method

Abstract

The present invention provides a method for preparing a population of genetically modified cells comprising a chimeric antigen receptor (CAR) or a transgenic T cell receptor (TCR), the method comprising the steps of: (i) providing a starting population of cells; (ii) depleting cells expressing a target antigen from the starting population to form a depleted starting population; (iii) sorting for CD4+ cells and CD8+ cells in the depleted starting population to form a sorted population of cells; and (iv) introducing into cells in the sorted population a nucleic acid sequence encoding a CAR or transgenic TCR against the target antigen. [Selection chart] None

Description

[Technical field]

[0001] FIELD OF THEINVENTION The present invention relates to a method for preparing a population of genetically modified cells comprising a chimeric antigen receptor (CAR) or a transgenic T cell receptor (TCR). The method comprises depleting cells expressing the target antigen of the CAR or transgenic TCR from a starting population to form a depleted starting population; then selecting for CD4+ and CD8+ cells in the depleted starting population to form a selected population of cells. Then, a nucleic acid sequence encoding the CAR or transgenic TCR against the target antigen is introduced into a cell or cell group in the selected population of cells. The present invention further relates to a genetically modified cell or a population of genetically modified cells obtainable by the method of the present invention. The present invention also provides pharmaceutical compositions comprising the genetically modified cells, and their use in treating and / or preventing diseases. [Background technology]

[0002] BACKGROUND OF THEINVENTION Adoptive cell therapy (ACT) is a personalized medicine that involves administering to a subject immune cells with activity against specific disease-associated antigens. ACT using naturally occurring tumor-reactive or tumor-infiltrating lymphocytes (TILs) has mediated durable complete regressions in patients with melanoma. However, melanoma appears to be the only cancer that reproducibly gives rise to TIL cultures capable of specific anti-tumor recognition and reactivity.

[0003] Subsequent approaches have explored the broader application of ACT to treat other diseases and cancers by genetically engineering cells to express antitumor receptors. For example, the TCR is composed of one α and one β chain. These receptors recognize antigens that have been processed and presented by MHC molecules. Normal circulating lymphocytes transduced with a retrovirus encoding a TCR that recognized the MART-1 melanoma-melanocyte antigen have been shown to mediate tumor regression.

[0004] Another approach is the administration of lymphocytes genetically expressed to express CAR. CAR is an artificial receptor that can be constructed by linking the variable region of the heavy chain and the variable region of the light chain of an antibody to the intracellular signaling chain alone or in combination with other signaling moieties. CAR recognizes antigens presented on the surface of tumor cells, but does not need to be MHC restricted. For example, CAR against B cell antigen CD19 has been shown to mediate the regression of advanced B cell lymphoma.

[0005] The key material in the manufacture of genetically modified cell therapy is the starting material, i.e. the cells to be genetically modified. These cells can be obtained from the patient in the case of autologous therapy, or from another donor in the case of allogeneic therapy. These cells can be obtained, for example, from peripheral blood or leukapheresate, derived from the patient to be treated (autologous) or from another donor (allogeneic). Summary of the Invention

[0006] There remains a need for improved methods for the generation of genetically modified cells that provide reproducible efficiency and / or enhanced safety, and / or avoid the production of insufficient cell populations and / or pharmaceutical compositions for administration to patients, thereby preventing delays in subsequent treatment. [Brief description of the drawings]

[0007] [Figure 1-1] Figure 1 - Comparison of current and next generation (NG) manufacturing processes (n=3). A: Total viable cell count; B: Total transduced cells. In the NG process, the dose was achieved 2-3 days earlier than in the current process (not including day -1); C: Transduction efficiency in the NG process is higher than in the current process in two of the three batches. [Figure 1-2] Same as above.

[0008] [Figure 2-1] Figure 2 - Comparison of T naive and TCM subsets in healthy volunteer leukapheresate (n=1) for current and NG manufacturing processes. The updated process shows an improved profile. A: percentage of T naive cells; B: percentage of TCM cells; C: ratio of CD3+, CD45RA+ and CCR7+ cells at different stages. When the current process reaches the target dose of 450 million cells (day 8), there are more TEMRA cells and fewer naive cells compared to when the NG process reaches that dose (day 5). [Figure 2-2] Same as above.

[0009] [Diagram 3] Figure 3 - Comparison of cell viability in healthy volunteer leukapheresate (n=1) between current (circles) and NG (squares) manufacturing processes. Between days 0 and 6, the NG process showed higher cell viability than the current process.

[0010] [Figure 4-1] FIG. 4 - Differentiation traits as a percentage of % of all T cells for the NG process. The NG process results in DPs with a less differentiated phenotype. [Figure 4-2] Same as above.

[0011] [Diagram 5]Figure 5 - Differentiation traits at time of harvest as a percentage of transduced cells. Naïve and central memory cells comprise 78% of all cells at harvest on day 7 of the NG process.

[0012] [Figure 6] Figure 6 - Comparison of differentiation traits of patient samples using the process of the present application (Next Generation Process) and a comparative process that does not include a sorting step. By day 10, cells in the NG process are formed with less differentiated cells than observed in the current process.

[0013] [Figure 7-1] Figure 7 - Memory population: clinical batch. For leukapheresate from patients treated with the invention described herein, the pre-collection memory profile and the cryopreserved product memory profile showed a predominantly early stage of the cells. [Figure 7-2] Same as above.

[0014] [Figure 8] FIG. 8 - Functional data: proliferation after killing. Using depletion and sorting of the starting leukapheresate material, there is greater proliferation of CAR+ cells in response to co-culture and killing of CAR antigen positive target cells compared to CAR+ cells generated from a process consisting of depletion alone. This is despite the fact that processes including and not including the present invention show similar killing of these target cells. A healthy donor sample is shown on the left and a patient sample is shown on the right. It can be observed that samples prepared with this new process show improved proliferation after killing of the target cells. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] (Summary of the Invention) We provide methods for preparing a population of genetically modified cells containing a CAR or a transgenic TCR.

[0016] The inventors have shown that the removal of cells expressing a target antigen from a population during production, combined with a subsequent CD4 / CD8 selection step, results in an improved genetically modified cell product, where the genetically modified cells express a CAR or transgenic TCR against the target antigen. An advantage of the present invention is that the genetically modified cells produced by this method are less differentiated and more naive than genetically modified cells produced without the removal of cells expressing the target antigen and without selection for CD4 and CD8 expression. Without wishing to be bound by theory, a sufficient percentage of undifferentiated cells indicates that the genetically modified cells of the present invention or the genetically modified cells produced by the method of the present invention have improved in vivo persistence after administration and / or improved cytolytic activity after administration.

[0017] Another advantage of the present invention is that the genetically modified cells produced by the method are more uniformly genetically modified, with less variation between methods performed on cell populations derived from different donors, and between cell preparations and / or pharmaceutical compositions.

[0018] Advantageously, when compared to methods that do not include CD4 / CD8 selection after the depletion step, the method according to the present invention produces a purer population of genetically modified cells that contain low or undetectable levels of cells expressing the target antigen. The method according to the present invention minimizes or eliminates the risk of genetically modifying cells expressing the target antigen. This may provide an additional safety advantage when the target cells (i.e., cells expressing the target antigen) are cancer cells.

[0019] Furthermore, methods according to the invention reduce transduction or transfection failures when a high proportion of cells expressing the target antigen are present in the source of cells (eg, the starting population of cells).

[0020] Without wishing to be bound by theory, removal of cells expressing a target antigen may reduce cellular differentiation and / or exhaustion due to fratricide during production (e.g., during the cell expansion phase) by cells expressing a CAR or transduced TCR against that target antigen.

[0021] In one aspect, the present invention provides a method for preparing a population of genetically modified cells comprising a chimeric antigen receptor (CAR) or a transgenic T cell receptor (TCR), the method comprising: (i) providing a starting population of cells; (ii) depleting cells expressing the target antigen from the starting population to form a depleted starting population; (iii) sorting for CD4+ cells and CD8+ cells in the depleted starting population to form a sorted population of cells; and (iv) introducing into cells in the selected cell population a nucleic acid sequence encoding a CAR or a transgenic TCR against the target antigen. Includes.

[0022] The CAR or transgenic TCR may be introduced into a cytolytic immune cell according to the present invention. Preferably, the CAR or transgenic TCR may be introduced into a T cell according to the present invention.

[0023] The starting population of cells in the methods of the invention may comprise a leukapheresate.

[0024] The starting population of cells in the methods of the invention may comprise peripheral blood mononuclear cells (PBMCs).

[0025] The depleted starting population in the methods of the invention may comprise PBMCs.

[0026] The depleted starting population in the methods of the invention may comprise cytolytic immune cells.

[0027] The depleted starting population in the methods of the invention may comprise T cells.

[0028] The selected population in the methods of the invention may comprise PBMCs.

[0029] The selected population in the methods of the invention may comprise cytolytic immune cells.

[0030] The selected population in the methods of the invention may comprise T cells.

[0031] The target antigen in the methods of the present invention may be TCR beta constant region 1 (TRBC1).

[0032] The target antigen in the methods of the present invention may be TCR beta constant region 2 (TRBC2).

[0033] In one embodiment, the percentage of CAR target antigen positive cells or transgenic TCR target antigen positive cells can be lower in the transduced or transfected cell population than in the starting population.Preferably, less than 10% of the transduced or transfected cells can express the target antigen of the CAR or transgenic TCR.Preferably, less than 5% of the transduced or transfected cells can express the target antigen of the CAR or transgenic TCR.Preferably, less than 1% of the transduced or transfected cells can express the target antigen of the CAR or transgenic TCR.

[0034] In one embodiment, the percentage of CAR target antigen positive cells or transgenic TCR target antigen positive cells can be lower in the population of genetically modified cells than in the starting population.Preferably, less than 10% of the genetically modified cells can express the target antigen of the CAR or transgenic TCR.Preferably, less than 5% of the genetically modified cells can express the target antigen of the CAR or transgenic TCR.Preferably, less than 1% of the genetically modified cells can express the target antigen of the CAR or transgenic TCR.

[0035] Suitably, the population of transduced or transfected cells may be prepared as a pharmaceutical composition.

[0036] Suitably, the population of genetically modified cells may be prepared as a pharmaceutical composition.

[0037] In a further aspect, the present invention provides a genetically modified cell comprising a CAR or a transgenic TCR obtainable (preferably obtained) by the method of the present invention.

[0038] Suitably, the present invention provides a population of genetically modified cells according to the present invention or a population of genetically modified cells obtainable (suitably obtained) by a method of the present invention.

[0039] Advantageously, the population of genetically modified cells according to the present invention may comprise cytolytic immune cells.

[0040] Suitably, the population of genetically modified cells according to the present invention may comprise T cells.

[0041] Advantageously, genetically modified cells according to the present invention may be less differentiated or more naive than genetically modified cells prepared without removing cells expressing the target antigen of the CAR or transgenic TCR and without selecting for CD4+ and CD8+ cells.

[0042] Genetically modified cells according to the present invention may have increased CD27 expression and / or CD62L expression compared to genetically modified cells prepared without removing cells expressing the target antigen of the CAR or transgenic TCR and without selecting for CD4+ and CD8+ cells.

[0043] Advantageously, genetically modified cells according to the present invention may be more naive than genetically modified cells prepared without removing cells expressing the target antigen of the CAR or transgenic TCR and without selecting for CD4+ and CD8+ cells.

[0044] Advantageously, the genetically modified cells according to the present invention may be non-exhausted compared to genetically modified cells prepared without removing cells expressing the target antigen of the CAR or transgenic TCR and without selecting for CD4+ and CD8+ cells.

[0045] Genetically modified cells according to the present invention may have reduced expression of one or more exhaustion markers compared to genetically modified cells prepared without removing cells expressing the target antigen of the CAR or transgenic TCR and without sorting for CD4+ and CD8+ cells.

