RU2772348C2 - Modified natural killer cells and natural killer cell lines with increased cytotoxicity - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to the field of biotechnology, in particular to a human cell - a natural killer (NK), genetically modified for the expression of the TRAIL variant, including the D269H/E195R mutation.

EFFECT: invention is effective for the treatment of cancer.

14 cl, 24 dwg, 1 tbl, 10 ex

Description

Introduction

The present invention relates to the modification of natural killer (NK) cells and NK cell lines to obtain their derivatives with a phenotype of increased cytotoxicity. In addition, the present invention relates to methods for obtaining modified NK cells and lines of NK cells, compositions containing these cells and their lines, as well as the use of these compositions for the treatment of cancer.

State of the art invention

Typically, immune cells require a target cell to introduce an antigen through the major histocompatibility complex (MHC) before provoking an immune response leading to the death of the target cell. This allows non-MHC class I cancer cells to avoid most immune responses.

At the same time, NK cells can recognize cancer cells in the absence of class I MHCs. As a result, they play a critical role in protecting the body from cancer.

On the other hand, in some circumstances, cancer cells demonstrate the ability to reduce the cytotoxic activity of NK cells through the expression of ligands that bind inhibitory receptors on the NK cell envelope. Cancer resistance may involve balancing these and other factors.

In this case, cytotoxicity refers to the ability of immune effector cells, such as NK cells, to induce the death of cancer cells, for example, through the production of cytolytic compounds or binding of cancer cell envelope receptors, as well as through apoptosis of these cancer cells. Cytotoxicity is influenced not only by signals that provoke the production of cytolytic compounds, but also by signals that inhibit their production. Therefore, increasing cytotoxicity will result in more efficient killing of cancer cells with less chance of reducing the cytotoxic activity of the NK cell by the cancer cell, as discussed above.

As a way to increase the cytotoxicity of NK cells against cancer cells that do not express MHC class I, but which can reduce the cytotoxicity of NK cells, a method of genetic modifications to eliminate the function of the inhibitory NK cell receptor has been proposed (Bodduluru et al. 2012) . NKG2A was chosen as an inhibitory receptor whose function should have been abolished under the circumstances, as certain cancer cells are known to produce MICA that binds NKG2A and inhibits NK cell cytotoxicity in the absence of MHC class I expression (Shook et al. 2011 WO 2006/023148).

In another way to reduce NKG2A expression, transfection of the gene encoding IL-15 in NK-92 cells has been shown to be associated with a decrease in NKG2A expression (Zhang et al. 2004). At the same time, despite the observed increase in NK cell cytotoxicity, it most likely resulted from a concomitant increase in the expression of the NKG2D activating receptor. This is supported by the observation that blocking NKG2A receptors in NK-92 cells was not associated with increased cytotoxicity against multiple myeloma cells (Heidenreich et al. 2012). However, it should be noted that the NK-92 cell line has high cytotoxicity with very low expression of inhibitory receptors. Therefore, any increase in cytotoxicity associated with reduced NKG2A expression might be too small to be detected.

Similar studies were carried out on mice. For example, mice secrete a receptor called Ly49 in NK cells that is analogous to human inhibitory KIR receptors. It has been demonstrated that when the Ly49 receptor is blocked by antibody fragments, NK cells have increased cytotoxicity and can kill murine leukemia cells in vitro and in vivo (Koch et al. 2001).

At the same time, it is precisely due to a decrease in the function of the inhibitory receptor that “normal” cells in the body also become more prone to the effects of modified NK cells, since the ability of these NK cells to distinguish “normal” cells from cancer cells is reduced. This is a significant disadvantage of reducing the "classic" function of the inhibitory receptor.

Another known method by which NK cells kill cancer cells is by expressing TRAIL on their surface. The TRAIL ligand can bind TRAIL receptors on cancer cells and induce their apoptosis. One proposed approach describes overexpressing TRAIL in NK cells to take advantage of this anti-cancer mechanism (EP 1621550). In addition, there is evidence that IL-12 causes an increase in TRAIL expression in NK cells (Smith et al. 2001).

However, cancer cells have evolved mechanisms to evade and protect against NK cells expressing TRAIL. TRAIL decoy receptors are often expressed on cancer cell walls, and binding of TRAIL to these decoy receptors cannot induce apoptosis. Ways to bypass such mechanisms have not yet been developed.

Acute myeloid leukemia (AML) is a hematopoietic malignancy involving the development of precursor cells in the bone marrow and accounts for a significant proportion of acute leukemia cases in both adults (90%) and children (15-20%) (Hurwitz, Mauns et al. 1995; Lowenberg, Downing et al. 1999). Although 80% of patients achieve remission with standard chemotherapy (Hurwitz, Mounce et al. 1995; Ribeiro, Razzuk et al. 2005), survival remains unsatisfactory due to the high relapse rate with minimal residual disease (MRD). Five-year survival depends on age. It is 60% for children (Rubnitz 2012), 40% for adults under 65 (Lowenberg, Downing et al. 1999) and 10% for adults over 65 (Ferrara and Schiffer 2013). These outcomes may be improved if patients have an appropriate hematopoietic cell donor, but many do not, highlighting the need for an alternative treatment approach.

Cells - natural killer (NK) - cytotoxic lymphocytes with pronounced phenotypes and effector functions that differ, for example, from T-cells-natural killer (NK-T). For example, while NK-T cells express both CD3 and T cell antigen receptors (TCRs), NK cells do not. In general, NK cells have been found to express CD16 and CD56 markers, with CD16 acting as an Fc receptor and mediating antibody-dependent cell-mediated cytotoxicity (ADCC), which will be discussed below. In this case, KHYG-1 is a significant exception. Despite the fact that NK cells are naturally cytotoxic, their lines with increased cytotoxicity have been isolated. NK-92 and KHYG-1 are two NK cell lines that have been extensively studied and hold promise in cancer treatment (Swift et al. 2011; Swift et al. 2012).

Adoptive cellular immunotherapy in the treatment of cancer often involves the introduction of natural and modified T cells into the patient. T cells can be modified in a number of ways, such as genetically, to express receptors and/or ligands that bind to specific target cancer cells. Transfection of T cells with high affinity T cell receptors (TCRs) and chimeric antigen receptors (CARs) associated with cancer cell antigens can induce a highly reactive T cell response to cancer. A major limitation of the present immunotherapeutic approach is that T cells must either be taken from the patient for autologous expansion in an ex vivo manner, or MHC matching T cells must be used to prevent immunological elimination immediately after cell transfer to the patient or, in some cases , before the development of graft rejection reaction (GVHD). In addition, successfully transferred T cells often survive in the circulation for extended periods of time, making it difficult to control long-term side effects due to treatment.

In haplotype transplantation, the graft-versus-leukemia effect is thought to be mediated by NK cells when inhibitory KIR receptors and ligand are mismatched, which may result in increased survival with AML treatment (Ruggeri, Capania et al. 2002; Ruggeri, Mancusi et al. 2005 ).

Moreover, rapid NK recovery is associated with better outcome and graft-versus-leukemia (GVL) effect in patients undergoing T-element-free haplotype hematopoietic cell (HCT) transplantation for AML (Savani, Melke et al. 2007). Other trials have used ex vivo expanded haploidentical NK cells to treat AML in adults (Miller, Soigner et al. 2005) and children (Rubnitz, Inaba et al. 2010).

Several permanent NK cell lines have been established, the most notable being NK-92 from a patient with non-Hodgkin's lymphoma, which expresses NK cell markers with the exception of CD16 (the Fc III gamma receptor). NK-92 has undergone extensive preclinical testing and demonstrates increased lysis against a wide range of tumors compared to activated NK cells and lymphokine-activated killer (LAK) cells (Gong, Mackie et al. 1994). NK-92 cells have been shown to be cytotoxic mainly to AML (Jan, Steinherz et al. 1998).

Another NK cell line, KHYG-1, has been identified as a potential candidate for clinical use (Sack et al. 2005), but has reduced cytotoxicity and therefore received less attention than NK-92. KHYG-1 cells are known to be preactivated. Unlike endogenous NK cells, KHYG-1 cells polarize from time to time, which increases their cytotoxicity and responds faster to external stimuli. NK-92 cells have greater baseline cytotoxicity than KHYG-1 cells.

Therefore, it is clear that current adoptive immunotherapy protocols are adversely affected by donor switching in terms of the number and quality of effector cells, variables that could be eliminated if current cell lines could provide more standardized therapy.

A significant amount of research on NK cell cytotoxicity has been carried out using mouse models. One example is the finding that perforin and granzyme B mRNAs are constitutively transcribed into mouse NK cells, but that minimal protein levels are observed prior to stimulation or activation of NK cells (Feniger et al. 2007). Although this work, as well as other work using murine NK cells, is of interest, it cannot be relied upon as conclusive evidence for the cytotoxicity of NK cells in humans. In contrast to the example above, human NK cells express high levels of perforin and granzyme B protein prior to stimulation (Leong et al. 2011). Result: When either mouse or human NK cells are recently isolated in culture, mouse NK cells have weak cytolytic activity while human NK cells show strong cytolytic abilities.

Mouse and human NK cells also differ greatly in expression markers, signaling cascades, and tissue distribution. For example, CD56 is used as a marker in human NK cells, while mouse NK cells do not express this marker at all. In addition, the well-established mechanism for regulating NK cell cytotoxicity is mediated through a ligand that binds NK cell activation and inhibitory receptors. Two of the most prominent receptors that activate human NK cells are NKp30 and NKp44, neither of which is expressed in mouse NK cells. With regard to inhibitory NK receptors, while human NK cells express KIRs that recognize MHC class I and reduce cytotoxic activity, KIRs are not expressed at all in mouse NK cells, but Ly49s is expressed instead (Trousdale et al. 2001). As a result, although mouse NK cells have come to perform the same function as human NK cells in the natural physiological environment, the mechanisms that provide this action differ significantly between species.

Thus, there is a need for an alternative and preferably improved human NK cells and cell lines, for example, with more cytotoxic properties.

The aim of the invention is to provide NK cells and their lines with a phenotype with increased cytotoxicity. An additional object is to provide methods for producing modified NK cells and NK cell lines, compositions containing these cells or lines thereof, as well as options for using these compositions for the treatment of cancer. Specific embodiments are directed to providing methods for treating certain cancers, for example, blood cancers such as leukemia. Specific embodiments of the invention are directed to the combination of two or more modifications of NK cells and their lines to further improve the cytotoxicity of the modified cells.

The essence of the invention

This document presents modified NK cells and their lines with a phenotype with increased cytotoxicity, as well as methods for obtaining these cells and their lines. Also provided are compositions of modified NK cells and their lines, as well as options for using these compositions for the treatment of cancer.

The present invention provides methods for modifying NK cells and cell lines by, for example, genetic engineering, to knock out genes encoding inhibitory receptors, to express genes encoding TRAIL ligands and variants, and to express genes encoding chimeric antigen receptors (CARs) and/or Fc receptors.

In addition, the compositions of the present invention include NK cells and their lines, which have two or more modifications, and multiple modifications further increase the cytotoxic activity of the composition.

In accordance with the present invention, there are additional methods for treating cancer, eg, blood cancer, with modified NK cell lines, eg, KHYG-1 cell derivatives. In this case, modified NK cell lines are obtained with the absence of inhibitory checkpoint receptors for the expression of TRAIL ligand variants and/or the expression of CARs and/or Fc receptors.

In particular, diseases treatable in accordance with the present invention include cancer, blood cancer, leukemia and, in particular, acute myeloid leukemia. Thus, human tumors and cancers can be treated. References to tumors in this document include references to neoplasms.

Detailed description of the invention

Accordingly, the present invention provides a natural killer (NK) cell or NK cell line that has been genetically modified to increase cytotoxicity.

As detailed in the examples below, NK cells and NK cell lines have been genetically modified to increase their anti-cancer cytotoxic activity.

Collectively, NK cells and NK cell lines will be referred to herein as NK cells (unless the context otherwise requires).

In certain embodiments, the present invention provides NK cells with or without reduced function of inhibitory checkpoint receptors. Thus, in the examples below, NK cells are formed by knocking out one or more checkpoint inhibitory receptor genes. Preferably, these receptors are certain inhibitory checkpoint receptors. It is also preferred that one, several or all of these inhibitory checkpoint receptors belong to CD96 (TACTILE), CD152 (CTLA4), CD223 (LAG-3), CD279 (PD-1), CD328 (SIGLEC7), SIGLEC9, TIGHT and /or TIM-3.

