BR112017015631B1 - USE OF A MODIFIED NK CELL, THERAPEUTIC COMPOSITION, METHODS FOR PRODUCING A UNIVERSAL NK CELL AND FOR PREPARING A MODIFIED NK CELL AND KIT - Google Patents

Abstract

UNIVERSAL EXTERMINATOR T CELL. The present invention relates to a modified natural killer (NK) cell and its use in personalized medicine. The modified NK cells of the present invention are non-immunogenic, which means that they can be administered to any recipient subject without being rejected by the host's immune system (they are 'universal'). In a first embodiment, non-immunogenic NK cells are modified to express CD3 to allow a T Cell Receptor (TcR) to be expressed. In another embodiment, non-immunogenic NK cells are further modified to express a TcR together with the CD3 coreceptor. Coexpression of CD3 with a specific TcR results in modified NK cells that show antigen-specific cytotoxicity towards target cells. Thus, universal NK cells can be directed against specific antigens and can therefore be used in personalized medicine, particularly in the field of oncology.

Description

[001] This invention relates to natural killer cells and their use in therapy, particularly in the treatment of cancer. Specifically, a modified natural killer cell has been developed, which has been modified to express CD3 and which can further be modified to coexpress an antigen receptor based on a T cell receptor specific for a cancer antigen or other target antigen. The engineered natural killer cell is non-immunogenic, for example, expresses low levels of MHC or is MHC-negative, meaning the cells are suitable for universal use regardless of the subject's MHC type. Thus, advantageously, the invention combines the characteristics of universality and personalization; The T cell receptor can be matched to both the disease condition (e.g. type of cancer) and the MHC type of the subject being treated, allowing the “universal cells” to be used in a treatment that is personalized to the subject to be discussed.

[002] Certain cells of the immune system demonstrate cytotoxic activity against specific target cells. Cytotoxic T lymphocytes express T cell receptors (TcRs) that are capable of specifically recognizing antigen-derived peptides bound to MHC class I molecules. In contrast, natural killer (NK) cells are not MHC restricted and do not require antigen presentation by MHC molecules to exert their killing effect. They are able to recognize stressed cells in the absence of peptide-loaded MHC and to kill cells lacking MHC. NK cells play an important role in innate immunity, as these “non-MHC” cells would otherwise not be detected and destroyed by other immune cells.

[003] Cytotoxic T cells (also known as cytotoxic T lymphocytes or killer T cells) are capable of recognizing infected or damaged cells in an antigen-specific manner through the TcR by binding to antigens (peptides) presented on the cell surface by molecules of MHC class I. Binding to peptide-MHC class I is mediated by the CD8 coreceptor, which increases the affinity of the binding interaction and increases signal transduction by the TcR. The TcRs of other types of T cells, especially helper T cells, recognize antigenic peptides presented by MHC class II molecules, mediated by the CD4 coreceptor. CD3 is required on T cells for localization of the TcR to the cell surface (Ahmadi et al. 2011. Blood 118, 3528-3537), and is required for activation of a T cell upon contact with an MHC protein loaded with a suitable antigen peptide. CD3 is a protein complex composed of four distinct chains: a CD3Y chain, a CD3δ chain, two CD3ε chains and the Z chain, which together with TcR, forms the TcR complex.

[004] NK cells (also defined as “large granular lymphocytes”) represent a cell lineage differentiated from the common lymphoid progenitor (which also gives rise to B lymphocytes and T lymphocytes). Unlike T cells, NK cells do not naturally comprise CD3 on the plasma membrane. Importantly, NK cells do not express a TCR and typically also lack other antigen-specific cell surface receptors (as well as TCRs and CD3, they also do not express immunoglobulin B cell receptors and instead typically express CD16 and CD56). Thus, NK cells are differentiated by their CD3-CD56+ phenotypes. The cytotoxic activity of NK cells does not require sensitization but is enhanced by activation with a variety of cytokines, including IL-2. NK cells are considered to lack appropriate or complete signaling pathways required for antigen receptor-mediated signaling and are therefore not thought to be capable of antigen receptor-dependent signaling, activation, and expansion.

[005] NK cells are cytotoxic and depend on activation of inhibitory receptor balance and signaling to modulate their cytotoxic activities. For example, NK cells expressing CD16 (the FCYRIII receptor) can bind to the Fc domain of antibodies bound to an infected cell, resulting in NK cell activation. Conversely, activity is reduced against cells expressing high levels of MHC class I proteins. Upon contact with a target cell, NK cells release proteins such as perforin and enzymes such as proteases (granzymes). Perforin can form pores in the cell membrane of a target cell, inducing apoptosis or cell lysis.

[006] Several T cell-based therapies have been developed for the treatment of cancer, and these treatments, known as adoptive cell transfer (ACT) have become increasingly attractive in recent years. Three main ACT strategies have been explored so far. The first of these, and the most developed, involves the isolation of the patient's own tumor-reactive T cells from peripheral or tumor sites (known as Tumor Infiltrating Lymphocytes (TILs)). These cells are expanded ex vivo and reinjected into a patient. However, this method can involve delays of several weeks while the cells are expanded and requires special cell production facilities.

[007] Two alternative therapies are available that involve modifying a patient's own T cells with receptors capable of recognizing a tumor. In one option, TcRs that have activity toward a cancer antigen can be isolated and characterized, and a gene encoding the TcR can be inserted into T cells and reinjected into a patient. This therapy has been shown to shrink solid tumors in some patients, but is associated with a significant disadvantage: the TcRs used must be adapted to the patient's immune type. Consequently, as an alternative to the use of TcRs, therapies involving the expression of Chimeric Antigen Receptors (CARs) on T cells have also been suggested. CARs are fusion proteins comprising an antibody linked to the signaling domain of the TcR complex and can be used to direct T cells against a tumor if a suitable antibody is selected. Unlike a TCR, a CAR does not need to be MHC compatible for the recipient. However, so far, few cancer-specific surface antigens have been identified that can be used as suitable targets for CARs, and therefore the use of CARs in cancer therapies is limited at present.

[008] All ACT approaches that involve modifying a T cell with a TcR or a CAR require the isolation and modification of T cells from a patient or tissue type-matched donor. The use of autologous T cells is necessary to avoid the risk of rejection if non-autologous cells are used. This increases the costs associated with ACT and may also increase the time scale required to prepare T cells for use in ACT.

[009] Alternative methods that seek to overcome the above limitations of ACT use cytotoxic NK cells, as described, for example, in WO 98/49268. NK cells are potent killer cells and are highly cytotoxic against many different malignant cells. Since NK cells will recognize target cells that express non-self HLA molecules but not self-HLA antigens, autologous NK cells are generally not effective and halogen NK cells (which require careful removal of T cells to avoid GvHD) or cell lines need to be used. Therapies based on irradiated cells from the NK cell line NK-92 have evolved into clinical trials in the treatment of leukemias and other hematological malignancies. Irradiation means that the cells are unable to proliferate and therefore the killing effect is of limited and defined duration. NK cells also do not attack healthy tissue. However, the natural “variety” of NK cells is limited, and although NK-92 cells have a wider variety than primary NK cells, since NK cells do not naturally comprise suitable receptors to specifically target an antigen in a target cell, a new modification of these cells is needed in order to broaden the variety of cancers that can be treated and to target, or redirect, cells against a specific or selected cancer. This redirection means, of course, that the requirement for halogenated NK cells is removed, but NK cell lines may still have advantages, e.g. improved cytotoxicity, compared to primary or autologous NK cells.

[010] To this end, NK cells that express CARs have been developed for use in cancer treatment. CARs that recognize antigens on the surface of cancer cells have been introduced into the continuously growing NK-92 cell line, and NK-92 cells comprising these CARs have been shown to have improved cytotoxicity to tumor cells relative to parental NK-92 cells (Uherek et al. 2002. Blood 200, 1265-1273). The first clinical trials are underway. However, as noted above, the lack of antigens available as targets for CARs currently limits the use of CAR-based therapies.

[011] The present invention is based on an alternative approach to providing specific cancer killer cells. More particularly, the present invention aims to provide specific cancer killer cells, based on NK cells, for universal use, meaning that the cells do not have to be matched to the immune type of the subject being treated, as is currently required to T cell-based therapies, but can, however, be tailored to the subject's specific type of immunity and cancer as needed. Thus, universal killer cells can be used for personalized medicine.

[012] The present invention thus proposes to modify an NK cell into a T cell and, therefore, make it capable of expressing a TcR and exterminating cells in a specific and targeted manner, like a T cell. potency and the inherent natural ability to kill NK cells with the specific targeting and cellular activation provided by TcR. Although CARs have previously been expressed on NK cells, it has not been considered possible until now that an NK cell would possess all of the necessary signaling pathways and machinery to allow a TcR to be successfully expressed in a manner that results in successful activation and NK cell-directed killing. Universality is provided by using an NK cell that is non-immunogenic, for example, that naturally expresses low levels of MHC, or that has reduced proliferative capacity, or by making the NK cell MHC negative (e.g., HLA), so so that the cell is not recognized as a foreign body by the recipient's immune system. Thus, the NK cell does not need to be adapted to the immune type (e.g., HLA-) of the subject to be treated and can be administered universally, regardless of the subject's immune type (i.e., to a subject with any type of immunity, e.g. example, any HLA profile/type).

