EA040420B1 - UNIVERSAL T-KILLER CELL - Google Patents

Description

The present invention provides natural killer cells and their use in therapy, in particular in the treatment of cancer. In particular, a modified natural killer cell has been developed that has been modified to express CD3 and that can be further modified to co-express a T-lymphocyte receptor-based antigen receptor specific for a cancer cell antigen or other target antigen. Said modified natural killer cell is non-immunogenic, for example, expresses MHC (major histocompatibility complex) at a low level or is MHC negative, which means that said cells are suitable for universal use regardless of the type of MHC of the subject. Thus, the present invention effectively combines the properties of versatility and personalization; the T-lymphocyte receptor can be tailored for both the pathological condition (e.g., depending on the type of cancer) and the type of MHC of the subject being treated, resulting in versatile cells that can be used in treatment that is personalized to the subject being treated. is required to be treated.

Certain cells of the immune system exhibit cytotoxic activity against specific target cells. Cytotoxic T-lymphocytes express T-lymphocyte receptors (TcR), which are able to specifically recognize antigen-derived peptides associated with MHC class I molecules. In contrast, natural killer cells (NK cells) are not limited by the need to recruit MHC and do not require antigen presentation by MHC molecules to exert their cytotoxic effects. They are able to recognize stressed cells in the absence of peptide-loaded MHC and kill cells that do not contain MHC. Thus, NK cells play an important role in innate immunity, since in no other way can these non-MCGS cells be detected and eliminated by other cells of the immune system.

Cytotoxic T cells (also called cytotoxic T lymphocytes or Tkiller cells) are able to recognize infected or damaged cells in an antigen-specific manner via TcR by binding to antigens (peptides) presented on the cell surface by class I MHC molecules. Binding to the class I MCGS peptide is mediated by the CD8 co-receptor, which enhances the affinity of the binding interaction and enhances signaling through TcR. TcRs of other types of T-lymphocytes, especially T-helpers, recognize antigenic peptides presented by MHC class II molecules, which is mediated by the CD4 co-receptor. CD3 is required for TcR localization on the cell surface in T-lymphocytes (Ahmadi et al. 2011. Blood 118, 3528-3537), and CD3 is required for T-lymphocyte activation upon contact with the MHC protein loaded with the corresponding antigenic peptide. CD3 is a protein complex consisting of four different chains: a CD3γ chain, a CD3δ chain, two CD3ε chains, and a ζ chain, which together with TcR form the TcR complex.

NK cells (also called large granular lymphocytes) are a cell line that differentiates from a common lymphoid progenitor (which also gives rise to B and T lymphocytes). Unlike T lymphocytes, NK cells do not normally contain CD3 on their plasma membrane. Importantly, NK cells do not express T-lymphocyte receptors and usually also lack other antigen-specific cell surface receptors (other than T-lymphocyte and CD3 receptors, these cells do not express B-lymphocyte immunoglobulin receptors, but instead usually express CD16 and CD56). Thus, NK cells differ in that they have a CD3(-), CD56(+) phenotype. The cytotoxic activity of NK cells does not require sensitization, but it is enhanced by the activation of a number of cytokines, including IL-2 (interleukin-2). It is generally assumed that NK cells lack the appropriate signaling pathways required for antigen receptor-mediated signaling, or such signaling pathways are not complete in them, and therefore these cells are considered to be incapable of antigen receptor-dependent signaling, activation and reproduction.

NK cells are cytotoxic, and in order to modulate their cytotoxic activity, these cells provide a balance in signaling from activating and inhibitory receptors. For example, NK cells expressing CD16 (FcyRIII receptor) can bind to the Fc domain of antibodies associated with the infected cell, resulting in NK cell activation. In contrast, activity against cells expressing MHC class I proteins at a high level is reduced. Upon contact with target cells, NK cells release proteins such as perforin and enzymes such as proteases (granzymes). Perforin can form pores in the target cell membrane, causing apoptosis or cell lysis.

A number of T-lymphocyte-based therapies have been developed for the treatment of cancer, and these methods, called adoptive cell transfer (ACT), have become very promising in recent years. To date, three main ACT strategies have been developed. The first of these, and the most developed, is based on the isolation of the patient's own tumor-active T-lymphocytes from peripheral blood or tumor foci (called tumor-infiltrating lymphocytes (TIL)). These cells are expanded ex vivo and reintroduced into the patient.

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However, this method may involve delays of several weeks while the cells are propagated, and it requires special facilities and specialists in obtaining cells.

There are two alternative treatments that are based on the modification of the patient's own T-lymphocytes, including the introduction of receptors that are able to recognize the tumor. In one embodiment, TcRs with activity against a cancer antigen can be isolated and characterized, and the gene encoding the TcR can be introduced into T-lymphocytes and these T-lymphocytes reintroduced into the patient. This treatment has been shown to shrink solid tumors in some patients, but has a significant disadvantage: the TcRs used must be matched to the patient's immune system. Accordingly, as an alternative to the use of TcR, treatments based on the expression of chimeric antigen receptors (CARs) in T lymphocytes have been proposed. CARs are recombinant proteins containing an antibody coupled to the signaling domain of the TcR complex and can be used to target T lymphocytes to a tumor if the appropriate antibody is chosen. Unlike TCR, CAR should not be tailored to the patient's MHC type. However, to date, very few cancer-specific surface antigens have been identified that can be used as suitable targets for CARs, and thus the use of CARs for cancer treatment is currently limited.

All ACT approaches based on T-lymphocyte modification using TcR or CAR require the isolation and modification of T-lymphocytes from the patient or from a histocompatible donor. If non-autologous cells are used, autologous T-lymphocytes should be used to eliminate the risk of rejection. This increases the costs associated with ACT and may also increase the time it takes to obtain T cells for use in ACT.

Alternative methods to overcome the limitations of ACT listed above are based on the use of cytotoxic NK cells, as described, for example, in WO 98/49268. NK cells are powerful killer cells and have high cytotoxicity against a number of cells of various malignant tumors. Because NK cells will recognize target cells that express foreign human lymphocytic antigen (HLA) molecules rather than self HLA antigens, autologous NK cells are usually ineffective and allogeneic NK cells (which require careful removal of T lymphocytes, to avoid graft-versus-host disease (GVHD) or cell lines. Therapies based on the irradiation of the NK-92 NK cell line have reached the stage of clinical trials for the treatment of leukemias and other hematological malignancies. Irradiation means that the cells are unable to proliferate, and therefore the elimination action has a limited and predetermined duration. Also, NK cells do not attack healthy tissues. However, the natural range of NK cells is limited, and although NK-92 cells have a wider spectrum than primary NK cells, since NK cells do not normally contain receptors suitable for specific antigen targeting on the target cell, further modification of these cells is required, to expand the range of cancers that can be treated and targeted or redirected to a specific or selected cancer. Of course, this redirection means that there is no need for allogeneic NK cells, but NK cell lines may still have advantages such as higher cytotoxicity compared to primary or autologous NK cells. To this end, CAR-expressing NK cells have been developed for use in cancer treatment. CARs that recognize antigens on the surface of cancer cells were introduced into a continuously growing culture of NK-92, and it was shown that NK-92 cells containing these CARs have increased cytotoxicity against tumor cells compared to the original NK-92 cells (Uherek et al 2002. Blood 200, 1265-1273). Early phase clinical trials are currently underway. However, as noted above, the lack of available antigens as targets for CAR currently limits the use of CAR-based therapies.

The present invention is based on an alternative approach to obtaining killer cells specific for a particular cancer. More specifically, the present invention is directed to providing NK-based cancer-specific killer cells for universal use, which means that the cells may not match the immune type of the subject to be treated, as is currently required for methods treatment based on T-lymphocytes, but nevertheless these cells can be modified taking into account the specific immune type and type of cancer of a particular subject, if necessary. Thus, universal killer cells can be used for personalized medicine.

Thus, according to the present invention, it is proposed to transform an NK cell into a T-lymphocyte and, therefore, to give it the ability to express TcR and kill cells specifically and directionally, as does a T-lymphocyte. The present invention combines the inherent power and elimination capacity of the NK cell with the specific targeting and cell activation provided by TcR. Although CARs have been previously expressed in NK cells, it has not been thought until now that it is not possible for NK cells to possess all the necessary signaling pathways and cellular machinery for successful TcR expression that would lead to successful activation and targeted elimination by NK cells. . Versatility is achieved through the use of NK cells that are non-immunogenic, for example, which normally express low levels of MHC or which have a reduced proliferative capacity, or said NK cells are negative for MHC (for example, for HLA) in such a way that these cells are not recognized immune system of the recipient as strangers. Thus, the NK cell may not match the immune type (eg, HLA type) of the subject to be treated and may be administered universally regardless of the subject's immune type (i.e., subject with any immune type, eg, any profile). HLA type).