[0046] The one or more exhaustion markers may be selected from the group consisting of PD1, Lag3 and Tim3.

[0047] In a further aspect, the present invention provides a pharmaceutical composition comprising a population of genetically modified cells according to the invention or a population of genetically modified cells obtainable (preferably obtained) by a method according to the invention.

[0048] In one aspect, the present invention provides a pharmaceutical composition according to the invention for use in treating and / or preventing a disease.

[0049] In another aspect, the present invention provides a method for treating and / or preventing a disease, the method comprising the step of administering a pharmaceutical composition according to the present invention to a subject in need thereof.

[0050] The method for treating and / or preventing the disease comprises: (i) providing a sample comprising a starting population of cells; (ii) depleting cells expressing the target antigen from the starting population to form a depleted starting population; (iii) sorting for CD4+ cells and CD8+ cells in the depleted starting population to form a sorted population of cells; and (iv) introducing into the cells in the selected population a nucleic acid sequence encoding a CAR or a transgenic TCR against the target antigen; and (v) administering cells derived from (iv) to a subject. It may further include.

[0051] Suitably, the method may further comprise a cell expansion step prior to administration to the patient, for example the cells may be cultured prior to administration to the patient.

[0052] The cells according to the invention may be of autologous origin. The cells according to the invention may be of allogeneic origin.

[0053] Cells according to the invention may be isolated from a subject.

[0054] In a further aspect the present invention relates to the use of a pharmaceutical composition according to the invention in the manufacture of a medicament for the treatment and / or prevention of a disease.

[0055] In one embodiment, the disease may be cancer, hi one embodiment, the cancer may be a solid tumor cancer.

[0056] In one embodiment, the disease may be a hematological malignancy. Preferably, the disease may be a leukemia. Preferably, the disease may be a lymphoma.

[0057] In a further aspect, the present invention provides a kit comprising: (i) a first nucleic acid sequence encoding a CAR or a transgenic TCR; (ii) a means for removing cells that express the target antigen for that CAR or transgenic TCR; and (iii) Means for selecting CD4+ cells and CD8+ cells Includes.

[0058] In one aspect, the present invention provides a method for reducing the number of cells in a pharmaceutical composition that express a target antigen and express a CAR or transgenic TCR to the target antigen, the method comprising: providing a starting population of cells; depleting cells expressing the target antigen from the starting population to form a depleted starting population; sorting for CD4+ cells and CD8+ cells in the depleted starting population to form a sorted population of cells; introducing into the cells in the selected population a nucleic acid encoding a CAR or a transgenic TCR for the target antigen; and Incorporating the cells into a pharmaceutical composition Includes.

[0059] The cells may be expanded prior to incorporation into the pharmaceutical composition.

[0060] The cells can be activated prior to introduction of the nucleic acid encoding the CAR or transgenic TCR.

[0061] The starting population of cells may be previously frozen and thawed prior to removal of cells expressing the target antigen.

[0062] The CAR or transgenic TCR can be introduced into the cells by transduction (e.g., using a retroviral or lentiviral vector), and the multiplicity of infection can be selected so that it is sufficient to transduce cells that do not express the target antigen, and insufficient to transduce cells that express the target antigen.

[0063] The percentage of CAR target antigen positive cells or transgenic TCR target antigen positive cells can be lower in the pharmaceutical composition than in the starting population of cells.Less than 10% of the cells in the pharmaceutical composition can express the target antigen of the CAR or transgenic TCR.Less than 5% of the cells in the pharmaceutical composition can express the target antigen of the CAR or transgenic TCR.Less than 1% of the cells in the pharmaceutical composition can express the target antigen of the CAR or transgenic TCR.

[0064] Less than 5% of the genetically modified cells in the pharmaceutical composition may express the target antigen. Less than 2% of the genetically modified cells in the pharmaceutical composition may express the target antigen. Less than 1% of the genetically modified cells in the pharmaceutical composition may express the target antigen.

[0065] (Detailed Description) The present invention provides a method for preparing a population of genetically modified cells, comprising chimeric antigen receptors or transgenic T cell receptors. The method includes removing cells expressing a target antigen, selecting for CD4+ and CD8+ cells, and introducing a nucleic acid sequence encoding a CAR or a transgenic TCR against the target antigen. These genetically modified cells are useful in methods for treating and / or preventing disease. By administering these genetically modified cells to a subject, these genetically modified cells cause the removal of cells expressing the target antigen.

[0066] (Chimeric Antigen Receptor (CAR)) Classical chimeric antigen receptors (CARs) are chimeric type I transmembrane proteins that link an extracellular antigen recognition domain (binder) with an intracellular signaling 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 that contain antibody-like antigen binding sites. A spacer domain is usually required to separate the binder from the membrane and orient it appropriately. A common spacer domain used is the Fc of IgG1. Smaller spacers (e.g., stalks from CD8α, and even IgG1 hinges alone) may be sufficient depending on the antigen. The transmembrane domain anchors the protein to the cell membrane and links the spacer to the endodomain.

[0067] Early CAR designs had endodomains derived from either the γ-chain of FcεR1 or the intracellular portion of CD3ζ. Consequently, these first generation receptors delivered immunological signal 1, which was sufficient to induce T cell killing of cognate target cells, but failed to sufficiently activate the T cell to proliferate and survive. To overcome this limitation, compound endodomains have been constructed: Fusing the intracellular portion of a T cell costimulatory molecule to the intracellular portion of CD3ζ results in second generation receptors that can simultaneously deliver activation and costimulatory signals after antigen recognition. The most commonly used costimulatory domain is that of CD28. This provides the most potent costimulatory signal (i.e., immunological signal 2) that induces T cell proliferation. Some receptors that contain TNF receptor family endodomains (e.g., the closely related OX40 and 41BB, which deliver survival signals) have also been described. Even more potent third generation CARs have now been described, with endodomains capable of delivering activation, proliferation and survival signals.

[0068] The nucleic acid encoding CAR can be transferred into T cells, for example, using retroviral vector.Lentiviral vector can be used.In this way, a large number of antigen-specific cells can be generated for adoptive cell transfer.When CAR binds to its target antigen, this results in transmitting activation signals to the T cells in which it is expressed.Therefore, the CAR directs the specificity and cytotoxicity of the T cells to the tumor cells that express the targeted antigen.

[0069] Thus, a CAR typically comprises: (i) an antigen-binding domain; (ii) a spacer; (iii) a transmembrane domain; and (iii) an intracellular domain that comprises or is associated with a signaling domain.

[0070] (Antigen-binding domain) The antigen binding domain is the portion of the CAR or transgenic TCR that recognizes the antigen.

[0071] Many antigen-binding domains are known in the art, including antigen-binding domains based on the antigen-binding site of antibodies, antigen-binding domains based on antibody mimetics, and antigen-binding domains based on 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; a single artificial binder (e.g., Darpin (designed ankyrin repeat protein)); or a single chain derived from a T cell receptor.

[0072] The antigen-binding domain may include a domain that is not based on the antigen-binding site of an antibody. For example, the antigen-binding domain may include a domain based on a protein / peptide (e.g., a soluble peptide, e.g., a cytokine or chemokine) that is a soluble ligand for a tumor cell surface receptor; or an extracellular domain of a membrane-anchored ligand or an extracellular domain of a receptor whose binding partner is expressed on tumor cells.

[0073] The antigen-binding domain may be based on a natural ligand of the antigen.

[0074] The antigen binding domain may comprise affinity peptides derived from a combinatorial library or de novo designed affinity proteins / peptides.

[0075] (Spacer domain) The CAR may contain a spacer sequence to link the antigen-binding domain to the transmembrane domain and to spatially separate the antigen-binding domain from the endodomain. A flexible spacer allows the antigen-binding domain to be oriented in various directions to facilitate binding.

[0076] The spacer sequence may, for example, comprise an IgG1 Fc region, an IgG1 hinge, or a human or mouse CD8 stalk. The spacer may alternatively comprise an alternative linker sequence having similar length and / or domain spacing characteristics as the IgG1 Fc region, the IgG1 hinge, or the CD8 stalk. The human IgG1 spacer may be altered to remove the Fc binding motif.

[0077] (Transmembrane domain) The transmembrane domain is the sequence of the CAR that spans the membrane.

[0078] The transmembrane domain can be any protein structure that is thermodynamically stable in the membrane. It is typically an alpha helix composed of several hydrophobic residues. The transmembrane domain of any transmembrane protein can be used to provide the transmembrane portion of the present invention.

[0079] The presence and extent 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 / ). Furthermore, considering that a transmembrane domain of a protein is a relatively simple structure (i.e., a polypeptide sequence that is predicted to form a hydrophobic α-helix of sufficient length to span the membrane), an artificially designed TM domain (e.g., as described in US7052906B1, which is incorporated herein by reference) can also be used.

[0080] The transmembrane domain may be derived from CD28, which provides good receptor stability.

[0081] (Activation endodomain) The endodomain is the signaling portion of the CAR. It can be part of or associated with the intracellular domain of the CAR. After antigen recognition, the receptors cluster, native CD45 and native CD148 are excluded from the synapse, and the signal is transmitted to the cell. The most commonly used endodomain component is that of CD3-ζ, which contains three ITAMs. It transmits an activation signal to antigen-bound T cells. CD3-ζ may not provide a fully qualified activation signal, and additional costimulatory signaling may be required. For example, chimeric CD28·OX40 may be used with CD3-ζ to transmit proliferation / survival signals, or all three may be used together.

[0082] When a CAR comprises an activating endodomain, it may comprise a CD3-zeta endodomain alone, a CD3-zeta endodomain and either a CD28 or OX40 endodomain, or a CD28 endodomain and both an OX40 and CD3-zeta endodomain.

[0083] Any endodomain containing an ITAM motif can act as an activating endodomain.

[0084] (Transgenic T cell receptor (TCR)) The T cell receptor (TCR) is a molecule found on the surface of T cells that is responsible for recognizing fragments of target antigens as peptides bound to major histocompatibility complex (MHC) molecules.

[0085] The TCR is a heterodimer composed of two different protein chains: in humans, in 95% of T cells the TCR consists of an alpha (α) chain (encoded by TRA) and a beta (β) chain (encoded by TRB), whereas in 5% of T cells the TCR consists of a gamma (γ) chain (encoded by TRG) and a delta (δ) chain (encoded by TRD).

[0086] Each chain is composed of two extracellular domains: a variable (V) region and a constant (C) region. The constant region is closest to the cell membrane, then the transmembrane region, then the short cytoplasmic tail, while the variable region binds to the peptide / MHC complex. The variable domains of the α and β chains of the TCR each have three hypervariable regions or complementarity determining regions (CDRs). CDR3 is the main CDR responsible for recognizing processed antigen. The constant domains of the TCR consist of short interlinking sequences in which cysteine ​​residues form disulfide bonds that form the link between the two chains.

[0087] When the TCR engages an antigenic peptide and MHC (peptide / MHC), the T lymphocyte is activated through signal transduction.

[0088] In contrast to conventional antibodies directing target antigens, antigens recognized by the TCR may include a whole host of possible intracellular proteins, which are processed and delivered to the cell surface as peptide / MHC complexes.

[0089] By artificially introducing TRA and TRB genes; or TRG and TRD genes into cells using vectors, cells can be engineered to express heterologous (i.e., non-native) TCR molecules. Such "heterologous" TCRs can also be referred to herein as "transgenic TCRs". For example, the gene of genetically modified TCR can be reintroduced into autologous T cells and transferred back into patients for T cell adoptive therapy.

[0090] (target antigen) As used herein, "target antigen" refers to an antigen for which a CAR or transgenic TCR has specificity, i.e., the antigen for which the antigen-binding domain of the CAR or transgenic TCR has been engineered to have specificity.

[0091] The target antigen may be a disease-associated antigen.

