduced in step (ii), step (i) may comprise disrupting endogenous expression of a TCR/CD3 complex component, and optionally CD5 and/or CD7. Preferably, the options set out for steps (i) and (ii) are combined as follows: (t) and (a); (t) and (d); (t) and (e); (t) and (g); (u) and (b); (u) and (d); (u) and (f); (u) and (g); (v) and (c); (v) and (e); (v) and (f); (v) and (g); (w) and (d); (w) and (g); (x) and (e); (x) and (g); (y) and (f); (y) and (g); or (z) and (g).
The invention provides a T cell expressing one or more CARs specific for CD3, CD5 and/or CD7 and having disrupted expression of one or more of an endogenous TCR complex component, CD5 and CD7. For instance, he invention provides a T cell expressing a CAR specific for CD3 and having disrupted expression of an endogenous TCR complex component (a 3CAR T cell). The invention also provides a T cell expressing a CAR specific for CD5 and having disrupted expression of endogenous CD5 (a 5CAR T cell). The invention also provides a T cell expressing a CAR specific for CD7 and having disrupted expression of endogenous CD7 (a 7CAR T cell).
The invention provides a T cell having disrupted endogenous expression of (a) a T cell receptor (TCR)/CD3 complex component (b) CD5, (c) CD7, (d) a T cell receptor (TCR)/CD3 complex component and CD5, (e) a T cell receptor (TCR)/CD3 complex component and CD7, (f) CD5 and CD7 or (g) a T cell receptor (TCR)/CD3 complex component, CD5 and CD7. The T cell may express (t) a CD3-specific CAR, (u) a CD5-specific CAR, (v) a CD7-specific CAR, (w) a CD3-specific CAR and a CD5-specific CAR, (x) a CD3-specific CAR and a CD7-specific CAR, (y) a CD5-specific CAR and a CD7-specific CAR or (z) a CD3-specific CAR, a CD5-specific CAR and a CD7-specific CAR.
The T cell may have disrupted endogenous expression of the target corresponding to the specificity of a CAR that it expresses. The T cell may also have disrupted endogenous expression of other targets. For example, if the T cell expresses a CD3-specific CAR, the T cell may have disrupted endogenous expression of a TCR/CD3 complex component, and optionally CD5 and/or CD7. Possible combinations of the recited CAR expressions and disruptions include e.g: (t) and (a); (t) and (d); (t) and (e); (t) and (g); (u) and (b); (u) and (d); (u) and (f); (u) and (g); (v) and (c); (v) and (e); (v) and (f); (v) and (g); (w) and (d); (w) and (g); (x) and (e); (x) and (g); (y) and (f); (y) and (g); or (z) and (g).
In any case, the T cells may express a suicide gene. CD3-specific, CD5-specific and CD7-specific CARS, TCR complex components and suicide genes are described in detail above.
The T cell of the invention may be produced using the method of the invention, as described above.
The T cell of the invention targets T cells. Therefore, the T cell of the invention may be used to target malignant T cells, or unwanted T cells, for example to treat cancer or ablate host immunity.
The 3CAR T cell of the invention has disrupted expression of an endogenous TCR complex component. Thus, the 3CAR T cell has a reduced ability to be recognised and/or attacked by T cells expressing an antigen receptor (such as a T cell receptor or a CAR) specific for an endogenous TCR complex component. Therefore, the 3CAR T cell has improved longevity, because it is not recognised and attacked by other T cell-targeted T cells. The 3CAR T cell-targeted T cell of the invention has a reduced susceptibility to fratricide.
The 5CAR T cell of the invention has disrupted expression of endogenous CD5. Thus, the 5CAR T cell has a reduced ability to be recognised and/or attacked by T cells expressing an antigen receptor (such as a T cell receptor or a CAR) specific for CD5. Therefore, the 5CAR T cell has improved longevity, because it is not recognised and attacked by other T cell-targeted T cells. The 5CAR T cell of the invention has a reduced susceptibility to fratricide.