Thus, the present invention provides means that allow the expression of functional, active TcRs in non-immunogenic NK cells. Thus, it is possible to obtain a universal T-killer, which does not depend on the compliance of the cell with MHC, but which has a controlled specificity dictated by the specificity of the TcR introduced into such a cell. Co-expression of CD3 and TcR in an NK cell has been shown to be sufficient for TcR to be localized to the surface of the NK cell, and unexpectedly, a TcR expressing NK cell exhibited antigen-specific cytotoxicity against target cells. In another contemplated method, the present invention provides methods for converting an NK cell into a cytotoxic T lymphocyte, with the added benefit of versatility, simply by co-expressing CD3 and TcR in a non-immunogenic NK cell. Thus, according to the present invention, a universal T-killer is provided, which can be used in personalized medicine.

Thus, according to the first aspect of the present invention, a modified NK cell is provided, said cell being non-immunogenic and modified to express CD3.

In particular, the cell is modified to be non-immunogenic, such as by irradiation to reduce proliferative capacity, or by other means of reducing proliferation, such that said cell does not survive long enough to elicit an immune response when administered to a subject, or by modifying the cell to become MHC-negative.

The cells of the present invention can be further modified to express TcRs that are specific for an antigen on a target cell (ie, target antigen). As will be defined in more detail below, the term TcR is used herein in its broadest sense and includes any antigen receptor that contains or is derived from or derived from TcR antigen recognition regions of TcR. Thus, both natural and synthetic TcRs are included, eg TcR variants or derivatives.

In one specific embodiment, the present invention provides a non-immunogenic NK cell expressing CD3 and a TcR, said TcR being specific for an antigen on a cancer cell. Again, the present invention provides a cancer-specific NK cell, said cell being non-immunogenic, and wherein cancer-specific specificity is provided by co-expression of cancer-specific TcR and CD3. As will be described in more detail below, in one preferred embodiment, TcR is independent of a co-receptor, in particular independent of CD4 and/or CD8.

According to a further aspect of the present invention, cells such as those described herein above are provided for use in therapy, more specifically for use in cancer therapy, or more generally for use in adoptive cell transfer therapy (e.g., in the treatment of any condition, which may respond to T-lymphocyte therapy).

According to another additional aspect, the use of modified NK cells, as described herein above, is provided in the preparation of a medicament for use in cancer therapy or for adoptive cell transfer therapy.

Also provided is a therapeutic composition comprising a modified NK cell as described herein above, 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.

According to another additional aspect, there is provided a method of treatment, more particularly a method of treating cancer, or a method of adoptive cell transfer therapy, which comprises administering to a subject in need thereof (e.g., a subject suffering from or diagnosed with cancer) an effective amount of modified NK- cells described above in the present description.

For use in therapy, the modified NK cells of the present invention must be modified to co-express TcR and CD3. Thus, obtaining a medicinal product or composition for use in therapy must include

- 3 040420 modification of NK cells such that they express TcR. TcRs must be designed or selected, firstly, so that they correspond to the immune type (i.e., MHC type) of the subject to be treated (i.e., recipient of modified NK cells), and, secondly, so that that they match or recognize cells in the body of the subject to be treated with said NK cells. That is, the TcR is designed or selected to recognize the desired target antigen in the subject. This may be a target antigen expressed (or presented) by a cancer cell in the subject, or any cell to be eliminated by adoptive cell transfer therapy, such as an infected cell, such as a cell expressing (or presenting) a viral antigen, or antigen of another pathogen. Thus, in order to carry out anti-cancer therapy or adoptive cell transfer therapy according to the present invention, or to obtain a modified NK cell for therapeutic use, the following steps can be carried out:

(a) obtaining a non-immunogenic NK cell that has been modified to express CD3;

(b) determining the subject's MCHS type; who needs to be treated;

(c) identifying a target antigen in the subject, such antigen being expressed or presented by cells in the subject's body, for example, by identifying or determining the type of cancer in the subject, and/or the expression of a particular cancer marker (e.g., antigen), or the presence of a particular mutation, and etc.;

(d) modifying the cell obtained in step (a) to express a TcR that is specific for the target antigen and matches the MHC type of the subject.

As will be discussed in more detail below, these steps can be carried out separately or simultaneously. Thus, for example, steps (a) and (b) can be performed simultaneously, more specifically said cell can be modified to express CD3 and TcR in one or more simultaneous steps. However, in another embodiment, the cell may be modified to express CD3 in a separate step prior to modification resulting in TcR expression.

In addition, according to the method of treatment or therapeutic application, the modified cell obtained in step (d) can be administered to a subject (for example, during additional step (e)).

According to another aspect of the present invention, a method is provided for obtaining a universal NK cell for use in obtaining a cell for personalized therapeutic use, namely for therapy based on adoptive cell transfer, and this method includes:

a) obtaining an NK cell that is non-immunogenic, more specifically, modifying the NK cell to make it non-immunogenic; And

b) modifying said cell such that it expresses CD3.

Additionally, in order to obtain a modified NK cell for use in adoptive cell transfer therapy, said cell is further modified in step (c) to express a TcR, and in particular, for personalized therapy, a TcR that matches the MHC type of the subject to be treated and which recognizes the target antigen expressed or presented by the target cell to which the modified NK cell is to be targeted.

In a preferred embodiment, there is provided a method for producing an NK cell specific for a particular cancer, said method comprising:

a) obtaining an NK cell that is non-immunogenic, for example, modifying the NK cell to make it non-immunogenic;

b) modifying said cell such that it expresses CD3;

c) modifying said cell such that it expresses a TcR specific for a particular cancer.

Steps (b) and (c) can be carried out together (simultaneously) or separately, for example, sequentially. However, according to preferred embodiments, as will be described in more detail below, it is possible to obtain a universal NK cell expressing CD3, and said cell can subsequently be used in separate steps to obtain personalized cells for therapy based on adoptive cell transfer, by further modifying these cells, such that they express a TcR that is selected based on the subject's diagnosis and type of MHC. Thus, a bank or library of TcR receptors can be obtained, including different TcRs (more specifically, nucleic acid molecules or vectors encoding TcR) according to the specificity of MHC (i.e. its type) and the specificity for the target antigen (for example, for a number of different known or previously identified target antigens, such as cancer antigens). Depending on the subject's diagnosis (e.g., a specific type of cancer, or a cancer known to express a specific antigen, or identified by a specific cancer assay in said subject), a specific TcR can be selected and can be used to modify the NK cell according to the present invention. Alternatively, you can get

- 4 040420 a bank or panel of modified NK cells expressing both CD3 and TcR with different specificities, from which the appropriate NK cell can be selected depending on the subject's diagnosis and MHC type.

Accordingly, according to another further aspect of the present invention, there is provided a combination product or kit, such as in particular a product or kit for use in adoptive cell transfer therapy or 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 expresses CD3; and (b) a panel of nucleic acid molecules, each encoding a TcR; moreover, these TcRs have different antigenic specificity and/or different specificity for MHC.

Conventionally, coding nucleic acid molecules can be provided as vectors, eg, vectors ready for introduction (ie, transfection or transduction, etc.) into a modified NK cell. In a preferred embodiment, the TcR is specific for a cancer antigen or for an antigen expressed (eg, preferentially or specifically) on cancer cells.

In another further embodiment, the present invention provides a panel, or library, of modified NK cells, wherein each NK cell is non-immunogenic and expresses CD3, e.g., an NK cell modified to be non-immunogenic and expresses CD3 and expresses ( ie modified so that it expresses) TcR, and TcR different NK cells have different antigenic specificity and/or different specificity for MHC. It will be understood that in such aspects there may be several TcRs recognizing the same antigen, but they may differ in specificity for MHC and vice versa.

The present invention is based on the use of cytotoxic cells of the immune system in the treatment of cancer. More specifically, the present invention relates to the expression of TcR in cytotoxic cells of the immune system, which may not otherwise express TcR, and methods that allow you to cause the expression of active, functional TcR in such cells.

The terms cytolytic and cytotoxic are used interchangeably herein and refer to a cell capable of causing the death of target cells by lysis or apoptosis.

The term target cell refers to any cell that is killed by the cytotoxic cells of the present invention. More specifically, a target cell is a cell that is targeted by NK cells, and therefore a cell that expresses or presents an antigen recognized by the TcR (target antigen). These cells may include cells of any type, and the cells according to the present invention are targeted due to the antigen present on the surface of the target cell.