[0092] Suitably, the target antigen may be associated with a chronic infection.

[0093] Advantageously, the target antigen may be associated with autologous immunity.

[0094] The target antigen can be a tumor-associated antigen (e.g., a cancer-associated antigen).

[0095] A variety of target antigens are known, as shown in the table below: The antigen-binding domain used in the present invention may be a domain capable of binding to the antigens shown in the table. [Table 1]

[0096] (cell) The present invention also relates to genetically modified cells comprising a CAR or a transgenic TCR obtainable (or obtained) by the method of the present invention.

[0097] As used herein, a "starting population of cells" refers to a sample of cells used to generate genetically modified cells containing a CAR or transgenic TCR.

[0098] The starting population of cells can be obtained from any source of blood cells or peripheral blood mononuclear cells (PBMCs). The source cells can be provided fresh or frozen prior to use. The starting population of cells can be used without any further manipulation or 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 can be obtained from whole blood by various methods, such as density gradient separation (e.g., using Ficoll-Paque density gradient media); magnetic bead separation (e.g., MACS Milteyni Biotec CD3, CD4 or CD8 beads); elutriation or any other method. Cell separation or isolation can be automated or performed manually.

[0099] The starting population of cells can be derived from blood (e.g., from a peripheral blood sample or biopsy). The starting population of cells can be peripheral blood mononuclear cells. The starting population of cells can be a leukapheresate.

[0100] Preferably, the starting population of cells can be obtained from a subject (first party). Preferably, the starting population of cells can be obtained from a donor (second party). Preferably, the starting population of cells can be obtained from an unrelated donor (third party).

[0101] Alternatively, the cells may be derived from inducible or embryonic precursor cells by ex vivo differentiation, e.g., to T cells. Alternatively, immortalized cell lines may be used that retain their lytic function and can act as therapeutic agents.

[0102] Preferably, the starting population may be whole blood obtained from the subject. Preferably, the starting population may be PBMCs obtained from the subject. Preferably, the starting population may be a leukapheresate obtained from the subject.

[0103] Preferably, the starting population may be whole blood obtained from a donor. Preferably, the starting population may be PBMCs obtained from a donor. Preferably, the starting population may be a leukapheresate obtained from a donor.

[0104] As used herein, a "depleted starting population" refers to a population of cells that remain after cells expressing the target antigen have been removed from the starting population. In other words, the starting population has been depleted of cells expressing the target antigen, i.e., the depleted starting population has been depleted of the target antigen.

[0105] As used herein, "depleted" means that a particular type of cell (e.g., a cell expressing a CAR target antigen or a transgenic TCR target antigen) has been selectively reduced in number or removed from a population of cells.

[0106] As used herein, "sorted population" refers to the population of cells remaining after the CD4+ and CD8+ sorting process. Any suitable method known in the art for sorting CD4+ and CD8+ cells can be used. For example, magnetic bead separation (e.g., MACS Milteyni Biotec CD4 and CD8 beads) can be used. Cell separation or isolation can be automated or performed manually.

[0107] In the methods of the invention, sorting of CD4+ and CD8+ cells is performed following removal of the target antigen from the starting population.

[0108] As used herein, "transduced or transfected cells" refers to a cell population that has undergone a transduction or transfection process. The cell population may include a mixture of cells that have been successfully genetically modified and cells that have not.

[0109] As used herein, "genetically modified cells" refers to cells that have been modified to contain or express a CAR or transgenic TCR. Methods for engineering cells are known in the art, including, but not limited to, genetic modification of cells by transduction (e.g., retroviral or lentiviral transduction), transfection (e.g., DNA-based or RNA-based transient transfection), including lipofection, polyethylene glycol, calcium phosphate, and electroporation. Any suitable method can be used to introduce the nucleic acid sequence encoding a CAR or transgenic TCR into a cell.

[0110] Preferably, the genetically modified cell is a cell whose genome has been modified, for example by transduction or transfection. Preferably, the genetically modified cell is a cell whose genome has been modified by retroviral transduction. Preferably, the genetically modified cell is a cell whose genome has been modified by lentiviral transduction.

[0111] As used herein, the term "introduced" refers to a method for inserting foreign DNA or foreign RNA into a cell.As used herein, the term introduced includes both transduction and transfection methods.Transfection is the process of introducing nucleic acid into a cell by non-viral methods.Transduction is the process of introducing foreign DNA or foreign RNA into a cell via a viral vector.

[0112] Genetically modified cells according to the invention can be made by introducing DNA or RNA encoding a CAR or transgenic TCR by one of a number of means, including transduction with a viral vector, transfection with DNA or transfection with RNA.

[0113] The cells can be activated and / or expanded prior to introduction of the nucleic acid sequence encoding the CAR or transgenic TCR, for example, by treatment with an anti-CD3 monoclonal antibody, or with both an anti-CD3 monoclonal antibody and an anti-CD28 monoclonal antibody.

[0114] Suitably, the genetically modified cells may be autologous.

[0115] Preferably, the cells may be of allogeneic origin.

[0116] In one embodiment, the genetically modified cells may be PBMCs.

[0117] Preferably, the genetically modified cell may be a B cell. Preferably, the genetically modified cell may be a NK cell. Preferably, the genetically modified cell may be a T cell.

[0118] The genetically modified cell can be a cytolytic immune cell.

[0119] As used herein, "cytolytic immune cells" are cells that directly kill other cells.Cytolytic cells can kill cancerous cells, virus-infected cells or other damaged cells.Cytolytic immune cells include T cells and natural killer (NK) cells.

[0120] Cytolytic immune cells can be T cells or T lymphocytes, which are a type of lymphocyte that plays a central role in cell-mediated immunity. T cells can be distinguished from other lymphocytes (e.g., B cells and NK cells) by the presence of TCR on their cell surface. There are various types of T cells, as summarized below.

[0121] Helper T cells (TH cells) assist other white blood cells in immunological processes, including maturation of B cells to plasma cells and memory B cells, and activation of cytotoxic T cells and macrophages. TH cells express CD4 on their surface. TH cells are activated when they are presented with peptide antigens by MHC class II molecules on the surface of antigen-presenting cells (APCs). These cells can be differentiated into one of several subtypes, including TH1, TH2, TH3, TH17, Th9, or THF, which secrete various cytokines to promote different types of immune responses.

[0122] Cytolytic T cells (TC cells, or CTLs) destroy virus-infected and tumor cells and are also involved in graft rejection. CTLs express CD8 on their surface. CTLs may be known as CD8+ T cells. These cells recognize their targets by binding to antigens 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, CD8+ cells can be inactivated into an anergic state, which prevents autoimmune diseases (e.g., experimental autoimmune encephalomyelitis).

[0123] Memory T cells are a subset of antigen-specific T cells that persist for a long time after an infection has cleared. They rapidly proliferate into large numbers of effector T cells when re-exposed to their cognate antigen, thus providing the immune system with a "memory" against past infections. Memory T cells include three subtypes: central memory T cells (TCM cells) and two types of effector memory T cells (TEM cells and TEMRA cells). Memory cells can be either CD4+ or CD8+. Memory T cells typically express the cell surface protein CD45RO.

[0124] Regulatory T cells (Treg cells), previously known as suppressor T cells, are important for the maintenance of immune tolerance. Their primary role is to shut down T cell-mediated immunity, terminating immune responses, and to suppress autoreactive T cells that have escaped the process of negative selection in the thymus.

[0125] Two major types of CD4+ Treg cells have been described: naturally occurring Treg cells and adaptive or inducible Treg cells.

[0126] Naturally occurring Treg cells (also known as CD4+CD25+FoxP3+ Treg cells) arise in the thymus and are associated with interactions between developing T cells and both myeloid (CD11c+) and plasmacytoid (CD123+) dendritic cells activated with TSLP. Naturally occurring Treg cells can be distinguished from other T cells by the presence of an intracellular molecule called FoxP3. Mutations in the FOXP3 gene can prevent the development of regulatory T cells and cause the fatal autoimmune disease IPEX.

[0127] As used herein, the term "natural Treg" refers to thymus-derived Treg. Natural Treg is CD4+CD25+FOXP3+Helios+Neuropilin1+. Compared with iTreg, natural Treg (nTreg) has increased expression of PD-1 (programmed cell death 1, pdcd1), neuropilin 1 (Nrp1), Helios (Ikzf2), and CD73. Natural Treg (nTreg) can be distinguished from iTreg based on the individual expression of Helios protein or neuropilin 1 (Nrp1).

[0128] Adaptive Treg cells (also known as Tr1 cells or Th3 cells) can arise during a normal immune response.

[0129] Peripherally generated Tregs may also be called inducible Treg (iTreg) cells.

[0130] As used herein, the term "induced regulatory T cells" (iTreg) refers to CD4+CD25+FOXP3+Helios-Neuropilin1- T cells that arise from mature CD4+conventional T cells outside the thymus. For example, iTreg can be induced in vitro from CD4+CD25-FOXP3- cells in the presence of IL-2 and TGF-β.

[0131] Preferably, the cell may be a T cell. Preferably, the cell may be a helper T cell. Preferably, the cell may be a cytolytic T cell. Preferably, the cell may be a memory T cell. Preferably, the cell may be a regulatory T cell (Treg). Preferably, the cell may be a naturally occurring Treg or an adaptive Treg.

[0132] Natural killer cells (or NK cells) are a type of cytolytic cell that forms part of the innate immune system. NK cells provide a rapid response to innate signals from virus-infected cells in an MHC-independent manner.

[0133] NK cells (belonging to the group of innate lymphoid cells) are defined as large granular lymphocytes (LGL) and constitute the third type of cell differentiated from a common lymphoid precursor that gives rise to B and T lymphocytes. NK cells are known to differentiate and mature in the bone marrow, lymph nodes, spleen, tonsils and thymus, after which they enter the circulation.

[0134] Preferably, the cell may be a natural killer cell.

[0135] Preferably, the cell may be a stem cell.

[0136] In one embodiment, the cell may be a progenitor cell.

[0137] As used herein, the term "stem cell" refers to an undifferentiated cell that can indefinitely give rise to more stem cells of the same type, from which other specialized cells can be generated by differentiation.Stem cells are pluripotent.Stem cells can be, for example, embryonic stem cells or adult stem cells.

[0138] As used herein, the term "progenitor cell" refers to a cell that can differentiate to form one or more types of cells but has limited self-renewal in vitro.

[0139] Suitably, the cell may be any cell capable of differentiating into a cytolytic immune cell.

[0140] Suitably, the cells may be capable of differentiating into T cells or NK cells.

[0141] Preferably, the cell may be an embryonic stem cell (ESC). Preferably, the cell may be a hematopoietic stem cell or hematopoietic progenitor cell. Preferably, the cell may be an induced pluripotent stem cell (iPSC). Preferably, the cell may be obtained from umbilical cord blood. Preferably, the cell may be obtained from adult peripheral blood.

[0142] In some embodiments, hematopoietic stem progenitor cells (HSPCs) can be obtained from umbilical cord blood. Umbilical cord blood can be collected according to techniques known in the art (e.g., U.S. Patent Nos. 7,147,626 and 7,131,958, which are incorporated herein by reference).

[0143] In one embodiment, HSPCs may be obtained from pluripotent stem cell sources, such as induced pluripotent stem cells (iPSCs) and embryonic stem cells (ESCs).

[0144] As used herein, the term "hematopoietic stem progenitor cell" or "HSPC" refers to cells expressing antigenic marker CD34 (CD34+) and populations of such cells. In certain embodiments, the term "HSPC" refers to cells that are identified by the presence of antigenic marker CD34 (CD34+) and the absence of lineage (lin) markers. The population of cells that includes CD34+ and / or Lin(-) cells includes hematopoietic stem cells and hematopoietic progenitor cells.