In other embodiments, NK cells are provided in which one or more inhibitory receptor signaling pathways are knocked out or exhibit reduced function - as a result, inhibitory receptor function is also reduced or absent. For example, signaling pathways mediated by SHP-1, SHP-2 and/or SHIP are knocked out by genetic modification of cells.

The resulting NK cells exhibit improved cytotoxicity and thus increased utility in cancer therapy, especially in blood cancer, in specific treatments for leukemia and multiple myeloma.

In one of the embodiments of the invention, the genetic modification occurs before the modification of the cell into an NK cell. For example, pluripotential stem cells (eg, iPSCs) can be genetically modified to lose the ability to express inhibitory checkpoint receptors. The modified iPSCs are then modified to produce genetically modified NK cells with increased cytotoxicity.

It is preferable to reduce the function of inhibitory checkpoint receptors in relation to other inhibitory receptors, due to the expression of the first mentioned, which occurs after the activation of NK cells. Normal or "classic" inhibitory receptors, such as most of the KIR family, NKG2A and LIR-2, bind class I MHCs and are therefore primarily involved in reducing the homing problem. Thus, it is preferable to knock out inhibitory checkpoint receptors. The reduced function of these receptors or their absence according to the present invention does not allow cancer cells to suppress the function of immune effectors (which may otherwise occur in the case of full functionality of the receptors). Thus, the key advantage of these embodiments of the present invention lies in NK cells that are less susceptible to the suppression of their cytotoxic activity by cancer cells, which leads to practical results in the treatment of cancer.

In the context of this document, references to inhibitory receptors generally refer to receptors expressed on the cytoplasmic envelope of an immune effector cell, such as an NK cell; after binding of its additional ligand, intracellular signals are responsible for reducing the cytotoxicity of the immune effector cell. These inhibitory receptors are expressed both in the "resting" and "active" state of immune effector cells, and are often associated with providing an immune system autotolerance mechanism that inhibits the cytotoxic response to body cells and tissues. An example is the inhibitory receptor of the KIR family, which is expressed on NK cells and recognizes MHC class I expressed on healthy body cells.

Also in the context of this document, inhibitory checkpoint receptors are generally considered to be a subset of the inhibitory receptors mentioned above. However, unlike other inhibitory receptors, inhibitory checkpoint receptors are expressed at higher levels with a prolonged period of activation and cytotoxicity of an immune effector cell, such as a NK cell. This phenomenon is useful for attenuating chronic cytotoxicity, for example, in the focus of inflammation. Examples include the inhibitory checkpoint receptors PD-1, CTLA-4, and CD96, all of which are expressed on NK cells.

The present invention also provides an NK cell without a checkpoint inhibitory receptor gene selected from CD96 (TACTILE), CD152 (CTLA4), CD223 (LAG-3), CD279 (PD-1), CD328 (SIGLEC7), SIGLEC9, TIGIT and TIM-3.

A NK cell without a gene may refer to a complete or partial deletion, mutation, or otherwise may result in a lack of expression of a functional gene product. In some embodiments, the NK cells do not contain genes responsible for two or more inhibitory receptors.

More specific embodiments of the invention include an NK cell without a gene responsible for inhibitory checkpoint receptors selected from CD96 (TACTILE), CD152 (CTLA4) and CD279 (PD-1). Preferred embodiments include an NK cell derived from KHYG-1.

In the examples described below, the present inventors sufficiently showed a cytotoxic effect when using small interfering RNA to knock down the expression of the inhibitory checkpoint receptor CD96 in KHYG-1 cells. Cell knockdown (KD) of KHYG-1 by CD96 demonstrated increased cytotoxicity against leukemia cells at various effector:target (E:M) ratios.

In other embodiments, the present invention provides NK cells expressing a TRAIL ligand or, preferably, a mutated (variant) TRAIL ligand. As described in the examples below, modifications that increase NK cell cytotoxicity also include increased expression of TRAIL ligand and/or mutated TRAIL ligand variants.

The resulting NK cells show increased binding to TRAIL receptors and thereby increased cytotoxicity against cancers, especially blood cancers, certain types of leukemia.

Preferably, the mutations/variants have low (or virtually no) affinity for decoy receptors compared to wild-type TRAIL binding to decoy receptors. Such decoy receptors are a class of TRAIL receptors that bind the TRAIL ligand but fail to initiate cell death and also, in some cases, antagonize the apoptosis signaling pathway. Mutated/variant TRAIL ligands can be prepared according to WO 2009/077857.

Mutations/variants can separately increase affinity for TRAIL receptors, eg DR4 and DR5. Wild-type TRAIL typically contains K _D >2 nM for DR4, >5 nM for DR5, and >20 nM for the decoy receptor DcR1 (WO 2009/077857; measured by surface plasmon resonance), or about 50-100 nM for DR4, 1-10 nM for DR5 and 175-225 nM for DcR1 (Truneh, A. et al. 2000; measured by isothermal titration calorimetry and ELISA). Therefore, increased affinity for DR4 is properly defined as K _D <2 nM or <50 nM, respectively, while increased affinity for DR5 is properly defined as K _D < 5 nM or <1 nM, respectively. Decreased affinity for the decoy receptor DcR1 is properly defined as K _D >50 nM or >225 nM, respectively. In any case, the increase or decrease in affinity that is elicited by the TRAIL variant/mutation refers to the base affinity that is elicited by wild-type TRAIL. Preferably, the affinity is increased by at least 10%, more preferably by at least 25%, compared to the wild-type TRAIL display.

Preferably, the TRAIL variant has an increased affinity for DR5 relative to its affinity for DR4, DcR1 and DcR2. Preferably, the affinity for DR5 is at least 1.5, 2, 5, 10, 100 or even 1000 times or more greater than the affinity for one or more of DR4, DcR1 and DcR2. More preferably, the affinity for DR5 is at least 1.5, 2, 5, 10, 100 or even 1000 times or more greater than the affinity for at least two and preferably all of DR4, DcR1 and DcR2.

The key advantage of these embodiments of the present invention is that NK cells are more efficient in killing cancer cells.

Further specific embodiments include an NK cell expressing a mutated TRAIL ligand that has reduced or no affinity for TRAIL decoy receptors. Preferably, the NK cell is derived from KHYG-1. Further, specific embodiments include an NK cell expressing a mutated TRAIL ligand that has reduced or absent affinity for TRAIL decoy receptors and increased affinity for DR4 and/or DR5.

In the examples of the present invention, described in more detail below, NK cells were genetically modified to express mutated TRAIL. Modified KHYG-1 cells expressed mutated TRAIL and NK-92 expressed mutated TRAIL. The modified KHYG-1 cells showed improved cytotoxicity against cancer cell lines by in vitro technology. KHYG-1 cells express TRAIL receptors (eg DR4 and DR5) but at lower levels. In other preferred embodiments of the modified NK cells, TRAIL receptors are not expressed at all or are underexpressed, or only at a low level—low enough that the viability of the modified NK cells is not adversely affected by expression of the mutated TRAIL.

In a further embodiment, treatment of cancer with modified NK cells expressing TRAIL or a TRAIL variant is enhanced by administering to the patient an agent capable of increasing the expression of TRAIL death receptors on cancer cells. This agent may be administered before, in combination with or after the administration of the modified NK cells. It is preferred that the agent be administered prior to the administration of the modified NK cells.

In a preferred embodiment, the agent causes an increase in DR5 expression on cancer cells. Additionally, a chemotherapeutic drug, such as Bortezomib, can be used as an agent, which is administered at a low dose capable of causing an increase in the expression of DR5 on cancer cells.

This invention is not limited to any specific agents capable of causing an increase in DR5 expression; the following agents containing DR5 are given as examples: Bortezomib, gefitinib, piperlongumin, doxorubicin, alpha-tocopherol succinate, and HDAC inhibitors.

According to a preferred embodiment of the present invention, the mutated/variant TRAIL ligand binds to one or more co-stimulatory domains of an NK cell, eg 41BB/CD137, CD3zeta/CD247, DAP12 or DAP10. Binding of the variant to the receptor on the target cell promotes apoptotic signals within the target cell and also stimulates cytotoxic signals in the NK cell.

In further preferred embodiments, the present invention provides NK cells that have a reduced function of inhibitory checkpoint receptors and also express a mutated TRAIL ligand, as described in more detail below, in relation to these respective NK cell modifications. In more preferred embodiments, NK cells express a mutated TRAIL ligand with reduced or no affinity for TRAIL decoy receptors and can be derived from KHYG-1, also without the gene encoding an inhibitory checkpoint receptor selected from CD96 (TACTILE), CD152 (CTLA4), CD223 (LAG-3), CD279 (PD-1), CD328 (SIGLEC7), SIGLEC9, TIGIT and TIM-3.

The present invention also contemplates NK cells and NK cell lines, preferably KHYG-1 cells, as well as derivatives thereof, modified to express one or more CARs.

For use in cancer therapy, CARs bind specifically to one or more ligands on cancer cells, for example, CS1 (SLAMF7) on myeloma cells. For use in the treatment of specific cancers, such as multiple myeloma, CAR can bind to CD38. For example, a CAR may include binding properties of different regions derived from, similar or identical to derivatives of a known monoclonal antibody (daratumumab). Such NK cells can be used in cancer therapy in combination with an angiogenesis inhibitory agent such as lenalidomide. For use in cancer therapy, especially leukemia and AML, CARs can bind CLL-1.

CAR-NKs can be bispecific, with their affinity applied to two separate ligands/antigens. Bispecific CAR-NKs can be used to either increase the number of potential binding sites on cancer cells or, alternatively, localize cancer cells to other immune effector cells that express ligands specifically for NK-CAR. For use in cancer therapy, a bispecific CAR can bind to a tumor target cell and an effector cell, such as a T cell, NK cell, or macrophagocyte. Thus, for example, in the case of multiple myeloma, the bispecific CAR may bind to a T cell antigen (eg, CD3, etc.) and a tumor cell marker (eg, CD38, etc.). Alternatively, the bispecific CAR can bind to two separate tumor cell markers, increasing the binding affinity of the NK cell for the target tumor cells. It can reduce the risk of cancer cells by developing resistance with one or more target antigens. As an example, in this case (the case of multiple meiloma), CAR binding to CD38 and CS-1/SLAMF7 can be given. Another tumor cell marker targeted by CAR is the "don't eat me" type marker on tumors, an example of which is CD47.

Additional features of the invention include the provision of further modifications to NK cells and their lines as described above, wherein, for example, the Fc receptor (which may be CD16, CD32 or CD64, including subtypes and derivatives) is expressed on the cell surface. When used, these cells can be characterized by increased recognition of antibody-coated cancer cells and improve the activation of the cytotoxic response.

Additional features of the invention include the adaptation of modified NK cells and their lines to improve targeting to specific target parts of the body. The NK cells of the present invention can be targeted to specific sites on cancer cells. In preferred embodiments for the treatment of blood cancer, the NK effectors of the present invention are adapted to target the bone marrow. Specific NK cells are modified by fucosylation and/or sialylation to target the bone marrow. This can be achieved by genetically modifying NK cells to express fucosyl transferase and/or sialyl transferase, respectively. Improved targeting of NK effector cells to tumor sites can also be achieved by disrupting the tumor vasculature, such as metronomic chemotherapy or using angiogenesis-targeting drugs (Melero et al. 2014) to normalize NK cell infiltration through cancer blood vessels.

Another additional feature of the invention is the provision of modified NK cells and their lines with increased endogenous ability of rapid growth and culture expansion. For example, this can be achieved by transfecting cells to overexpress the growth-promoting cytokines IL-2 and IL-15. In addition, this additional change provides a cost-effective alternative to replenishing growth media with cytokines on an ongoing basis.

The present invention also provides a method for producing a modified NK cell or line thereof, including genetic modification of the cell or line thereof, as described herein, in order to increase its cytotoxicity. This genetic modification can be a stable gene knockout, such as CRISPR, or a temporary gene knockdown, such as small interfering RNA.

In a preferred embodiment, a stable genetic modification method, such as CRISPR, is used to generate a new NK cell line with enhanced cytotoxicity, such as a KHYG-1 cell derivative.