Thus, a target cell can be any cell in the subject's body from which it is desired to get rid of, for example, to remove, destroy, inactivate, etc. The preferred target cell is a cancer cell, but it can be any cell resulting from a disease or clinical condition. Thus, said disease or condition can be any condition for which adoptive cell transfer therapy, or more specifically, T-lymphocyte therapy targeting cells to kill or remove them, etc. is useful. In addition to cancer cells, other cells that may be desirable to remove from a clinical point of view include infected cells, ie cells infected with a pathogen. Typically, such cells will include virus-infected cells, but they may also be infected by another pathogenic organism, such as a microorganism, such as a bacterium, fungus, mycoplasma, protozoa, prions. Alternatively, the target cell may be an apoptotic or pre-apoptotic cell, or a stressed cell (ie that expresses stress related markers on its surface), or may be a mutant cell, eg expressing a particular mutation.

A target antigen is (or more specifically is or contains) a molecule that is recognized by a TcR when presented on the surface of a target cell. Thus, the target antigen, or more specifically the peptide derived from the target antigen, is presented on the surface of the target cell in a manner characteristic of MHC-I or MHC-II, as required for its TcR recognition. Accordingly, as discussed above, said target antigen may be a cancer antigen, i.e. an antigen that is expressed specifically, selectively, or preferentially by the cancer cell, or that is characteristic of the cancer cell, or that is known or used as a cancer cell marker. Said cancer antigen may be said cancer antigen, i. e. an antigen often found in a wide range of different cancers, or may be specific to a particular type of cancer. Alternatively, it may be an antigen from an infectious pathogen that is present on the surface of the target cell, or any other

- 5 040420 an antigen expressed or presented by any other cell that it is desirable to abolish.

The term MHC refers to the major histocompatibility complex protein and includes both MHC I and MHC II proteins. MHC II proteins are usually found only on the surface of antigen-presenting cells such as dendritic cells, mononuclear phagocytes, some endothelial cells, and thymic epithelial cells. MHC II proteins are also expressed by B-lymphocytes and therefore can be expressed by malignant tumors from B-lymphocytes. Also, MHC II proteins are often expressed by melanoma cells. The term MHC encompasses human lymphocytic antigen (HLA), and thus, where the subject is a human, these terms can be used interchangeably.

According to the present invention, any NK cell can be used. The term NK cell refers to a large granular lymphocyte, which is a cytotoxic lymphocyte derived from a common lymphoid progenitor that does not inherently contain an antigen-specific receptor (eg, T-lymphocyte receptor or B-lymphocyte receptor). NK cells can be distinguished by their CD3 ^- , CD56 ⁺ phenotype. Thus, as used herein, the term includes any known NK cell, or any NK-like cell, or any cell with the characteristics of an NK cell.

In one embodiment, NK cells may be obtained from a subject and grown in vitro to obtain a population of NK cells for use in accordance with the present invention. NK cells may be autologous (i.e., derived from the body of the subject to be treated with the cells and methods of the present invention), or may be heterologous (i.e., the cells may be obtained from the body of a donor subject and may be intended for the treatment of a second subject (i.e., they may be allogeneic, isogenic, or xenogeneic)). Thus, primary NK cells can be used. According to an alternative implementation according to the present invention, you can use an NK cell known in the art, which has previously been isolated and cultured. Thus, an NK cell line can be used. A number of different NK cells are known and described in the literature, and any of them can be used, or a primary NK cell can be cultured, 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, such as being easier to grow and maintain in culture, easier to propagate, and more readily available in standardized quality for adoptive cell transfer therapy when needed. Certain cell lines, such as the NK-92 line, may also exhibit higher cytotoxic activity, and in their unmodified state (i.e., not modified according to the present invention), they may have a wider range of target cells, such as cancer cells. -targets (this may be due to the absence or reduction in the number of inhibitory receptors). In addition to NK-92, suitable NK cells include (but are by no means limited to) the NK-YS, NK-YT, MOTN-1, NKL, KHYG-1, HANK-1, or NKG cell lines.

In a preferred embodiment, the cell is NK-92 (Gong et al., 1994, Leukemia 8, 652-658) or a variant thereof. A number of different variants of the parent NK-92 have been produced, described or available that are non-immunogenic. Any such variations may be used and are included within the term NK-92. However, any NK-92, or indeed any NK cell for use according to the present invention, must not be modified to express any antigen-specific receptor other than TcR that is introduced or intended to be introduced into an NK cell modified according to the present invention. For example, the cell should not express CAR (a monoclonal antibody coupled to an intracellular signaling domain from the TcR complex). More broadly, the modified NK cell of the present invention does not express an antigen-specific receptor based on or containing antibody recognition regions. Normally, the NK cell does not contain antigen-specific receptors, and thus the only antigen-specific receptor present in the cells of the present invention is the TcR that is introduced or intended to be introduced into said NK cell.

According to the present invention, the modified NK cell is non-immunogenic. From a functional point of view, this means that the specified cell is not recognized by the immune system of any subject into which it is introduced - it passes unnoticed by the immunological defense system and is not rejected or not recognized by the body of the recipient subject as foreign. In other words, there is no clinically significant immunological response to the modified NK cells from the immune system of the subject to whom said modified NK cells are administered. Thus, a cell may be non-immunogenic when it, when administered to a subject, does not generate an immune response that impairs, interferes with, or interferes with the use of said cells in therapy.

Thus, in the present description, the term non-immunogenic is used in a broad sense and means that when a cell is administered to a subject by injection or otherwise, there is essentially no fuss.

- 6 040420 kay or there is no clinically significant immune response to the specified cell. More specifically, the cell does not (or is not capable of) eliciting an immune response sufficient to result in rejection of said cell and/or impair the function of said cells, e.g., the immune response is not sufficient to abolish or substantially or significantly reduce a function or effect ( for example, benefit) of these cells. Thus, the cells retain cytotoxic activity against the target cell. In a specific 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 negligible) immune response to a NK cell (eg, a small immune response) may be tolerable if the function or benefit of said cells is not significantly impaired (i.e., if said cells still perform their function).

Therefore, it can be seen that an autologous cell (which is a cell that is administered or intended to be administered to the same subject from which it is derived) will be non-immunogenic (because it is self). Similarly, a non-autologous MHC-matched NK cell will be non-immunogenic or substantially non-immunogenic. Thus, the term non-immunogenic includes functional non-immunogenicity. However, in certain embodiments, and as noted above, a non-immunogenic NK cell of the present invention is non-immunogenic regardless of the recipient of said cell, i. regardless of the subject into which it is administered or intended to be administered (i.e., it should not elicit a clinically significant immune response if administered to a non-autologous subject). In specific embodiments, the NK cell may be treated to become non-immunogenic (ie, it may be modified to become non-immunogenic). For example, the treatment may be physical, chemical, or biological, or may be a structural or physicochemical modification of cells (e.g., a treatment that causes a structural or physical change or modification of cells), such as irradiation, or genetic modification, such as to reduce or eliminate the expression of MHC cell. Accordingly, in some embodiments, the NK cell is not only not functionally immunogenic, but has or contains a modification that renders it non-immunogenic.

Accordingly, NK cells may be inherently non-immunogenic or may be modified to become non-immunogenic. Inherently non-immunogenic NK cells will not express the MHC molecule, or show only weak expression of the MHC molecule, or may express a non-functional MHC molecule that cannot stimulate an immunological response. NK cells, which may be immunogenic, can be modified to eliminate the expression of the MHC molecule or to only express the MHC molecule at a low level on their surface. Alternatively, such cells can be modified to express a non-functional MHC molecule.

Any such cells (either non-immunogenic in nature or modified in some way) do not contain sufficient amounts of MHC on their surface to trigger a clinically significant immunological response at the expense of the subject's immune response and may be considered MHC-negative. In other words, an MHC-negative cell does not express on its surface an MHC molecule that is immunologically functional, or does not show a significantly high level of expression of the MHC molecule on its surface so that it can be recognized by another immune cell, in particular, not its own immune cell or an immune cell from organism of the intended recipient subject. The cell may not contain an MHC molecule, or may not express MHC on its surface, or only weakly express MHC on its surface, or any MHC molecule that is expressed may be non-functional, for example, it may be mutated or otherwise modified so to become non-functional. Any methods that disrupt the expression of a functional MHC molecule are included. Therefore, they may include inactivation (knockdown) or knockout of the gene molecule from the MHC complex and/or they may include a modification that prevents adequate transport and/or correct expression of the MHC molecule, or the entire complex, on the cell surface.

In particular, the expression of one or more class I MHC proteins on the cell surface of the present invention may be impaired. In one embodiment, said cells may be human cells that are HLA-negative and, accordingly, cells that are de-expressed (e.g., inactivated) by one or more HLA molecules, such as HLA class I MHC complex I molecules.