[0145] HSPCs can be obtained or isolated from adult bone marrow, including femur, hipbone, rib, sternum and other bones.Bone marrow aspirate containing HSPCs can be obtained or isolated directly from hipbone using 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 certain embodiments, collecting sufficient amount of HSPCs for use in therapeutic applications may require the recruitment of the stem and progenitor cells in the subject.

[0146] As used herein, the term "induced pluripotent stem cell" or "iPSC" refers to non-pluripotent cells that are reprogrammed into a pluripotent state.Once the cells of interest are reprogrammed into a pluripotent state, they can then be programmed into a desired cell type, such as hematopoietic stem cells (HSCs) or hematopoietic progenitor cells (HPCs).

[0147] As used herein, the term "reprogramming" refers to a method of increasing the differentiation potential of a cell to a less differentiated state.

[0148] As used herein, the term "programming" refers to a method of reducing the differentiation potential of a cell or causing the cell to differentiate towards a more differentiated state.

[0149] The cells of the invention can be any of the cell types described above.

[0150] As used herein, a "population of genetically modified cells" refers to one or more genetically modified cells according to the present invention.

[0151] Suitably, a population of genetically modified cells as used herein may refer to two or more (or a plurality) of genetically modified cells according to the present invention.

[0152] The genetically modified cells or populations of genetically modified cells of the present invention include (i) providing a starting population of cells; (ii) depleting cells expressing the target antigen from the starting population to form a depleted starting population; (iii) sorting for CD4+ cells and CD8+ cells in the depleted starting population to form a sorted population of cells; and (iv) introducing into cells in the selected population a nucleic acid sequence encoding a CAR or a transgenic TCR against the target antigen. It can be generated by:

[0153] In one embodiment, the cell or population of cells may be activated before the CAR or transgenic TCR is introduced into the cell (iv).

[0154] In another embodiment, the cell or population of cells may not be activated before the CAR or transgenic TCR is introduced into the cell (iv).

[0155] Optionally, the method may further comprise the step of isolating a cell containing sample from the subject, which cell containing sample may be used as the starting population of cells.

[0156] Optionally, cells for use in the present invention may be activated and / or expanded prior to the introduction of the nucleic acid sequence encoding a CAR or transgenic TCR.

[0157] Any method known in the art for activating and / or expanding cells can be used in the methods of the present invention.For example, cells (e.g., T cells) for use in the present invention can be activated and / or expanded by treatment with anti-CD3 monoclonal antibody, or treatment with both anti-CD3 monoclonal antibody and anti-CD28 monoclonal antibody.

[0158] Preferably, interleukin 7 (IL-7) and / or interleukin 15 (IL-15) may be used to grow cells (e.g., T cells) in vitro. Preferably, interleukin 2 (IL-2) may be used for the growth of cells in vitro.

[0159] NK cells for use in the present invention may be activated and / or expanded by treatment with cytokines, such as interleukin 2 (IL-2) and / or interleukin 15 (IL-15). Incubation with accessory cells, such as monocytes, B lymphoblastoid cells or cell lines expressing stimulatory molecules, may be used to provide additional signals for proliferation of the NK cells.

[0160] As used herein, "activated" means that a cell has been stimulated to cause the cell to proliferate, differentiate, or initiate an effector function.

[0161] Methods for measuring cell activation are known in the art and include, for example, measuring the expression of activation markers (e.g., expression of CD69, CD25, CD71, CD38 or HLA-DR) by flow cytometry or measuring intracellular cytokines.

[0162] As used herein, "expanded" means that a cell or population of cells has been induced to proliferate.

[0163] The proliferation of a population of cells can be measured, for example, by counting the number of cells present in the population. The phenotype of the cells can be determined by methods known in the art, such as flow cytometry.

[0164] In one embodiment, a method according to the invention generates a population of engineered cells (e.g., genetically modified cells) that contain a chimeric antigen receptor or a transgenic T cell receptor.

[0165] Suitably, a genetically modified cell or a population of genetically modified cells according to the invention may be produced by a method according to the invention.

[0166] In one embodiment, a population of genetically modified cells according to the invention or a population of genetically modified cells obtainable (e.g., obtained) by a method according to the invention is less differentiated than genetically modified cells not prepared according to a method of the invention (i.e., prepared without removing cells expressing the target antigen of the CAR or transgenic TCR and without sorting for CD4+ and CD8+ cells).

[0167] As used herein, "differentiated" refers to a developmental stage of a particular cell within the linear progression of differentiation of that cell type. For example, CD4+ T cells and CD8+ T cells can be classified into distinct memory subsets based on their differentiation stage. CD4+ T cells and CD8+ T cells can be differentiated into central and effector memory cell populations (terminally differentiated effector memory cells (T EMRA ). The differentiation state of CD8+ T cells is inversely related to their ability to proliferate and persist.

[0168] Preclinical studies suggest that improved antitumor responses are achieved when genetically modified T cells are at an early differentiation stage (e.g., naive cells or central memory cells). Central memory cells can be classified as effector memory cells or T EMRA It has improved in vivo persistence compared to cells.

[0169] In one embodiment, the population of genetically modified cells according to the invention or the population of genetically modified cells obtainable by the method according to the invention is more naive than genetically modified cells that were not prepared according to the method of the invention (i.e., prepared without removing cells expressing the target antigen of the CAR or transgenic TCR and without sorting for CD4+ and CD8+ cells).

[0170] As used herein, "naive" refers to a cell that is not fully differentiated. Naive T cells may not have encountered an antigen.

[0171] Naive T cells can be characterized by surface expression of L-selection (CD62L), the absence of activation markers CD25, CD44, or CD69, and the absence of memory CD45RO isoforms, e.g., naive T cells express CD62L Hi CD25 Lo CD44 Lo CD69 Lo Naive T cells also express a functional IL-7 receptor, consisting of the subunits IL-7 receptor alpha, CD127, and the common gamma chain, CD132.

[0172] In one embodiment, the naive cell subset may be defined as CCR7+ / CD45RA+ cells. Suitably, the naive cell subset may be further defined as CCR7+ / CD45RA+ / CD62L+ / CD27+ cells.

[0173] Suitably, genetically modified cells according to the present invention or genetically modified cells obtainable (e.g. obtained) by a method according to the present invention may have increased CD27 expression and / or CD62L expression compared to genetically modified cells not prepared according to a method of the present invention (i.e. prepared without removing cells expressing the target antigen of the CAR or transgenic TCR and without selecting for CD4+ and CD8+ cells).

[0174] 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 increased in vivo persistence and increased cytolytic activity when compared to cells with a more differentiated phenotype.

[0175] Advantageously, the method according to the present invention can generate more naive or central memory cells than the method that does not remove cells expressing the target antigen of CAR or transgenic TCR and does not select for CD4+ and CD8+ cells.Advantageously, at least 75% of the genetically modified cells can be naive or central memory cells.Advantageously, at least 80% of the genetically modified cells can be naive or central memory cells.Advantageously, at least 85% of the genetically modified cells can be naive or central memory cells.

[0176] Advantageously, the method according to the present invention can generate fewer effector cells and effector memory cells than a method that does not remove cells expressing the target antigen of the CAR or transgenic TCR. Advantageously, less than 25% of the genetically modified cells can be effector cells or effector memory cells. Advantageously, less than 20% of the genetically modified cells can be effector cells or effector memory cells. Advantageously, less than 15% of the genetically modified cells can be effector cells or effector memory cells.

[0177] In one embodiment, a population of genetically modified cells according to the present invention or a population of genetically modified cells obtainable (or obtained) by a method according to the present invention is not exhausted compared to genetically modified cells not prepared according to a method of the present invention (i.e., prepared without removing cells expressing the target antigen of the CAR or transgenic TCR and without selecting for CD4+ and CD8+ cells).

[0178] As used herein, "exhaustion" or "exhausted" means that cells exhibit reduced effector function and / or altered phenotype.Immune cell exhaustion describes the state of dysfunction of immune cells, usually in the context of tumors or chronic infections.Exhaustion can be accompanied by phenotypic changes, epigenetic modifications and altered transcriptional profiles.

[0179] Effector functions can include the production of effector cytokines and direct cytotoxic activity.

[0180] Suitably, a population of genetically modified cells according to the invention or a population of genetically modified cells obtainable (or obtained) by a method according to the invention may have reduced expression of one or more exhaustion markers compared to genetically modified cells not prepared according to a method of the invention (i.e. prepared without removing cells expressing the target antigen of the CAR or transgenic TCR and without sorting CD4+ and CD8+ cells).

[0181] Preferably, the one or more exhaustion markers may be two exhaustion markers. Preferably, the one or more exhaustion markers may be three exhaustion markers. Preferably, the one or more exhaustion markers may be four exhaustion markers. Preferably, the one or more exhaustion markers may be five exhaustion markers. Preferably, the one or more exhaustion markers may be six exhaustion markers. Preferably, the one or more exhaustion markers may be seven exhaustion markers.

[0182] For example, in the context of NK cells, effector functions can include the production of interferon gamma (IFN-γ). Other effector functions of NK cells include direct cytotoxic activity (e.g., perforin- and granzyme-dependent activity) or the induction of target cell apoptosis by tumor necrosis factor alpha (TNF-α), Fas ligand (FasL), and TNF-related apoptosis-inducing ligand (TRAIL).

[0183] Suitably, exhausted NK cells may produce reduced amounts of effector cytokines (e.g., IFN-γ) compared to non-exhausted NK cells. Suitably, exhausted NK cells may have reduced cytolytic activity compared to non-exhausted NK cells, e.g., produce reduced amounts of CD107a and / or granzyme B and / or perforin compared to non-exhausted NK cells.

[0184] Suitably, the one or more exhaustion markers may be selected from the group consisting of IFN-γ, TNF-α, FasL, TRAIL, CD107a, granzyme B and perforin. Suitably, the one or more exhaustion markers may be selected from the group consisting of IFN-γ, TNF-α, FasL, TRAIL, CD107a, granzyme B and perforin, and the genetically modified cells are NK cells.

[0185] Suitably, the one or more exhaustion markers may include decreased IFN-γ production. Suitably, the one or more exhaustion markers may include decreased TNF-α production. Suitably, the one or more exhaustion markers may include decreased expression of FASL. Suitably, the one or more exhaustion markers may include decreased expression of TRAIL. Suitably, the one or more exhaustion markers may include decreased expression of CD107a. Suitably, the one or more exhaustion markers may include decreased production of granzyme B. Suitably, the one or more exhaustion markers may include decreased production of perforin.

[0186] For example, in the context of T cells, exhaustion may be defined by poor effector function, persistent expression of inhibitory receptors and / or a transcriptional state that differs from that of functional effector or memory T cells. For example, exhausted T cells may express high levels of PD1, Tim3, Lag3, CD43 (1B11), CD69 and inhibitory receptors, but low levels of CD62L and CD127, and may produce reduced interleukin-2 (IL-2), TNF-α and IFN-γ.

[0187] Suitably, the one or more exhaustion markers may include increased (e.g. high) expression of PD1. Suitably, the one or more exhaustion markers may include increased (e.g. high) expression of Tim3. Suitably, the one or more exhaustion markers may include increased (e.g. high) expression of Lag3. Suitably, the one or more exhaustion markers may include increased (e.g. high) expression of CD43 (1B11). Suitably, the one or more exhaustion markers may include increased (e.g. high) expression of CD69. Suitably, the one or more exhaustion markers may include increased (e.g. high) expression of an inhibitory receptor. Suitably, the one or more exhaustion markers may include decreased (e.g. low) expression of CD62L. Suitably, the one or more exhaustion markers may include decreased (e.g. low) expression of CD127. Suitably, the one or more exhaustion markers may include decreased (e.g., low) IL-2 production upon target engagement. Suitably, the one or more exhaustion markers may include decreased (e.g., low) TNF-α production upon target engagement. Suitably, the one or more exhaustion markers may include decreased (e.g., low) IFN-γ production upon target engagement.