In some embodiments of the invention, the method is provided for obtaining an NK cell or its line, which has been modified to reduce the function of an inhibitory receptor. Preferably, these inhibitory receptors are inhibitory checkpoint receptors.

More specific embodiments of the invention include a method for producing an NK cell or line thereof with reduced inhibitory receptor function, wherein the inhibitory checkpoint receptors are selected from CD96 (TACTILE), CD152 (CTLA4), CD223 (LAG-3), CD279 (PD-1 ), CD328 (SIGLEC7), SIGLEC9, TIGIT and TIM-3.

In preferred embodiments, the method includes modifying NK cells to reduce the function of two or more inhibitory receptors.

The present invention also provides a method for producing a modified NK cell or line thereof, including genetic modification of the cell or line thereof to express a TRAIL ligand or a mutated (variant) TRAIL ligand.

In some embodiments, the method includes modifying the NK cell or cell line to express a mutated TRAIL ligand with increased affinity for TRAIL receptors. Preferably, the TRAIL receptors are DR4 and/or DR5. Preferred embodiments of the invention provide a method for modifying NK cells or cell lines to express a mutated TRAIL ligand with reduced affinity for TRAIL decoy receptors.

In additional preferred embodiments, the method comprises modifying the NK cell or cell line to eliminate inhibitory checkpoint receptor function as well as expression of a mutated TRAIL ligand with reduced or no binding affinity for TRAIL decoy receptors.

Additional standard embodiments provide a method for producing an NK cell or cell line wherein the function of one or more inhibitory checkpoint receptors is omitted and/or a mutated TRAIL ligand with reduced or no binding affinity for TRAIL decoy receptors is expressed and the cell is further modified to expression of a CAR or a bispecific CAR. The CAR properties are optional as shown above.

In some embodiments of the invention, the method includes obtaining an NK cell or line thereof, in which the function of one or more inhibitory checkpoint receptors is excluded and/or a mutated TRAIL ligand with reduced or no binding affinity for TRAIL decoy receptors is expressed, and the cell is further modified to express a CAR or a bispecific CAR, and the cell is further modified to express one or more Fc receptors. The respective Fc receptors are selected from CD16 (FcRIII), CD32 (FcRII) and CD64 (FcRI).

Preferred embodiments of the invention from the above include a method for obtaining NK cells and their lines derived from KHYG-1.

According to the objectives of the present invention, a modified NK cell, a line or composition thereof with increased cytotoxicity is provided for use in the treatment of cancer in a patient, in particular blood cancer.

In preferred embodiments, the modified NK cell, line or composition thereof is for use in the treatment of blood cancers, including acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), Hodgkin's lymphoma, non-Hodgkin's lymphoma, including T-cell and B-cell lymphomas, asymptomatic myeloma, indolent multiple myeloma (SMM), active myeloma or light chain myeloma.

In more preferred embodiments, the invention provides an NK cell line derived from KYHG-1 by reducing the function of inhibitory checkpoint receptors in a KHYG-1 cell or the expression of a mutated TRAIL ligand in a KHYG-1 cell, or both, for use in the treatment of blood cancer.

The modified NK cells, their lines and compositions described herein above and below are suitable for the treatment of cancer, in particular human cancers, for example, for the treatment of blood cancer or solid cancer. Preferably, the NK cells and derivatives are human NK cells. Human NK cells are preferably used to treat a patient.

Various administration methods are well known to those skilled in the art for administering active substances and combinations thereof to a patient. Embodiments of the present invention are provided for the treatment of blood cancer. The introduction of modified NK cells and/or their lines can be systemic or local, for example, intraperitoneal administration.

In other embodiments of the present invention, the active substance is administered directly. Thus, administration can be intratumoral, particularly suitable for solid tumors.

Generally, NK cells are considered suitable for the methods, uses and compositions of the present invention. According to the cells used in certain examples, the NK cell may be a cell derived from a cancer cell line. Preferably, an NK cell that has been treated to reduce tumorigenicity, for example by producing a mortal cell and/or a cell that cannot divide, can be obtained from a blood cancer cell line and can be used in the methods of the invention to treat blood cancer.

To obtain a cancer cell that is more suitable for therapeutic use, it is usually processed or pre-treated in a certain way to reduce or eliminate the possibility of tumor formation in the patient. The specific lines of modified NK cells used in the examples are safe because they cannot divide; they are irradiated and retain their cytotoxic activity, but die within approximately 3-4 days. Thus, specific cells and their lines cannot proliferate, for example, as a result of irradiation. Methods for treating potential NK cells for use in the methods described herein include irradiation to avoid their division and tumor formation in vivo, and genetic modification to reduce tumorigenicity, for example, for a sequence encoding a "suicide" gene that can be activated during avoid cell division and tumor formation in vivo. "Suicide" genes can be activated by exogenous, for example, circulating substances that cause cell death in cells expressing the gene. Another alternative is to use monoclonal antibodies that target specific NK cells for therapy. For example, CD52 is expressed on KHYG-1 cells, and binding of monoclonal antibodies to this marker can cause antibody-dependent cell-mediated cytotoxicity (ADCC) and death of KHYG-1 cells.

As stated in the article published by Sack et al. 2006, NK cancer cells and their lines are easily irradiated using irradiators such as the Gammacell 3000 Elan. A source of caesium-137 is used to control the dose of irradiation, and a dose-response curve between 1 Gy and 50 Gy, for example, can be used to determine the optimal dose to eliminate cell proliferative capacity while maintaining the benefits of increased cytotoxicity. This is achieved by analyzing cells for cytotoxicity after each dose of radiation.

There are significant advantages to using an irradiated NK cell line for adoptive cellular immunotherapy over the established approach of autologous cells or MHC-matched T cells. First, the use of a high proliferative NK cell line implies that the growth of modified NK cell lines can be more easily achieved commercially. Irradiation of the modified NK cell line may be performed before the cells are introduced into the patient. These irradiated cells, while retaining their beneficial cytotoxicity, have a limited life cycle and, unlike modified T cells, do not circulate for long periods of time, causing long-term side effects.

In addition, the use of allogeneic modified NK cells and their lines implies that MHC class I expressing cells in the patient's body cannot inhibit the cytotoxic NK cell response in the same way as for the cytotoxic response of autologous NK cells. The advantage of using allogeneic NK cells and their lines to kill cancer cells is the previously mentioned effect of GVL and, unlike T cells, allogeneic NK cells and their lines do not stimulate the development of the GVHD response, which makes them the preferred option for cancer treatment using adoptive cellular immunotherapy.

As indicated in the claims and elsewhere herein, the present invention contemplates the following embodiments:

1. A natural killer (NK) cell or NK cell line that has been genetically modified to increase its cytotoxicity.

2. An NK cell or lineage thereof according to embodiment 1 modified to provide reduced function of one or more inhibitory receptors.

3. The NK cell or lineage thereof according to embodiment 2, wherein the inhibitory receptors are inhibitory checkpoint receptors.

4. An NK cell or lineage thereof according to embodiment 3, wherein the inhibitory checkpoint receptors are selected from CD96 (TACTILE), CD152 (CTLA4), CD223 (LAG-3), CD279 (PD-1), CD328 (SIGLEC7 ), SIGLEC9, TIGIT and TIM-3.

5. An NK cell or lineage thereof according to any of embodiments 2-4 modified to provide reduced function of two or more inhibitory receptors.

6. An NK cell or line thereof according to any of embodiments 1-5 modified to express a TRAIL ligand.

7. An NK cell or lineage thereof according to embodiment 6, wherein the TRAIL ligand is a mutated TRAIL ligand.

8. An NK cell or lineage thereof according to embodiment 7, wherein the mutated TRAIL ligand has increased affinity for TRAIL receptors, eg DR4 and/or DR5.

9. An NK cell or lineage thereof according to any one of embodiments 7-8, wherein the mutated TRAIL ligand has reduced affinity for TRAIL decoy receptors.

10. An NK cell or lineage thereof according to any of the previous embodiments modified to eliminate inhibitory checkpoint receptor function as well as expression of a mutated TRAIL ligand with reduced or no binding affinity for TRAIL decoy receptors.

11. An NK cell or line thereof according to any of the previous embodiments expressing a chimeric antigen receptor (CAR).

12. An NK cell or lineage thereof according to embodiment 11, wherein CAR is a bispecific CAR.

13. An NK cell or lineage thereof according to embodiment 12, wherein the bispecific CAR binds two ligands for one cell type.

14. An NK cell or lineage thereof according to embodiment 12, wherein the bispecific CAR binds one ligand on either of two distinct cell types.

15. An NK cell or lineage thereof according to embodiments 11 and 12, wherein the ligand(s) for CAR or bispecific CAR are expressed on the cancer cell.

16. An NK cell or lineage thereof according to embodiment 13, characterized in that the ligands for the bispecific CAR are expressed on the cancer cell.

17. The NK cell or lineage thereof according to embodiment 14, wherein the ligands for the bispecific CAR are expressed on the cancer and immune effector cell.

18. An NK cell or lineage thereof according to any of the previous embodiments, modified to express one or more Fc receptors.

19. An NK cell or lineage thereof according to embodiment 18, wherein the Fc receptors are selected from CD16 (FcRIII), CD32 (FcRII) and CD64 (FcRI).

20. An NK cell or line thereof according to any of the previous embodiments, characterized in that the cell line is derived from the KHYG-1 cell line.

21. NK cell lacking an inhibitory checkpoint receptor gene selected from CD96 (TACTILE), CD152 (CTLA4), CD223 (LAG-3), CD279 (PD-1), CD328 (SIGLEC7), SIGLEC9, TIGIT and TIM-3.

22. The NK cell of Embodiment 21 without genes responsible for two or more inhibitory checkpoint receptors selected from CD96 (TACTILE), CD152 (CTLA4), CD223 (LAG-3), CD279 (PD-1), CD328 ( SIGLEC7), SIGLEC9, TIGIT and TIM-3.

23. The NK cell of embodiment 21 or 22, wherein the inhibitory checkpoint receptor is selected from CD96 (TACTILE), CD152 (CTLA4) and CD279 (PD-1).

24. An NK cell according to any one of embodiments 21-23 derived from KHYG-1.

25. NK cell expressing a mutated TRAIL ligand that has reduced or no affinity for TRAIL decoy receptors.

26. NK cell of Embodiment 25 derived from KHYG-1.

27. The NK cell of embodiment 25 or 26 without a checkpoint inhibitory receptor gene selected from CD96 (TACTILE), CD152 (CTLA4), CD223 (LAG-3), CD279 (PD-1), CD328 (SIGLEC7 ), SIGLEC9, TIGIT and TIM-3.

28. An NK cell or lineage thereof according to any of the previous embodiments, which cannot proliferate, for example, as a result of irradiation.

29. A method for producing a modified NK cell or line thereof, including genetic modification of a cell or line thereof in order to increase its cytotoxicity.

30. The method of embodiment 29 wherein the NK cell or lineage thereof is modified to reduce the function of an inhibitory receptor.

31. The method of embodiment 30 wherein the inhibitory receptors are inhibitory checkpoint receptors.

32. The method of embodiment 31 wherein the inhibitory checkpoint receptors are selected from CD96 (TACTILE), CD152 (CTLA4), CD223 (LAG-3), CD279 (PD-1), CD328 (SIGLEC7), SIGLEC9, TIGIT and TIM-3.

33. The method of any one of embodiments 29-32, comprising modifying NK cells to reduce the function of two or more inhibitory receptors.

34. The method of any one of embodiments 29-33, comprising modifying an NK cell or line thereof to express a TRAIL ligand or a mutated TRAIL ligand.

35. The method of embodiment 34 wherein the mutated TRAIL ligand has increased affinity for TRAIL receptors.

36. The method of embodiment 35 wherein the TRAIL receptors are DR4 and/or DR5.

37. The method of any one of embodiments 34-36 wherein the mutated TRAIL ligand has reduced affinity for TRAIL decoy receptors.

38. The method of any one of embodiments 29-37, wherein the NK cell or lineage thereof is modified to eliminate inhibitory checkpoint receptor function as well as expression of a mutated TRAIL ligand with reduced or no binding affinity for TRAIL decoy receptors.

39. The method of embodiment 38, wherein the NK cell or lineage thereof is modified to express a CAR or a bispecific CAR.

40. The method of Embodiment 39 wherein the bispecific CAR binds two ligands on the same cell type.

41. The method of embodiment 39 wherein the bispecific CAR binds one ligand on either of two separate cell types.

42. The method of embodiment 39, wherein the ligand(s) for the CAR or the bispecific CAR are expressed on the cancer cell.