In a preferred embodiment, disruption of class I MHC expression can be accomplished by inactivating the gene encoding _β2 -microglobulin, a component of the mature MHC class I complex. Expression e ₂ m. can be eliminated by targeted disruption of the β ₂ microglobulin (v2sh) gene, for example by site-directed mutagenesis of the e ₂ m promoter. (to inactivate the promoter), or in the gene encoding the 2sh protein to introduce an inactivating mutation that prevents the expression of the _2sh protein, for example, a frameshift mutation or the introduction of an early temporary STOP codon in the specified gene. Alternatively, site-directed mutagenesis can be used to generate a non-functional e ₂ m protein that is unable to form an active MHC protein on the cell surface. In this way, the e ₂ m MHC protein may remain inside the cell or may appear on the surface but be non-functional.

Alternatively, the NK cell may be irradiated prior to administration to a subject. NK-92s that have been irradiated have been shown to be non-immunogenic in clinical studies and have been approved for use in immunotherapy (Ton T. et al. 2013 Cytotherapy 15 1563-70).

Without wishing to be bound by theory, it is believed that irradiation of cells results in such cells being present in the subject's body only temporarily, and thus reducing the time during which the subject's immune system generates an immunological response to the modified NK cells. Although such cells may express a functional MHC molecule on their surface, they may also be considered non-immunogenic.

Thus, the NK cell according to the present invention can be modified to be non-immunogenic by reducing its ability or potential to proliferate, that is, by reducing its proliferative ability.

The NK cell of the present invention can be modified to express CD3. As mentioned above, the NK cell of the present invention does not express CAR, or indeed any antigen-specific receptor other than TcR. Thus, in particular, with the exception of the TcR-CD3 fusion, CD3 is not expressed as part of the chimeric receptor. More specifically, CD3 is not expressed as part of a chimeric antigen receptor (CAR), or any chimeric receptor based on (or containing) an antigen-binding site(s) derived from an antibody, or a binding domain derived from any binding molecule or component that are not TcRs, eg from any other affinity binding partner, eg from a non-TcR ligand or receptor. Thus, CD3 is not expressed as part of a chimeric receptor that contains an antigen-binding domain (ie, binding portion or component) 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 single molecule or chain, or as part of a chimeric protein with a component that is not or part of the CD3 chain, or a linker or spacer molecule), and/or it is expressed as part of (or in the composition) of the TcR-CD3 hybrid. Accordingly, in particular, the NK cell of the present invention does not express a CAR or other chimeric receptor that contains a CD3 chain or molecule other than a CD3-TcR fusion. In one embodiment, when CD3 is used, or as part of a fusion (ie, fusion protein), it is a CD3 fusion or a CD3-TcR fusion, as described in more detail below.

Normally (or by nature) NK cells do not express CD3 and, therefore, to render said cell capable of expressing CD3, a nucleic acid molecule encoding CD3 is introduced into the cell. Thus, the cell is modified to recombinantly express CD3. In other words, the cell is modified to allow expression of a heterologous nucleic acid molecule encoding CD3. CD3 can be matched to the cell species (ie, the same species from which the cell is derived), but CD3 from other species can also be used. Thus, for a human NK cell, it is possible to express human CD3, or CD3 of other mammalian species, e.g., mouse, rat, rabbit, etc. In a preferred embodiment, all of the CD3 chains (i.e., CD3γ chain, CD3δ chain, two CD3ε chains and a ζ chain). However, it is also contemplated that only one CD3 chain is present (eg, only CD3γ chain, or only CD3δ chain, or only CD3ε chain, or only CD3ε chain), or any combination thereof. Thus, the NK cell can be modified to express any CD3 chain alone, or in combination with other CD3 chains, up to and including the entire CD3 molecule. Without wishing to be bound by theory, it is hypothesized that it may be possible to direct TcR to the cell surface and/or initiate signaling when one or more of the CD3 chains are not expressed on the NK cell. It is also contemplated that one or more of the CD3 chains may be present in the form of a fusion protein in which at least two chains are expressed as a single polypeptide, so long as CD3 localization and signaling functions are maintained.

Also, one or more CD3 strands can be encoded by one nucleic acid molecule or by more than one nucleic acid molecule, for example by different molecules. Conventionally, a construct will be created that contains a nucleic acid molecule encoding all three CD3 strands, eg under the control of a single promoter, or equally linked to each other.

The NK cell of the present invention can be further modified to express TcR. A TcR can be considered any receptor containing 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 containing a TcR antigen recognition domain, an antigen recognition sequence, or it may comprise a TcR antigen recognition domain, an antigen recognition sequence, or a fragment thereof, fused to an amino acid sequence, and /or protein domains (ie, as part of a chimeric molecule or fusion protein) that are not naturally found together with TcR (ie, non-native context). Thus, in the present description, the term TcR is used in a broad sense and includes any antigen receptor in which the antigen recognition site is represented by an antigen recognition domain or sequence of a TcR, for example, an antigen recognition site from a native TcR, or derived from a native TcR. Accordingly, the term TcR includes native and non-native TcRs, that is, synthetic or artificial TcR molecules or constructs, TcR variants or derivatives derived from or based on natural TcR.

Thus, a TcR-expressing NK cell has antigen-specific (ie, targeted) cytotoxic activity against the target cell. Said TcR may be any TcR having specificity for the antigen on the target cell in question (target antigen). The specificity of the NK cell (ie the antigen it binds to) will be determined by the specificity of the TcR. In other words, to give the NK cell the desired specificity (ie, specificity for a specific antigen on the target cell), an appropriate TcR must be selected. Thus, in one embodiment, the present invention provides a NK cell modified to express CD3 and further modified to express a TcR having specificity for an antigen on the target cell.

In one preferred embodiment, the TcR binds to an antigen on the surface of the target cell with high affinity (ie, the TcR is a high affinity TcR). In such an advantageous embodiment, the TcR is able to bind to the MHC antigen complex on the surface of the target cell with a high enough affinity that the cell expressing the TcR can undergo activation in the absence of the CD8/CD4 co-receptors that are normally required to activate the TcR in a cytotoxic cell or T-helper. , respectively. Thus, the present invention provides a NK cell modified to express CD3 and further modified to express a TcR having specificity for an antigen on a target cell, wherein the TcR binds to said target cell with high affinity. and undergoes activation in the absence of CD8 and/or CD4 co-receptors. Alternative expressed, TcR, which is introduced into the NK cell according to the present invention, does not depend on co-receptors (cofactors); that is, it does not require an additional receptor or cofactor for cell activation (ie, for cell signaling to occur due to TcR binding). Thus, TcR can bind to an antigen (MHC-antigen complex) with a _koff value in the normal physiological range. In particular, it is independent of CD8 and/or CD4, eg, independent of CD8, and more specifically, independent of CD8 and CD4. However, in an alternative embodiment, the NK cell may be further modified to express CD8 and/or CD4.

TcR recognizes an antigen (peptide) presented on the cell surface by a protein from the MHC family I or II class, and binds to such an antigen. Thus, antigen recognition depends on both the MHC cell type and the peptide (more specifically, the peptide sequence) (antigen). In general, the target antigen is known and the TcR is selected according to the specificity of that antigen. In a preferred embodiment, the peptide sequence of the antigen presented by the target cell may be known. In addition, the MHC target cell type may also be known. Thus, in a preferred embodiment, the TcR may be chosen to be able to specifically bind to the antigen-MHC complex on the surface of the target cell. Thus, according to a preferred embodiment of the present invention, there is provided a NK cell modified to express CD3 and further modified to express a TcR having specificity for an antigen on the target cell, said TcR being able to specifically bind to the antigen-MHC complex on the target cell. target cell surface.

According to a preferred embodiment of the present invention, a TcR can be selected from a cytotoxic T lymphocyte that has been shown to specifically exhibit cytotoxicity against a target cell or T helper. Alternatively, an antigen-recognizing domain or sequence of such a TcR or fragment thereof may be selected. Said T-lymphocyte may be obtained from the body of the patient to be treated, or may be obtained from the body of another subject (ie, a TcR donor). Thus, a TcR from a T-lymphocyte that has been shown to have proven activity against the target cell can be selected as the TcR. As an example, T-lymphocytes, such as tumor-infiltrating lymphocytes, can be isolated from the body of a patient suffering from cancer. By their nature, these cells will be or include T-lymphocytes that are active against the cancer of the specified patient. The genes encoding the TcRs of such cells can be identified and cloned and used to express a TcR, or an antigen recognition domain or sequence of such a TcR, or a fragment thereof, in an NK cell of the present invention. T-lymphocytes can

- 9 040420 obtained in the body of a patient or subject by vaccination, for example, by administering a vaccine containing a cancer antigen. It is then possible to select those T-lymphocytes induced by vaccination that are most active against a cancerous tumor in the subject's body or against cancer cells. In this way, the most potent or efficient TcRs can be selected, for example, TcRs with maximum affinity, or which are independent of co-receptors. To obtain an optimal TcR, affinity maturation can be performed.