[013] The present invention thus provides means that allow a functional and active TcR to be expressed in a non-immunogenic NK cell. In this way, a universal killer T cell can be provided that is not dependent on the cell's MHC matching, but that has controlled specificity dictated by the specificity of the TcR that is introduced into the cell. Coexpression of CD3 and a TcR on NK cells was sufficient for the TcR to localize to the surface of NK cells, and surprisingly, the NK cell expressing the TcR demonstrated antigen-specific cytotoxicity toward target cells. Viewed another way, the present invention provides a means for converting an NK cell into a cytotoxic T cell with the additional benefits of universality, simply through the co-expression of CD3 and a TcR on a non-immunogenic NK cell. The present invention thus provides a “universal” killer T cell, which can be used in personalized medicine.

[014] Thus, in a first aspect, the present invention provides a modified NK cell, wherein said cell is non-immunogenic and is modified to express CD3.

[015] In particular, the cell is modified to be non-immunogenic, for example, by irradiation to reduce proliferative capacity or by some other means of reducing proliferation, so that the cell does not persist long enough to mount an immune response when introduced into a subject or by modifying the cell to be MHC negative.

[016] The cells of the invention can further be modified to express a TcR with specificity in relation to an antigen on a target cell (i.e., a target antigen). As further defined below, the term “TcR” is used broadly herein to include any antigen receptor that comprises or is based on TcR antigen recognition regions or is derived from a TcR. Thus, native and synthetic TcRs, e.g., TcR variants or derivatives, are included.

[017] In a particular embodiment, the present invention provides a non-immunogenic NK cell that expresses CD3 and a TcR, wherein said TcR is specific for an antigen on a cancer cell. Stated differently, the present invention provides a cancer-specific NK cell, wherein the cell is non-immunogenic and wherein cancer specificity is provided by co-expression of a cancer-specific TcR and CD3. As will be described in more detail below, in a preferred embodiment, the TcR is coreceptor independent, particularly, independent of CD4 and/or CD8.

[018] In another aspect, the present invention provides cells as previously defined for use in therapy, more particularly, for use in cancer therapy or, more generally, in adoptive cell transfer therapy (for example, in the treatment of any condition that may respond to T-cell therapy).

[019] In yet another additional aspect, it provides the use of a modified NK cell as previously defined in the manufacture of a medicine for use in cancer therapy or for adoptive cell transfer therapy.

[020] Also provided is a therapeutic composition comprising a modified NK cell as previously defined, together with at least one pharmaceutically acceptable carrier or excipient, and such a therapeutic composition for use in cancer therapy or adoptive cell transfer therapy.

[021] Another additional aspect provides a method of treatment, more particularly, a method of treating cancer, or a method of adoptive cell transfer therapy, which method comprises administering to a subject in need thereof (for example, a subject suffering from or diagnosed with cancer) an effective amount of a modified NK cell as defined earlier herein.

[022] For use in therapy, the modified NK cells of the invention will be modified to co-express a TcR with CD3. Thus, the preparation of a medicament or composition for use in therapy will comprise modifying the NK cell to express a TcR. The TcR will be designed or selected firstly to match the immune type (i.e. MHC) of the subject to be treated (i.e. the recipient of the modified NK cells) and secondly to match or recognize the cells in the subject to be treated. be targeted by NK cells. This is the TcR designed or selected to recognize a desired target antigen in the subject. This may be a target antigen expressed (or presented) by a cancer cell in the subject or by any cell that is to be abrogated by adoptive cell transfer therapy, e.g., an infected cell, e.g., a cell that expresses (or presents) a virus or other pathogenic antigen.

[023] Thus, to carry out cancer therapy or adoptive cell transfer therapy according to the invention or to prepare a modified NK cell for therapeutic use, the following steps can be performed: (a) providing a non-immunogenic NK cell that has been modified to express CD3; (b) determine the MHC type of a subject to be treated; (c) identifying a target antigen in the subject, which antigen is expressed or presented by cells in the subject, e.g., identifying or determining the subject's type of cancer and/or the expression of a specific cancer marker (e.g., antigen ) or presence of a particular mutation, etc.; (d) modifying the cell from step (a) to express a TcR with specificity for the target antigen and matching the subject's MHC type.

[024] As will be discussed later, the steps can be performed separately or sequentially, or simultaneously. Thus, for example, steps (a) and (d) can be carried out simultaneously, more particularly, the cell can be modified to express CD3 and a TcR in one or more simultaneous steps. However, in another embodiment, the cell can be modified to express CD3 in a separate step prior to modification to express a TcR.

[025] Finally, in a treatment method or therapeutic use, the modified cell from step (d) can be administered to the subject (for example, in another step (e)).

[026] In another aspect of the present invention, there is provided a method for producing a universal NK cell (which cell is suitable for (or capable of) therapeutic use), or more particularly, a universal NK cell for use in the preparation of a cell for personalized therapeutic use, i.e., for adoptive cell transfer therapy, said method comprising: a) providing an NK cell that is non-immunogenic, more specifically, modifying an NK cell to make it non-immunogenic; and b) modifying said cell to express CD3.

[027] To further provide a modified NK cell for use in adoptive cell transfer therapy, the cell is further modified in step (c) to express a TcR and, in particular, for personalized therapy, a TcR that is compatible with the MHC type of the subject to be treated and which recognizes a target antigen expressed or presented by a target cell to be targeted by the modified NK cell.

[028] In a preferred embodiment, there is provided a method for producing a cancer-specific NK cell, said method comprising: a) providing an NK cell that is non-immunogenic, for example, modifying an NK cell to make it non-immunogenic; b) modifying said cell to express CD3; c) modifying said cell to express a cancer-specific TcR.

[029] Steps (b) and (c) can be carried out together (simultaneously) or separately, for example, sequentially. However, in advantageous embodiments, as will be described later, a universal NK cell can be prepared to express CD3, which cell can subsequently be used in separate steps to prepare personalized cells for adoptive transfer therapy by further modifying the cells to express CD3. a TcR that is selected based on the subject's diagnosis and MHC type. Thus, a bank or library of TcR receptors can be prepared containing different TcRs (more specifically, nucleic acid molecules or vectors encoding TcRs) according to MHC specificity (i.e., type) and antigen-specificity. target (e.g., to a variety of different known or previously identified target antigens, e.g., cancer antigens). Depending on the subject's diagnosis (e.g., a specific type of cancer or cancer known to express a particular antigen or identified by specific analysis of the subject's cancer), a particular TcR can be selected and used to modify the NK cell according to the invention. Alternatively, a bank or panel of modified NK cells can be prepared expressing CD3 and TcRs of varying specificities, from which an appropriate NK cell can be selected depending on the subject's diagnosis and MHC type.

[030] Accordingly, another additional aspect of the present invention provides a combined product or kit, particularly, said product or kit for use in adoptive cell transfer therapy, or in cancer therapy, said product or kit comprising: (a) a modified universal NK cell that is non-immunogenic and expresses CD3, more particularly an NK cell that is modified to be non-immunogenic and express CD3; and (b) a panel of nucleic acid molecules each encoding a TcR, wherein the TcRs have different antigen specificity and/or different MHC specificity.

[031] Conveniently, the encoding nucleic acid molecules can be provided in vectors, for example, vectors ready for introduction (i.e., transfection or transduction, etc.) into the modified NK cell. In a preferred embodiment, the TcRs have specificity for a cancer antigen or for an antigen expressed (e.g., preferentially or specifically) on cancer cells.

[032] Another additional aspect of the invention provides a panel or library of modified NK cells, wherein each NK cell being non-immunogenic and expressing CD3, for example, modified to be non-immunogenic and to express CD3, and expressing (i.e. , being modified to express) a TcR, wherein the TcRs of different NK cells have different antigen specificity and/or different MHC specificity.

[033] It should be understood that, in such aspects, there may be multiple TcRs that recognize the same antigen, but may differ in their MHC specificity and vice versa.

[034] The present invention employs cytotoxic cells of the immune system in the treatment of cancer. Specifically, the present invention relates to the expression of TcRs in cytotoxic cells of the immune system that would not otherwise express a TcR, and methods that allow the expression of active and functional TcRs in such cells.

[035] The terms “cytolytic” and “cytotoxic” are used interchangeably herein to refer to a cell capable of inducing cell death by lysis or apoptosis in a target cell.

[036] The term “target cell” refers to any cell that is killed by the cytotoxic cells of the invention. More particularly, the target cell is the targeted cell of NK cells and therefore a cell that expresses or presents an antigen recognized by the TcR (a target antigen). These can include any type of cell and are targeted by a cell of the invention via an antigen displayed on the surface of the target cell.