43. The method of embodiment 40 wherein the ligands for the bispecific CAR are expressed on the cancer cell.

44. The method of embodiment 41 wherein the ligands for the bispecific CAR are expressed on the cancer and immune effector cell.

45. The method of any one of embodiments 29-44, wherein the NK cell or lineage thereof is modified to express one or more Fc receptors.

46. The method of embodiment 45 wherein the Fc receptors are selected from CD16 (FcRIII), CD32 (FcRII) and CD64 (FcRI).

47. The method of any one of embodiments 29-46, wherein the cell line is derived from the KHYG-1 cell line.

48. An NK cell or cell line thereof, obtained by the method of any of embodiments 29-47.

49. KHYG-1 obtained by the method of any one of embodiments 29-48.

50. Modified NK cell, line or formulation thereof with increased cytotoxicity for use in the treatment of cancer in a patient.

51. An NK cell or cell line thereof according to any of embodiments 1-28, or obtained according to any of embodiments 29-49, for use in embodiment 50.

52. Modified NK cell, line or formulation thereof for use in embodiment 50 or 51, characterized in that the type of cancer is blood cancer.

53. Modified NK cell, line or composition thereof for use in embodiment 52, characterized in that the type of blood cancer is acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), Hodgkin's lymphoma, non-Hodgkin's lymphoma, including T-cell and B-cell lymphomas, asymptomatic myeloma, indolent multiple myeloma (SMM), active myeloma or light chain myeloma.

54. An NK cell line derived from KYHG-1 by reducing the function of inhibitory checkpoint receptors in a KHYG-1 cell or the expression of a mutated TRAIL ligand in a KHYG-1 cell, or both, for use in the treatment of blood cancer.

Examples

The present invention has a more detailed and specific description of obtaining derivatives of KHYG-1 NK cells, modified to exhibit greater cytotoxic activity and, thus, the ability to lead to the death of a leukemia cell in clinical practice.

This invention is illustrated by means of specific embodiments with reference to the accompanying drawings, where:

In FIG. 1 shows the DNA sequence of the target region of the LIR2 gene and the flanking regions of the "guide" RNA are marked;

In FIG. 2 shows the DNA sequence of the target region of the CTLA4 gene and the flanking regions of the guide RNA are marked;

In FIG. 3 shows the structure of the RNA (expression vector) used for transfection;

In FIG. 4 shows gel electrophoresis bands for primary and mutated LIR2 DNA before and after transfection;

In FIG. 5 shows gel electrophoresis bands for primary and mutated CTLA4 DNA before and after transfection;

Fig. 6A is a FACS plot showing successful knockdown of CD96 by electropulse opening of cell pores;

Fig. 6B is a FACS plot showing successful knockdown of CD96 by electropulse opening of cell pores;

Fig. 7 is a bar graph showing the increased cytotoxicity of knockdown of KHYG-1 cells by CD96 compared to K562 cells at various E:M ratios;

In FIG. 8 shows CD328 (Siglec-7) knockdown in NK-92 cells;

In FIG. 9 shows NK cell cytotoxicity enhanced by CD328 (Siglec-7) knockdown;

In FIG. 10 shows a FACS plot of baseline TRAIL expression on KHYG-1 cells;

In FIG. 11 shows a FACS plot of TRAIL and TRAIL variant expression after transfection with KHYG-1 cells;

In FIG. 12 shows a FACS plot of CD107a expression after transfection with KHYG-1 cells;

In FIG. 13 shows the effect of transfecting KHYG-1 cells with TRAIL and the TRAIL variant on cell viability;

In FIG. 14 shows a FACS plot of baseline expression of DR4, DR5, DcR1 and DcR2 on both KHYG-1 cells and NK-92 cells;

In FIG. 15, 16 and 17 show the effect of TRAIL or TRAIL variant expression in KHYG-1 cells on apoptosis of three target cell populations: K562, RPMI8226 and MM1.S, respectively;

In FIG. 18 shows two FACS plots of DR5 expression in RPMI8226 cells and MM1.S cells, respectively; in addition, the effect of treatment with Bortezomib on DR5 expression was shown;

In FIG. 19 shows FACS plots of apoptosis in Bortezomib pretreated/non-treated MM1.S cells cultured with KHYG-1 cells with/without TRAIL variant.

In FIG. 20 shows a FACS plot of the expression of perforin levels in KHYG-1 cells treated with 100 nM CMA for 2 hours;

In FIG. 21 shows FACS plots of KHYG-1 cell viability after treatment with 100 nM CMA or vehicle;

In FIG. 22 shows FACS plots of apoptosis in MM1.S cells cultured with KHYG-1 cells, with/without TRAIL variant, pretreated/untreated CMA;

In FIG. 23 shows FACS plots of apoptosis in K562 cells cultured with KHYG-1 cells with CD96 small interfering RNA and/or TRAIL variant expression; as well as

In FIG. 24 shows FACS plots of apoptosis in MM1.S cells cultured with KHYG-1 cells with CD96 small interfering RNA and/or TRAIL variant expression;

DNA, RNA and amino acid sequences are given below, where:

SEQ ID no: 1 is the complete DNA sequence of LIR2;

SEQ ID NO: 2 is the amino acid sequence of LIR2;

SEQ ID no: 3 is the sequence of the "guide" RNA g9 LIR2;

SEQ ID no: 4 is the sequence of the "guide" RNA g18 LIR2;

SEQ ID NO: 5 is the sequence of the LIR2 forward primer;

SEQ ID no: 6 is the sequence of the reverse primer LIR2;

SEQ ID NO: 7 is the complete DNA sequence of CTLA4;

SEQ ID NO: 8 is the amino acid sequence of CTLA4;

SEQ ID NO: 9 is the sequence of the "guide" RNA g7 CTLA4;

SEQ ID NO: 10 is the sequence of the "guide" RNA g15 CTLA4;

SEQ ID NO: 11 is the sequence of the CTLA4 forward primer; as well as

SEQ ID NO: 12 is the CTLA4 reverse primer sequence.

Example 1 Knockout of Inhibitory Receptor Function

CRISPR/Cas9

Cells were prepared as follows after removal of inhibitory receptor function. Guide RNA structures were designed and prepared to interact with the genes encoding the "classical" inhibitory receptor LIR2 and the inhibitory "checkpoint" receptor CTLA4 in the human NK cell genome. Thereafter, CRISPR/Cas9 genome editing was used to knock out the LIR2 and CTLA4 target genes.

For each target gene, two candidate RNA guide RNAs were selected and their cleavage activity in K562 was determined. Candidate guide RNA sequences are shown in Table 1, and the protospacer adjacent motif (PAM) refers to the last 3 sequence backbones. The flanking regions of the guide RNA sequences on the LIR2 gene (SEQ ID no: 1) and the CTLA4 gene (SEQ ID no: 7) are shown in Figures 1 and 2, respectively.

Table 1. Sequences and "guide" RNA candidates

K562 cells were transfected with prepared guide RNA structures (Figure 3) and sequentially selected for PCR amplification. The presence of GFP expression has been used to report the successful introduction of the "guide" RNA structure into K562 cells. Thus, the expression of the Cas9 gene and, consequently, the possibility of knockout of the expression of the LIR2 and CTLA4 genes were confirmed.

The cleavage activity of the guide RNA structures was determined in vitro using an incompatibility assay. T7E1 endonuclease I recognizes and cleaves non-ideally matched DNAs, thereby allowing comparison of the original LIR2 and CTLA4 genes with mutated genes after CRISPR/Cas9 transfection and non-homologous end joining (NHEJ).

Figure 4 shows bands obtained by agarose gel electrophoresis after LIR2 gene knockout with g9 and g18 guide RNA sequences. The three bands corresponding to each mutation refer to each original gene and two resulting structures after the detection of incompatibility in the DNA sequence at the completion of transfection. The g9 guide RNA sequence resulted in a transfection success rate of 11%, while the g18 guide RNA sequence resulted in a success rate of 10%.

Figure 5 shows bands obtained by agarose gel electrophoresis after CTLA4 gene knockout with g7 and g15 guide RNA sequences. As a result of the g7 guide RNA sequence, the transfection success rate was 32%, while as a result of the g15 guide RNA sequence, the success rate was 26%.

After successful knockout of LIR2 and CTLA4 in K562 cells, KHYG-1 cells were transfected with guide RNA structures.

Clones derived from KHYG-1 having homozyotic deletions were identified. For this purpose, the expression of Cas9 / puromycin acetyltransferase (RAS) was used. Successfully transfected cells were determined based on their resistance to the antibiotic puromycin.

Cas9RNP

Another protocol used to knock out inhibitory NK checkpoint receptors is Cas9 RNP transfection. The advantage of using this protocol was that a similar transfection efficiency was achieved, but with a significantly lower level of toxicity, compared to using CRISPR/Cas9 protocol DNA plasmids.

KHYG1 1×10 ⁶ cells were selected for each transfection experiment. Cells were washed with PBS and spun in a centrifuge. After that, the supernatant was removed. Then, CRISPR RNP (RNA binding protein) materials were prepared as follows:

(1) a 20 µM solution of the required synthesized crRNAs and tRNAs (purchased from Dharmacon) was prepared.

(2) 4 μl of crRNA (20 μM) and 4 μl of RNA (20 μM) were mixed together.

(3) The mixture was then added to 2 μl of Cas9 protein (5 μg/μl).

(4) All components were mixed and kept at room temperature for 10 minutes.

After treatment in the Neon® transfection system, the cells were mixed with Cas9 RNP and the cell pores were electrically pulsed opened using the following parameters:

Voltage: 1450V

Pulse duration: 30ms

Number of impulses: 1

The cells were then transferred to one well of a 12-well plate containing growth medium (including IL-2 and IL-15).

Cells were sampled after 48-72 hours to confirm gene editing efficiency by T7 endonuclease assay and/or Sanger's method. The presence of inserts was confirmed, indicating successful knockout of CTLA4, PD1 and CD96 in KHYG1 cells.

Site-specific nucleases

Another protocol used to knock out inhibitory NK checkpoint receptors is XTN TALEN transfection. The advantage of using this protocol was that it was possible to achieve a particularly high level of specificity compared to wild-type CRISPR.

Stage 1: Preparation of reagents

KHYG-1 cells were analyzed for certain characteristics, including transfection efficiency, single cell cloning efficiency, and chromosome set/copy number. Thereafter, the cells were cultured according to the supplier's recommendations.

Depending on the inhibitory checkpoint receptor that was knocked out, nucleases were prepared according to individual requirements from at least two XTN TALEN pairs. The step of determining individual requirements includes the assessment of the gene locus, the number of copies and functional assessment (ie assessment of homologues, off-target assessment).

Step 2: Obtaining a cell line

Cells were transfected with step 1 nucleases; this step was repeated 3 times to achieve high cutting levels, the cultures were separated and the intermediate cultures were maintained in a certain state until transfection began.

Preselection occurred a few days after each transfection; cell populations were tested for cutting efficiency by Cel-1 assay. Upon reaching acceptable levels or flat cutting characteristics after repeated transfections, the cells were considered ready for single cell cloning.

Pooled cells were sorted one cell per well of a 96-well plate; the number of plates for each population depended on the efficiency of cloning one cell, which is determined in step 1. The plates were incubated for 3-4 weeks.

Stage 3 - Selection and expansion

Upon completion of cell fusion in a 96-well plate, the cultures were pooled and divided into three 96-well plates; one plate was frozen as a backup, another plate was replanted to continue growing clones, and the last plate was used for genotype confirmation.

Each clone in the genotype confirmation plate was analyzed for quantitative PCR signal loss, thus confirming that all allelomorphs had been altered. All negative clones were subjected to PCR amplification and cloned to determine the nature of the inserts and the absence of any wild-type or in-frame inserts.

Knockout-confirmed clones were pooled on no more than 24 well plates and then further expanded; typically, one knockout results in 5 to 10 cryotubes containing 1×10 ⁶ cells per tube for 5 separate clones.

Stage 4 - Approval

Cells were stored under aseptic conditions.

The main selection criteria for all cells that were stored included the number of viable cells (before freezing and after thawing), confirmation of identification by STR, assurance of total sterility, and testing for the presence of microplasma; other release criteria were applied as needed (chromosomal recruitment, surface marker expressivity, high level sterility, matrix or protein evaluation by knockout, etc.).