It will be advantageous if the HLA profile or MHC profile of the subject from which the TcR is obtained (TcR donor) is the same as the HLA profile or MHC profile of the subject to be treated. As noted above, a TcR in a T-lymphocyte having specificity for a target cell can be identified and characterized, and the amino acid sequence of said TcR and the nucleotide sequence of the gene encoding the TcR can be obtained. To reiterate, a cytotoxic T lymphocyte or T helper containing a TcR having specificity for a certain 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 encoding the receptor can be obtained. The DNA sequence or gene can be used to provide a nucleic acid molecule or construct for insertion into an NK cell so that the TcR can be expressed in the host (ie, recipient) NK cell.

In an alternative embodiment of the present invention, an artificial TcR (ie, a TcR not identified from a cytotoxic T-lymphocyte or T-helper) can be used. The TcR may be an artificially generated alloreactive TcR. The TcR binding domain can be generated based on a combinatorial method such as phage display or ribosomal display, and a chimeric protein can be generated containing an artificial binding domain specific for a particular combination of antigen and MHC (eg, HLA).

The TcR may be proposed as a construct or fusion containing other protein domains.

One or more CD3 chains (or an entire CD3 construct) may be expressed as a fusion protein along with the TcR. In other words, one or more CD3 chains may be present on the same polypeptide chain as the TcR. In one preferred embodiment, the CD3 molecule, or one or more CD3 chains (e.g., CD3γ chain, CD3δ chain, CD3ε chain, or CD3ζ chain, or any combination thereof) may be present as a TcR-CD3 hybrid construct that will directly was directed to the plasma membrane, i.e. without the need for an additional CD3 molecule to also be present in the cell (because the fusion construct retains the transmembrane domain, eg provided by the CD3 chain) (Willemsen RM 2000 Gene Therapy 7:13691377). Thus, in such an embodiment, a cell can be modified to simultaneously express CD3 and TcR as part of the same construct or fusion. Although there may not be a need for a separately expressed CD3, it can nonetheless be proposed, for example, to improve signaling and therefore increase cell activity.

As noted above, a panel of different TcRs can be designed, with each TcR in the panel having specificity for (eg, specific affinity for) a particular antigen-MHC complex. Thus, different antigens and/or different epitopes on the target antigen may be recognized and/or said TcRs may have different affinity and/or specificity for the antigen. In addition, for a particular target antigen, said panel may contain TcRs with different MHC specificity (or MHC type). In this way, a specific TcR capable of binding to a specific antigen-HLA complex can be selected from the panel for use in the methods of the present invention, i.e. a TcR that matches both the target antigen and the subject's MHC type. Thus, in a particular preferred embodiment, a specific TcR may be selected depending on the nature of the antigens presented by particular target cells and their type of MHC (eg, HLA). It is contemplated that such a panel can be used to enable the rapid generation of NK cells having specific cytotoxic activity against the target cell. Accordingly, the method of the present invention for producing an NK cell having specificity for a target cell may include:

a) modifying the NK cell such that it expresses CD3;

b) determining the MHC profile in said target cell and the nature of the antigen presented by said target cell; And

c) modifying the NK cell such that it expresses a TcR, said TcR being selected from a panel of TcRs each having specificity for a different antigen and/or type of MHC, said TcR having specificity for MHC and the antigen presented on specified target cell.

TcRs can be presented in the panel in the form of nucleic acid molecules containing nucleotide sequences encoding TcRs. For convenience, the nucleic acid molecules may be contained in vectors in NK cells. The nucleic acid molecule can be DNA or RNA. For example, TcRs can be presented in the form of mRNA (messenger RNA) or viral vectors, for example

- 10 040420 measures, retroviral vectors.

In one embodiment, the NK cell is modified to express TcR after the cell has been modified to express CD3. Thus, as noted above, it is possible to obtain a universal NK cell by modifying a non-immunogenic NK cell to express CD3. Such modified NK cells can be grown or maintained in culture or stored for future use. Accordingly, in one embodiment, said cell may be allowed to proliferate to form a population of CD3-expressing NK cells before it is modified to express TcR. NK cells can grow in culture or multiply. In an alternative implementation, it is contemplated that the cell may be modified to express CD3 and TcR at the same time, or substantially at the same time. In one embodiment, a single vector or construct containing genes for both CD3 and TcR can be used to modify a cell to express both CD3 and TcR, and in other embodiments, separate vectors can also be used. As discussed above, a fusion protein containing both TcR and CD3 functionality in the same polypeptide molecule can also be expressed in a NK cell, and thus a vector or construct encoding such a fusion protein can be used to modify the cell. NK cells modified to express CD3 and TcR may be allowed to reproduce, for example, they may be grown in culture, or expanded prior to administration of said cells to a subject. Thus, according to one embodiment of the present invention, a population of NK cells expressing CD3 can be generated, and a subpopulation of NK:CD3 cells can be modified to express a TcR having specificity for an antigen on the surface of a target cell.

The modified NK cells of the present invention can also be modified 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 or localization receptor that is designed to direct or improve cell localization in a particular tissue or site in the body. Also, cells can be modified to express one or more components of the T lymphocyte signaling pathway to enhance the cytotoxic response exhibited by NK cells. Any such modification can be carried out before, after or simultaneously with the modification according to the present invention.

The cell according to the present invention can be modified so as to change its ability to reproduce in vivo and/or in vitro. In one embodiment, the ability of a cell to reproduce (i.e., the ability of a cell to reproduce, i.e., proliferate) can be increased. According to one preferred implementation variant, this can be achieved by modifying cells so that they increase the expression of a cytokine, such as IL-2 (interleukin-2). Normally, IL-2 is required by the NK cell for growth and maintenance of cytolytic function, and thus cells expressing IL-2 do not require additional IL-2 as additions to the growth medium and retain cytotoxic activity when administered. subject. Cytokine expression can be enhanced by introducing a heterologous (non-native) vector or construct containing the cytokine-encoding nucleic acid molecule into the cell, or alternatively, expression of an endogenous cytokine-encoding gene can be enhanced. The expression of a cytokine may be constitutive (ie, the promoter that controls the expression of a gene encoding a cytokine may be constitutively active, or turned on), or may be inducible by an external stimulus.

In one embodiment, the cell may be modified to have an increased ability to reproduce before the cell is modified to express CD3 and/or TcR. According to another implementation variant, the cell can be modified so that it has an increased ability to reproduce, after the cell has been modified to express CD3 and/or TcR. In an alternative implementation, a cell may be modified to have increased reproducibility after the cell has been modified to express CD3 but before it has been modified to express TcR (i.e., such Thus, it is possible to obtain a large population of CD3-expressing NK cells before the cells are modified to express TcR). Alternatively, any two or more of the modifications listed above may be carried out at essentially the same time. According to one implementation variant, all three modifications can be carried out essentially at the same time.

The proliferative capacity and viability (survival) of the NK cells according to the present invention can also be reduced before they are administered to a patient. As noted above, this can be done to make the cell non-immunogenic. In a preferred embodiment, the reproducibility and/or viability of a cell can be reduced by irradiation. The ability to reproduce and/or the viability of the cell can be reduced (ie, reproduction may become slower) or eliminated, depending on the dose and nature of the radiation affecting the cells. Irradiation can be from any source of α, β or γ radiation, or it can be

- 11 040420 x-ray or ultraviolet light. Radiation doses of 5-10 Gy may be sufficient to abolish proliferation, however other suitable radiation doses may include 1-10, 2-10, 3-10, 4-10, 6-10, 7-10, 8-10 or 9 -10 Gy, or higher doses of 11, 12, 13, 14, 15, or 20 Gy. Alternatively, NK cells can be modified to express a suicide gene that allows the cells to be inducibly killed or prevented from reproducing in response to an external stimulus.

The cell can be modified so that A) it has an increased ability to reproduce and B) it has a reduced ability to reproduce and/or viability. The two modifications may be made sequentially in any order (ie, A and then B, or B and then A), or may be made at substantially the same time (A and B together). In such an embodiment, a cell can be modified to express a cytokine (e.g., IL-2) to enhance cell growth in vitro, before, concurrently, or after the cell is modified to express CD3 and/or TcR, and subsequently modified so that its ability to reproduce and/or viability is reduced prior to the introduction of the cell into the body of the subject.