[037] Thus, the target cell can be any cell in the subject's body that is desired to be nullified, for example, to remove, kill, leave inactive, etc. A preferred target cell is a cancer cell, but it can be any cell resulting from a disease or clinical condition. The disease or condition can therefore be any condition that could benefit from adoptive cell transfer therapy, or more particularly, T cell therapy that targets cells for killing or removal, etc. In addition to cancer cells, other cells that may be clinically desirable to remove include infected cells, which are cells infected with any pathogen. Typically, such cells will be virus-infected cells, but they may also be infected with any other pathogenic organism, for example, any microorganism, for example, bacteria, fungi, mycoplasma, protozoa, prions. Alternatively, the target cell may be apoptotic or pre-apoptotic, or be in a stressed state (i.e., expressing stress-related markers on its cell surface), or may be a mutant cell, for example, expressing a particular mutation.

[038] The “target antigen” is (or, more specifically, provides or comprises) the molecule that is recognized by the TcR when presented on the target cell. Thus, the target antigen, or more particularly, a peptide derived from the target antigen, is presented on the target cell in an MHC-I or MHC II-restricted manner, such as is required to be recognized by a TcR. Accordingly, as discussed above, the target antigen may be a cancer antigen, that is, an antigen that is expressed specifically, selectively or preferentially by a cancer cell, or that is characteristic of a cancer cell or known or used as a marker of cancer cells. The cancer antigen may be a universal cancer antigen, that is, an antigen commonly found in a wide variety of different cancers, or it may be specific to a particular type of cancer. Alternatively, it may be an antigen from an infecting pathogen that is presented by the target cell, or any other antigen expressed or presented by any other cell that is desired to be nullified.

[039] An “MHC” refers to a protein of the major histocompatibility complex and includes MHCI and MHCII proteins. MHCII proteins are generally only found on the surface of antigen-presenting cells, such as dendritic cells, mononuclear phagocytes, some endothelial cells, and thymic epithelial cells. MHCII proteins are also expressed by B cells, and therefore can be expressed by B cell malignancies. MHCII proteins are also frequently expressed by melanoma cells. The term MHC encompasses human leukocyte antigen (HLA) and therefore, where the subject is human, these terms may be used interchangeably.

[040] Any NK cell can be used according to the present invention. The term “NK cell” refers to a large granular lymphocyte, a cytotoxic lymphocyte derived from the common lymphoid progenitor that does not naturally comprise a specific antigen receptor (e.g., a T-cell receptor or a B-cell receptor). NK cells are differentiated by their CD3-CD56+ phenotypes. The term used herein includes any known NK cell or any NK-like cell or any cell having the characteristics of an NK cell.

[041] In one embodiment, NK cells can be derived from an individual and cultured in vitro to provide a population of NK cells for use in the present invention. NK cells may be autologous (i.e., derived from the subject being treated using the cells and methods of the present invention), or they may be heterologous (i.e., the cells may be derived from a donor individual and may be intended for treatment from a second subject (i.e., they can be halogenic, syngeneic or xenogeneic). Thus, primary NK cells can be used. In an alternative embodiment, an NK cell known in the art that has been previously isolated and cultured can be used in the present invention. Thus, an NK cell line can be used. Several different NK cells are known and reported in the literature and any of these can be used or a cell line can be prepared from a primary NK cell, for example by viral transformation (Vogel B et al. 2014 Leukemia 28: 192-195). Cell lines can have a number of advantages over primary NK cells, for example, they can be easier to grow and maintain in culture, more easily expanded, and readily available on-demand in standardized quality for adoptive cell transfer therapy. Certain cell lines, for example the NK-92 cell line, may also exhibit higher cytotoxic activity, and in their unmodified state (which is not modified in accordance with the present invention) may have a broader spectrum of cellular targets. , for example, cancer cell targets (this may be due to an absence or reduction in the number of inhibitory receptors). Suitable NK cells include (but are by no means limited to), in addition to NK-92, the NK-YS, NK-YT, MOTN-1, NKL, KHYG-1, HANK-1 or NKG lines.

[042] In a preferred embodiment, the cell is an NK-92 cell (Gong et al., 1994, Leukemia 8, 652-658), or a variant thereof. Several different variants of the original NK-92 cells have been prepared and are described or available, including NK-92 variants that are not immunogenic. Any such variants can be used and are included under the term “NK-92”. Variants from other cell lines can also be used. However, any NK-92 or indeed any NK cell for use in accordance with the present invention must not be modified to express any specific antigen receptor other than the TcR which is or which is to be introduced into the NK cell modified in accordance with the present invention. invention. For example, the cell must not express a CAR (a monoclonal antibody linked to an intracellular signaling domain of the TcR complex). More generally, the NK cell modified according to the invention does not express a specific antigen receptor based on or comprise antibody recognition regions. An NK cell naturally does not comprise antigen-specific receptors and, therefore, the only antigen-specific receptor present in the cells of the invention is the TcR that is or is to be introduced into the NK cell.

[043] According to the present invention, the modified NK cell is not immunogenic. Functionally speaking, this means that the cell is not recognized by the immune system of any subject it is introduced into - it passes under the immunological radar and is not rejected or recognized as a foreign body by a receiving subject. That is, no clinically significant immune response is raised against the modified NK cells by the immune system of the subject to whom the NK cells are administered. Thus, a cell may be non-immunogenic if it does not, when administered to a subject, generate an immune response that affects, interferes with, or prevents the use of the cells in therapy.

[044] The term “non-immunogenic” is thus used broadly in this document to mean that, when the cell is injected or otherwise administered to an individual, there is no significant or clinically significant immune response to the cell. More particularly, the cell does not raise (or is not capable of raising) an immune response sufficient to lead to rejection of the cell and/or to affect the function of the cells, e.g., the immune response is not sufficient to nullify or substantially reduce or significantly the function or effect (i.e., usefulness) of cells. Thus, the cells retain cytotoxic activity in the subject, more particularly, a significant or substantial or measurable cytotoxic activity against a target cell. In a particular embodiment, the cell is not rejected as foreign. As with any biological system, the absence of an immune response may not be absolute (or 100%), a small (or mild or minor) immune response to the NK cell (e.g., a de minimis immune response) may be tolerated, as long as the function or utility of the cells is not substantially affected (i.e., while the cells can still perform their function).

[045] Therefore, it will be observed that an autologous cell (which is a cell administered or for administration to the same subject from whom it was obtained) will not be immunogenic (because it is “self”). Similarly, a non-autologous MHC-compatible NK cell will be non-immunogenic or substantially non-immunogenic. The term “non-immunogenic” thus includes functional non-immunogenicity. However, in certain embodiments, and as mentioned above, a non-immunogenic NK cell of the invention is non-immunogenic regardless of the cell's receptor, i.e., regardless of the subject into which it is or should be introduced (i.e., it would not induce a response clinically significant immune response if administered to a non-autologous subject). In particular embodiments, the NK cell may be subjected to a treatment that renders it non-immunogenic (i.e., it may be modified to be non-immunogenic). For example, the treatment may be a physical, chemical or biological treatment, or a structural or physicochemical modification of the cells (e.g., a treatment that causes a structural or physical change or alteration of the cells), for example, irradiation or a genetic modification, for example, to reduce or eliminate the expression of MHC by the cell. Accordingly, in some embodiments, the NK cell is not simply functionally immunogenic, but has or comprises a modification that renders it non-immunogenic.

[046] Consequently, NK cells can be naturally non-immunogenic or can be modified to be non-immunogenic. Naturally non-immunogenic NK cells will not express the MHC molecule or only weakly express the MHC molecule, or they may express a non-functional MHC molecule that does not stimulate an immune response. NK cells that would be immunogenic can be modified to eliminate the expression of the MHC molecule or only weakly express the MHC molecule on their surface. Alternatively, such cells can be modified to express a non-functional MHC molecule.

[047] Any such cell (naturally non-immunogenic or modified in some way) does not comprise MHC on its surface at a level sufficient to trigger a clinically significant immune response by a subject's immune response and can be considered as MHC-negative. In other words, an MHC-negative cell does not express on its surface any MHC molecule that is immunologically functional or does not express on its surface MHC molecule at a sufficiently high level that it can be recognized by another immune cell, particularly an immune cell. non-autoimmune or an immune cell of an intended recipient. The cell may lack an MHC molecule, or may not express MHC on its cell surface, or only weakly express MHC on its cell surface, or any MHC molecule that is expressed may be nonfunctional, e.g. mutated or otherwise modified so that it is non-functional. Any means by which expression of a functional MHC molecule is disrupted is covered. Therefore, this may accomplish knock-out or knock-down of the MHC complex molecule and/or may include a modification that prevents the proper transport and/or correct expression of an MHC molecule, or the entire complex, on the surface. cell phone.

[048] In particular, the expression of one or more functional MHC class I proteins on the surface of a cell of the invention can be interrupted. In one embodiment, the cells may be human cells that are HLA-negative and, consequently, cells in which the expression of one or more HLA molecules is disrupted (e.g., eliminated), e.g., HLA class I complex molecules. HLA MHC.