[0188] Suitably, the one or more exhaustion markers may be selected from the group consisting of PD1, Lag3 and Tim3. 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, and the genetically modified cell is a T cell.

[0189] Without wishing to be bound by any one theory, the inventors believe that the inclusion of the depletion and selection steps in the method according to the invention advantageously significantly reduces or eliminates cytokine production during the manufacturing process compared to a comparable manufacturing process that does not include a depletion step of target antigen-expressing cells.

[0190] Cytokine analysis of media from terminal cultures during CAR-T cell production (i.e., days 6-10 after activation) may show production of low levels of IL-2, TNFα, IFNγ and granzyme B by cells that have had their target antigens removed.

[0191] The cells according to the invention or cells obtainable (e.g. obtained) by the method according to the invention may, when used in a cytotoxicity functional assay, show significantly increased production of the cytokines IFN-γ, IL-2 and TNF-α after co-culture with target cells (i.e. cells expressing a target antigen) compared to cells produced by a comparable manufacturing process that does not include a target antigen removal step.

[0192] In one embodiment, 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 invention or the population of genetically modified cells obtainable (or obtained) by the method according to the invention can be naive (CCCR7+ / CD45RA+) and CD62L+ / CD27+. Preferably, more than 10% of the genetically modified cells according to the invention can be naive (CCCR7+ / CD45RA+) and CD62L+ / CD27+. Preferably, more than 15% of the genetically modified cells according to the invention can be naive (CCCR7+ / CD45RA+) and CD62L+ / CD27+. Preferably, more than 20% of the genetically modified cells according to the invention can be naive (CCCR7+ / CD45RA+) and CD62L+ / CD27+. Advantageously, greater than 30% of the genetically modified cells according to the invention may be naive (CCCR7+ / CD45RA+) and CD62L+ / CD27+. Advantageously, greater than 40% of the genetically modified cells according to the invention may be naive (CCCR7+ / CD45RA+) and CD62L+ / CD27+.

[0193] Suitably, the production of naive cells may be measured in the CD8+ T cell subset.

[0194] In one embodiment, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 5% of the population of genetically modified cells according to the present invention or the population of genetically modified cells obtainable (or obtained) by the method according to the present invention may express multiple exhaustion markers. Preferably, less than 30% of the genetically modified cells according to the present invention may express multiple exhaustion markers. Preferably, less than 25% of the genetically modified cells according to the present invention may express multiple exhaustion markers. Preferably, less than 20% of the genetically modified cells according to the present invention may express multiple exhaustion markers. Preferably, less than 15% of the genetically modified cells according to the present invention may express multiple exhaustion markers.

[0195] Suitably, the multiple exhaustion markers may be selected from increased (e.g., high levels) expression of PD1, Tim3, Lag3, CD43 (1B11), CD69 and inhibitory receptors, and decreased (e.g., low levels) expression of CD62L and CD127, and decreased (e.g., low) production of interleukin-2 (IL-2), TNF-α and IFN-γ. Suitably, the multiple exhaustion markers may be selected from increased (e.g., high levels) expression of Lag3, PD1 and Tim3.

[0196] In one embodiment, a population of genetically modified cells according to the present invention or a population of genetically modified cells obtainable (or obtained) by a method according to the present invention has an increased level of transduction efficiency of the CAR or transgenic TCR compared to genetically modified cells not prepared according to the method of the present invention (i.e., prepared without removing cells expressing the target antigen of the CAR or transgenic TCR and without sorting CD4+ and CD8+ cells).

[0197] Suitably, the level of transduction efficiency in a population of genetically modified cells according to the invention or a population of genetically modified cells obtainable (or obtained) by a method according to the invention may be at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% higher than the transduction efficiency of genetically modified cells not prepared according to a method of the invention (i.e., prepared without removing cells expressing the target antigen of the CAR or transgenic TCR and without selecting CD4+ and CD8+ cells).

[0198] Advantageously, the level of transduction efficiency in a population of genetically modified cells according to the present invention or a population of genetically modified cells obtainable (or obtained) by a method according to the present invention may be at least 50% higher than the transduction efficiency of genetically modified cells not prepared according to the method of the present invention (i.e., without removing cells expressing the target antigen of the CAR or transgenic TCR and without selecting CD4+ and CD8+ cells). Advantageously, the level of transduction efficiency in a population of genetically modified cells according to the present invention or a population of genetically modified cells obtainable (or obtained) by a method according to the present invention may be at least 60% higher than the transduction efficiency of genetically modified cells not prepared according to the method of the present invention (i.e., without removing cells expressing the target antigen of the CAR or transgenic TCR and without selecting CD4+ and CD8+ cells). Advantageously, the level of transduction efficiency in a population of genetically modified cells according to the present invention or a population of genetically modified cells obtainable (or obtained) by a method according to the present invention may be at least 70% higher than the transduction efficiency of genetically modified cells not prepared according to the method of the present invention (i.e., without removing cells expressing the target antigen of the CAR or transgenic TCR and without selecting CD4+ and CD8+ cells). Advantageously, the level of transduction efficiency in a population of genetically modified cells according to the present invention or a population of genetically modified cells obtainable (or obtained) by a method according to the present invention may be at least 80% higher than the transduction efficiency of genetically modified cells not prepared according to the method of the present invention (i.e., without removing cells expressing the target antigen of the CAR or transgenic TCR and without selecting CD4+ and CD8+ cells).Suitably, the level of transduction efficiency in a population of genetically modified cells according to the invention or a population of genetically modified cells obtainable (or obtained) by a method according to the invention may be at least 90% higher than the transduction efficiency of genetically modified cells not prepared according to a method of the invention (i.e. prepared without removing cells expressing the target antigen of the CAR or transgenic TCR and without selecting CD4+ and CD8+ cells).

[0199] In one embodiment, 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 transduced or transfected cells comprise a CAR or transgenic TCR. Preferably, at least 40% of the population of transduced or transfected cells comprise a CAR or transgenic TCR. Preferably, at least 50% of the population of transduced or transfected cells comprise a CAR or transgenic TCR. Preferably, at least 60% of the population of transduced or transfected cells comprise a CAR or transgenic TCR.

[0200] Preferably, the percentage of cells expressing the target antigen that are transduced or transfected to express a CAR or transgenic CAR is less than 5%. Preferably, the percentage of cells expressing the target antigen that are transduced or transfected to express a CAR or transgenic CAR is less than 3%. Preferably, the percentage of cells expressing the target antigen that are transduced or transfected to express a CAR or transgenic CAR is less than 2%. Preferably, the percentage of cells expressing the target antigen that are transduced or transfected to express a CAR or transgenic CAR is less than 1%. Preferably, the percentage of cells expressing the target antigen that are transduced or transfected to express a CAR or transgenic CAR is undetectable.

[0201] (method) Methods are provided for preparing a population of genetically modified cells that contain a chimeric antigen receptor (CAR) or a transgenic T cell receptor (TCR).

[0202] Methods for preparing a population of genetically modified cells for cell therapy are generally known in the art. A method for preparing a population of genetically modified cells for cell therapy may include some or all of the following steps:

[0203] The starting material may be initially frozen, for example, the starting population of cells may be frozen once it is obtained from a donor (e.g., a source of cells). If frozen material is used, there may be a thawing and optional rest period before proceeding to the next step. Alternatively, fresh starting material may be used. The starting material may be subjected to an initial purification / enrichment for white blood cells (e.g., Ficoll gradient) or T cells.

[0204] The starting material can then be activated, e.g., T cells can be activated. This can be done by any method 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 will be appreciated in the art, the length of the activation step can vary, e.g., from less than 1 hour to more than 72 hours, before proceeding to the next step.

[0205] These activated cells can then be transduced with a viral vector (e.g., a retroviral or lentiviral vector). This can be done in the presence of a transduction enhancer (e.g., retronectin or polybrene), or by spinoculation, or by simple incubation. Non-viral vectors can also be used for this genetic modification step (e.g., using RNA electroporation or DNA-based transfer).

[0206] The cells may then be subjected to a proliferation step which may last from a few hours to a few days depending on the final dose of cells required. Generally, the more cells required, the longer the proliferation step.

[0207] The cells at the end of the manufacturing process can be used fresh, or preferably frozen before use.

[0208] Therefore, the entire process may take between 2 and 18 days. Typically, the entire process takes between 6 and 10 days.

[0209] During the process, the cells may be cultured in a cell growth medium that may contain additional supplements, such as human serum, fetal bovine serum, human serum albumin, and / or cytokines (e.g., IL2, IL7 and / or IL15, IL21).

[0210] A method for preparing a population of genetically modified cells comprising a chimeric antigen receptor (CAR) or a transgenic T cell receptor (TCR) is provided, the method comprising: (i) providing a starting population of cells; (ii) depleting cells expressing the target antigen from the starting population to form a depleted starting population; (iii) sorting for CD4+ cells and CD8+ cells in the depleted starting population to form a sorted population of cells; and (iv) introducing into cells in the selected population a nucleic acid sequence encoding a CAR or a transgenic TCR against the target antigen. Includes.

[0211] Optionally, the method can include performing (iv) (introducing into cells in the sorted population a nucleic acid sequence encoding a CAR or transgenic TCR against the target antigen) before (ii) (removing cells expressing the target antigen from the starting population), i.e., a nucleic acid sequence encoding a CAR or transgenic TCR against the target antigen can be introduced into cells before the cells expressing the target antigen are removed from those cells.

[0212] As used herein, "MOI" / "multiplicity of infection" refers to the number of vector infectious particles per cell used in transduction. For example, an MOI of 1 is 10 4 10 to 10 cells 4 The number of infectious particles is obtained by titration of the viral vector on a permissive cell line.

[0213] Preferably, the transduced cell type will be the same cell type used for the titer determination, in which case an MOI of 1 will result in an average of 1 vector integration per cell, as estimated by quantitative PCR or other suitable methods.

[0214] Suitably, the depleted starting population may be transduced at a low multiplicity of infection (MOI), e.g., a low MOI may be used in the methods of the invention to achieve a particular cell transduction level, as compared to the MOI required to transduce cells prepared by methods that do not deplete target antigen positive cells.

[0215] Advantageously, transducing a population of cells at a lower MOI may prevent transduction of cells that express the target antigen while allowing transduction of cells that do not express the target antigen. A preferred target MOI is 1.0.

[0216] Preferably, the MOI is selected so that the cells that do not express the target antigen are transduced and the cells that express the target antigen are not transduced. Preferably, the MOI is selected so that the cells that do not express the target antigen are transduced to a higher degree than the cells that express the target antigen. The population of genetically modified cells can have a higher ratio of cells that do not express the target antigen: cells that express the target antigen than the cells before transduction.

[0217] Suitably, the MOI is selected such that at least 5% of cells that do not express the target antigen are transduced, and that cells that express the target antigen are not transduced.

[0218] Preferably, the MOI is selected such that at least 10% of cells that do not express the target antigen are transduced, and cells that express the target antigen are not transduced. Preferably, the MOI is selected such that at least 15% of cells that do not express the target antigen are transduced, and cells that express the target antigen are not transduced.

[0219] Preferably, the MOI is selected such that at least 20% of cells that do not express the target antigen are transduced, and that cells that express the target antigen are not transduced.

[0220] Cells expressing the target antigen may be "untransduced" in that such cells are undetectable or are transduced at minimal or very low levels (e.g., 1% or less).

[0221] Advantageously, the method of the present invention may advantageously allow the use of a low MOI for transduction. Advantageously, the MOI required to achieve transduction of 20% to 30% of the target antigen negative cells may be lower in the method of the present invention than in a corresponding method that does not remove the target antigen positive cells. A preferred target MOI is 1.0.