Example 2 - Knockdown of Inhibitory Checkpoint Receptor CD96 Function by RNA Interference

Knockdown of CD96 in KHYG-1 cells using small interfering RNA was performed by electropulse opening of cell pores. The Nucleofection T kit was used in combination with Lonza's Amaxa Nucleofector II as this device is suitable for use with cell lines and can transfect both replicating and non-replicating cells and achieves transfection efficiencies of up to 90%.

Control small interfering RNA (catalog number: sc-37007) and small interfering RNA CD96 (catalog number: sc-45460) were obtained from Santa Cruz Biotechnology. Antibiotic-free RPMI-1640 medium containing 10% FBS and 2 mM L-glutamine was used for culturing after nucleofection. Mouse antibody CD96-APC (catalog number: 338409) was obtained from Biolegend for staining.

A 20 μM small interfering RNA stock solution was prepared. The lyophilized siRNA duplex was re-dissolved in 33 µl of water without RNase (SIRNA dilution buffer: sc-29527) for FITC control/control siRNA, in 165 µl of water without RNase for siRNA of the target gene (small interfering RNA CD96). The tube was heated to 90°C for 1 minute and then kept at 37°C for 60 minutes. Thereafter, the small interfering RNA stock solution was stored at -20° C. until needed.

KHYG-1 cells were passaged one or two days before nucleofection, as the cells must be in the logarithmic growth phase.

The Nucleofector solution was warmed to room temperature (100 µl per sample).

An aliquot of culture medium containing serum and supplements was also preheated at 37° C. in a 50 ml tube. 1.5 ml culture medium containing serum and supplements was added to 6-well plates. The plates were previously kept in a CO ₂ incubator with 5% humidity at 37°C.

100 µl solution for nucleofection with 2×10 ⁶ cells was mixed with 4 µl solution containing 20 µM small interfering RNA (1.5 µg small interfering RNA). The formation of air bubbles during mixing was avoided. The mixture was transferred to Amaxa certified cuvettes placed in the Nucleofector cuvette holder and program U-001 was selected.

The completion of the program was confirmed, after which the samples were immediately withdrawn from the cuvettes. Then, 500 µl of pre-weighed culture medium was added to each cuvette. Thereafter, the samples in each cuvette were carefully transferred to the appropriate well of the prepared 6-well plate to obtain a 2 ml final volume in each well.

The cells were then kept in a CO ₂ incubator, 5% humidity, at 37° C., until the start of the transfection assay. Flow cytometry analysis was performed 16-24 hours after electropulse opening of cell pores in order to measure CD96 expression levels. This protocol of electropulse opening of cell pores was performed several times and was found to always result in CD96 knockdown in KHYG-1 cells (see, for example, Figures 6A and 6B).

Example 3 NK Cell Cytotoxicity Enhanced by CD328 Knockdown

KHYG-1 cells, both with and without CD96 knockdown, were co-cultured with K562 cells at different :target (E:M) effectors.

Cytotoxicity was measured 4 hours after co-cultivation using the DELFIA EuTDA Cytotoxicity Test Kit from PerkinElmer (Catalog Number: AD0116).

K562 target cells were cultured in RPMI-1640 medium containing 10% FBS, 2 µl L-glutamine and antibiotics. 96-well V-bottom plates (part number: 83.3926) were purchased from SARSTEDT. A 581 OR Eppendorf centrifuge (with plate rotor) was used to pellet the plate. VARIOSKAN FLASH (with Scanlt software version 2.4.3) was used to measure the fluorescence signal produced by dissolved K562 cells.

The K562 cells were washed with culture medium, and with its help the number of cells was brought to 1×10 ⁶ cells/ml. From 2 to 4 ml of cells were added to 5 μl of BATDA reagent and incubated for 10 minutes at 37°C. Ester bonds in cells undergo hydrolysis and form a ligand that easily absorbs water and dissolves in water, which no longer leaves the membrane. Cells were centrifuged at 1500 rpm for 5 minutes to wash the loaded K562 cells. The procedure was repeated 3 to 5 times using medium containing 1 mM probenecid (Sigma P8761). After the final wash, the cell pellet was redissolved in the culture medium and adjusted to approximately 5×10 ⁴ cells/ml.

Wells were prepared for background detection, spontaneous releases and maximum release. 100 µl of loaded target cells (5000 cells) were transferred to the wells of a V-bottom plate and 100 µl of effector cells (KHYG-1 cells) were added at various cell concentrations to give a target effector of 1:1 to 20:1. The tablet was centrifuged at 100 x g for 1 minute, and kept for 4 hours in a CO ₂ atmosphere with a humidity of 5%, at 37°C. 15 minutes before medium collection, 10 µl of lysis buffer was added to each maximum release well. The plate was centrifuged at 500 xg for 5 minutes.

20 µl of the supernatant was transferred to a 96-well flat bottom plate, after which 200 µl of prewarmed europium solution was added. The plate was kept at room temperature for 15 minutes using a plate shaker. After dissolution of K562 cells with KHYG-1 cells, they release ligand into the medium. This ligand then reacts with the europium solution to form a fluorescent chelate that is directly related to the number of dissolved cells.

Fluorescence was measured in a time resolved fluorometer using VARIOSKAN FLASH. Specific release was calculated using the following formulas:

% Specific Release = Experimental Release - Spontaneous Release/Maximum Release - Spontaneous Release

Statistical analysis was performed using Graphpad Prism 6.04 software. A paired t-test was used to compare the difference between CD96 knockdown in KHYG-1 cells using small interfering RNA and control groups (n=3).

A significant increase in specific release was found in co-cultures containing CD96 knockdown in KHYG-1 cells. This happened with all targets E : M (see figure 7).

Since fluorescence is directly related to cell dissolution, it has been confirmed that knockdown of CD96 expression in KHYG-1 cells leads to an increase in their ability to kill K562 cancer target cells.

Example 4 - NK Cell Cytotoxicity Enhanced by CD328 Knockdown (Siglec-7)

CD328 Knockdown in NK-92 Cells Using Small Interfering RNA Medium

Materials, reagents and instruments

Control small interfering RNA (catalog number: sc-37007) and small interfering RNA CD328 (catalog number: sc-106757) were purchased from Santa Cruz Biotechnology. To achieve transfection efficiency up to 90% with high cell viability (>75%) in NK-92 cells, the Nucleofector™ device (Nucleofector II, Lonza) and the Nucleofector™ T kit from Lonza were used. RPMI-1640 containing 10% FBS and 2 mM L-glutamine, without antibiotics, was used for culture after nucleofection. Mouse antibody CD328-APC (catalog number: 339206) was purchased from Biolegend.

Protocol

To prepare 10 µM small interfering RNA stock solution

Перерастворить лиофилизированный дуплекс малой интерферирующей РНК в 66 мкл воды без РНКазы (буфер для разведения малой интерферирующей РНК: sc-29527) для контроля FITC/контрольной малой интерферирующей РНК, в 330 мкл воды без РНКазы для малой интерферирующей РНК гена-мишени (малая интерферирующая РНК CD328).

Reconstitute the lyophilized siRNA duplex in 66 µl of RNase-free water (SIRNA dilution buffer: sc-29527) for FITC control/Small interfering RNA control, in 330 µl of RNase-free water for the target gene's SIRNA (small interfering RNA). CD328).

Нагреть трубку до 90°С за 1 минуту.

Heat the tube to 90°C for 1 minute.

Выдерживать при 37°С в течение 60 минут.

Maintain at 37°C for 60 minutes.

Хранить основной раствор малой интерферирующей РНК при -20°С, если не используется сразу после получения.

Store SIRNA stock solution at -20°C if not used immediately upon receipt.

Один образец Nucleofection содержит (на 100 мкл стандартных кювет)

One Nucleofection sample contains (per 100 µl standard cuvettes)

Число клеток: 2×10⁶ клеток

Number of cells: 2×10 ⁶ cells

Малая интерферирующая РНК: 4 мкл от 10 мкМ основного раствора

Small interfering RNA: 4 µl from 10 µM stock solution

Раствор Nucleofector: 100 мкл

Nucleofector solution: 100 µl

Nucleofection

Культивировать требуемое число клеток. (Пассировать один или два дня перед нуклеофекцией, так как клетки должны находиться в фазе логарифмического роста).

Cultivate the required number of cells. (Passage one or two days prior to nucleofection as the cells must be in a logarithmic growth phase).

Подготовить малую интерферирующую РНК для каждого образца.

Prepare small interfering RNA for each sample.

Предварительно нагреть раствор Nucleofector до комнатной температуры (100 мкл на образец).

Pre-warm the Nucleofector solution to room temperature (100 µl per sample).

Предварительно нагреть аликвоту культурной среды, содержащей сыворотку и добавки, при 37°С в 50 мл трубке. Подготовить 6-луночные планшеты, наполнив их 1,5 мл культурной среды, содержащей сыворотку и добавки, а также предварительно выдерживать в инкубаторе СО₂ с уровнем влажности 5%, при 37°.

Preheat an aliquot of culture medium containing serum and supplements at 37°C in a 50 ml tube. Prepare 6-well plates by filling them with 1.5 ml of culture medium containing serum and supplements, and pre-incubating in a CO ₂ incubator with a humidity level of 5%, at 37°.

Взять аликвоту культуры клеток и подсчитать клетки, чтобы определить их плотность.

Take an aliquot of the cell culture and count the cells to determine their density.

Центрифугировать необходимое число клеток при 1500 об/мин в течение 5 мин. Полностью удалить супернатант, чтобы никакая остаточная среда не покрывала клеточный осадок.

Centrifuge the required number of cells at 1500 rpm for 5 min. Completely remove the supernatant so that no residual medium covers the cell pellet.

Повторно растворить клеточный осадок в растворе Nucleofector при комнатной температуре, чтобы получить окончательную концентрацию 2×10⁶ клеток/100 мкл. Избегать выдерживания суспензии клеток в растворе Nucleofector больше 15-20 мин, так как это снижает жизнеспособность клеток и эффективность переноса генов.

Redissolve the cell pellet in Nucleofector's solution at room temperature to obtain a final concentration of 2x10 ⁶ cells/100 µl. Avoid keeping the cell suspension in the Nucleofector solution for more than 15-20 min, as this reduces cell viability and the efficiency of gene transfer.

Смешать 10 мкл суспензии клеток с малой интерферирующей РНК.

Mix 10 µl of cell suspension with small interfering RNA.

Переместить образец в кювету, сертифицированную для работы с Amaxa. Убедиться, что образец покрывает днище кюветы, избегать образования пузырей воздуха при отмеривании пипеткой. Закрыть кювету синим колпачком.

Transfer the sample to an Amaxa-certified cuvette. Make sure that the sample covers the bottom of the cuvette, avoid the formation of air bubbles when pipetting. Close the cuvette with a blue cap.

Выбрать соответствующую программу в устройстве Nucleofector (А-024 для клеток NK-92). Вставить кювету в держатель кювет (Nucleofector II: необходимо повернуть карусель по часовой стрелке в крайнее положение) и нажать кнопку «х» для запуска программы.

Select the appropriate program in the Nucleofector device (A-024 for NK-92 cells). Insert the cuvette into the cuvette holder (Nucleofector II: you must turn the carousel clockwise to the end position) and press the "x" button to start the program.

Во избежание повреждения клеток, необходимо изъять образцы из кюветы сразу по завершению работы программы (на дисплее будет отображаться «ОК»). Добавить 500 мкл предварительно нагретой культурной среды в кювету и переместить образец на подготовленный 6-луночный планшет.

To avoid damaging the cells, it is necessary to remove the samples from the cuvette as soon as the program ends (the display will show “OK”). Add 500 µl of prewarmed culture medium to the cuvette and transfer the sample to the prepared 6-well plate.

Выдерживать клетки в инкубаторе СО₂ с влажностью 5%, при 37°С. Через 16-24 часа провести проточный цитометрический анализ и взять пробу на цитотоксичность.

Incubate the cells in a CO ₂ incubator with 5% humidity, at 37°C. After 16-24 hours, perform a flow cytometric analysis and take a sample for cytotoxicity.

Results: The above protocol was followed and flow cytometric analysis of CD328 expression level in NK-92 cells was performed. The results of one of the illustrative experiments are shown in Fig. 8, which confirms a successful knockdown.