The 7CAR T cell of the invention has disrupted expression of endogenous CD7. Thus, the 7CAR T cell has a reduced ability to be recognised and/or attacked by T cells expressing an antigen receptor (such as a T cell receptor or a CAR) specific for CD7. Therefore, the 7CAR T cell has improved longevity, because it is not recognised and attacked by other T cell-targeted T cells. The 7CAR T cell of the invention has a reduced susceptibility to fratricide.
The T cell may be any type of T cell. The T cell may be a CD4+ T-cell, or helper T-cell (Tx cell), such as a TH1, TH2, TH3, TH17, TH9, or TFH cell. The T cell may be a regulatory T-cell (Treg). The T cell is preferably a CD8+ T-cell, or cytotoxic T-cell. The T cell may be a naive, effector, memory, effector memory, central memory, memory stem T cell. The T cell may be a cord blood cell. The T cell may be a peripheral lymphocyte. The T cell may be expanded from PBMNCs. The T cell may be autologous with respect to an individual into which it is to be administered. The T cell may be allogeneic with respect to an individual into which it is to be administered. The T cell may be partially HLA-mismatched with respect to an individual into which it is to be administered.
A T cell expressing a CD3-specific CAR (3CAR) may comprise one or more, such as two or more, three or more, four or more, five or more or ten or more, nucleic acid sequences encoding a 3CAR. When the 3CAR T cell comprises two or more sequences encoding a 3CAR, the sequences may encode the same 3CAR or different 3CARs.
A T cell expressing a CD5-specific CAR (5CAR) may comprise one or more, such as two or more, three or more, four or more, five or more or ten or more, nucleic acid sequences encoding a 5CAR. When the 5CAR T cell comprises two or more sequences encoding a 5CAR, the sequences may encode the same 5CAR or different 5CARS.
A T cell expressing a CD7-specific CAR (7CAR) may comprise one or more, such as two or more, three or more, four or more, five or more or ten or more, nucleic acid sequences encoding a 7CAR. When the 7CAR T cell comprises two or more sequences encoding a 7CAR, the sequences may encode the same 7CAR or different 7CARs.
The 3CAR T cell may have reduced or completely eliminated expression of one or more of the TCR complex components described above. Accordingly, the 3CAR T cell may have a reduced or completely eliminated capacity to induce off-target effects such as GVHD following administration to a HLA-mismatched recipient or patient.
The 3CAR T cell may have reduced or completely eliminated expression of MHC class I and/or MHC class II. Accordingly, the 3CAR T cell may be subject to minimal amount of rejection when administered to a HLA-mismatched recipient or patient.
The invention provides a T cell of the invention for use in a method of treatment of the human or animal body.
The invention also provides a T cell of the invention for use in a method of treating cancer, the method comprising administering the T cell to an individual in need thereof. The cancer may be the cancer a T cell malignancy. The T cell malignancy may be T-cell acute lymphoblastic leukaemia (T-ALL) or T cell lymphoma. The cancer may be primary cancer or secondary cancer.
The invention also provides a T cell of the invention for use in a method of treating an autoimmune or autoinflammatory condition, the method comprising administering the T cell to an individual in need thereof. The autoimmune condition may be alopecia areata, autoimmune encephalomyelitis, autoimmune hemolytic anemia, autoimmune hepatitis, dermatomyositis, diabetes (type 1), autoimmune juvenile idiopathic arthritis, glomerulonephritis, Graves' disease, Guillain-Barré syndrome, idiopathic thrombocytopenic purpura, myasthenia gravis, autoimmune myocarditis, multiple sclerosis, pemphigus/pemphigoid, pernicious anemia, polyarteritis nodosa, polymyositis, primary biliary cirrhosis, psoriasis, rheumatoid arthritis, scleroderma/systemic sclerosis, Sjögren's syndrome, systemic lupus erythematosus, autoimmune thyroiditis, uveitis or vitiligo. The autoinflammatory condition may be Familial Mediterranean fever (FMF), Hyperimmunoglobulinemia D with recurrent fever (BIDS), TNF receptor associated periodic syndrome (TRAPS), Muckle-Wells syndrome (urticaria deafness amyloidosis), Neonatal onset multisystem inflammatory disease (NOMID), or Deficiency of the interleukin-1-receptor antagonist (DIRA).