[0222] Without wishing to be bound by theory, the method of the present invention may provide a safety advantage for the production of genetically modified cells because a lower MOI is required for transduction. Advantageously, cells expressing the target antigen may not be transduced by the method of the present invention or may be transduced at minimal / very low levels.

[0223] As known in the art, the use of transduction enhancers can change the MOI required for transduction, for example, lowering the MOI required for transduction. Transduction enhancers are known in the art (e.g., VectoFusin or RetroNectin).

[0224] Preferably, the MOI may be selected to achieve about 10% to about 50% transduction. Preferably, the MOI may be selected to achieve about 15% to about 40% transduction. Preferably, the MOI may be selected to achieve about 20% to about 30% transduction. Alternatively, the MOI may be selected to achieve the highest possible transduction efficiency (e.g., greater than 50%).

[0225] Preferably, the MOI may be about 0.05, about 0.1, about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1, about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 2.0, about 2.5, about 3.0, about 3.5, about 4.0, about 4.5, or about 5.0. Preferably, the MOI may be in the range of about 0.1 to about 5.0. Preferably, the MOI may be in the range of about 0.2 to about 1.0. Preferably, the MOI may be in the range of about 0.3 to about 0.8. Preferably, the MOI may be in the range of about 0.4 to about 0.6.

[0226] Preferably, the MOI for achieving about 20% to about 30% transduction may be about 0.05, about 0.1, about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1, about 1.1, or about 1.2. Preferably, the MOI for achieving about 20% to about 30% transduction may be in the range of about 0.1-1.2. Preferably, the MOI for achieving about 20% to about 30% transduction may be in the range of about 0.2-1.0. Preferably, the MOI for achieving about 20% to about 30% transduction may be in the range of about 0.3-0.8. Preferably, the MOI for achieving about 20% to about 30% transduction may be in the range of about 0.4-0.6.

[0227] Suitably, the percentage of CAR target antigen positive cells or transgenic TCR target antigen positive cells may be lower in the depleted starting population or population of genetically modified cells than in the starting population.

[0228] Preferably, the percentage of target antigen positive cells can be reduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or 100% in the removed starting population or the population of genetically modified cells when compared to the percentage of target antigen positive cells in the starting population. Preferably, the percentage of target antigen positive cells can be reduced by at least 90%. Preferably, the percentage of target antigen positive cells can be reduced by at least 95%. Preferably, the percentage of antigen positive cells can be reduced by at least 98%. Preferably, the percentage of antigen positive cells can be reduced by at least 99% in the removed starting population or the population of genetically modified cells when compared to the percentage of target antigen positive cells in the starting population.

[0229] Suitably, less than 40%, less than 30%, less than 20%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1% of the depleted starting population may express the target antigen of the CAR or transgenic TCR.

[0230] Suitably, less than 10% of the depleted starting population may express the target antigen of the CAR or transgenic TCR.

[0231] Suitably, less than 5% of the depleted starting population may express the target antigen of the CAR or transgenic TCR.

[0232] Suitably, less than 3% of the depleted starting population may express the target antigen of the CAR or transgenic TCR.

[0233] Suitably, less than 2% of the depleted starting population may express the target antigen of the CAR or transgenic TCR.

[0234] Suitably, less than 1% of the depleted starting population may express the target antigen of the CAR or transgenic TCR.

[0235] Suitably, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1% of the genetically modified cells may express the target antigen of the CAR or transgenic TCR.

[0236] Suitably, less than 10% of the genetically modified cells may express the target antigen of the CAR or transgenic TCR.

[0237] Suitably, less than 5% of the genetically modified cells may express the target antigen of the CAR or transgenic TCR.

[0238] Suitably, less than 1% of the genetically modified cells may express the target antigen of the CAR or transgenic TCR.

[0239] Pharmaceutical Composition 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.

[0240] In one aspect, a pharmaceutical composition is provided comprising a population of genetically modified cells according to the invention or a population of genetically modified cells obtainable by a method according to the invention.

[0241] Advantageously, the pharmaceutical composition may comprise a cryopreserved genetically modified cell according to the invention or a cryopreserved genetically modified cell obtainable by a method according to the invention.

[0242] The pharmaceutical composition may further comprise a pharma- ceutically acceptable carrier, diluent or excipient.The pharmaceutical composition may optionally comprise one or more additional pharma- ceutically active polypeptides and / or compounds.Such preparations may be, for example, in a form suitable for intravenous infusion.

[0243] In one aspect, the present invention provides a method for reducing the number of cells in a pharmaceutical composition that express a target antigen and express a CAR or transgenic TCR to the target antigen, the method comprising: providing a starting population of cells; depleting cells expressing the target antigen from the starting population to form a depleted starting population; sorting for CD4+ cells and CD8+ cells in the depleted starting population to form a sorted population of cells; introducing into the cells in the depleted starting population a nucleic acid encoding a CAR or a transgenic TCR for the target antigen; and Incorporating the cells into a pharmaceutical composition Includes.

[0244] Preferably, the number of cells that express the target antigen and express a CAR or transgenic TCR to the target antigen is reduced compared to a pharmaceutical composition produced without removing cells that express the target antigen from the starting population and without selecting for CD4+ and CD8+ cells.

[0245] In one aspect, the present invention provides a method for preparing a pharmaceutical composition, the method comprising: providing a starting population of cells; depleting cells expressing the target antigen from the starting population to form a depleted starting population; sorting for CD4+ cells and CD8+ cells in the depleted starting population to form a sorted population of cells; introducing into the cells in the depleted starting population a nucleic acid encoding a CAR or a transgenic TCR for the target antigen; and Incorporating the cells into a pharmaceutical composition Includes.

[0246] Suitably, the cells may be expanded prior to incorporation into a pharmaceutical composition, for example, the cells may be expanded following introduction of a nucleic acid encoding a CAR or transgenic TCR against the target antigen.

[0247] Suitably, the cells can be activated prior to the introduction of a nucleic acid encoding a CAR or transgenic TCR, for example, the cells can be activated prior to the introduction of a nucleic acid encoding a CAR or transgenic TCR against its target antigen.

[0248] The cells can be activated and / or expanded by any method known in the art (e.g., by treatment with an anti-CD3 monoclonal antibody, or by treatment with both an anti-CD3 monoclonal antibody and an anti-CD28 monoclonal antibody).

[0249] Suitably, the starting population of cells may be previously frozen. If frozen cells are used, they may be thawed and, if necessary, allowed to recover in culture before being processed.

[0250] The method may further comprise enriching the starting population for leukocytes. Any method known in the art for isolating or enriching leukocytes may be used. For example, leukocytes or PBMCs may be obtained from whole blood by various methods, such as density gradient separation (e.g., using Ficoll-Paque density gradient medium); magnetic bead separation (e.g., MACS Milteyni Biotec CD3 beads, CD4 beads or CD8 beads); elutriation or any other method. Cell separation or isolation may be automated or performed manually.

[0251] Preferably, the CAR or transgenic TCR can be introduced into the cells by transduction or transfection. Preferably, the CAR or transgenic TCR can be introduced into the cells by transduction, and the multiplicity of infection can be selected so that it is sufficient to transduce cells that do not express the target antigen, and insufficient to transduce cells that express the target antigen.

[0252] Successful transduction or transfection of cells with a nucleic acid encoding a CAR or transgenic TCR can be identified by methods known in the art (e.g., by flow cytometry).

[0253] Suitably, the method according to the invention reduces the number of genetically modified cells expressing the target antigen of the CAR or transgenic TCR, i.e., the method reduces the number of genetically modified target cells in the pharmaceutical composition.

[0254] Advantageously, the percentage of CAR target antigen positive cells or transgenic TCR target antigen positive cells can be lower in the pharmaceutical composition than in the starting population of cells.Advantageously, 10% of the cells in the pharmaceutical composition can express the target antigen of CAR or transgenic TCR.Advantageously, less than 5% of the cells in the pharmaceutical composition can express the target antigen of CAR or transgenic TCR.Advantageously, less than 1% of the cells in the pharmaceutical composition can express the target antigen of CAR or transgenic TCR.

[0255] Preferably, less than 10% of the cells in the pharmaceutical composition can express the CAR or transgenic TCR and the target antigen of the CAR or transgenic TCR. Preferably, less than 5% of the cells in the pharmaceutical composition can express the CAR or transgenic TCR and the target antigen of the CAR or transgenic TCR. Preferably, less than 1% of the cells in the pharmaceutical composition can express the CAR or transgenic TCR and the target antigen of the CAR or transgenic TCR.

[0256] In one embodiment, the population of genetically modified cells according to the invention is the active ingredient of the pharmaceutical composition.

[0257] As will be understood by those skilled in the art, pharmaceutical compositions can further comprise impurities.In the context of the present invention, impurities can be cells that express the target antigen.Preferably, in the context of the present invention, impurities can be cells that express the target antigen and express the CAR or transgenic TCR.In the context of the present invention, impurities can be cells that do not express the CAR or transgenic TCR.

[0258] The present invention provides methods for reducing impurities (e.g., cells expressing the target antigen of the CAR or transgenic TCR) in a pharmaceutical composition.

[0259] Preferably, the pharmaceutical composition according to the invention comprises a population of cells, where less than 10% of the cells are impurities. Preferably, the pharmaceutical composition according to the invention comprises a population of cells, where less than 5% of the cells are impurities. Preferably, the pharmaceutical composition according to the invention comprises a population of cells, where less than 3% of the cells are impurities. Preferably, the pharmaceutical composition according to the invention comprises a population of cells, where less than 2% of the cells are impurities. Preferably, the pharmaceutical composition according to the invention comprises a population of cells, where less than 1% of the cells are impurities.

[0260] (Method of treatment) The genetically modified cells of the present invention may be capable of killing target cells (eg, cancer cells, virally infected cells or other damaged cells).

[0261] The genetically modified cells of the present invention can be used in a therapeutic method. The genetically modified cells of the present invention can be used for the treatment and / or prevention of a disease. Preferably, the pharmaceutical composition comprising the genetically modified cells according to the present invention can be used in a therapeutic method. Preferably, the pharmaceutical composition comprising the genetically modified cells according to the present invention can be used for the treatment and / or prevention of a disease.

[0262] It is understood that the target antigen of the CAR or transgenic TCR is selected based on the required treatment.For example, if the CAR or transgenic TCR is for treating cancer, the target antigen of the CAR or transgenic TCR can be an antigen associated with cancer.

[0263] The genetically modified cells of the present invention can be used for the treatment of infectious diseases (eg, viral infections).

[0264] The genetically modified cells of the present invention can also be used for the control of pathogenic immune responses (eg, in autoimmune diseases, allergies and graft-versus-host rejection).

[0265] The present invention provides a method for treating and / or preventing a disease, the method comprising the step of administering to a subject a genetically modified cell of the present invention.

[0266] The present invention provides a method for treating and / or preventing a disease, the method comprising the step of administering to a subject a pharmaceutical composition of the present invention.

[0267] The present invention also provides the genetically modified cells of the present invention for use in treating and / or preventing disease.

[0268] The present invention also provides a pharmaceutical composition of the present invention for use in treating and / or preventing disease.

[0269] The present invention also relates to the use of a genetically modified cell according to the invention in the manufacture of a medicament for treating and / or preventing a disease.

[0270] Suitably, the method of treatment may involve administration of a pharmaceutical composition of the present invention to a subject.

[0271] Preferably, the present invention provides a method of treatment, the method comprising: (i) providing a sample comprising a starting population of cells; (ii) depleting cells expressing the target antigen from the starting population to form a depleted starting population; (iii) sorting for CD4+ cells and CD8+ cells in the depleted starting population to form a sorted population of cells; (iv) introducing into the cells in the selected population a nucleic acid sequence encoding a CAR or a transgenic TCR against the target antigen; and (v) administering cells derived from (iv) to a subject. Includes.