[049] In a preferred embodiment, interruption of MHC class I can be accomplished by knocking out the gene encoding β2-microglobulin, a component of the mature MHC class I complex. Expression of β2m can be eliminated through a targeted disruption of the β2-microglobulin (β2m) gene, for example by site-directed mutagenesis of the β2m promoter (to inactivate the promoter) or within the gene encoding the β2m protein to introduce an inactivating mutation that prevents expression of the β2m protein, for example, a frameshift mutation or premature 'STOP' codon within the gene. Alternatively, site-directed mutagenesis can be used to generate non-functional β2m that is not capable of forming an active MHC protein on the cell surface. In this way, the β2m protein or MHC may be retained intracellularly, or may be present, but not functional, on the cell surface.

[050] NK cells can alternatively be irradiated before being administered to a subject. NK-92 cells that were irradiated were found to be non-immunogenic in clinical trials and were licensed for use in immunotherapy (Ton T. et al. 2013 Cytotherapy 15 1563-70). Without intending to be bound by theory, it is thought that irradiation of cells results in cells being only present transiently in a subject, thus reducing the time available for a subject's immune system to mount an immune response against the modified NK cells. . Although such cells can express a functional MHC molecule on their cell surface, they can also be considered non-immunogenic.

[051] Thus, an NK cell according to the invention can be modified to be non-immunogenic by reducing its ability, or ability, to proliferate, that is, reducing its proliferative capacity.

[052] An NK cell of the invention is modified to express CD3. As mentioned above, an NK cell of the invention does not express a CAR, or even any specific antigen receptor other than a TcR. Thus, in particular, except in the case of a TcR-CD3 fusion, CD3 is not expressed as part of a chimeric receptor. More particularly, CD3 is not expressed as part of a chimeric antigen receptor (CAR), or any chimeric receptor based on (or comprising) antibody-derived antigen-binding region(s) or a binding domain derived from any molecule or binding moiety that is not a TcR, e.g., of any other affinity binding partner, e.g., of a ligand or receptor other than a TcR. Thus, CD3 is not expressed as part of a chimeric receptor that comprises an antigen-binding domain (i.e., a binding moiety or moiety) or any binding domain or ligand that is not a TcR or that is not derived from a TcR. In other words, CD3 is expressed independently (i.e., as a separate molecule or chain, or not as part of a fusion with a moiety that is not a CD3 chain or part thereof, or a spacer molecule or linker) and /or is expressed as part of (or within) a TcR-CD3 fusion. Accordingly, in particular, an NK cell of the invention does not express a CAR or other chimeric receptor comprising a CD3 chain or molecule, other than a CD3-TcR fusion. In an embodiment where CD3 is provided as or as part of the fusion (i.e., a fusion protein), it is a CD3 fusion or a CD3-TcR fusion, as described further below.

[053] NK cells normally (or natively) do not express CD3 and, therefore, to make the cell capable of expressing CD3, a nucleic acid molecule that encodes CD3 is introduced into the cell. Thus, the cell is modified to express CD3 recombinantly. In other words, the cell is modified to allow expression of a heterologous nucleic acid molecule that encodes CD3. CD3 can be matched to the cell species (i.e., from the same species as the cell), however, CD3 from a different species can also be used. Thus, for a CD3 from human NK cells or CD3 from other mammalian species such as mouse, mouse, rabbit, etc. can be expressed. In a preferred embodiment, all CD3 chains (i.e., one CD3Y chain, one CD3δ chain, two CD3ε chains and the Z chain) are expressed on an NK cell. However, it is also contemplated that only a single CD3 chain (e.g., just the CD3Y chain, or just the CD3δ chain, or just the CD3Z chain, or just the CD3ε chain, or any combination thereof, is present. Thus, a NK cell can be modified to express any of the CD3 chains alone, or in combination with other CD3 chains, up to and including the complete CD3 molecule. Without wishing to be bound by theory, it is hypothesized that it may be possible to target a TcR to the cell surface and/or initiate signaling where one or more of the CD3 chains are not expressed on an NK cell. It is also anticipated that one or more of the CD3 chains may be provided as a fusion protein, wherein at least two of the chains are expressed as a single polypeptide as long as CD3 localization and signaling functions are maintained.

[054] One or more CD3 chains can also be encoded by one nucleic acid molecule or more than one nucleic acid molecule, for example, in separate molecules. Conventionally, a construct will be created comprising a nucleic acid molecule encoding all three CD3 chains, for example, under the control of the same promoter or linked together in some way.

[055] An NK cell of the invention can also be modified to express a TcR. A TcR can be viewed as any receptor comprising a TcR antigen recognition domain, an antigen recognition sequence, or a fragment thereof that selectively binds to an antigen on the surface of a target cell. Thus, in the context of the present invention, a TcR may be a canonical TcR that comprises a TcR antigen recognition domain in its native (or natural) context, or may comprise a TcR antigen recognition domain, a antigen or a fragment thereof linked to an amino acid sequence and/or protein domains (i.e., as part of a chimeric molecule or fusion protein) that are not naturally found together in a TcR (i.e., a non-native context ). The term “TcR” is thus used broadly herein to include any antigen receptor in which the antigen recognition region is provided by a TcR antigen recognition domain or sequence, e.g., an antigen recognition region of a TcR. native or derived from a native TcR. The term “TcR”, accordingly, includes native and non-native TcRs, which are synthetic or artificial TcR molecules or constructs, variants or derivatives of TcR, or a TcR molecule derived from or based on a native TcR.

[056] An NK cell that expresses a TcR thus has an antigen-specific (i.e. targeted) cytotoxic activity towards a target cell. The TcR can be any TcR with specificity toward an antigen on a target cell of interest (a target antigen). The specificity of the NK cell, (i.e. the antigen to which the cell binds) will be determined by the specificity of the TcR. In other words, selection of a suitable TcR is necessary to provide an NK cell with the desired specificity (i.e., specificity toward a specific antigen on a target cell). Thus, in one embodiment, the present invention provides an NK cell modified to express CD3 and further modified to express a TcR with specificity for an antigen on a target cell.

[057] In a preferred embodiment, the TcR binds to an antigen on the surface of a target cell with high affinity (that is, the TcR is a high affinity TcR). In said advantageous embodiment, the TcR is capable of binding to the MHC antigen complex on the surface of the target cell with sufficiently high affinity that the cell expressing the TcR is able to undergo activation in the absence of CD8/coreceptors. CD4 typically required for activation of a TcR on a cytotoxic or helper T cell, respectively. Thus, the present invention provides an NK cell modified to express CD3 and further modified to express a TcR with specificity toward an antigen on a target cell, wherein said TcR binds to said target cell with high affinity and undergoes activation in the absence of CD8 and/or CD4 coreceptors. Alternatively expressed, the TcR that is introduced into the NK cell in accordance with the present invention is coreceptor (or cofactor) independent; that is, it does not require another receptor or cofactor for the cell to be activated (i.e., for signaling in the cell induced by TcR ligation). Thus, the TcR can bind antigen (the MHC-antigen complex) with a koff within the normal physiological range. In particular, it is independent of CD8 and/or CD4, for example, independent of CD8 and, more particularly, independent of CD4 and CD8. However, in an alternative embodiment, the NK cell can be further modified to express CD4 and/or CD8.

[058] A TcR recognizes and binds to an antigen (a peptide) presented on the cell surface by an MHC class I or II family protein. Antigen recognition thus depends on both the MHC type of the cell and the peptide (more specifically, the peptide sequence) (antigen). Generally, the target antigen is known and the TcR is selected according to its antigen specificity. In a preferred embodiment, the sequence of the peptide antigen presented by a target cell may be known. Furthermore, the MHC type of a target cell can also be known. Thus, in a preferred embodiment, one can select a TcR that is capable of specifically binding to an antigen-MHC complex on the surface of a target cell. Thus, in a preferred embodiment, the present invention provides an NK cell modified to express CD3 and further modified to express a TcR with specificity for an antigen on a target cell, wherein said TcR is capable of specifically binding to an antigen complex. -MHC on the surface of a target cell.

[059] In a preferred embodiment of the invention, a TcR can be selected from a cytotoxic T cell that has been identified as specifically showing cytotoxicity to a target cell or a helper T cell. Alternatively, the antigen recognition domain or sequence of this TcR or fragment thereof can be selected. Said T cell can be obtained from a patient who will be the subject of treatment, or can be obtained from a second subject (i.e., TcR donor). Thus, a TcR can be selected from a T cell identified as having "proven" activity against a target cell. In other words, a TcR can be selected that has been shown to be effective against the target cell. By way of example, T cells, e.g., tumor-infiltrating lymphocytes, can be isolated from a cancer patient. By their nature, these will be, or include, T cells that are active against the patient's cancer. The genes encoding the TcR of these cells can be identified, cloned and used to express the TcR or the antigen recognition domain or sequence of this TcR or fragment thereof, in an NK cell according to the invention. T cells can be produced in the patient or in a subject by vaccination, for example, by administering a vaccine comprising a cancer antigen. Those vaccination-induced T cells that are most active against a cancer in the subject, or against cancer cells, can then be selected. In this way, the most potent or effective TcRs can be selected, for example, TcRs with higher affinity or that are coreceptor independent. Affinity maturation can be performed to obtain an optimal TcR.