As will be appreciated from the foregoing, the cell of the present 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 enable the expression of such a heterologous gene, a nucleic acid molecule corresponding to or containing the gene in question is introduced into the cell. For convenience, the nucleic acid molecule can be introduced into the cell as part of a vector or recombinant construct. Methods for expressing heterologous genes are known in the art, both in terms of obtaining constructs/vectors, and in terms of introducing a nucleic acid molecule (vector or construct) into a cell. Thus, promoters and/or other expression control sequences suitable for use with mammalian cells, in particular lymphoid cells or NK cells, and corresponding vectors (eg viral vectors) are well known in the art.

The vectors or constructs (nucleic acid molecules) can be introduced into the cell of the present invention by a variety of routes, including chemical transfection agents (such as calcium phosphate, branched chain organic compounds, liposomes, or cationic polymers), electroporation, cell contraction, pore formation by ultrasound, hydrodynamic delivery or viral transduction. In a preferred embodiment, the vector or construct is introduced via viral transduction. Heterologous nucleic acid molecules introduced into a cell may be expressed episomally, or may be integrated into the cell's genome at the appropriate locus.

Cell culture methods and reagents suitable for NK cells are also well known in the art. Any desired cell culture method may be used, depending on choice and convenience, etc. For example, Teflon bags for large scale cultures, or automated cell culture systems can be used.

The target cell according to the present invention may be a cancer cell. As used herein, cancer is defined broadly and includes any neoplasm, whether malignant, precancerous, or non-cancerous. However, it can usually be cancer. Solid and non-solid tumors are contemplated, and the term cancer cell may be considered as a synonym for tumor cell.

Any type of cancer is meant, including both solid cancers and hematopoietic cancers. Typical cancers include acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), adrenocortical carcinoma, AIDS-associated cancer (eg, Kaposi's sarcoma and lymphoma), anal cancer, appendix cancer, astrocytomas, atypical teratoma/rhabdoid tumor, basal -cell carcinoma, bile duct cancer, extrahepatic gallbladder cancer, bone cancer (eg, Ewing's sarcoma, osteosarcoma, and malignant fibrous histiocytoxanthoma), brainstem glioma, brain cancer, breast cancer, bronchial tumors, Burkitt's lymphoma, carcinoid tumors, cardiac tumors , central nervous system cancer (including atypical teratoma/rhabdoid tumor, fetal tumors, geminogenic tumor, lymphoma), cervical cancer, chordoma, chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), chronic myeloproliferative disease, colon cancer, cancer colon and rectum, 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 bones, gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor (GIST), germ cell tumor, gestational trophoblastic disease, glioma, hairy cell leukemia, head and neck cancer, heart cancer, hepatocellular (liver) cancer , Langerhans cell histiocytosis, Hodgkin's lymphoma, hypopharyngeal cancer, intraocular melanoma, islet cell tumors

- 12 040420 current, pancreatic neuroendocrine tumors, Kaposi's sarcoma, kidney cancer (including renal cell tumor and Wilms' tumor), Langerhans cell histiocytosis, laryngeal cancer, leukemia (including acute lymphoblastic (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL) , chronic myeloid leukemia (CML), lip and mouth cancer, liver cancer (primary), lobular carcinoma in situ (LCIS), lung cancer, lymphoma, Waldenström macroglobulinemia, melanoma, Merkel cell carcinoma, mesothelioma, metastatic squamous cell carcinoma of the neck with occult primary cancer, NUT median tract carcinoma, oral cavity cancer, multiple endocrine neoplasia syndromes, childhood, multiple myeloma/plasma cell tumor, mycosis fungoides, myelodysplastic syndromes, myelodysplastic/myeloproliferative neoplasms, multiple myeloma, myeloproliferative disorders, nasal cancer, and paranasal sinuses, nasopharyngeal cancer, neuro blastoma, non-Hodgkin's lymphoma, non-small cell lung cancer, mouth cancer, oral cancer, oropharyngeal cancer, ovarian cancer, pancreatic cancer, pancreatic neuroendocrine tumors (islet cell tumors), papillomatosis, cancer of the nasal cavity and paranasal sinuses, parathyroid cancer , penile cancer, pharyngeal cancer, pheochromocytoma, pituitary tumor, plasma cell tumor/multiple myeloma, pleuropulmonary blastoma, pregnancy and breast cancer, primary central nervous system (CNS) lymphoma, prostate cancer, rectal cancer, renal cell cancer (kidney), transitional cell carcinoma of the kidney, pelvis and ureter, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma, erythroderma reticularis, skin cancer, small cell lung cancer, small intestine cancer, soft tissue sarcoma, squamous cell carcinoma, squamous cell carcinoma of the neck with occult primary cancer, metastatic, gastric cancer, T-cell lymphoma, testicular cancer, throat cancer, thymoma and thymus cancer, thyroid cancer, transitional cell carcinoma of the kidney, pelvis and ureter, cancer of the uterus, endometrium, uterine sarcoma, vaginal cancer, vulvar cancer, Waldenström's macroglobulinemia, and Wilms' tumor.

As noted above, a number of cancers have been shown to express particular or specific cancer antigens, or should be characterized by the expression of a particular or specific cancer antigen. Thus, a known or recognized cancer antigen can be used. As is known in the art, certain antigens may appear in a number of different types of cancer, while 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 the subject and identify a cancer antigen suitable for use. Thus, any cancer can be treated with a TcR directed to the universal cancer antigen, and such TcRs have been identified. Alternatively, the cancer may be any cancer currently being treated or contemplated to be treated with adoptive T-lymphocyte transfer therapy, eg, common cancers such as melanoma, hematologic cancers, lung cancer, colon and rectal cancer. As another alternative, the cancer may be a rare cancer for which few treatment options are currently available (eg, with orphan drug status), such as for pancreatic cancer or sarcoma.

In other embodiments, the target cell may be an infected cell, and in particular a cell infected with a virus. The virus can be HIV (human immunodeficiency virus), but it will usually be a pathogenic virus. As an example, the virus can be HIV, hepatitis virus (for example, HBV (hepatitis B virus) or HVC (hepatitis C virus)), HPV (human papillomavirus), CMV (cytomegalovirus) or EBV (Epstein-Barr virus), HHV-8 (human herpes virus type 8), HTLV-1 (human T-cell leukemia virus), SV40 (monkey virus 40), enterovirus. Other possible infectious agents or pathogens also include bacteria, such as Helicobacter pylori, Chlamydia pneumoniae, and parasites, such as Schistosoma haematobium, liver fluke, Opisthorchis viverrini, Clonorchis sinensis, and malaria.

According to one implementation variant of the present invention, the cells can be administered to the subject directly into the vein. In an alternative implementation, the cells can be injected directly into the tumor via intratumoral injection.

The dose of cells administered to a subject may vary depending on the nature of the target cell. In a preferred embodiment, when the target cell is a cancer cell, the dose may be calculated based on the type of cancer being treated. In a preferred embodiment, a dose of approximately 10 ⁹ cells may be administered to a subject, however this may vary depending on the type and grade of cancer. It is possible that the dose administered is approximately 10 ⁶ , 10 ⁷ or 10 ⁸ cells. Alternatively, you can enter a higher dose of approximately 10 ¹⁰ , 10 ¹¹ or 10 ¹² cells. Also, the dose of cells administered may vary depending on the size of the patient's body, and thus a dose of 105, 10 ⁶ , 10 ⁷ , 10 ⁸ , 10 ⁹ , 10 ¹⁰ , 10 ¹¹ or 10 ¹² cells per m ² of the patient's body surface can be administered. or per kilogram of patient weight.

It is also contemplated that multiple infusions may be required to effectively treat a subject. For example, a patient may receive 2, 3, 4, 5, 6 or more separate infusions 24 or 48 hours apart, or every 3, 4, 5, 6 or 7 days. Also, the intervals between infusions can be a week,

- 13 040420 two weeks or a month, or intervals of 6 weeks or 2, 3, 4, 5 or 6 months. It is also possible to carry out infusions once a year.

The subject to be treated with the methods and cells of the present invention may be a mammal of any species. For example, the subject may be a domestic animal of any kind, such as a mouse, rat, gerbil, rabbit, guinea pig, hamster, cat, or dog, or a farm animal such as a goat, sheep, pig, cow, or horse. According to a further preferred embodiment of the present invention, the subject may be a primate such as a monkey, gibbon, gorilla, orangutan, chimpanzee, and bonobo. However, according to a preferred embodiment of the present invention, the subject is a human.

It is contemplated that NK cells for use in accordance with the present invention may be derived from any species of mammal, however, in a preferred embodiment, NK cells will be derived from a mammal of the same species as the subject to be treated. In addition, as noted above, in a preferred embodiment, the cell can be modified with genes expressing proteins from a mammal of the same species (eg, human CD3 and TcR would be used for a human subject). However, it is possible that CD3 and TcR from another species would also be used, for example, the cell could be modified using genes expressing mouse CD3 and human TcR.