[0272] Suitably, the method may further comprise a cell expansion step prior to administration to the patient, for example the cells may be cultured prior to administration to the patient.

[0273] The genetically modified cells or pharmaceutical compositions of the present invention may be used for the treatment and / or prevention of: cancerous diseases, such as hematological malignancies, bladder cancer, breast cancer, colon cancer, uterine cancer, renal cancer (renal cell), lung cancer, melanoma, pancreatic cancer, prostate cancer and thyroid cancer, oral and pharyngeal cancer (including tongue, mouth and pharynx cancer); digestive system cancer (including esophageal cancer, stomach cancer and colorectal cancer); liver and bile duct cancer (including hepatocellular carcinoma and cholangiocarcinoma); respiratory system cancer (including bronchogenic carcinoma and laryngeal cancer); bone and joint cancer (including osteosarcoma); skin cancer (including melanoma); breast cancer; reproductive organ cancer (including uterine, ovarian and cervical cancer in women, prostate and testicular cancer in men); urinary system tract cancers (including renal cell carcinoma, and transitional cell carcinoma of the utterer or bladder); brain cancers (including glioma, glioblastoma multiforme and medulloblastoma); endocrine system cancers (including thyroid cancer, adrenal cancer and cancers associated with multiple endocrine neoplasia syndrome); and other and non-site specific cancers (including neuroblastoma).

[0274] Suitably, the genetically modified cells or pharmaceutical compositions of the present invention may be used for the treatment and / or prevention of hematological malignancies.

[0275] As used herein, "hematologic malignancies" refers to cancers that affect the blood and lymphatic system, including leukemia, lymphoma, myeloma, and related blood disorders.

[0276] Suitably, the genetically modified cells or pharmaceutical compositions of the present invention may be used for the treatment and / or prevention of hematological malignancies.

[0277] Advantageously, the genetically modified cells or pharmaceutical compositions of the present invention may be used in the treatment and / or prevention of: both acute and chronic myeloid or lymphoid leukemias (acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), acute promyelocytic leukemia (APL), and B-cell acute lymphoblastic leukemia (B-ALL) or T-cell acute lymphoblastic leukemia (T-ALL), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), chronic myelomonocytic leukemia (CMML), hairy cell leukemia (HCL) and large granular lymphocytic leukemia (LGL). lymphomas, including Hodgkin's lymphoma and non-Hodgkin's lymphoma (NHL) (both low-grade NH and high-grade NHL); myelomas, including multiple myeloma (MM) (including smoldering or indolent myeloma, and symptomatic myeloma), and other conditions associated with blood cancers, such as monoclonal gammopathy of undetermined significance (MGUS), myelodysplastic syndromes (MDS), isolated plasmacytoma, and myeloproliferative neoplasms (MPN), including essential thrombocythemia (ET), myelofibrosis (MF), and polycythemia vera (PV).

[0278] Preferably, the genetically modified cells or pharmaceutical compositions of the present invention can be used in the treatment and / or prevention of T-cell lymphoma. Preferably, the genetically modified cells or pharmaceutical compositions of the present invention can be used in the treatment and / or prevention of T-cell leukemia.

[0279] Methods for treating T-cell lymphoma and / or T-cell leukemia relate to therapeutic uses of the genetically modified cells of the present invention. The genetically modified cells can be administered to a subject with an existing disease of T-cell lymphoma and / or T-cell leukemia to attenuate, reduce, or ameliorate at least one symptom associated with the disease, and / or to slow, reduce, or prevent the progression of the disease.

[0280] Advantageously, the method of the present invention can be used for the treatment of any lymphoma and / or leukemia associated with the clonal proliferation of cells expressing a T cell receptor (TCR) comprising a β constant region. Thus, the present invention can relate to a method for treating a disease involving malignant T cells expressing a TCR comprising TRBC (e.g., TRBC1 or TRBC2).

[0281] Advantageously, the method of the present invention can be used to treat T cell lymphoma, in which malignant T cells express TCR, including TRBC. "Lymphoma" is used herein according to its standard meaning to refer to cancer, which typically develops in lymph nodes, but can also affect spleen, bone marrow, blood and other organs. Lymphoma typically appears as a solid tumor of lymphocytes. The main symptom associated with lymphoma is lymphadenopathy, while secondary (B) symptoms can include fever, night sweats, weight loss, loss of appetite, fatigue, shortness of breath and itching.

[0282] The methods of the invention can be used to treat T cell leukemia in which malignant T cells express TCRs, including TRBCs. "Leukemia" is used herein according to its standard meaning to refer to cancer of the blood or bone marrow.

[0283] The following is an exemplary, non-exhaustive list of diseases that can be treated by the methods of the present invention.

[0284] Preferably, the T cell lymphoma or T cell leukemia may be peripheral T cell lymphoma, not otherwise specified (PTCL-NOS). Preferably, the T cell lymphoma or T cell leukemia may be angioimmunoblastic T cell lymphoma (AITL). Preferably, the T cell lymphoma or T cell leukemia may be anaplastic large cell lymphoma (ALCL). Preferably, the T cell lymphoma or T cell leukemia may be enteropathy-associated T cell lymphoma (EATL). Preferably, the T cell lymphoma or T cell leukemia may be hepatosplenic T cell lymphoma (HSTL). Preferably, the T cell lymphoma or T cell leukemia may be extranodal NK / T cell lymphoma, nasal type. Preferably, the T cell lymphoma or T cell leukemia may be cutaneous T cell lymphoma (CTCL). Preferably, the T cell lymphoma or leukemia may be primary cutaneous (ALCL). Preferably, the T cell lymphoma or leukemia may be T cell prolymphocytic leukemia. Preferably, the T cell lymphoma or leukemia may be T cell acute lymphoblastic leukemia.

[0285] Treatment with genetically modified cells of the invention or pharmaceutical compositions according to the invention may help prevent the escape or release of tumor cells that often occurs with standard approaches.

[0286] The term "treat / treatment / treating" refers to the administration of 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 to attenuate, reduce, or ameliorate at least one symptom associated with the disease and / or to slow, reduce, or prevent the progression of the disease.

[0287] As used herein, references to "prevention" / "preventing" (or prophylaxis) refer to delaying or preventing the onset of disease symptoms. Prevention may be absolute (so that the disease does not develop at all) or may be effective in only some individuals or for a limited time.

[0288] In a preferred embodiment of the 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.

[0289] (Administration) Administration of the pharmaceutical composition can be accomplished using any of a variety of routes that make the genetically modified cells contained in the pharmaceutical composition bioavailable to the subject. For example, the composition can be administered by oral and parenteral routes, intraperitoneally, intravenously, subcutaneously, transdermally, intramuscularly, or by local delivery (e.g., by catheter or stent).

[0290] Preferably, the genetically modified cells according to the invention or the pharmaceutical composition according to the invention are administered intravenously.

[0291] Those skilled in the art will understand that, for example, the route of delivery (e.g., oral, intravenous, subcutaneous, etc.) can affect the dosage and / or the required dosage can affect the delivery route. For example, if a particularly high concentration of the drug is of interest in a particular site or location, focused delivery may be desirable and / or useful. Other factors to consider when optimizing the route and / or administration schedule for a given treatment regimen may include, for example, the disease to be treated (e.g., type or stage, etc.), the clinical condition of the subject (e.g., age, general health, etc.), the presence or absence of concomitant therapy, and other factors known to medical practitioners.

[0292] The amount administered is such that it is sufficient to stabilize or ameliorate symptoms of the disease.

[0293] Typically, a physician will determine the actual dosage that will be most appropriate for an individual subject, which will vary with the age, weight, and response of that particular patient, that dosage being that which is sufficient to reduce the number of cells expressing the target antigen or to eliminate cells expressing the target antigen.

[0294] (use) The present invention also provides a pharmaceutical composition or a population of genetically modified cells according to the invention for use in treating a disease. The pharmaceutical composition or the population of genetically modified cells may be any as defined above.

[0295] The present invention also relates to the use of a population of genetically modified cells of the invention as defined above in the manufacture of a medicament for the treatment of a disease.

[0296] (kit) The present invention also provides a kit, the kit comprising: (i) a first nucleic acid sequence encoding a CAR or a transgenic TCR; (ii) a means for selecting CD4+ cells and CD8+ cells; and (ii) a means for removing cells expressing a target antigen for that CAR or transgenic TCR. Includes.

[0297] As used herein, "means for removing cells expressing a target antigen" refers to any product known in the art suitable for removing or separating specific cells from a mixed population of cells.

[0298] As used herein, "means for selecting CD4+ and CD8+ cells" refers to any product known in the art that is suitable for removing or separating CD4+ and CD8+ cells from a mixed population of cells.Any method known in the art that is suitable for selecting CD4+ and CD8+ cells can be used.For example, magnetic bead separation (e.g., MACS Milteyni Biotec CD4 and CD8 beads) can be used.Cell separation or isolation can be automated or performed manually.

[0299] Suitably, the kit may include an antibody specific for the target antigen.

[0300] Suitably, the kit may include a depletion antibody. A "depletion antibody" is an antibody that binds to an antigen present on a target cell and mediates the death of the target cell. Thus, administration of a depletion antibody to a population of cells results in a reduction / reduction in the number of cells in the population that express the target antigen.

[0301] Suitably, the kit may include a solid matrix coated with an antibody, and cells expressing the target antigen may be removed by adsorption to the antibody-coated solid matrix.

[0302] Advantageously, the kit may include a fluorescent antibody specific for the target antigen. Cells containing the target antigen may be isolated by flow cytometry.

[0303] Suitably, the kit may include an immunomagnetic product (ie, an antibody specific for a target antigen attached to a magnetic nanoparticle or bead). EXAMPLES

[0304] The present invention will now be further described by way of examples, which are intended to serve to assist those skilled in the art in practicing the present invention and are not intended to limit the scope of the invention in any way. (Example) Example 1 - Preparation of a Genetically Modified Autologous Cell Gene Therapy Product for the Treatment of T-Cell Lymphoma

[0305] The standard CAR-T cell manufacturing process can be summarized by the following five steps: Introducing a TRBC1+ cell depletion step and a CD4 / CD8 selection step prior to the activation step (herein referred to as the next generation process or NG process) was evaluated and compared with a method without a CD4 / CD8 selection step (herein referred to as the current process). 1. Isolation of PBMCs from the leukapheresate to provide the starting material for the manufacturing process. 2. Activation of patient-derived T cells using anti-CD3 and anti-CD28 monoclonal antibodies in the presence of cytokines. 3. Transduction of patient-derived T cells with retroviral supernatant. 4. Proliferation of the transduced T cells using cytokines (drug substance [DS]). 5. Formulation and filling of cells into bags and cryopreservation (DP).

[0306] Proof-of-principle (PoP) development experiments (capturing TRBC1+, CD4+ and CD8+ cells labeled with anti-TRBC1, anti-CD4 or anti-CD8 antibodies using MACS microbeads and columns (Miltenyi)) demonstrated the feasibility for efficient cell sorting as well as manufacturing. In summary, the incorporation of a CD4 / CD8 sorting step resulted in a more robust process that allowed the target dose of 450 million transduced cells to be reached in a shorter time frame (Figure 1), all the while with improved cell viability (Figure 3). Cells produced by the NG process also showed improved differentiation profiles (Figures 2 and 4). In particular, naïve and central memory cells accounted for 78% of the transduced cells at harvest on day 7 of the NG process (Figure 5).

[0307] Further comparison of the NG process with previous processes using patient samples confirmed that the NG process produced improved differentiation profiles, particularly at the time of collection (Figure 6).

[0308] This study establishes the feasibility of deleting cells expressing a target antigen and selecting for CD4+ and CD8+ cells as part of a genetically modified cell manufacturing process, without compromising the ability of those depleted and selected cells to be activated, transduced, and expanded in culture.