The invention also provides a T cell of the invention for use in a method of treating HIV infection, the method comprising administering the T cell to an individual in need thereof.
The invention also provides a T cell of the invention for use in a method of treating GVHD, the method comprising administering the T cell to an individual in need thereof.
Any of the aspects above, the method may comprise administering an allogeneic transplant to the individual. The allogeneic transplant may be a haematopoietic stem cell transplant, such as a bone marrow, peripheral blood or cord blood transplant.
The invention also provides a method for treating disease, comprising administering a cell produced according to a method the invention to an individual in need thereof. The disease may be cancer. The disease may be an autoimmune condition. The disease may be an autoinflammatory condition. Exemplary cancers, autoimmune conditions and autoinflammatory conditions are described above. The disease may be HIV infection. The disease may be GVHD.
The invention further provides a T cell of the invention for use in a method of depleting immune cells and/or bone marrow cells in an individual. The individual may be a patient preparing for a transplant. The transplant may be from an allogeneic or HLA-mismatched (or partially mismatched) donor. The transplant may be of an organ, a tissue, or cells. The method of depleting immune cells and/or bone marrow cells may be performed prior to transplantation of the organ, tissue or cells into the individual. In this way, the individual is “conditioned” prior to receiving the transplant. By providing 3CAR, 5CAR or 7CAR T cells targeted against host T cells, host immunity is depleted. Thus, the subsequent transplant is less likely to be rejected.
The T cells of the invention may be provided as a pharmaceutical composition. The pharmaceutical composition preferably comprises a pharmaceutically acceptable carrier or diluent. The pharmaceutical composition may be formulated using any suitable method. Formulation of cells with standard pharmaceutically acceptable carriers and/or excipients may be carried out using routine methods in the pharmaceutical art. The exact nature of a formulation will depend upon several factors including the cells to be administered and the desired route of administration. Suitable types of formulation are fully described in Remington's Pharmaceutical Sciences, 19th Edition, Mack Publishing Company, Eastern Pennsylvania, USA.
The T cells or pharmaceutical composition may be administered by any route. Suitable routes include, but are not limited to, intravenous, intramuscular, intraperitoneal or other appropriate administration routes. The T cells or pharmaceutical composition are preferably administered intravenously.
Compositions may be prepared together with a physiologically acceptable carrier or diluent. Typically, such compositions are prepared as liquid suspensions of cells. The cells may be mixed with an excipient which is pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients are, for example, water, saline, dextrose, glycerol, of the like and combinations thereof.
In addition, if desired, the pharmaceutical compositions of the invention may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, and/or adjuvants which enhance effectiveness. The composition preferably comprises human serum albumin.
One suitable carrier or diluents is Plasma-Lyte A®. This is a sterile, nonpyrogenic isotonic solution for intravenous administration. Each 100 mL contains 526 mg of Sodium Chloride, USP (NaCl); 502 mg of Sodium Gluconate (C6H11NaO7); 368 mg of Sodium Acetate Trihydrate, USP (C2H3NaO2.3H2O); 37 mg of Potassium Chloride, USP (KCl); and 30 mg of Magnesium Chloride, USP (MgCl2.6H2O). It contains no antimicrobial agents. The pH is adjusted with sodium hydroxide. The pH is 7.4 (6.5 to 8.0).
The T cells are administered in a manner compatible with the dosage formulation and in such amount will be therapeutically effective. The quantity to be administered depends on the subject to be treated, the disease to be treated, and the capacity of the subject's immune system. Precise amounts of T cells required to be administered may depend on the judgement of the practitioner and may be peculiar to each subject.
Any suitable number of T cells may be administered to a subject. For example, at least, or about, 0.2×106, 0.25×106, 0.5×106, 1.5×106, 4.0×106 or 5.0×106 cells per kg of patient may administered. For example, at least, or about, 105, 106, 107, 108, 109 cells may be administered. As a guide, the number of cells of the invention to be administered may be from 105 to 109, preferably from 106 to 108. Typically, up to 2×108 3CAR, 5CAR or 7CAR T cells are administered to each patient. In such cases where cells are administered or present, culture medium may be present to facilitate the survival of the cells. In some cases the cells of the invention may be provided in frozen aliquots and substances such as DMSO may be present to facilitate survival during freezing. Such frozen cells will typically be thawed and infused or placed in a buffer or medium either for maintenance or for administration.