The present invention may be better understood with reference to the Examples section below and with reference to the drawings, which include the following.

In FIG. 1 shows that CD3 can be expressed in NK-92 cells. The CD3-IRES-GFP retroviral construct was introduced into NK-92. Fluorescence was monitored by the FITC (fluorescein isothiocyanate) channel. The labeled area shows GFP+ cells. Black color: untransfected cells, gray color: transfected cells.

In FIG. 2 shows that CD3 can be detected on the surface of NK-92 expressing CD3IRES-GFP in the presence of TcR. CFP+ sorted NK-92 (NK-92-CD3) were superinfected with four different TcRs, namely Radium-1 (TGFbRII frameshift-specific, GCHC-I), and the cysteine variant -Pαguy-1 _cys , DMF-5 (MART-1-specific, MHC-I), Radium-3 (hTERT-specific, MHC-II), and cells without superinfection underwent the procedure as a control. Cells were stained with anti-CD3 antibody to detect D3 on the cell surface, and fluorescence was measured by FITC (GFP) and APC (anti-CD3) channels. Cells in the upper right quadrant express CD3 on their surface.

In FIG. 3 shows that TcR Radium-1 and DMF-5 can be specifically detected on the surface of NK-92 expressing CD3-GFP. Cells were transfected to express CD3 and two different TcRs: Radium-1 (TGFbRII frameshift specific), DMF-5 (MART-1) and CD3 only (no TcR). Cells were stained with V-beta specific antibody that detects Radium-1 V-beta chain (not DMF5, top row) or MART-1 multimer to detect DMF-5 TcR on the cell surface (bottom row). Fluorescence was measured by FITC (GFP) and APC (anti-Vb and M1-multimer) channels.

In FIG. 4 shows that NK-92 modified to express CD3 and TcR Radium-1 exhibit antigen-specific cytotoxic activity. NK-92 transfected with CD3-IRES-GFP (GFP+ - upper frame - CD3_IRES_GFP) and cells that have not been transfected (GFP^- - lower frame - NTr), were sorted into two separate populations. SupT1 cells were separated from NK cells by determining the expression of the NK marker - CD56. Grit loss was monitored by detecting the CD107 marker in NK cells after incubation with SupT1 cells in GFP+ and GFP populations. , and they are plotted together (histogram, grey: GFP^-, dotted line: GFP+).

In FIG. 5 shows that a pure population of NK-92 CD3 superinfected with TcR DMF-5 or Radium-1 and sorted is specifically activated by T2 lymphocytes loaded with the respective peptide. Upper panel: CD3 signal and GFP are used to separate two cell lines. The CD3+ population is then assessed for CD107a expression (loss of granularity). Histogram: NK-92-CD3-TcR shown was incubated with T2 lymphocytes loaded with O/N 1 μM peptides (gray shading: no peptide, dark gray outline: MART-1, light gray outline: TGFbRII).

In FIG. 6 shows the calculation of EC ₅₀ values (half maximum effective concentration) for TcR Radium-1 and DMF-5.

In FIG. 7 shows the results of a phospho-flow cytometric analysis of NK92(CD3/Radium-1) cell activation upon contact with α-CD3 and α-CD28 antibodies. The shift of the flow cytometry spectrum to the right indicates an increase in the level of phosphorylation of a significant protein, which in turn indicates the activation of the TCR complex.

In FIG. 8 shows the specificity of stimulation of NK-92(CD3/Radium-1) and NK-92(CD3/DMF-5) cells. NK-92 was incubated with antigen presenting cells that presented either a specific, related peptide for each receptor (TGFbRII for cells expressing Radium-1, MART-1 for cells expressing DMF-5) or a random peptide. Stimulation of signal transduction through the TCR complex was measured at different time points according to the level of phosphorylation of different

- 14 040420

TcR/CD3-related proteins. These phosphorylation levels were measured by phosphoflow cytometry. In each graph, the solid line shows cell activation by a specific, related peptide; the dotted line shows cell activation by a random peptide.

In FIG. 9 shows the kinetics of stimulation of NK-92(CD3) cells transduced with either TcR Radium5 or TcR Radium-6. Both of these receptors are derived from CD4+ T lymphocytes and specifically bind to the TGFbRII peptide with a frameshift; Radium-5 binds specifically to it in the context of HLA-DR7, Radium-6 in the context of HLA-DR4. EC ₅₀ values for Radium-5 and Radium-6 are calculated using tissue from donor patients (patients H, S and B). Patient H had the HLA-DR7 haplotype; patients S and B had the HLA-DR4 haplotype.

Example 1

Expression of CD3 in NK92.

The codon-optimized pMP71CD3ζ-CD3ε-CD3γ-CD3δ-IRES-GFP (CD3-GFP) vector (Ahmadi et al. 2011. Blood 118, 3528-3573) was used to transfect NK92 with the CD3 receptor, and the cells were transfected by retroviral transduction. A retrovirus containing the CD3-GFP construct was generated in a packaging cell line (Hek-Phoenix) and NK cells were centrifuged (0.3 M cells incubated with viral supernatant and spun at 900xg for 1 h at 32°C on plates coated with retronectin (Tanaka Biotech)). GFP (Green Fluorescent Protein) was used as a marker for successful transduction.

Successful transduction of NK-92 by CD3 receptors (NK-92(CD3)) was confirmed by flow cytometry using a FACS Canto flow cytometer (BD Biosciences). Fluorescence was monitored by the SSC and FITC channels and successful transfection was observed by monitoring the fluorescence by the GFP channel (see FIG. 1).

A transfection efficiency of approximately 50% was achieved. Alternatively, GFP+ cells were sorted to obtain a pure population.

Example 2

Expression of TcR in NK-92(CD3).

The ability of NK-92(CD3) to co-express CD3 and TcR was assessed. Both CD3 and TcR require co-expression of another member of the TcR complex in order to be targeted to the cell surface. Thus, it was possible to use the expression of CD3 on the surface of NK cells as a marker of successful co-transfection of the cell with both CD3 and TcR.

NK-92(CD3) was superinfected with four different TcRs using a retroviral supernatant (cysteine variant Radium-1 _cys , Radium-1-specific (TGFbRII frameshift), DMF-5, MART-1-specific DMF-5 (Johnson , LA et al., 2006 J Immunol 177:65486559)) and Radium-3 (hTERT, MHC-II)), and cell surface CD3 expression was monitored for each TcR using an anti-CD3 antibody multimer labeled with APC (allophycocyanin). CD3 expression was determined by monitoring the expression of GFP cells. Fluorescence was determined by the FITC and APC channels. Cells expressing GFP are displayed in the right area of the scatter plots, and cells expressing CD3 on their surface are displayed in the upper area of the scatter plots. Cells co-expressing CD3 and TcR (ie, cells with a CD3-TcR complex located on the cell surface) are displayed in the upper right area of the scatter plots.

In cells modified to express each of the TcRs, cell surface expression of CD3 was determined (FIGS. 2A-D) (cells in the upper right quadrant of each scatter plot). In particular, cells modified to express the Radium-1 TcR showed CD3 expression on the cell surface (FIG. 2B). Also in FIG. 2D shows a slight increase in fluorescence through the APC channel for GFP+ cells, indicating a low level of expression of the Radium-3 TcR. For cells lacking TcR, CD3 expression on the cell surface was not detected (FIG. 2E).

Cell surface TcR expression was also determined in NK-92(CD3) modified to express Radium-1 and DMF-5 TcRs. The presence of TcR on the cell surface was monitored using either a V-beta specific antibody that detects the V-beta chain of Radium-1, or a MART-1 multimer to detect TcR DMF-5 on the cell surface (bottom row) labeled with APC. Fluorescence was determined by FITC (GFP) and APC (anti-Vb and M1-multimer) channels. No increase in signal was observed across the APC channel for NK-92(CD3) not modified to express TcR (FIG. 3A). Cells modified to express the Radium-1 TcR showed an increase in APC channel signal when stained with an anti-Vp antibody but not with the MART-1 multimer (FIG. 3B). Cells modified to express TcR DMF-5 showed an increase in APC channel signal when stained with the MART-1 multimer (FIG. 3C).

Example 3

NK-92(CD3) expressing TcR are functional.

To test the functionality of our TcR when it is expressed in an NK cell, we assessed whether the TcR expressed in an NK cell is capable of specifically recognizing target cells.

NK-92(CD3) expressing TcR were incubated with target cells expressing a single chain trimer (SCT) molecule containing the correct target peptide (TGFbRII) or non-specific peptide (irr) for 5 hours, or in the absence of target cells (without APC), and expression of the granule loss marker CD107 was monitored as a marker of the ability of NK cells to be stimulated by target cells. (SCT is an MHC class I antigen presenting protein and consists of an antigenic peptide, in ₂ -microgloblin and h-chain expressed as a single polypeptide chain, see US Patent 2010/015954 and Yu, YY et al. (2002 ) J Immunol 168: 3145-3149).