CD328 knockdown increases cytotoxicity

Materials, reagents and instruments

The DELFIA EuTDA Fluorescence Enhancement Ligand Cytotoxicity Assay Kit (Catalog Number: AD0116) was purchased from PerkinElmer. K562 target cells were cultured in RPMI-1640 medium containing 10% FBS, 2 µl L-glutamine and antibiotics. 96-well V-bottom plates (part number: 83.3926) were purchased from SARSTEDT. A 581 OR Eppendorf centrifuge (with plate rotor) was used to pellet the plate. VARIOSKAN FLASH (with Scanlt software version 2.4.3) was used to measure the fluorescence signal produced by dissolved K562 cells.

Protocol

Нагрузить клетки K562 реагентом BATDA DELFIA лиганда, увеличивающего флуоресценцию.

Load K562 cells with the BATDA DELFIA fluorescence enhancing ligand reagent.

Промыть клетки K562 культурной средой и с ее помощью довести число клеток до 1×10⁶ клеток/мл. Добавить от 2 до 4 мл клеток в 5 мкл реагента BATDA, выдерживать в течение 10 минут при 37°С.

Rinse the K562 cells with culture medium and adjust the cell count to 1×10 ⁶ cells/ml with this. Add 2 to 4 ml of cells to 5 µl of BATDA reagent, incubate for 10 minutes at 37°C.

Производить осаждение при скорости 1500 об/мин в течение 5 минут для промывки нагруженных клеток K562 от 3 до 5 раз с использованием среды, содержащей 1 мМ пробенецида (Sigma Р8761).

Precipitate at 1500 rpm for 5 minutes to wash loaded K562 cells 3 to 5 times using media containing 1 mM probenecid (Sigma P8761).

После окончательной промывки, повторной растворить клеточный осадок в культурной среде и довести приблизительно до 5×10⁴ клеток/мл.

After the final wash, re-dissolve the cell pellet in the culture medium and bring to approximately 5×10 ⁴ cells/ml.

Sampling for cytotoxicity

Подготовить лунки для обнаружения фона, спонтанных высвобождений и максимального высвобождения.

Prepare wells for background detection, spontaneous releases, and maximum release.

Отобрать пипеткой 100 мкл нагруженных клеток (5000 клеток) в планшет с V-образным днищем.

Pipette 100 µl of loaded cells (5000 cells) into a V-bottomed plate.

Добавить 100 мкл эффекторных клеток (NK-92) различных концентраций клеток. Получить эффектор для мишеней от 1:1 до 20:1.

Add 100 µl effector cells (NK-92) of various cell concentrations. Get an effector for targets from 1:1 to 20:1.

Производить осаждение планшета при 100 х г RCF в течение 1 минуты.

Precipitate the plate at 100 x g RCF for 1 minute.

Выдерживать в течение 2 часов в атмосфере СО₂ с влажностью 5%, при 37°С. За 15 минут до сбора среды, в каждую лунку максимального высвобождения добавить 10 мкл лизирующего буфера.

To withstand for 2 hours in an atmosphere of CO ₂ with a humidity of 5%, at 37°C. 15 minutes before medium collection, add 10 µl of lysis buffer to each maximum release well.

Производить осаждение планшета при 500 х г в течение 5 минут.

Produce tablet deposition at 500 x g for 5 minutes.

Переместить 20 мкл супернатанта на 96-луночный планшет с плоским днищем, добавить 200 мкл предварительно нагретого раствора европия, выдерживать при комнатной температуре, в течение 15 минут, с использованием планшетного шейкера.

Transfer 20 µl of supernatant to a 96 well flat bottom plate, add 200 µl of prewarmed europium solution, incubate at room temperature for 15 minutes using a plate shaker.

Измерить флуоресценцию в флуорометре с временным разрешением, с использованием VARIOSKAN FLASH. Специфическое высвобождение рассчитывалось с помощью следующих формул:

Measure fluorescence in a time resolved fluorometer using VARIOSKAN FLASH. Specific release was calculated using the following formulas:

% специфическое высвобождение = Экспериментальное высвобождение - Спонтанное высвобождение/Максимальное высвобождение - Спонтанное высвобождение

% Specific Release = Experimental Release - Spontaneous Release/Maximum Release - Spontaneous Release

Results: The above protocol was followed to determine the effect of CD328 knockdown on cytotoxicity. The results of one illustrative experiment are shown in Figure 9. As can be seen, cytotoxicity against target cells increased in CD328 knockdown cells.

Example 5 Knockdown/Knockout of Inhibitory Checkpoint Receptor Therapy Protocol for Blood Cancer

As shown in the above examples, knockdown or knockout of the inhibitory checkpoint receptor function can be provided in various ways. The following protocol has been developed for use in the treatment of patients with blood cancer:

Once a patient has been diagnosed with a cancer to be treated according to the present invention, an aliquot of the modified NK cells may be thawed and cultured before administration to the patient.

Alternatively, temporary mutation can be provided, for example, using small interfering RNA for one or two days, as described above. The MaxCyte Flow Electroporation platform provides the appropriate solution to enable fast, large-scale transfections in the clinic.

Exclusion of certain inhibitory checkpoint receptors may be more effective than other methods. It probably depends on the patient and the type of cancer. For this reason, the method of diagnosing cancer is not always biopsy, and cancer cells are grown in ex vivo culture. Thus, a range of NK cells with various modifications of inhibitory checkpoint receptors can be tested for cytotoxicity against a particular type of cancer. This step can be used to select the most appropriate NK cell or derivative thereof for therapy.

After successful modification, cell redissolution is performed in an appropriate vehicle (eg, saline) for intravenous and/or intratumoral administration to the patient.

Example 6 - Knockin KHYG-1 TRAIL / TRAIL variant

KHYG-1 cells were transfected with TRAIL and the TRAIL variant to assess viability and ability to kill cancer cells after transfection.

The TRAIL variant used is the variant described in WO 2009/077857. It is encoded by the wild-type TRAIL gene, which includes the D269H/E195R mutation. This mutation significantly increases the affinity of the TRAIL variant for DR5 while decreasing the affinity for both decoy receptors (DcR1 and DcR2).

Basic expression TRAIL

Baseline expression of TRAIL (CD253) in KHYG-1 cells was analyzed using flow cytometry.

Mouse antibody CD253-APC (Biolegend part number: 308210) and isotype control antibody (Biolegend part number: 400122) were used to stain cell samples and analyzed on a BD FACS Canto II flow cytometer.

KHYG-1 cells were cultured in RPMI 1640 medium containing 10% FBS, 2 mM L-glutamine, penicillin (100 U/ml)/streptomycin (100 mg/ml) and IL-2 (10 ng/ml). 0.5-1.0×10 ⁶ cells/samples were collected by centrifugation (1500 rpm×5 minutes) and the supernatant was aspirated. Cells (single cell suspension) were washed with 4 ml of chilled FACS buffer (PBS, 0.5-1% BSA, 0.1% NaN3 sodium azide). Cells were resuspended in 100 µl of chilled FACS buffer, 5 µl of antibody was added to each tube and kept frozen for 30 minutes. Cells were washed 3 times by centrifugation at 1500 rpm for 5 minutes. The cells were then resuspended in 500 μl of chilled FACS buffer and temporarily kept frozen in the dark.

Subsequently, cells were analyzed using a flow cytometer (BD FACS Canto II) and data processing was performed using FlowJo 7.6.2 software.

In FIG. 10 shows that the FACS analysis revealed a weak baseline expression of TRAIL on the surface of the KHYG-1 cell.

Knockin TRAIL / TRAIL variant by electropulse opening of cell pores

Wild type TRAIL mRNA and TRAIL variant mRNA (D269H/195R) were synthesized by TriLink Bio Technologies, aliquoted and stored at -80°C. Mouse CD253-APC (Biolegend part number: 308210) and isotype control antibody (Biolegend part number: 400122) and mouse CD107a-PE antibody (eBioscience part number: 12-1079-42) and isotype control antibodies ( eBioscience part number: 12-4714) were used to stain cell samples and analyzed on a BD FACS Canto II flow cytometer. SYTOX-Green DNA dye was used (Life Technologies catalog number: S7020; 5 mM solution in DMSO). To achieve transfection efficiency up to 90% with high cell viability in KHYG-1 cells, the Nucleofector™ device (Nucleofector II, Lonza) and the Nucleofector™ T kit from Lonza were used. Antibiotic-free RPMI-1640 medium containing 10% FBS, L-glutamine (2 mM) and IL-2 (10 ng/ml) was used for cultivation after nucleofection.

KHYG-1 and NK-92 cells were passaged one or two days before nucleofection, as the cells must be in the logarithmic growth phase. The Nucleofector solution was prewarmed to room temperature (100 µl per sample) with an aliquot of culture medium containing serum and supplements at 37° C. in a 50 ml tube. 1.5 ml of culture medium containing serum and supplements was added to 6-well plates and pre-incubated in a CO ₂ incubator with 5% humidity at 37°C. An aliquot of cell culture was prepared and cell counts were performed to determine cell density. Before complete removal of the supernatant, the required number of cells was centrifuged at 1500 rpm for 5 minutes. The cell pellet was re-dissolved in room temperature Nucleofector solution to a final concentration of 2×10 ⁶ cells/100 μl (maximum residence time in suspension = 20 minutes). 100 μl of cell suspension was mixed with 10 μg of mRNA (RNA volume<10 μl). The sample was transferred to Amaxa certified cuvettes (making sure the sample covered the bottom of the flask and avoiding air bubbles). An appropriate program was selected in the Nucleofector device (ie U-001 for KHYG-1 cells). The cuvettes were then inserted into the cuvettes holder. 500 µl of prewarmed culture medium was added to the cuvette, and the sample was transferred to the prepared 6-well plate immediately after the program ended to avoid cell damage. The cells were kept in a CO ₂ incubator with a humidity of 5% at 37°C. Flow cytometric analysis and sampling for cytotoxicity were performed 12-16 hours after electropulse opening of cell pores. Flow cytometric staining was performed as above.

In FIG. 11 and 12 show that expression of TRAIL/TRAIL variant and CD107a (NK cell activation marker) increased post-transfection, confirming successful knocking of the TRAIL genes in KHYG-1 cells.

In FIG. 13 shows proof of viability of KHYG-1 cells before and after transfection by electropulse opening of cell pores. It can be seen that there are no statistically significant differences in cell viability after cell transfection with TRAIL/TRAIL variant, confirming the non-toxicity of wild-type TRAIL or TRAIL variant expression on cells. This observation contradicts the corresponding findings regarding NK-92 cells, according to which the TRAIL variant gene knockin is toxic to cells (data not shown). However, this is likely due to the relatively high expression levels of the TRAIL DR4 and DR5 receptors on the surface of NK-92 cells (see Fig. 14).

Effect of TRAIL/TRAIL Variant on Cytotoxicity of KHYG-1 Cells The murine CD2-APC antibody (BD Pharmingen catalog number: 560642) was used. The Annexin V-FITC antibody was used (Immuno Tools part number: 31490013). SYTOX-Green DNA dye (Life Technologies catalog number: S7020) was used. A 24-well cell culture plate was used (SARSTEDT AG catalog number: 83.3922). Myeloid leukemia cell line K562, multiple myeloma cell line RPMI8226 and MM1.S were used as target cells. K562, RPMI8226, MM1.S were cultured in RPMI 1640 medium containing 10% FBS, 2 mM L-glutamine and penicillin (100 U/ml)/streptomycin (100 mg/ml).

As described above, KHYG-1 cells were transfected with TRAIL/TRAIL variant.

Target cells were washed and pelleted by centrifugation at 1500 rpm for 5 minutes. The transfected KHYG-1 cells were diluted to 0.5×10 ⁶ /ml. The target cell density was then adjusted in prewarmed RPMI 1640 medium to give an effector:target (E:M) ratio of 1:1.

0.5 ml of KHYG-1 cells and 0.5 ml of target cells were mixed in a 24-well culture plate and placed in a 5% humidity CO ₂ incubator at 37° C. for 12 hours. A flow cytometric assay was then provided to sample the cytotoxicity of KHYG-1 cells; cells cultured together (at different times) were washed and then stained with CD2-APC antibody (5 µl/sample), Annexin V-FITC (5 µl/sample) and SYTOX-Green (5 µl/sample) using buffer Annexin V solution.