The following Examples illustrate the invention.
First, a CD3-specific CAR (3CAR) was derived from the antigen binding elements of the mouse anti-human CD3 monoclonal antibody, OKT3. The variable heavy chain and variable light chain published by Pfishershammer et al. were codon optimized (by GeneArt) to generate a single chain variable fragment (scFv). The scFv was linked to activation domains derived from 41BB and CD3ζ. The resultant 3CAR construct was cloned into a lentiviral vector (pCCL) under the control of a PGK promoter using the BamHI and Mlu1 sites (
This example demonstrates that disrupting endogenous expression of a TCR complex component in a T cell prior to expressing an introduced nucleic acid encoding a 3CAR to the cell was advantageous to the amplification of 3CAR T cells. In contrast, the traditional approach (where a nucleic acid sequence encoding a 3CAR is introduced and expressed in a T cell prior to disrupting endogenous expression of a TCR complex component) resulted in ‘T on T’ killing in the cultures.
This reduced propagation of CAR-expressing T cells is specific to T cells expressing 3CAR (
Furthermore, using the traditional approach to generate 3CAR T cells after having disrupted endogenous expression of a TCR complex component did not produce a sufficient yield of 3CAR T cells for use in a patient (
In contrast, significantly improved yields of 3CAR positive, TCR negative T cells were obtained when the order of CAR introduction and TCR complex component disruption were reversed (
In contrast, control LV CAR19 vector shows persistence of CD3+ CAR+ T cells (b, right panels). Therefore, the 3CAR T cells produced using this method appear to “self-select” and become enriched for 3CAR T cells that do not express CD3 (TCR complex) (b lower middle pane;). Both CD4 and CD8 subsets persisted (c).
Self-selection for 3CAR T cells that do not express CD3 (TCR complex) surprisingly abrogated any need for additional processing (e.g. by CliniMacs) to eliminate residual CD3+ T cells before therapeutic use, as demonstrated in
T cells engineered to express chimeric antigen receptors (CARs) against B cell antigens are being investigated as cellular immunotherapies. Similar approaches designed to target T cell malignancies have been hampered by the critical issue of ‘T on T’ cytotoxicity, whereby fratricide or self-destruction of healthy T cells prohibits cell product manufacture. To date, there have been no reports of T cells engineered to target the definitive T cell marker, CD3. Recent improvements in gene editing now provide access to efficient disruption of such molecules on T cells and this has provided a route to generation of 3CAR, CD3 specific CAR T cells. T cells were transduced with a lentiviral vector incorporating an anti-CD3ϵ CAR derived from OKT3, either before or after TALEN mediated disruption of the endogenous TCRαβ/CD3 complex. Only transduction after disrupting assembly of TCRαβ/CD3 yielded viable 3CAR T cells and these cultures were found to undergo self-enrichment for 3CAR+TCR-CD3− T cells without any further processing. Specific cytotoxicity against CD3ϵ was demonstrated against primary T cells and against childhood acute lymphoblastic leukemia (T-ALL). 3CAR T cells mediated potent anti-leukemic effects in a human:murine chimeric model, supporting the application of cellular immunotherapy strategies against T cell malignancies. 3CAR provides a ‘bridging’ strategy to achieve T cell eradication and leukemic remission ahead of conditioned allogeneic stem cell transplantation.