[060] Advantageously, the HLA profile or MHC profile of the subject from which the TcR is derived (TcR donor) will be the same as the HLA or MHC profile of the subject to be treated. As noted above, a TcR on a T cell with specificity for a target cell can be identified and characterized, and the amino acid sequence of the TcR and the nucleic acid sequence of the gene encoding the TcR can be obtained. Stated another way, a cytotoxic T cell or a helper T cell that comprises a TcR with specificity for a particular antigen on a target cell can be identified in a subject and the amino acid sequence of the receptor and the DNA sequence of the gene they encode the receiver can be obtained. The DNA sequence or gene can be used to prepare a nucleic acid molecule or construct for introduction into an NK cell to allow the TcR to be expressed in the host (i.e., recipient) NK cell.

[061] In an alternative embodiment of the invention, an artificial TcR (that is, an unidentified TcR from a cytotoxic or helper T cell) can be used. The TcR may be an artificially generated alloreactive TcR. The binding domain of a TcR can be generated by a combinatorial-based method, such as phage display or ribosome display, and a chimeric protein comprising an artificial binding domain specific for a particular combination of antigen and MHC (e.g. example, HLA), can be obtained.

[062] The TcR can be provided as a construct, or fusion, comprising other protein domains.

[063] One or more of the CD3 chains (or the entire CD3 construct) can be expressed as a fusion protein with the TcR. In other words, one or more of the CD3 chains may be present on the same polypeptide chain as the TcR. In a preferred embodiment, the CD3 molecule, or one or more CD3 chains (e.g., CD3Y chain, CD3δ chain, CD3Z chain, or CD3ε chain, or any combination thereof) may be present as a TcR fusion construct. -CD3 that will be targeted directly to the plasma membrane, i.e. without the need for another CD3 molecule to also be present in the cell (as long as the fusion construct retains a transmembrane domain, for example, provided by a CD3 chain (Willemsen RM 2000 Gene Therapy 7: 1369-1377). Thus, in this embodiment, the cell can be modified to express CD3 and a TcR simultaneously, as part of the same construct or fusion. Although separately expressed CD3 is not necessary, it can be provided, in the However, for example, to improve signaling and therefore cellular activity.

[064] As noted above, a panel of different TcRs can be developed, each TcR in the panel having specificity for (e.g., specific affinity toward) a specific antigen-MHC complex. Thus, different target antigens may be recognized and/or different epitopes on a target antigen and/or TcRs may have different affinity and/or selectivity for an antigen. Furthermore, for a given target antigen, the panel may comprise TcRs of different MHC specificity (or MHC type). In this way, a particular TcR that is capable of binding to a specific antigen-HLA complex can be selected from the panel for use in the methods of the invention, i.e., a TcR that corresponds to both the target antigen and the MHC type of a subject. Thus, in a particularly preferred embodiment, a specific TcR can be selected based on the nature of the antigens presented by a specific target cell and its MHC type (e.g., HLA). It is envisioned that such a panel could be used to enable the rapid generation of an NK cell with target cell-specific cytotoxic activity. Accordingly, a method of the present invention for producing an NK cell with specificity for a target cell may comprise: a) modifying an NK cell to express CD3; b) determining the MHC profile of said target cell and the identity of an antigen displayed by said target cell; and c) modifying said NK cell to express a TcR, wherein said TcR is selected from a panel of TcRs, each having specificity for a different antigen and/or MHC type, and wherein said TcR has specificity for MHC and antigen displayed on said target cell.

[065] TcRs can be provided in the panel in the form of nucleic acid molecules comprising nucleotide sequences that encode TcRs. Conveniently, the nucleic acid molecules may be comprised within vectors or constructs, particularly vectors or constructs suitable directly for introduction into an NK cell. The nucleic acid molecule can be DNA or RNA. For example, TcRs can be delivered as mRNA or as viral vectors, e.g., retroviral vectors.

[066] In one embodiment, an NK cell is modified to express a TcR after the cell is modified to express CD3. Thus, as mentioned above, a universal NK cell can be prepared by modifying a non-immunogenic NK cell to express CD3. These modified NK cells can be grown and maintained in culture, or stored for future use. Accordingly, in one embodiment, the cell can be replicated to produce a population of CD3-expressing NK cells, before modifying the cells to express a TcR. NK cells can be grown in culture or expanded. In an alternative embodiment, it is contemplated that the cell can be modified to express CD3 and a TcR at the same time or substantially the same time. In one embodiment, a single vector or construct comprising genes for CD3 and TcR can be used to modify a cell to express CD3 and TcR, and in other embodiments, separate vectors can also be used. As discussed above, the fusion protein comprising the functionalities of TcR and CD3 in a single polypeptide molecule can also be expressed in NK cells and therefore a vector or construct encoding a fusion protein can be used to modify a cell. . NK cells modified to express CD3 and TcR can be replicated, for example, they can be grown in culture or expanded before introducing the cells into a subject. Thus, in one embodiment of the present invention, a population of CD3-expressing NK cells can be generated and a subpopulation of NK:CD3 cells can be modified to express TcR with specificity for an antigen on the surface of a target cell.

[067] The modified NK cells of the invention may also be subject to modification in other ways, for example, to alter or modify other aspects of cell function or behavior and/or to express other proteins. For example, cells can be modified to express a homing receptor, or homing receptor, which acts to direct or enhance the localization of cells to a specific tissue or location in the body. Cells can also be modified to express one or more of the components of the T cell signaling pathway in order to enhance the cytotoxic response exhibited by NK cells. Any such modification may occur before, after or simultaneously with the modification according to the present invention.

[068] The cell of the invention can be modified to alter its ability to replicate in vivo and/or in vitro. In one embodiment, the cell's replication capacity (the ability of a cell to replicate, i.e., to proliferate) can be improved. In a preferred embodiment, this can be achieved by modifying the cells so that they have enhanced expression of a cytokine, such as IL-2. IL-2 is generally required for NK cells to grow and maintain cytolytic function, and therefore IL-2-expressing cells do not require additional IL-2 to be supplemented in growth medium and retain cytotoxic activity when introduced into a subject. Expression of a cytokine can be enhanced by introducing a heterologous (non-native) vector or construct comprising a nucleic acid molecule encoding a cytokine into a cell or, alternatively, expression of an endogenous gene encoding a cytokine can be enhanced. Expression of a cytokine may be constitutive (i.e., the promoter that controls expression of the gene encoding a cytokine may be constitutively active, or 'turned on'), or it may be inducible by an external stimulus.

[069] In one embodiment, the cell can be modified to have enhanced replicative capacity before the cell is modified to express CD3 and/or TcR. In another embodiment, the cell can be modified to have enhanced replicative capacity after the cell is modified to express CD3 and/or TcR. In an alternative embodiment, the cell may be modified to have an enhanced replicative capacity after the cell is modified to express CD3, but before the cell is modified to express TcR (i.e., it may therefore be possible to produce a large population of CD3-expressing NK cells, before modifying the cells to express TcR). Alternatively, any two or more of the above modifications may be carried out substantially at the same time. In one embodiment, all three modifications can be performed substantially at the same time.

[070] The proliferative capacity and viability (survival) of the NK cells of the present invention can also be reduced before the cell is introduced into a patient. As noted above, this can be done to render the cell non-immunogenic. In a preferred embodiment, the replicative capacity and/or viability of the cell can be reduced by irradiation. A cell's ability to replicate and/or its viability may be reduced (i.e. replication may occur more slowly) or eliminated depending on the dose and nature of the irradiation delivered to the cells. The radiation may be from any source of α, β, or Y radiation, or may be X-ray radiation or ultraviolet light. A radiation dose of 5-10 Gy may be sufficient to nullify proliferation, however, other suitable radiation doses may be 1-10, 2-10, 3-10, 4-10, 6-10, 7-10 , 8-10 or 9-10 Gy, or higher doses such as 11, 12, 13, 14, 15 or 20 Gy. Alternatively, NK cells can be modified to express a "suicide gene", which allows the cells to be inducibly eliminated or prevented from replicating in response to an external stimulus.

[071] A cell can be modified a) to have enhanced replication capacity and b) to have reduced replication capacity and/or viability. The two modifications can be performed sequentially in any order (i.e., A then B, or B then A), or they can be performed essentially at the same time (A and B together). In this embodiment, the cell may be modified to express a cytokine (such as IL-2) to enhance cell growth in vitro before, at the same time, or after modifying the cell to express CD3 and/or a TcR and may be, subsequently modified to have its replicative capacity and/or viability reduced before introducing the cell into a subject.

[072] It will be clear from the foregoing that a cell of the invention can be modified to alter the level of expression of one or more genes or, in particular, to allow the expression of one or more genes that are not normally expressed by the cell . To allow expression of this heterologous gene, a nucleic acid molecule that corresponds to or comprises the gene in question is introduced into the cell. Conveniently, the nucleic acid molecule can be introduced into the cell in a vector or recombinant construct. Methods of expression of heterologous genes are known in the art, both in terms of preparing the construct/vector and in terms of introducing the nucleic acid molecule (vector or construct) into the cell. Thus, promoters and/or other expression control sequences suitable for use with mammalian cells, in particular, lymphoid cells or NK cells, and appropriate vectors, etc. (e.g., viral vectors) are well known in the art.