The data were further analyzed using FlowJo 7.6.2 software. CD2 positive and negative gates were provided, which represent populations of KHYG-1 cells and target cells, respectively. Cells positive for Annexin V-FITC and SYTOX-Green in the CD2 negative population were then analyzed for TRAIL-induced apoptosis.

In FIG. 15, 16 and 17 show the effect of both KHYG-1 cells expressing TRAIL or the TRAIL variant on apoptosis of three target cell lines: K562, RPMI8226 and MM1.S, respectively. Clearly, for all target cell populations, TRAIL expression on KHYG-1 cells increased the rate of apoptosis when compared to normal KHYG-1 cells (not transfected with TRAIL). In addition, expression of the TRAIL variant on KHYG-1 cells further increased the rate of apoptosis in all target cell lines when compared to KHYG-1 cells transfected with wild-type TRAIL.

The cells of the present invention expressing the TRAIL variant have a significant advantage in cancer therapy due to higher affinity for the DR5 death receptor. When the cells of the present invention are involved, the development of protective strategies of cancer cells to prevent their death in a certain way is excluded. Thus, cancers cannot effectively prevent TRAIL-induced cell death by increasing the amount of TRAIL decoy receptors because the cells of the present invention are modified to remain cytotoxic under the circumstances.

Example 7 - NK Cell Therapy Protocol for Blood Cancer Using Knockin Variants of TRAIL

KHYG-1 cells were transfected with the TRAIL variant as described in Example 6 above. The following protocol has been developed for use in the treatment of patients with blood cancer:

Once a patient has been diagnosed with a cancer to be treated according to the present invention, a DR5-containing agent such as Bortezomib is administered prior to the administration of the modified NK cells, and therefore is used in small doses to increase the expression of DR5 on cancer cells, making therapy with using modified NK cells more efficient.

An aliquot of the modified NK cells is then thawed, cultured, and injected into the patient's body.

Because the TRAIL variant that is expressed by NK cells used in therapy has a low affinity for decoy receptors compared to wild-type TRAIL, increased death receptor binding is observed on the surface of cancer cells, and therefore more cancer cell apoptosis.

Before implementing the above protocol, another option is a biopsy method for diagnosing cancer and culturing cancer cells ex vivo. This step can be used to identify cancers expressing particularly high levels of decoy receptors and/or low levels of death receptors to determine if a DR5 containing agent is appropriate for a given patient. This step may also be considered during therapy using the above protocol, since this cancer may adapt, for example, to reduce the expression of DR5, and therefore may be suitable for treatment with a DR5 containing agent during therapy.

Example 8 A Low Dose of Bortezomib Sensitizes Cancer Cells to NK Cells Expressing the TRAIL Variant

Bortezomib (Bt) is a proteasome inhibitor (chemotherapeutic drug) useful in the treatment of multiple myeloma (MM). It is known that Bortezomib can increase the expression of DR5 in some types of cancer cells, including MM cells.

Before being used to target MM cells with and without Bortezomib, KHYG-1 cells were transfected with the TRAIL variant as described in Example 6 above.

DR5 expression by Bortezomib

Bortezomib was purchased from Millennium Pharmaceuticals. Mouse antibody DR5-AF647 (catalog number: 565498) was purchased from BD Pharmingen. Stained cell samples were analyzed on BD FACS Canto II.

(1) MM RPMI8226 and MM1.S cell lines were grown in RPMI1640 medium (Sigma, St. Louis, MO, USA) supplemented with 2 mM L-glutamine, 10 mM HEPES, 24 mM sodium bicarbonate, 0.01% antibiotics and 10% fetal calf serum (Sigma, St. Louis, Missouri, USA) in an atmosphere with 5% CO ₂ at 37°C.

(2) MM cells were seeded into 6-well plates 1×10 ⁶ /ml, 2 ml/well.

(3) MM cells were then treated with different doses of Bortezomib for 24 hours.

(4) Thereafter, DR5 expression in Bortezomib-treated/untreated MM cells was analyzed by flow cytometry (FIG. 18).

A low dose of Bortezomib treatment was found to increase DR5 expression in both MM cell lines (FIG. 18). An increase in DR5 was associated with a weak induction of apoptosis (data not shown). At the same time, it was found that DR5 expression could not be increased by high doses of Bortezomib due to high toxicity, leading to the death of most MM cells.

Cancer cell sensitization by Bortezomib

KHYG-1 cells were transfected with the TRAIL variant (D269H/E195R TRAIL) as described in Example 6 above.

(1) Bortezomib treated/untreated MM1.S cells were used as target cells. MM1.S cells were treated with 2.5 nM Bortezomib or vehicle (control) for 24 hours.

(2) 6 hours after electropulse opening of the cell pores of the TRAIL variant, mRNA KHYG-1 cells were cultured with MM cells in 12-well plates. After washing, cell concentrations were adjusted to 1×10 ⁶ /ml before mixing KHYG-1 and MM1.S cells in a 1:1 ratio to grow for 12 hours.

(3) A flow cytometric analysis of the cytotoxicity of KHYG-1 cells was performed. Cells cultured together were washed and then stained with CD2-APC (5 µl/sample), AnnexinV-FITC (5 µl/sample), and SYTOX-Green (5 µl/sample) antibody using AnnexinV buffer.

(4) Data were further analyzed using FlowJo 7.6.2 software. MM1.S cells are represented by a CD2 negative population.

KHYG-1 cells show a markedly positive reaction for CD2. Finally, AnnexinV-FITC and SYTOX-Green positive cells were analyzed in the CD2 negative population.

Apoptosis flow cytometric analysis was performed on MM1.S cells with/without Bortezomib pretreatment, co-cultured with KHYG-1 cells, electropulsed cell pore opening with/without the TRAIL variant (FIG. 19).

Bortezomib was found to sensitize MM cells to KHYG-1 cells expressing the TRAIL variant. Therefore, the data indicated that the agent that caused the expression of DR5 was effective in a model for increasing cytotoxicity against cancer cells, and thus may be useful in expanding the cancer therapy of the present invention.

Example 9 Confirmation of Apoptosis Induced by Variant TRAIL

Despite the overwhelming evidence for increased cytotoxicity of NK cells as a result of the expression of the TRAIL variant in the previous examples, it also needed to be tested whether the increased cytotoxicity resulted from the induction of apoptosis of cancer cells (most likely) or from involuntary activation of NK cells, which exhibited a more cytotoxic phenotype and therefore thereby destroying cancer cells through the secretion of perforin.

It has been demonstrated that concanamycin A (CMA) can induce perforin-mediated cytotoxic activity of NK cells, mainly due to the accelerated degradation of perforin by increasing the pH of lytic granules. A study was made as to whether the cytotoxicity of KHYG-1 cells expressing the TRAIL variant could be isolated if perforin-mediated cytotoxicity was partially attenuated by CMA.

Decreased expression of perforin. caused by SMA

Mouse anti-perforin antibody AF647 (catalog number: 563576) was purchased from BD Pharmingen. Concanamycin A (catalog number:

SC-202111) was purchased from Santa Cruz Biotechnology. Stained cell samples were analyzed using BD FACS Canto II.

KHYG-1 cells were cultured in RPMI 1640 medium containing 10% FBS (fetal calf serum), 2 mM L-glutamine, penicillin (100 U/mL)/streptomycin (100 mg/mL) and IL-2 (10 ng/mL). ).

(2) Next, KHYG-1 cells were treated with 100 nM CMA or vehicle (DMSO) of the same volume for 2 hours (cultured in RPMI1640 medium without penicillin/streptomycin 6 hours after electric pulse opening of cell pores).

(3) Cells were collected (1.0×10 ⁶ cells/sample) by centrifugation (1500 rpm×5 minutes) and the supernatant was aspirated.

(4) Cells were fixed in paraformaldehyde 4% in PBS at room temperature for 15 minutes.

(5) Cells were washed twice in 4 ml of FACS buffer (phosphate-buffered saline, 0.5-1% BSA, 0.1% sodium azide).

(6) Cells were permeabilized in 1 ml PBS/saponin buffer 0.1% for 30 minutes at room temperature.

(7) Cells were washed in 4 ml PBS/saponin buffer 0.1%.

(8) Cells were resuspended in 100 µl PBS/saponin buffer 0.1% before adding 5 µl of antibodies and freezing for 30 minutes to each tube.

(9) Cells were washed 3 times with PBS/saponin buffer 0.1% by centrifugation at 1500 rpm for 5 minutes.

(10) Prior to analysis, cells were resuspended in 500 µl of chilled FACS buffer and kept briefly in the dark on ice or at 4° C. in a refrigerator.

(11) Cells were analyzed on a flow cytometer (BD FACS Canto II). The data were processed using the Flow J software o 7.6.2.

CMA treatment significantly reduced the level of perforin expression in KHYG-1 cells (FIG. 20) and did not adversely affect the viability of KHYG-1 cells (FIG. 21).

Cytotoxicity of TRAIL NK cell variants in the presence of SMA

KHYG-1 cells were transfected with the TRAIL variant (D269H/E195R TRAIL) as described in Example 6 above.

(1) MM1.S cells were used as target cells.

(2) 6 hours after electropulse opening of cell pores of TRAIL mRNA, KHYG-1 cells were treated with 100 mM CMA or vehicle of the same volume for 2 hours.

(3) KHYG-1 cells were washed in RPMI1640 medium by centrifugation and resuspended in RPM11640 medium containing IL-2 with cell concentration adjusted to 1×10 ⁶ /ml.

(4) MM1.S cells were resuspended in RPMI1640 medium containing IL-2, with cell concentration adjusted to 1×10 ⁶ /ml.

(5) KHYG-1 and MM1.S cells were mixed at a ratio of 1:1 and co-cultured for 12 hours.

(6) A flow cytometric analysis of the cytotoxicity of KHYG-1 cells was performed. Cells cultured together were washed and stained with CD2-APC antibody (5 μl/sample).

(7) After washing, additional staining was performed using AnnexinV-FITC (5 µl/sample) and SYTOX-Green (5 µl/sample) using AnnexinV buffer solution.

(8) Data were further analyzed using FlowJo 7.6.2 software. MM1.S cells are represented by a CD2 negative population. KHYG-1 cells show a markedly positive CD2 count. Cells positive for AnnexinV-FITC and SYTOX-Green were then analyzed in the CD2 negative population.

Again, NK cells expressing the TRAIL variant were found to exhibit higher cytotoxicity than control cells lacking expression of the TRAIL variant (FIG. 22). However, in the present example, it was demonstrated that CMA was unable to significantly reduce the cytotoxic activity of NK cells expressing the TRAIL variant, in contrast to the result established in relation to control NK cells treated with CMA.

NK cells without the TRAIL variant (control or dummy NK cells) were found to cause 48% cancer cell death in the absence of SMA and 35.9% in those with SMA (FIG. 22). NK cells expressing the TRAIL variant were able to kill more cancer cells than control NK cells in both the presence and absence of SMA. In fact, even in the presence of SMA, NK cells expressing the TRAIL variant caused more cancer cells to die than control NK cells in the absence of SMA.

Thus, the present data demonstrate the importance of the TRAIL variant in increasing NK cell cytotoxicity to cancer cells through a mechanism less susceptible to perforin-related downregulation of cellular components. Since perforin is primarily used by NK cells to kill target cells, and many cancer cells have developed mechanisms to reduce the expression of NK cell perforin, the NK cells of the present invention provide a good alternative to prevent cytotoxic effects, less subject to attenuation by cancer cells.

Example 10 - Combined Expression of a Mutated TRAIL Variant and Knockdown of the Inhibitory Checkpoint Receptor CD96 in KHYG-1 Cells

Upon knockdown of the expression of the inhibitory checkpoint receptor CD96, as well as upon expression of the TRAIL variant, an increase in cytotoxicity in NK cells was observed. A study was also conducted with a combination of genetic modifications to provoke a synergistic effect on the cytotoxicity of NK cells.

CD96 expression was knocked down in KHYG-1 cells as shown in Example 2.

KHYG-1 cells were transfected with the TRAIL variant (D269H/E195R TRAIL) as described in Example 6 above.

(1) 12 hours after electropulse opening of cell pores, KHYG-1 cells were co-cultured with target cells (K562 or MM1.S) at a concentration of 1×10 ⁶ /ml in 12-well plates (2 ml/well) for 12 hours. The E : M ratio was 1:1.