There has been notable progress in the development of engineered T cell therapies against B cell malignancies, with chimeric antigen receptors (CARs) against B cell antigens including CD19, CD20, CD22 and other targets have proven highly effective against refractory B cell leukemia [1]. CARs usually comprise an extracellular domain derived from an antibody single-chain variable fragment (scFv), linker, transmembrane moiety and activation domains derived from combinations of CD28, 41BB, and CD3 [2]. Most of these therapies have been generated in a bespoke manner using autologous or HLA-matched allogeneic peripheral blood mononuclear cell harvests, although recently non-matched ‘universal’ donor CAR19 T cells manufactured using TALEN (Transcription activator-like effector nuclease) gene editing have been reported [3, 4]. The development of similar approaches against T cell malignancies has been extremely challenging, not least because of fratricidal T-on-T cytotoxicity during cell manufacture. Certain cell surface antigens such as CD5 [5] have been targeted using specific CARs and downregulation of their expression in activated T cells has allowed effector cells to expand and yield a product. Similarly, CD4 has been targeted by CD8 T cells expressing a CD4 specific CAR [8] and T cells directed against one or other of the T cell receptor constant regions (TRBC1 or TRBC2) have also been developed and rely on eradicating whichever of the two compartments any leukaemic blasts are derived from [7]. Alternatively, the disruption of the expression of the target ligand in T cells has recently been reported for another T cell receptor, CD7 [8]. In that case, electroporation of CRISPR (Clustered regularly interspaced short palindromic repeat)/Cas9 reagents was used to disrupt CD7 expression in T cells transduced to express anti-CD7 CAR. There was no evidence of detrimental effects on T cell function or cytotoxicity, including in a human murine tumour model. We report anti-T cell immunotherapy using 3CAR, a chimeric antigen receptor against the CD3ϵ chain of the T cell receptor complex, generated by fusing the scFv regions of OKT3, a widely used anti-CD3ϵ monoclonal antibody, with a transmembrane stalk and intracellular activation domains from 41BB and CD3ζ (
T-cell acute lymphoblastic leukemias (T-ALL) account for around 15% of paediatric, and 25% of adult acute lymphoblastic leukemias (ALL), and although outcomes have improved over time, most relapsing subjects face high levels of morbidity and mortality [9]. To date there have been no reports of CAR T cell therapy targeting the definitive T cell marker CD3. Previous attempts to generate anti-CD3 CAR cells been reported using CD3− natural killer (NK) cells, which were then able to specifically eliminate CD3+ lymphoma lines and primary T cell targets in vitro and CD3+Jurkat leukemia in human:murine chimeras [10]. We generated 3CAR by fusing the scFv fragment of OKT3 (a clinical phase anti-CD3ϵ monoclonal antibody) to a CD8 transmembrane region linked to 41BB and CD3ζ signalling domains. Lentiviral expression of 3CAR (
To assess scalability, cells were activated, edited and transduced and 2×106 3CAR T cells cultured further in a gas-permeable static cell culture flask (G-Rex). Cells expanded by 100 fold over 11 days (
3CAR T cells mediated specific cytotoxicity against CD3+leukemic targets. This was assessed in vitro using multiple approaches. Firstly, 51Cr− labelled TCR/CD3+ or TCR/CD3− Jurkat leukemia cells [12] were cocultured with either 3CAR T cells or non-transduced control T cells. 3CAR+ cells mediated specific high level cytotoxicity of targets expressing TCR/CD3+, but not TCR/CD3− targets (
3CAR T cells also mediated cytotoxic effects against primary CD3+ T cells purified from healthy donor peripheral blood mononuclear cells (PBMC) and loaded with CSFE fluorescent dye. Only around 4% of these cells survived compared to 42% in control cultures. TCR/CD3− populations were not eliminated by 3CAR cells, confirming specificity against CD3ϵ (
To further evaluate the function of 3CAR T cells cultures were established against viable tissue bank samples from children with T cell acute lymphoblastic leukemia (T-ALL). Flow cytometry verified CD3, CD7, CD19 and CD34 expression upon thawing in these targets (
In vivo functionality of 3CAR T cells was assessed in a humanized murine model of leukemic T cell clearance. NSG mice were inoculated intravenously with either 10×106 TCR/CD3+eGFP+LUC+or TCR/CD3− eGFP+LUC+ Jurkat T cell targets and imaged after 3 days to confirm leukemia establishment, and were then injected with effector cells. These comprised either 3CAR or untransduced effector T cells. Serial bioluminescence imaging on days 3-18 showed rapid clearance of TCR/CD3+ Jurkats in the cohort receiving 3CAR T cells with negligible signal detected by day 11, in contrast to mice receiving non-transduced T cells or PBS where leukemia progressed (
The gene editing platform used in this iteration of 3CAR was based on TALENs, and derived from our experience with similar reagents. Previously we have reported minimal evidence of off-target effects using high throughput NGS interrogation of in silico predicted sites of TALEN activity. Alternative editing platforms may be equally effective, although are likely to need to operate within similarly well defined window of activity to ensure target antigen removal before 3CAR expression.