[073] The vectors or constructs (nucleic acid molecules) can be introduced into a cell of the invention by a variety of means, including chemical transfection agents (such as calcium phosphate, branched organic compounds, liposomes or cationic polymers), electroporation, cell compression, sonoporation, optical transfection, hydrodynamic delivery or viral transduction. In a preferred embodiment, a vector or construct is introduced by viral transduction. Heterologous nucleic acid molecules introduced into a cell can be expressed episomally, or they can be integrated into the cell's genome at an appropriate locus.

[074] Cell culture methods and reagents suitable for NK cells are also well known in the art. Any desired method of cell culture can be used, according to choice and convenience, etc. For example, Teflon bags can be used for large-scale culture or automated cell culture systems.

[075] The target cell of the invention may be a cancerous cell. Cancer is defined broadly in this document to include any malignant, premalignant, or nonmalignant neoplastic condition. Generally, however, it can be a malignant condition. Both solid and non-solid tumors are included and the term "cancer cell" can be considered synonymous with "tumor cell".

[076] Any type of cancer is covered, including solid and hematopoietic cancers. Representative cancers include acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), adrenocortical carcinoma, AIDS-related cancers (e.g., Kaposi's sarcoma and lymphoma), anal cancer, appendiceal cancer, astrocytomas, atypical teratoid rhabdoid tumor, basal cell carcinoma, bile duct cancer, extrahepatic bladder cancer, bone cancer (e.g., Ewing sarcoma, osteosarcoma, and malignant fibrous histiocytoma), brainstem glioma, brain cancer, breast cancer, bronchial tumors , Burkitt's lymphoma, carcinoid tumor, cardiac (heart) tumors, Central Nervous System cancer (including Atypical Teratoid/Rhabdoid Tumor, Embryonic Tumors, Germ Cell Tumor, Lymphoma), Cervical Cancer, Chordoma, Chronic Lymphocytic Leukemia (CLL), Chronic Myelogenous Leukemia (CML), Chronic Myeloproliferative Disorder, Colon Cancer, Colorectal Cancer, Craniopharyngioma, Cutaneous T-cell Lymphoma, Bile Duct Cancer, Extrahepatic Ductal Carcinoma In Situ (DCIS), Embryonic Tumors, Endometrial Cancer, Ependymoma , Esophageal Cancer, Esthesioneuroblastoma, Ewing's Sarcoma, Extracranial Germ Cell Tumor, Extragonadal Germ Cell Tumor, Extrahepatic Bile Duct Cancer, Eye Cancer (including Intraocular Melanoma and Retinoblastoma), Fibrous Histiocytoma of Bone, Gall Bladder Cancer , Gastric Cancer (Stomach), Gastrointestinal Carcinoid Tumor, Gastrointestinal Stromal Tumors (GIST), Germ Cell Tumor, Gestational Trophoblastic Disease, Glioma, Hairy Cell Leukemia, Head and Neck Cancer, Heart Cancer, Hepatocellular Cancer (Liver) , Histiocytosis, Langerhans Cell, Hodgkin's Lymphoma, Hypopharyngeal Cancer, Intraocular Melanoma, Islet Cell Tumors, Pancreatic Neuroendocrine Tumors, Kaposi's Sarcoma, Kidney Cancer (including renal cell and Wilms Tumor), Langerhans Cell Histiocytosis, Laryngeal Cancer, Leukemia (including Acute Lymphoblastic (ALL), Acute Myeloid (AML), Chronic Lymphocytic (CLL), Chronic Myelogenous (CML)), Lip and Oral Cavity Cancer, Liver Cancer (Primary), Lobular Carcinoma In Situ (LCIS), Lung Cancer, Lymphoma, Macroglobulinemia, Waldenstrom, Melanoma, Merkel Cell Carcinoma, Mesothelioma, Metastatic Squamous Cell Neck Cancer with Occult Primary, Midline Tract Carcinoma involving NUT gene, mouth cancer, syndromes of multiple endocrine neoplasms, childhood, multiple myeloma/plasma cell neoplasia, mycoses fungoides, myelodysplastic syndromes, myelodysplastic/myeloproliferative neoplasms, multiple myeloma, myeloproliferative disorders, Cancer of the Nasal Cavity and Paranasal Sinuses, Nasopharyngeal Cancer, Neuroblastoma, Non-Hodgkin's Lymphoma, Non-small cell lung cancer, oral cancer, cancer of the oral cavity, oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, pancreatic neuroendocrine tumors (Islet cell tumors), papillomatosis, paraganglioma, paranasal sinusitis and cancer nasal cavity, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pituitary tumor, plasma cell neoplasm/multiple myeloma, pleuropulmonary blastoma, pregnancy and breast cancer, primary central nervous system (CNS) lymphoma, cancer Prostate Cancer, Rectal Cancer, Renal (Kidney) Cancer, Transitional Cell Cancer Renal, Pelvis and Ureter, Retinoblastoma, Rhabdomyosarcoma, Salivary Gland Cancer, Sarcoma, Sézary Syndrome, Skin Cancer, Small Cell Lung Cancer, Cancer of small intestine, Soft tissue sarcoma, Squamous cell carcinoma, Squamous cell neck cancer with occult primary, Metastatic and stomach (gastric) cancer, T-cell lymphoma, testicular cancer, throat cancer, thymoma and thymic carcinoma, breast cancer thyroid, transitional cell cancer Renal, Pelvis and Ureter, Urethral Cancer, Uterine Cancer, Endometrial, Uterine Sarcoma, Vaginal Cancer, Vulvar Cancer, Waldenstrom Macroglobulinemia and Wilms Tumor

[077] As mentioned above, several cancers have been identified to express specific or particular cancer antigens, or to be characterized by the expression of specific or particular cancer antigens. Thus, a known or recognized cancer antigen can be used. As is known in the art, certain antigens may occur in several different types of cancer, others may be specific to a particular type of cancer. However, in other cases, it may be appropriate or necessary to characterize the cancer in a subject and identify a suitable cancer antigen for use. Thus, any cancer can be treated using a TcR directed against a universal cancer antigen and such TcRs have been identified. Alternatively, the cancer may be any cancer that is currently treated, or proposed for treatment, by adoptive T cell therapy, for example, common cancers such as melanoma, hematological cancer, lung cancer, colorectal cancer. Furthermore, the cancer may be a rare cancer where there are few treatment options currently available (e.g., with orphan drug status) such as pancreatic cancer or sarcoma.

[078] In other embodiments, the target cell may be an infected cell and, in particular, a cell infected with a virus. The virus can be any virus, but will generally be a pathogenic virus. By way of example, the virus may be HIV, hepatitis virus (e.g., HBV or HCV), HPV, CMV or EBV, HHV-8, HTLV-1, SV40, enterovirus. Other possible infectious agents or pathogens also include bacteria, e.g. Helicobacter pylori, Chlamydia pneumoniae, and parasites, e.g. Schistosoma haematobium and liver flukes, Opisthorchis viverrini, Clonorchis sinensis, and malaria.

[079] In one embodiment of the present invention, cells can be administered to a subject directly intravenously. In an alternative embodiment, cells can be administered directly into a tumor via intratumoral injection.

[080] The dose of cells administered to a subject will vary depending on the nature of the target cell. In a preferred embodiment of the invention, where the target cell is a cancer cell, the dose can be calculated based on the type of cancer that is to be targeted. In a preferred embodiment, a dose of approximately 10 cells may be administered to the subject, however, this may vary depending on the type and extent of the cancer. It is possible that a dose of approximately 106, 107 or 108 cells could be administered. Alternatively, a higher dose of approximately 1010, 1011 or 1012 cells can be administered. The dose of cells administered may also vary according to the size of the patient's body and, 5 6 7 8 9 10 11 12 therefore, one dose can be administered per m2 of the area of patient's body surface or per kg of the patient's weight.

[081] It is also anticipated that multiple infusions may be necessary to treat a subject effectively. For example, 2, 3, 4, 5, 6 or more separate infusions may be administered to a patient, at intervals of 24 or 48 hours, or every 3, 4, 5, 6 or 7 days. Infusions can also be spaced at weekly, biweekly, or monthly intervals, or 6-week or 2-, 3-, 4-, 5-, or 6-month intervals. It is also possible that annual infusions could be given.

[082] The subject to be treated using the methods and cells of the present invention can be any species of mammal. For example, the subject can be any species of domestic pet, such as a mouse, rat, gerbil, rabbit, guinea pig, hamster, cat or dog, or livestock, such as a goat, sheep, pig, cow, or horse. In another preferred embodiment of the invention, the subject may be a primate, such as a monkey, gibbon, gorilla, orangutan, chimpanzee or bonobo. However, in a preferred embodiment of the invention, the subject is a human being.

[083] It is contemplated that the NK cells to be used in the present invention can be obtained from any species of mammal, however, in a preferred embodiment, the NK cells will be from the same species of mammal as the subject to be treated. Furthermore, and as noted above, in a preferred embodiment, the cell will be modified using genes expressing proteins from the same mammalian species (e.g., human CD3 and TcR will be used for a human subject). However, it is possible that a CD3 and TcR from different species could also be used, for example, a cell could be modified using genes expressing a mouse CD3 and a human TcR.