NK-92(CD3) was first stained with anti-CD56 Tx Red (CD56 is a NK marker) and monitored for TxRed and GFP fluorescence (Figure 4A). Based on CFP expression, distinct populations of NK-92 were shown. Both GFP+ cells (upper region) and NK GFP ⁻ cells (lower region) were determined. The expression of CD107a was monitored for each cell population, and ^GFP cells were used as a negative control (ie cells not expressing TcR).

NK-92(CD3) expressing a Radium-1 specific TcR were incubated with SupT1 expressing a single chain trimer (SCT) molecule with a non-specific peptide (FIG. 4C) or a target peptide for Radium-1 (FIG. 4D). Cells incubated in the absence of an antigen presenting cell were also monitored (FIG. 4B). CD3 expression was monitored for the NK-92 cell line and cells were sorted as described above. Each histogram shows populations of GFP+ (dashed line) and GFP ^- (solid line).

When incubated with SupT1 cells expressing the Radium-1 SCT (Fig. 4D), only NK-92(CD3) expressing the Radium-1-specific TcR showed an increase in CD107 expression, as indicated by a shift of the dotted line to the right compared to the solid line ( negative control GFP ^- ). For these cells, when they were incubated with SupT1 cells expressing a non-specific peptide (Fig. 4C), or for cells incubated in the absence of antigen presenting cells, no increase in CD107 expression was observed.

Taken together, these data indicate that NK-92(CD3) expressing the Radium-1 specific TcR are able to be activated by target cells expressing the target peptide for Radium-1. This demonstrates that the TcR expressed in NK-92(CD3) is active and functional. Thus, NK-92(CD3) expressing TcRs have target-cell-specific cytotoxicity.

Example 4

Activation of NK-92(CD3) expressing TcR by T2 lymphocytes loaded with a significant peptide.

NK-92(CD3) were modified to express TcR Radium-1 or DMF-5, and CD3 expression on the cell surface was determined using an APC-labeled anti-CD3 antibody. CD3+ cells were selected for grain loss analysis (FIG. 5, top panel) and sorted.

NK-92(CD3) modified to express the TcR Radium-1 or DMF-5 were incubated with T2 lymphocytes (Fig. 5, bottom panel) loaded with either TGFbRII peptides (light gray outline) or MART1 (dark outline). gray outline) that are target antigens for TcR Radium-1 and DMF-5, respectively. CD107a expression was measured in NK-92(CD3) TcR-expressing Radium-1 (FIG. 5, lower left panel) and DMF-5 (FIG. 5, lower right panel) incubated with T2 lymphocytes in the presence of either peptide. NK-92(CD3) modified to express the Radium-1 TcR showed activation in the presence of the TGFbRII peptide, and cells expressing the DMF-5 TcR showed activation in the presence of MART-1. Nonspecific activation was observed.

A similar experiment was also performed to determine the EC ₅₀ for each TcR. NK92(CD3) modified to express TcR Radium-1 (circles) and DMF-5 (squares) were incubated with T2 lymphocytes in the presence of TGFbRII or MART-1 peptides, respectively, at a range of different peptide concentrations. Activation was measured by determining the expression of CD107a, as described above. CD107a expression was measured at each concentration and relative activation was calculated (CD107a as a percentage of maximum CD107a expression). Calculations showed EC ₅₀ values of 2 nM (Radium-1) and 7 nM (DMF-5) (FIG. 6).

Example 5

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

NK-92(CD3) transfected with TcR Radium-1 was evaluated using phospho-flow cytometry to study their signal transduction capability. First, the overall signaling ability of the TCR complex was analyzed by stimulating cells with anti-CD3 and anti-CD28 antibodies that stimulate activation through the TCR. As shown in FIG. 7, clustering of TcR and CD3 leads to activation of the signaling cascade, similar to how it is observed in T-lymphocytes. This is shown by an increase in the level of phosphorylation of several TcR/CD3-related proteins, including ZAP-70, SLP-76 and CD3Z.

- 16 040420

The cells were then assessed for stimulation with specific antigens using antigen presenting cells and tracking the signaling activity of the TCR complexes at different time points by phospho-flow cytometry. Both NK-92(CD3)-Radium-1 and NK-92(CD3)-DMF-5 were stimulated by their cognate peptides. As shown in FIG. 8, only specific stimulation leads to phosphorylation of the signaling molecule. It was possible to distinguish between early and late signals, and their character was similar to that observed in T-lymphocytes. Taken together, these data indicate that NK-92(CD3-TCR) respond like T-lymphocytes when they are in contact with their substrates.

Example 6

Stimulation of NK-92(CD3) by TcRs derived from CD4+ T lymphocytes The TcRs derived from CD4+ T lymphocytes, Radium-5 and Radium-6, were transduced into NK-92(CD3). These TcRs were able to retarget cells specifically to target MHC class II peptides. Both Radium-5 and Radium-6 specifically target the frameshift mutant peptide

TGFbRII (KSLVRLSSCVPVALMSAMT);

Radium-5 targets this peptide specifically in the context of HLA-DR7, and Radium-6 in the context of HLA-DR4. The kinetics of stimulation of NK-92(CD3)-Radium-5/6 cells is shown in FIG. 9. NK-92(CD3) stimulation is measured as described in Example 4 above. EC50 values were calculated and are shown in FIG. 9. EC50 for Radium-5 in a sample from a patient with an HLA-DR7 haplotype, calculated to be 19 μM; The EC50 for Radium-6 was calculated to be 4 μM in one patient's sample and 0.4 μM in the second patient's sample. Samples from both patients were with the HLA-DR4 haplotype.

Example 7

Analysis of protein expression in NK-92 with and without CD3/CD3-TcR expression Protein expression profiles in NK-92 were compared with those of NK-92(CD3/CD3-TcR). There were no differences between the two cell groups (except of course for the presence of CD3 in NK-92(CD3/CD3TcR)). The comparison is shown in the table below (PBMC=peripheral blood mononuclear cells).

surface markers NK-92 NK-92 652 (CD3 transduced) ICPC CD3 - (+) + CD4 - - + CD8 - - + CD16 - - + CD25 + + + CD28 + + + CD45RA + + + CD45RO + + + CD56 + + + CD57 + + + CD62L - - - CD134 (0X40) + + + CD152 (CTLA-4) + + (+) CD336 (NKp44) - - - CD337 (NKp30) + + + PD1 + + + HLA cl I + + + HLA-DR - - - CXCR3 + + + CXCR4 + + + NKG2D + + +

CLAIM

Claims (14)

CLAIM 1. A modified NK cell to destroy a target cell, wherein said NK cell expresses CD3 CD3y, CD3δ, CD3ε and CD3Z and TCR chains, wherein said CD3 chains and said TCR form a functional CD3-TcR complex that is localized on the surface the specified NK cell. 2. An NK cell according to claim 1, characterized in that said target cell is a cancer cell and said modified NK cell is intended for cancer therapy. 3. An NK cell according to claim 1 or 2, characterized in that said NK cell is a modified NK-92 cell. - 17 040420 4. An NK cell according to claim 1 or 2, characterized in that said NK cell is a modified primary NK cell. 5. An NK cell according to any one of claims 1 to 4, characterized in that said TCR is independent of co-receptors. 6. An NK cell according to any one of claims 1 to 5, characterized in that said NK cell is modified to disrupt or prevent the expression of β ₂ microglobulin. 7. An NK cell according to any one of claims 1 to 6, characterized in that said NK cell is a human cell and is negative for HLA. 8. An NK cell according to any one of claims 1 to 7, characterized in that the proliferative capacity of said NK cell is reduced by irradiation. 9. A therapeutic composition for the treatment of cancer, comprising a modified NK cell according to any one of claims 1 to 8 and at least one pharmaceutically acceptable carrier or excipient. 10. A method of treating cancer by adoptive cell transfer therapy, said method comprising administering to a subject in need of such treatment an effective amount of a modified NK cell according to any one of claims 1 to 8. 11. A kit for obtaining an NK cell as defined in claim 1, said kit including: (a) an NK cell that expresses the CD3 chains CD3y, CD3δ, CD3ε and CD3Z, and (b) a panel of nucleic acid molecules each encoding a TcR, said TcRs having specificity for different antigens and/or different specificity for a major histocompatibility complex. 12. The kit according to claim 11, characterized in that said nucleic acid molecules are contained in vectors. 13. The set according to claim 12, characterized in that said vectors are viral vectors. 14. The kit according to any one of claims 11-13, characterized in that said NK cell is a modified primary NK cell.