The inventors have found that gene editing to disrupt TCR expression can be exploited to enable 3CAR T cells to be generated through avoidance of ‘T on T’ fratricide or self destruction. In previous campaigns the inventors had found that the generation of universal TCR-CAR19+ T cells was most efficient when TRAC disruption was performed after lentiviral transduction. However, a similar schedule for 3CAR resulted in poor yields, presumably because prior expression of 3CAR mediated detection of CD3ϵ. A strictly ordered sequence of TRAC disruption ahead of 3CAR expression was found to be essential for the generation of 3CAR, and this event precedence was incorporated into scaled production of effector cells. The resulting 3CAR cells were potent mediators of T cell specific cytotoxity, both against primary T cells in vitro and against human leukemia in chimeric models.
The expression of CD3 on leukemic T cells can be variable but critically 3CAR mediated cytotoxicity of CD3+ targets was near complete in 6/6 samples evaluated. It is likely, that for effective elimination of heterogenous leukemias, a strategy targeting multiple T cell antigens will be required, akin to combinational antibiotic therapy for mycobacterial disease or antiviral therapy against HIV. Based on our analysis, a combination of CARs targeting CD3 and CD7 could be highly effective against childhood T-ALL. Applications in patients with CD3+ ALL who are refractory to conventional chemotherapy is envisaged, most likely as a ‘bridge to transplant’ after achieving molecular remission. In this setting, pre-manufactured 3CAR T cells from a healthy allogeneic donor could be used in an ‘off the shelf’ manner, similar to ‘universal’ CAR19 T cells against B cell malignancies. However, whereas suppression of the normal B cell compartment by persisting CAR T cells can be addressed by administration of replacement immunoglobulin therapy, long-term T cell lymphopenia mediated by 3CAR could be prohibitively immunosuppressive. Once remission was secured, additional conditioning and serotherapy would be used to eradicate 3CAR, a step essential for subsequent donor derived T cell reconstitution to proceed. Such a strategy could also lead to broader 3CAR mediated conditioning strategies for allogeneic stem cell transplantation providing an alternative to intensive chemotherapy regimens.
Cell lines: Clonal expansion of TCR/CD3+ and TCR/CD3− expressing Jurkats cells (Acute T Cell Leukemic cell line) maintained in culture in RPMI-1640 (Gibco-BRL, San Francisco, Calif., USA) supplemented with 10% fetal bovine serum (Sigma-Aldrich).
Generation of anti-CD3 CAR: CD3-specific CAR (3CAR) was derived by codon optimisation (geneart) of variable heavy chain and variable light chain antigen binding elements of the mouse anti-human CD3 monoclonal antibody, OKT3. (Pfishershammer et al). The scFv was linked to activation domains derived from 41BB and CD3ζ as previously described. The resultant 3CAR construct was cloned into a lentiviral vector (pCCL) under the control of a PGK promoter (
Generation of CAR-modified T cells: Peripheral blood mononuclear cells were obtained from healthy donors and were cultured in 48-well plates at a density of 1×106/mL in TexMACS (Miltenyi Biotec), 3% Human Serum (Seralab)+20 ng/mL Human Recombinant interleukin (IL)-2 (Miltenyi Biotec) and activated with TransAct Reagent (Miltenyi Biotec). Activated T cells were electroporated with TALEN_TCR_2a Right and Left mRNA (TRAC mRNA) {Berdien, 2014 #461} (TriLink Biotechnologies) on day 2, cells were divided into untransduced controls or transduced with CD3-CAR vector at a MOI of 5.