[0309] The analyses described herein demonstrate that depletion of TRBC1+ cells, combined with CD4+ and CD8+ sorting, provides distinct advantages for the generation of cellular gene therapy formulations comprising T cells retrovirally transduced to express anti-TRBC1-specific CARs.

[0310] (Experimental approach) Optimal TRBC1+ cell labeling and depletion / sorting conditions were established using Ms (mouse) anti-TRBC1 (JOVI-1) antibody (unconjugated and biotinylated) in combination with MACS microbeads (anti-Ms and anti-biotin) and columns. A series of PoP development experiments (performed on healthy donor or patient leukapheresate at various scales) were followed to evaluate the incorporation of depletion and sorting steps into the CAR-T cell manufacturing process, including adaptation to the automated CliniMACS Prodigy CAR-T cell manufacturing protocol for GMP compliance. Data were collected to determine the efficiency of depletion throughout the process, as well as to evaluate transduction efficiency, cell proliferation and viability, and memory and activation phenotypes.

[0311] Cytotoxicity assays allow for functional assessment of the generated CAR-T cells. This involves co-culturing those CAR-T cells with TRBC1+ Raji cells or TRBC+ Jurkat cells (lymphoblastoid tumor cell lines transduced to express the TRBC1 tumor antigen). FACS was used to determine target cell killing after 48-72 hours. Furthermore, cell proliferation and interferon gamma cytokine release analyses provide measures of CAR-T cell survival, cytotoxicity and differentiation capacity.

[0312] (Protocol Overview) For small-scale and large-scale experiments, 1.5 × 10 TRBC1+ cells were used. 7 pieces~10×10 8 The small-scale and large-scale experiments were performed using 0.3 × 10 cells. 6 pieces~6×10 8 T cells were transduced. Additionally, some large-scale experiments can be automated on the CliniMACS Prodigy starting from day 0. All experiments follow the same basic protocol. On day -1, frozen donor leukapheresis samples were thawed and allowed to recover by overnight incubation in serum-supplemented cell culture medium. In some cases, frozen leukapheresate was also thawed on day 0 before being treated with TRBC1 depletion.

[0313] In some cases, fresh leukapheresis was immediately processed for TRBC1 depletion on day 0. For depletion on day 0, TRBC1+ cells were cultured at 10 6 The cells were labeled with 0.02 μg–0.125 μg of anti-TRBC1 antibody (either unconjugated or biotinylated) per cell, then depleted / sorted on a MACS column, and then cultured for 10 min at 4°C for 10 min. 7Depletion / sorting with 7.5 μl-10 μl of MACS microbeads (anti-Ms or anti-biotin) per cell. FACS was then used to determine the TRBC1+ cell content in the pre-depletion / sorting and post-depletion / sorting samples to provide an assessment of the sorting efficiency. Cells are then sorted for CD4 and CD8 expression using MACS columns after labeling with anti-CD4 and anti-CD8 antibodies. Cells from those sorted and unsorted subsets are seeded in cell culture medium supplemented with serum, IL-7, IL-15 and TransAct reagent (Miltenyi) for T cell activation.

[0314] (CAR transduction) On day 2 (48 h after activation), cells are transduced with anti-TRBC1 CAR vectors at a specific MOI along with Retronectin (Takara) or Vectofusin (Miltenyi) used as transduction enhancers. Cells are allowed to transduce for 18-22 h, after which the cultures are washed and re-seeded in fresh cell culture medium supplemented with serum, IL-7 and IL-15, and the cells are then cultured until day 5-10.

[0315] (cell purity) Analyses performed at the end of the manufacturing process consist of FACS to determine the % TRBC1+ cell content and transduction efficiency in CD3+, CD4+, and CD8+ cell subsets, along with activation phenotype (CD25, CD69, and CD71) and memory phenotype (CCR7 expression versus CD45RA expression). Cell counts are also performed to assess the proliferation of the cultures.

[0316] These data demonstrate that the novel process of the present invention produces improved preparations from both healthy donor and patient samples.

[0317] All publications mentioned in the above specification are incorporated herein by reference. Various modifications and variations of the described methods and systems of the present invention will be apparent to those skilled in the art without departing from the scope and spirit of the present invention. Although the present invention has been described in terms of certain preferred embodiments, it should be understood that the claimed invention should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention that are obvious to those skilled in the art of molecular biology or related fields are intended to be within the scope of the appended claims.

Claims

1. 1. A method for preparing a population of genetically modified cells comprising a chimeric antigen receptor (CAR) or a transgenic T cell receptor (TCR), comprising: (i) providing a starting population of cells; (ii) depleting cells expressing a target antigen from the starting population to form a depleted starting population; (iii) sorting for CD4+ cells and CD8+ cells in the depleted starting population to form a sorted population of cells; and (iv) introducing into cells in the selected population a nucleic acid sequence encoding a CAR or a transgenic TCR against the target antigen. A method comprising:

2. 2. The method of claim 1, wherein the CAR or transgenic TCR is introduced into a cytolytic immune cell.

3. 10. The method of claim 1, wherein the starting population of cells comprises a leukapheresis product.

4. 2. The method of any preceding claim, wherein the starting population of cells comprises peripheral blood mononuclear cells (PBMCs).

5. 2. The method of any preceding claim, wherein the depleted starting population comprises PBMCs.

6. 2. The method of any preceding claim, wherein the depleted starting population comprises cytolytic immune cells.

7. 2. The method of any preceding claim, wherein the depleted starting population comprises T cells.

8. 2. The method of any of the preceding claims, wherein the target antigen is TCR beta constant region 1 (TRBC1).

9. 8. The method of any one of claims 1 to 7, wherein the target antigen is TCR beta constant region 2 (TRBC2).

10. 2. The method of any of the preceding claims, wherein the percentage of CAR target antigen positive cells or transgenic TCR target antigen positive cells is lower in the population of genetically modified cells than in the starting population.

11. 2. The method of any preceding claim, wherein less than 10% of the genetically modified cells express the target antigen of the CAR or transgenic TCR.

12. 2. The method of any preceding claim, wherein less than 5% of the genetically modified cells express the target antigen of the CAR or transgenic TCR.

13. 2. The method of any preceding claim, wherein less than 1% of the genetically modified cells express the target antigen of the CAR or transgenic TCR.

14. 2. The method of any preceding claim, wherein the genetically modified cells are formulated as a pharmaceutical composition.

15. A genetically modified cell comprising a CAR or a transgenic TCR obtainable by any of the preceding claims.

16. A population of genetically modified cells according to claim 15.

17. 17. The population of genetically modified cells of claim 16, wherein the cells are cytolytic immune cells.

18. 18. The population of genetically modified cells of claim 16 or 17, wherein the cells are T cells.

19. 19. The population of genetically modified cells of any one of claims 16-18, wherein the genetically modified cells are less differentiated than genetically modified cells prepared without removing cells expressing a target antigen of the CAR or transgenic TCR and without sorting for CD4+ and CD8+ cells.

20. 20. The population of genetically modified cells of any one of claims 16 to 19, wherein the genetically modified cells have increased CD27 expression and / or CD62L expression compared to genetically modified cells prepared without removing cells expressing a target antigen of the CAR or transgenic TCR and without sorting for CD4+ and CD8+ cells.

21. 21. The population of genetically modified cells of any one of claims 16 to 20, wherein the genetically modified cells are more naive than genetically modified cells prepared without removing cells expressing a target antigen of the CAR or transgenic TCR and without sorting for CD4+ and CD8+ cells.

22. 22. The population of genetically modified cells of any one of claims 16-21, wherein the genetically modified cells are not exhausted compared to genetically modified cells prepared without removing cells expressing a target antigen of the CAR or transgenic TCR and without sorting for CD4+ and CD8+ cells.

23. 23. The population of genetically modified cells of any one of claims 16-22, wherein the genetically modified cells have reduced expression of one or more exhaustion markers compared to genetically modified cells prepared without removing cells expressing a target antigen of the CAR or transgenic TCR and without sorting for CD4+ and CD8+ cells.

24. 24. The population of genetically modified cells of claim 23, wherein the one or more exhaustion markers are selected from the group consisting of PD1, Lag3 and Tim3.

25. A pharmaceutical composition comprising a population of genetically modified cells according to any one of claims 16 to 24.

26. 26. A pharmaceutical composition according to claim 25 for use in treating and / or preventing a disease.

27. A method for treating and / or preventing a disease, comprising: Administering the pharmaceutical composition of claim 25 to a subject in need thereof. A method comprising:

28. 28. The method of claim 27, (i) providing a sample comprising a starting population of cells; (ii) removing cells expressing the target antigen from the starting population; (iii) introducing into cells in the depleted starting population a nucleic acid sequence encoding a CAR or a transgenic TCR against the target antigen; and (iv) administering cells derived from (iii) to a subject. A method comprising:

29. 30. The method of claim 28, wherein the cells are autologous.

30. 29. The method of claim 28, wherein the cells are of allogeneic origin.

31. 26. Use of the pharmaceutical composition according to claim 25 in the manufacture of a medicament for the treatment and / or prevention of a disease.

32. The pharmaceutical composition for use according to claim 26, the method according to any one of claims 27 to 30, or the use according to claim 31, wherein the disease is cancer.

33. 33. The pharmaceutical composition, method or use according to claim 31 or claim 32, wherein the disease is a hematological malignancy.

34. 34. The pharmaceutical composition, method or use according to claim 32 or claim 33, wherein the disease is leukemia or lymphoma.

35. (i) a first nucleic acid sequence encoding a CAR or a transgenic TCR; and (ii) a means for removing cells expressing a target antigen for said CAR or transgenic TCR. Including the kit.

36. 1. A method for reducing the number of cells in a pharmaceutical composition that express a target antigen and express a CAR or a transgenic TCR to the target antigen, comprising: Providing a starting population of cells; removing cells expressing a target antigen from said starting population; introducing into cells in the depleted starting population a nucleic acid encoding a CAR or a transgenic TCR against the target antigen; and Incorporating the cells into a pharmaceutical composition. A method comprising:

37. 37. The method of claim 36, wherein the number of cells that express a target antigen and express a CAR or transgenic TCR to the target antigen is reduced compared to a pharmaceutical composition produced without removing cells that express the target antigen from the starting population and without sorting for CD4+ and CD8+ cells.

38. 38. The method of claim 36 or 37, wherein the cells are expanded prior to incorporation into the pharmaceutical composition.

39. 39. The method of any one of claims 36 to 38, wherein the cells are activated prior to introduction of a nucleic acid encoding a CAR or a transgenic TCR against the target antigen.

40. 40. The method of any one of claims 36 to 39, wherein the starting population of cells has been previously frozen and thawed prior to removal of cells expressing a target antigen.

41. 41. The method of any one of claims 36 to 40, wherein the CAR or transgenic TCR is introduced into the cells by transduction, and wherein the multiplicity of infection is sufficient to transduce cells that do not express the target antigen and insufficient to transduce cells that express the target antigen.

42. 42. The method of any one of claims 36 to 41, wherein the percentage of cells expressing the target antigen is lower in the pharmaceutical composition than in the starting population of cells.

43. The method according to one of claims 36 to 40, wherein less than 10% of the cells in the pharmaceutical composition express the target antigen.

44. The method of any one of claims 36 to 41, wherein less than 5% of the cells in the pharmaceutical composition express the target antigen.

45. The method of any one of claims 36 to 42, wherein less than 1% of the cells in the pharmaceutical composition express the target antigen.

46. The method of any one of claims 36 to 43, wherein less than 5% of the cells in the pharmaceutical composition express a CAR or a transgenic TCR against the target antigen and the target antigen.

47. The method of any one of claims 36 to 43, wherein less than 1% of the cells in the pharmaceutical composition express a CAR or a transgenic TCR against the target antigen and the target antigen.

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