[084] The present invention can be better understood from the Examples below and reference to the figures, in which: Figure 1 shows that CD3 can be expressed on NK-92 cells. A CD3-IRES-GFP retroviral construct was transduced into NK-92 cells. Fluorescence was monitored in the FITC channel. GFP+ cells are indicated in the marked region. Black: untransfected cells, gray: transfected cells. Figure 2 shows that CD3 can be detected on the surface of NK-92 cells expressing CD3-IRES-GFP, in the presence of a TcR. NK-92 GFP+ ordered (NK-92-CD3) were superinfected with four different TcRs, namely Radium-1 (TGFbRII frameshift-specific MHC-I) and a cysteine variant, Radium-1Cis, DMF-5 (MHC-1 specific of MART-1), Radium-3 (hTERT-specific MHC-II) and non-superinfected control cells were run. Cells were stained with anti-CD3 to detect CD3 on the cell surface and fluorescence was measured in the FITC (GFP) and APC (anti-CD3) channels. Cells in the upper right quadrant express CD3 on the cell surface. Figure 3 shows that Radium-1 and DMF-5 TcRs can be specifically detected on the surface of NK-92 cells expressing CD3-GFP. Cells were transfected to express CD3 and two different TcRs: Radium-1 (TGFbRII frameshift), DMF-5 (MART-1), and CD3 alone (NoTcR). Cells were stained with a V-beta specific antibody that can detect the V-beta chain of Radium-1 (not DMF5, top row) or MART-1 multimer to detect DMT-5 TcR on the cell surface (bottom row). Fluorescence was measured in FITC (GFP) and APC (anti-Vb and M1-multimer) channels. Figure 4 shows that NK-92 cells modified to express CD3 and Radium-1 TcR demonstrate antigen-specific cytotoxic activity. NK-92 cells transfected with CD3-IRES-GFP (GFP - upper box - CD3_IRES_GFP) and cells that were not transfected (GFP- - lower box - NTr) were gated into two separate populations. NK cells were incubated with SupT1 cells expressing single-chain trimer with the TGFbRII target peptide and irrelevant peptide or alone. SupT1 cells were separated from NK cells by detecting expression of the NK cell marker CD56. Degranulation was monitored by detection of the CD107 marker on NK cells after incubation with SupT1 cells in the GFP+ and GFP-e populations are represented together (histogram, gray: GFP-, dashed: GFP+). Figure 5 shows that a pure CD3 population of NK-92 cells superinfected with DMF-5 or Radium-1 TcRs and sorted are specifically activated by T2 cells loaded with the relevant peptide. Start: activation strategy: CD3 signal and GFP are used to separate the two cell lineages. Then, the CD3+ population is tested for CD107a expression (degranulation). Histograms: Indicated NK-92-CD3-TcR was incubated with T2 cells loaded O/N with 1 μM peptides (gray shading: no peptide, dark gray outline: MART-1, light gray outline: TGFbRII). Figure 6 shows the calculation of EC50 values for TcRs Radio-1 and DMF-5. Figure 7 shows the results of flow cytometry analysis (phospho-flow) of the activation of NK-92 cells (CD3/Radio-1) after contact with the α-CD3 and α-CD28 antibodies. Right shifts of flow cytometry spectra indicate increased levels of phosphorylation of the relevant protein, which in turn indicate activation of the TCR complex. Figure 8 shows the specificity of stimulation of NK-92 (CD3/Radium-1) and NK-92 (CD3/DMF-5) cells. NK-92 cells were incubated with antigen-presenting cells presenting either the specific or cognate peptide for each receptor (TGFbRII for cells expressing Radium-1, MART-1 for cells expressing DMF-5) or a random peptide. The stimulation of signaling through the TCR complex was measured at various time points according to the phosphorylation level of several TcR/CD3-related proteins. These phosphorylation levels were measured by flow cytometry (phospho-flow). In each graph, the solid line defines cell activation by the specific, cognate peptide; a dotted line defines cell activation by the random peptide. Figure 9 shows the kinetics of stimulation of NK-92 (CD3) cells transduced with Radium-5 or Radium-6 TcR. Both receptors are derived from CD4+ T cells and specifically bind a frameshift peptide of TGFbRII; Radio-5 binds it specifically in the context of HLA-DR7, Radio-6 in the context of HLA-DR4. EC50 values for Radium-5 and Radium-6 are calculated using tissue from donor patients (patients H, S and B). Patient H has the HLA-DR7 haplotype; patients S and B have HLA-DR4 haplotype.

Example 1. CD3 expression on NK92 cells.

[085] A codon-optimized pMP71-CD3Z-CD3ε-CD3Y-CD3δ-IRES-GFP (CD3-GFP) vector (Ahmadi et al. 2011. Blood 118, 3528-3573) was used to transfect NK92 cells with CD3, and cells were transfected using retroviral transduction. The retrovirus containing the CD3-GFP construct was produced in the packaging cell line (Hek-Phoenix) and NK cells were spinoculated (0.3 M cells with incubated with viral supernatant and centrifuged at 900xg for 1 hour at 32C on coated plates with retronectin (Tanaka Biotech)). GFP was used as a marker for successful transduction.

[086] Successful transduction of NK-92 cells with CD3 (NK-92(CD3)) was confirmed by flow cytometry using a FACS Canto flow cytometer (BD Biosciences). Fluorescence was monitored in the SSC and FITC channels and successful transfection was observed by monitoring fluorescence in the GFP channel (see Figure 1). A transfection efficiency of approximately 50% was achieved. Alternatively, GFP+ cells were sorted to obtain a pure population.

Example 2. TcR expression in NK-92(CD3) cells

[087] The ability of NK-92(CD3) cells to coexpress CD3 and a TcR was tested. CD3 and TcRs require the coexpression of another member of the TcR complex to be targeted to the cell surface. Thus, it was possible to use CD3 expression on the surface of NK cells as a marker for successful cotransfection of a cell with CD3 and TcR.

[088] NK-92(CD3) cells were superinfected with four different TcRs using retroviral supernatants (a cysteine variant Radio-1Cis, Radio-1-specific (frameshift TGFbRII), DMF-5, MART-1 specific (DMF- 5 (Johnson, L.A. et al. 2006 J Immunol 177: 6548-6559)) and Radium-3 (hTERT, MHC-II)) and cell surface CD3 expression was monitored for each TcR using an anti-CD3 antibody multimer marked with APC. CD3 expression was detected by monitoring cells for GFP expression. Fluorescence was detected in the FITC and APC channels. Cells that express GFP appear in the right region of the dotplot graphs, and cells that express CD3 on their cell surface appear in the upper region of the dotplot graphs. Cells that coexpress CD3 and TcR (i.e., cells with the CD3-TcR complex located on the cell surface) appear in the upper right region of the dotplot graphs.

[089] CD3 expression on the cell surface was detected in cells modified to express each TcRs (Figure 2 A-D) (cells in the upper right quadrant in each dotplot graph). In particular, cells engineered to express Radium-1 TcR showed expression of CD3 on the cell surface Figure 2B. Figure 2D also shows a small increase in fluorescence in the APC channel for GFP+ cells, indicating a low level of Radio-3 TcR expression. No CD3 expression is detected on the cell surface for cells lacking TcR (Figure 2E).

[090] The expression of TcRs on the cell surface was also detected in NK-92(CD3) cells modified to express Radio1 and DMF-5 TcRs. The presence of TcR on the cell surface was monitored using a specific V-beta antibody that can detect the V-beta chain of Radium-1 or a MART-1 multimer to detect DMF-5 TcR on the cell surface (bottom row), labeled with APC. Fluorescence was measured in FITC (GFP) and APC (anti-Vb and M1- multimer) channels. No increase in signal in the APC channel was observed for NK-92(CD3) cells not modified to express TcR (Figure 3A). Cells modified to express Radium-1 TcR showed an increase in signal in the APC channel when stained with anti-Vβ antibody but not with the MART-1 multimer. (Figure 3B). Cells engineered to express DMF-5 TcR showed an increase in signal in the APC channel when stained with the MART-1 multimer (Figure 3C).

Example 3. NK-92(CD3) expressing TcR are functional

[091] To validate the functionality of our TcR when expressed on an NK cell, we tested whether TcR expressed on NK cells was capable of specifically recognizing target cells.

[092] NK-92(CD3) cells expressing a TcR were incubated with target cells expressing a single-chain trimer (SCT) molecule comprising the correct target peptide (TGFbRII) or a non-specific peptide (irr) by 5 hours or, in the absence of target cells (no APC), and expression of the degranulation marker marker CD107 was monitored as a marker for the ability of NK cells to be stimulated by target cells. (SCTs represent an MHC class I protein that displays an antigen and consist of an antigen peptide, β2-microglobulin and h-chain expressed as a single polypeptide chain, see US 2010/015954 and Yu, Y. Y. et al. ( 2002) J Immunol 168: 3145-3149.