Flow cytometry: Cells were stained with the following primary anti-human antibodies CD2, CD3, TCRαβ, CD4, CD8, CD19, CD20, CD56 and programmed death 1 (PD-1) (from Miltenyi Biotec) and mCD11b. To assess the efficiency of CD3-CAR transduction, cells were stained using a Biotin-SP (long spacer) AffiniPure F(ab′) Fragment Goat Anti-Mouse immunoglobulin (Ig)G, F(ab′) Fragment Specific antibody (Jackson Immunoresearch, Stratech Scientific Limited, Suffolk, UK) followed by Streptavidin-APC or Streptavidin-Fluorescein isothicyanate (FITC) (Biolegend). Cells were acquired on a Cyan or on a BD LSRII (BD Biosciences, Oxford, UK) and analysis performed using FlowJo v10 (TreeStar Inc. Ashland Oreg., USA).
Chromium release assay of cytotoxicity: Cytotoxic activity of 3CAR cells was assessed by chromium 51 (51Cr) release assay. For this 5×103 51Cr-labeled TCR/CD3-positive Jurkats or TCR/CD3-negative Jurkats target cells were incubated with 3CAR or untransduced control effector cells at increasing effector-to-target (E:T) ratios in 96-well microplates for 4 h at 37° C. 51Cr release was then measured in a microplate scintillation counter (Wallac 1450 MicroBeta TriLux).
Flow based cytotoxicty assay: CD3 CART cells or control non-transduced T cells were cocultured with TCR/CD3-positive Jurkats or TCR/CD3-negative Jurkats target cells that were loaded with CFSE dye (Cell Trace, Invitrogen), at the effector to target ratio 1:1 (100,000 of both effector and target cells) for 24 h. Dot plots show representative frequency of gated CFSE+ tumor cells at the end of coculture. CD3 CART cells or control non-transduced T cells also were cocultured with healthy donor PBMC, CD3+ or CD3− cells (MicroBeads, Miltenyi, Bergisch-Gladbach, Germany).
Cytokine production: 3CAR-transduced cells were thawed, washed and re-suspended in RPMI 10% FCS at a concentration of 1×106/mL. TCR/CD3-negative Jurkat cells, TCR/CD3− positive Jurkat cells and healthy donor PBMCs were all counted and also adjusted to a concentration of 1×106/mL. Cells were mixed at a 1:1 ratio in 24-well tissue culture plates and incubated at 37° C. After 24-h supernatants were harvested and stored at −80° C. Levels of cytokine in the stored supernatants were quantified using the TH1/TH2/TH17 cytometric bead array kit (CBA; BD Biosciences) as per the manufacturer's protocol.
Mixed leukocyte reaction and 3H-thymidine incorporation: To assess allogeneic T-cell proliferation in a MLR, 3CAR T cells were mixed with target PBMCs or PBMCs depleted of CD3 using CD3 MicroBeads, (Miltenyi, Bergisch-Gladbach, Germany), in RPMI 10% FCS at a 1:1 ratio in 96-well plates. To assess a one-way MLR, targets cells were irradiated (3,000 rads) prior to coculture. Targets alone were used as negative controls, and all cultures were run in triplicate. After co-incubation for 5 days, 3H-thymidine (1 μCi per well) was added for 18 h before harvesting onto filter mats for radioactivity measurement in a scintillation counter (Wallac 1450 MicroBeta TriLux).
In vivo anti-tumor activity: NOD/SCID/c−/− (NSG) mice, were inoculated IV with 10×106 TCR+ve or TCR−ve Jurkat T cell tumour targets by tail vein injection on day 0. The Jurkat T cell targets had been stably transduced to express both enhanced green fluorescent protein (EGFP) and Luciferase and following TALEN-mediated TCRαβ disruption had been sorted for TCR+ and TCR− expressers. Tumour engraftment was confirmed by in vivo imaging of bioluminescence using an IVIS Lumina III In Vivo Imaging System (PerkinElmer, Massachusetts, USA, live image version 4.5.18147) on day 3. TCR+ or TCR− injected mice were further injected on day 4 with either PBS (n=2), 10×106 untransduced T cells (n=4) or 10×106 3CAR T cells (n=6) per tumour group. Analysis of tumor clearance was performed by serial bioluminescent imaging on days 3, 7, 11, and 18 and processing of bone marrow for the monitoring of tumor progression vs clearance was carried out on day 18. Bone marrow samples were processed by a red blood cell lysis followed by staining for flow cytometry. All animal studies were approved by the University College London Biological Services Ethical Review Committee and licensed under the Animals (Scientific Procedures) Act 1986 (Home Office, London, United Kingdom).
Primary T-ALL blasts: Primary T-ALL patient samples are all from the ALL2003 trial where peripheral blood was often sent at diagnosis instead of bone marrow, with information on CD3+ status, and other surface antigens (see table) (From Bloodwise Childhood Leukaemia Cell Bank). To phenotype patient cells, cells were stained with the following primary antibodies CD3, CD7, CD19 and CD34. For FACS-Based Killing assay, CD3 CART cells, control CAR19 T cells or control non-transduced T cells were cocultured with T-ALL patient sample target cells that were loaded with CFSE dye (Cell Trace, Invitrogen), at the effector to target ratio 1:1(100,000 of both effector and target cells) for 24 h.
For in vitro studies, data points from individual donors are shown in all figures. Statistical significance in pairwise comparisons was determined by unpaired two-tailed Student t test. For in vivo studies, a two-tailed Man-Whitney U test was used for non-parametric comparison of grouped data and values are presented as mean percentages of three or more samples with standard error of the mean (SEM) or standard deviation (SD) or as a median with the 25th and 75th percentiles stated. Linear Regression was used for the comparison of serial measurements, taking each Y value as an individual point. All statistical analysis was performed using GraphPad Prism software version 5.01.
1. Riviere, I. and M. Sadelain, Chimeric Antigen Receptors: A Cell and Gene Therapy Perspective. Mol Ther, 2017. 25 (5): p. 1117-1124.
2. Sadelain, M., R. Brentjens, and I. Riviere, The basic principles of chimeric antigen receptor design. Cancer Discov, 2013. 3 (4): p. 388-98.
3. Poirot, L., et al., Multiplex Genome-Edited T-cell Manufacturing Platform for “Off-the-Shelf” Adoptive T-cell Immunotherapies. Cancer Res, 2015. 75 (18): p. 3853-64.
4. Qasim, W., et al., Molecular remission of infant B-ALL after infusion of universal TALEN gene-edited CAR T cells. Sci Transl Med, 2017. 9 (374).
5. Mamonkin, M., et al., A T-cell-directed chimeric antigen receptor for the selective treatment of T-cell malignancies. Blood, 2015. 126 (8): p. 983-92.
6. Pinz, K., et al., Preclinical targeting of human T-cell malignancies using CD4-specific chimeric antigen receptor (CAR)-engineered T cells. Leukemia, 2016. 30 (3): p. 701-7.
7. Maciocia, P. M., et al., Targeting the T cell receptor beta-chain constant region for immunotherapy of T cell malignancies. Nat Med, 2017.
8. Gomes-Silva, D., et al., CD7-edited T cells expressing a CD7-specific CAR for the therapy of T-cell malignancies. Blood, 2017. 130 (3): p. 285-296.
9. Goldberg, J. M., et al., Childhood T-cell acute lymphoblastic leukemia: the Dana-Farber Cancer Institute acute lymphoblastic leukemia consortium experience. J Clin Oncol, 2003. 21 (19): p. 3616-22.
10. Chen, K. H., et al., Novel anti-CD3 chimeric antigen receptor targeting of aggressive T cell malignancies. Oncotarget, 2016. 7 (35): p. 56219-56232.
11. Alam, S. M., I. N. Crispe, and N. R. Gascoigne, Allelic exclusion of mouse T cell receptor alpha chains occurs at the time of thymocyte TCR up-regulation. Immunity, 1995. 3 (4): p. 449-58.
12. Schneider, U., H. U. Schwenk, and G. Bornkamm, Characterization of EBV-genome negative “null” and “T” cell lines derived from children with acute lymphoblastic leukemia and leukemic transformed non-Hodgkin lymphoma. Int J Cancer, 1977. 19 (5): p. 621-6.
| Number | Date | Country | Kind |
|---|---|---|---|
| 1622044.4 | Dec 2016 | GB | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/GB2017/053888 | 12/22/2017 | WO | 00 |