[093] NK-92(CD3) cells were initially stained with anti-CD56 Tx Red (CD56 is a marker for NK cells) and Tx Red and GFP fluorescence were monitored (Figure 4A). Separate populations of NK-92 cells were observed based on GFP expression. GFP+ cells (upper region) and GFP- NK cells (lower region) were detected. CD107a expression was monitored for each cell population and GFP- cells were used as a negative control (i.e., cells expressing non-TcR).

[094] NK-92(CD3) cells expressing Radio1-specific TcR were incubated with SupT1 expressing a single-chain trimer molecule (SCT) with non-specific peptide (Figure 4C) or Radium-1 target peptide TGFbRII (Figure 4D). Cells incubated in the absence of an antigen-presenting cell were also monitored (Figure 4B); CD3 expression was monitored for the NK-92 cell line and cells were gated as described above. The GFP+ (dotted line) and GFP- (solid line) populations are visible in each histogram.

[095] Only NK-92(CD3) cells expressing Radio-1-specific TcR showed an increase in CD107 expression when incubated with SupT1 cells expressing Radio-1 SCT (Figure 4D), as indicated by the shift to the right of the dotted line compared to solid line (GFP- negative control). No increase in CD107 expression was observed for these cells when incubated with SupT1 cells expressing the nonspecific peptide (Figure 4C) or cells incubated in the absence of antigen-presenting cells.

[096] Together, these data indicate that NK-92(CD3) that express Radio-1-specific TcR may be capable of being activated by target cells that express the Radio-1 target peptide. This demonstrates that the TcR expressed on NK-92(CD3) cells is active and functional. NK-92(CD3) cells expressing a TcR are thus shown to have target cell-specific cytotoxicity.

Example 4. Activation of NK-92(CD3) cells expressing TcRs by T2 cells loaded with the relevant peptide

[097] NK-92(CD3) cells were modified to express Radium-1 or DMF-5 TcRs and cell surface CD3 expression was detected using APC-labeled anti-CD3. CD3+ cells were selected for degranulation analysis (Figure 5, top panel) and were sorted.

[098] NK-92(CD3) cells modified to express Radium-1 or DMF-5 TcRs were incubated with T2 cells (Figure 5, lower panels) loaded with TGFbRII (light gray outline) or MART1 (dark gray outline) peptides ) which are the target antigens for Radium-1 and DMF-5 TcRs, respectively. Expression of CD107a by NK-92(CD3) cells expressing Radium-1 TcRs (Figure 5, lower left panel) and DMF-5 (Figure 5, lower right panel) with T2 cells in the presence of either peptide was measured. NK-92(CD3) cells modified to express Radium-1 TcR exhibited activation in the presence of the TGFbRII peptide, and cells expressing DMF-5 TcR exhibited activation in the presence of MART-1. No non-specific activation was observed.

[099] A similar experiment was also carried out to determine the EC50 for each TcR. NK-92(CD3) cells modified to express Radium-1 (circles) and DMF-5 (squares) TcRs were incubated with T2 cells in the presence of TGFbRII or MART-1 peptides, respectively, in a range of different concentrations. of peptides. Activation was measured by detecting CD107a expression as described above. CD107a expression was measured at each concentration and relative activation (CD107a as a percentage of maximum CD107a expression) was calculated. EC50 values of 2nM (Radium-1) and 7nM (DMF-5) were calculated (Figure 6).

Example 5. TcR/CD3-mediated signaling in NK-92(CD3) cells

[100] NK-92(CD3) cells transfected with Radium-1 TcR were tested using flow cytometry (phospho-flow) to investigate their signaling capacity. Initially, the general signaling capacity of the TCR complex was analyzed by stimulating cells with anti-CD3 and anti-CD28 antibodies, which simulate activation through the TCR. As shown in Figure 7, the clustering of TcR and CD3 leads to the activation of a signaling cascade similar to that observed in T cells. This is demonstrated by increased phosphorylation levels of several TcR/CD3-related proteins, including ZAP-70, SLP-76 and CD3Z.

[101] The cells were then tested for stimulation by specific antigens using antigen-presenting cells and monitoring the signaling activity of the TCR complexes at different time points by flow cytometry (phospho-flow). NK-92(CD3)-Radium-1 and NK-92(CD3)-DMF-5 were stimulated with their cognate peptides. As shown in Figure 8, only specific stimulation resulted in phosphorylation of the signaling molecule. Early and late signals can be distinguished and the pattern was similar to that observed in T cells. Taken together, these data demonstrate that NK-92(CD3-TCR) react like T cells when in contact with their substrate.

Example 6. Stimulation of NK-92(CD3) cells by TcRs Derived from CD4+ T Cells

[102] Radio 5 and Radio 6 of TcRs derived from CD4 + T cells were transduced into NK-92(CD3) cells. These TcRs were able to redirect cells specifically against MHC Class II peptide targets. Radium-5 and Radium-6 specifically target a TGFbRII frameshift mutant peptide (KSLVRLSSCVPVALMSAMT); Radium-5 targets this peptide specifically in the context of HLA-DR7, Radium-6 in the context of HLA-DR4. The kinetics of NK-92(CD3)-Radium-5/6 cell stimulation are shown in Figure 9. NK-92(CD3) cell stimulation is measured as in Example 4 above. The EC50 values of both TcRs were calculated, as shown in Figure 9. The EC50 of Radium-5 in a patient sample of HLA-DR7 haplotype was calculated as 19 μM; the EC50 of Radium-6 was calculated as 4 μM in one patient sample and 0.4 μM in a second. Both patient samples were HLA-DR4 haplotype.

Example 7. Analysis of NK-92 protein expression with and without CD3/CD3-TcR expression

[103] The protein expression profiles of NK-92 cells were compared with those of NK-92(CD3/CD3-TcR) cells. No change was observed between the two groups of cells (except, of course, the presence of CD3 in NK-92 cells (CD3/CD3-TcR). The comparison is shown in the table below (PBMC = peripheral blood mononuclear cells).

Claims (16)

1. Use of a modified NK cell, characterized by the fact that it is for the manufacture of a medicine for use in adoptive cell transfer therapy, in which said modified NK cell expresses a TcR and the CD3 CD3Y, CD3δ, CD3ε and CD3Z chains , and the CD3 chains and the TcR form a functional CD3-TcR complex located on the NK cell surface. 2. Use according to claim 1, characterized by the fact that said TcR is independent of CD8 and/or CD4. 3. Use according to claim 1 or 2, characterized in that said modified NK cell is a modified NK-92 cell. 4. Use according to claim 1 or 2, characterized in that said modified NK cell is a primary modified NK cell. 5. Use according to any one of claims 1 to 4, characterized by the fact that said NK cell is modified to be non-immunogenic, preferably universally non-immunogenic. 6. Use according to any one of claims 1 to 5, characterized by the fact that said NK cell is: i) human and HLA-negative; ii) modified to interrupt or prevent the expression of β2 microglobulin; or iii) irradiated. 7. Use according to any one of claims 1 to 6, characterized by the fact that said therapy is cancer therapy. 8. Use according to any one of claims 1 to 6, characterized by the fact that said TcR has specificity against an antigen on a target cell, and said target cell is an infected cell. 9. Use according to any one of claims 1 to 8, characterized in that the TcR is specific for both an antigen expressed by a target cell in an individual to be treated by administration of the cell and for the MHC type of the individual. 10. Therapeutic composition, characterized by the fact that it comprises a modified NK cell defined in any one of claims 1 to 6, together with at least one pharmaceutically acceptable carrier or excipient. 11. Method for producing a universal NK cell for use in preparing a cell for adoptive cell transfer therapy, wherein said method is characterized by the fact that it comprises: (a) providing a universally non-immunogenic NK cell; (b) modifying said cell to express the CD3 CD3Y, CD3δ, CD3ε and CD3Z chains and a TcR, such that the CD3 chains and the TcR form a functional CD3-TcR complex located on the surface of the NK cell. 12. Method according to claim 11, characterized by the fact that said TcR has specificity for a cancer antigen or an antigen expressed by an infected cell. 13. Method for preparing a modified NK cell for therapeutic use, wherein said method is characterized by the fact that it comprises: (a) providing an NK cell that expresses the CD3 CD3Y, CD3δ, CD3ε and CD3Z chains; (b) determine the MHC type of an individual to be treated; (c) identifying a target antigen in the individual, which antigen is expressed or presented by cells in the individual, (d) modifying the cell from step (a) to express a TcR having specificity for the target antigen and matching the MHC type of the individual, so that the CD3 chains and the TcR form a functional CD3-TcR complex located on the surface of the NK cell. 14. Method according to claim 13, characterized by the fact that the TcR is specific for a cancer antigen. 15. Kit for use in adoptive cell transfer therapy, wherein said kit is characterized by the fact that it comprises: (a) an NK cell that expresses the CD3 CD3Y, CD3δ, CD3ε and CD3Z chains; and (b) a panel of nucleic acid molecules each encoding a TcR, wherein the TcRs have different antigen specificity and/or different MHC specificity. 16. Kit according to claim 15, characterized by the fact that said nucleic acid molecules are contained in vectors, preferably in viral vectors.