(2) 12 hours after co-culture, cells were harvested, washed, stained with CD2-APC antibody, washed again, and further stained with AnnexinV-FITC (5 µl/sample) and SYTOX-Green (5 µl/sample) using AnnexinV buffer solution .

(3) Cell samples were analyzed using a BD FACS Canto II flow cytometer. The data were further analyzed using FlowJo 7.6.2 software. MM1.S cells are represented by a CD2 negative population. KHYG-1 cells show a markedly positive reaction for CD2. Cells positive for AnnexinV-FITC and SYTOX-Green were then analyzed in the CD2 negative population.

Simultaneous knockdown of CD96 expression and expression of the TRAIL variant in KHYG-1 cells were found to synergistically increase cell cytotoxicity against both K562 target cells (FIG. 23) and MM1.S target cells (FIG. 24). This was indicated by the fact that in both groups of target cells, most of the cases of death occurred as a result of simultaneous gene modification, and not due to individual modifications in isolation.

At the same time, there was additional evidence that CD96 knockdown increased NK cell cytotoxicity (Figs. 23 and 24), in addition to evidence that expression of mutated/variant TRAIL also increased NK cell cytotoxicity (Figs. 23 and 24). 24).

Thus, the present invention provides NK cells and cell lines and their products for use in blood cancer therapy.

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SEQUENCE LISTING

<110> Onkimmune Limited

<120> MODIFIED NATURAL KILLER CELLS AND NATURAL KILLER CELL LINES

HAVING INCREASED CYTOTOXICITY

<130> P40153EP

<160> 12

<170>PatentIn version 3.5

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ggagccccat ttaacacgat gcctccttct ctcctaggag cctacccaaa acccaccctc 2160

tcagcccagc ccagccctgt ggtgacctca ggaggaaggg tgaccctcca gtgtgagtca 2220

caggtggcat ttggcggctt cattctgtgt aaggaaggag aagatgaaca cccacaatgc 2280

ctgaactccc agccccatgc ccgtgggtcg tcccgcgcca tcttctccgt gggccccgtg 2340

agcccgaatc gcaggtggtc gcacaggtgc tatggttatg acttgaactc tccctatgtg 2400

tggtcttcac ccagtgatct cctggagctc ctggtcccag gtgagaaatt cacagcattg 2460

tctggagttc cctgagtctc cctgagtctc caggcaggtg gggagcagcc gtgtctcagg 2520

gcagttccag gtgggatgat gttggggcga gagggctcag gggtcctggg gccagagaca 2580

caggaagatc agcagtggtg aggcaccggg ggagagggag ggtttgtggg gaagcctgag 2640

ggtcggctcc tggaaaccat gagcaccttt tcccaggtgt ttctaagaag ccatcactct 2700

cagtgcagcc gggtcctgtc atggcccctg gggaaagcct gaccctccag tgtgtctctg 2760

atgtcggcta tgacagattt gttctgtaca aggaggggga acgtgacctt cgccagctcc 2820

ctggccggca gccccaggct gggctctccc aggccaactt caccctgggc cctgtgagcc 2880

gctcctacgg gggccagtac agatgctacg gtgcacacaa cctctcctct gagtgctcgg 2940

cccccagcga ccccctggac atcctgatca caggtgagga gcccagcggg ttcagtcagg 3000

gacccagact ctgcacaggc cctgccgggg gaatccaatt agtgatggcc aggatgaggc 3060

ggggggtggt cccaagggag ggagagacag agagagagac aggggatgggg tggggagggg 3120

aagactcaga gaaaacagag acagaggctc ctagagaggc ctggggaggt ctcagctcag 3180

agcaaggtgg ggcagcccct cacccatcct tcttctctcc aggacagatc cgtggcacac 3240

ccttcatctc agtgcagcca ggccccacag tggcctcagg agagaacgtg accctgctgt 3300

gtcagtcatg gcggcagttc cacactttcc ttctgaccaa ggcgggagca gctgatgccc 3360

cactccgtct aagatcaata cacgaatatc ctaagtacca ggctgaattc cccatgagtc 3420

ctgtgacctc agccccgcg gggacctaca ggtgctacgg ctcactcaac tccgacccct 3480

acctgctgtc tcaccccagt gagcccctgg agctcgtggt ctcaggtggg ggccttgacc 3540

ctgtcctctc tgagctcaaa ggctcagctc aggccctgcc ccccaggaga gctctgggct 3600

gggatggagt gagcgggggt ctgagcgggg ctcagccagt gggagactca ccctcagagg 3660

gaaggaggac aacaggccct cccaggcctg cgcacactca gcggcatcgc cagcatcatg 3720

gacaggagag gcgggtggag ggaggggcct ggggaggcca cagggcccat gtagagaaat 3780

ttggtttgag gtggagactt caggaaagcc ccagctcctc accctcctct cattctttca 3840

cccaggaccc tccatggggtt cgcccccc acccaccggt cccatctcca cacctggtga 3900

gtccctgagg cctctggctc gaagggagcg cagcgacccc cagggcagct ttgagtgtcc 3960

aggaggatcc cattcccttc agggactcaa tcaagggctt ctgtccaggg agctgggcag 4020

agccagagga ggggccacag ggtccccagg gctctgaggc tgggctggtg aggggtgggg 4080

ggtcaaggca gagagaaatg ttggggccca gcctggggga ggagcagccg ggctgatgtg 4140

gggagcaggg cagccccagc ccctcacctcc ccgtcctgac ccagcaggcc ctgaggacca 4200

gcccctcacc cccactgggt cggatcccca aagtggtgag tgaggggctc tgagtgggag 4260

gtgggcgggg tcccggggag gcaggggtgg gttctgtcct aggttcaggc tcctctggag 4320

gtggtgatgt agacaggctc ctcccctgcc tgggcctcag tttctccaag tgtaaaggag 4380

agaggcctgc aggtgggaaa gttcctttca gctctcactc ccagctgtga cctcctggga 4440

gaggaggccc ctcagggaag actccaagac tcgattccgc gggggcctgt cccgtcccac 4500

ctgcagcaga gacggtgacc tggggcaggg gaggggagca gagtcgtggt tcaggacggt 4560

aaggctcttt ccctgcagct ccggggctcg gctctggtgc aggaacaagg gctgcaggtc 4620

agactcccag gctcccttcc cagctctgcc gcttcctggc tgggggcccg gggcaggcga 4680

ttcccctctc tgagcgtcag ttttttcatct gtagagtggg tggggtggat gtttgtgtgc 4740

tgcacgactg ttgtgggggt tggaggtggt gaacagaagg tccagcagtc acctgcacac 4800

agtaggcgct catttcaatg acatcacccc catccctgac atcatcgtgc tcaaggtctg 4860

ggaaggcacc tgggggttgt gatcggcatc ttggtggccg tcgtcctact gctcctcctc 4920

ctcctcctcc tcttcctcat cctccgacat cgacgtcagg gcaaacactg gacatcgagt 4980

gagtagggaa ggggaaaccc tgtgggccga ccgagggtgg gctcagggca cagccaaaga 5040

gaatccaaac cactgggcaa atgcagcttt gagaaactgt tccagcattt ctcaccaggt 5100

gaatggagaa agcacttaac gtcagtccca tctacaaata taaagtgtcc tcggggctca 5160

gtcccatcta caaatgtaaa gtgtccttcg gactctgtcc atctcatgag gcatttggaa 5220

catggaggca ggagtgtttt taggtttcct tccttacctt cgagctgtgt gtgcaggggca 5280

gggggctcca atgttcccag ggctgaggct ctgtccttct tcccccagcc cagagaaagg 5340

ctgatttcca acatcctgca ggggctgtgg ggccagagcc cacagacaga ggcctgcagt 5400

ggaggtaatt ctgcccgaag accccagact cccacctgct cgtggcccat acactgcccc 5460

taaagctccc attcctcccc caggtccagc ccagctgccg acgccgga agaaaacctc 5520

tgtgagtgag aggaagaggt gaccagccag gagggagata ggggccccga agtttccgta 5580

gcaatgggga aaggggcacc ggctggaaag ggtctggggc tcagggtgag atcatctcac 5640

cccacactgt gggacctcag ggacattgca gcccctccct gcatctcagt agccccatct 5700

gggagcaggg caggggctgg caggactcag aggtcccagg gaaccttccc aagagacgaa 5760

ccccttgctc tgccccagca gatgctgccg tgaaggacac acagcctgaa gatggggtgg 5820

agatggacac tcgggtgaga ccccgcccct gtcccaggca ccaaaggcct cctggtgcca 5880

gatctaatcc agcaggactt ctctgtcctc cttcccccgg ctctcagcat cgtcacggtg 5940

gacccctcct tgtccagcac gctgcctccc gcctgctgtg acctcactct ctcctgctgt 6000

ccctgggacct cgtggggcctc ctcccggggtc cccttcctgc tcctcatcct ctgtttggcc 6060

gtctggttgt tagagcgctc cccaggcctc tggaggatga ggaataaatg aaccaccccg 6120

gtcccctggg ctccccttca ttcattcaac cagtgagtgt tcccagggag ctcactgtgg 6180

atcaggctcc ccatgggagc tgcagacaca gcagggagca aagccgcccc cgcctcctga 6240

gctcacctca tggtgggaga caaaatgcaa ataaatgcgc catgtccagg agtgcaacgt 6300

6360

atggaagggg agggctgtct gctcagctgt catctgagaa gcctggacag agtggggcac 6420

acgatcctct aatggacgag cccctgcagg cagaggaaac agccgtgcaa aggccccgag 6480

gcagcagcga gctcttgcgg gaaggcccat gaggctgcag ccaaatgggc aaggtcaaag 6540

tgaggagcag aggccagaac cacaggaagg gagcggccag accctccacg gccttagggc 6600

6660

attgactcca ggaggtcagg gggactcaag gacacccccc accactgtct ctctccagca 6720

gagcccacat gatgaagacc cccaggcagt gacatatgcc ccggtgaaac actccagacc 6780

taggagagaa atggcctctc ctccctcccc actgtccggg gaattcctgg acacaaagga 6840

cagacaggca gaagaggaca gacagatgga cactgagaga gtcctttcct ctccaggccc 6900

ccaggcctcc ccccacccca ccacgttcct tacctctcac tctcccccgc tgcaggctgc 6960

tgcatctgaa gccccccagg atgtgaccta cgcccagctg cacagcttga ccctcagacg 7020

7080

cgccaccctg gccatccact agcccgggagg gtacgcagac tccacactca gtagaaggag 7140

actcaggact gctgaaggca cgggagctgc ccccagtgga caccaatgaa ccccagtcag 7200

ccctggaccc taacaaagac catgaggaga tgctgggaac tttgggactc acttgattct 7260

gcagtcgaaa taactaatat ccctacattt tttaattaaa gcaacagact tctcaataat 7320

caatgagtta accgagaaaa ctaaaatcag aagtaagaat gtgctttaaa ctgaatcaca 7380

atataaatat tacacatcac acaatgaaat tgaaaaagta caaaccacaa atgaaaaaag 7440

tagaaacgaa aaaaaaaaac taggaaatga atgacgttgg ctttcgtata aggaatttag 7500

aaaaagaata accaattatt ccaaatgaag gtgtaagaaa gggaataaga agaagaagag 7560

ttgctcatga ggaaaaacca aaacttgaaa attcaacaaa gccaatgaag ctcattcttg 7620

aaaatattaa ttacagtcat aaatcctaac tacattgagc aagagaaaga aagagcaggc 7680

acgcatttcc atatgggagt gagccagcag acagcccagc agatcctaca cacattttca 7740

caaactaacc ccagaacagg ctgcaaacct ataccaatat actagaaaat gcagattaaa 7800

tggatgaaat attcaaaact ggagtttaca taatgaacgt aagagtaatc agagaatctg 7860

actcatttta aatgtgtgtg tatgtgtgtg tatatatatg tgtgtgtgtg tgtgtgtgtg 7920

tgtgtgtgaa aaacattgac tgtaataaaa atgttcccat cgtatcaact ccagttcagg 7980

8040

agagcatcac aaatgtttct tgagaataga aaaagaggca atgtgcccgg gtgcggtggc 8100

tcacgcctgt aatctcaaca cctaggggagg cagaggccac agattacttg aggccggggag 8160

ttcaagacca gcatggccaa caaggcaaaa ccccatctct actaaaaata caaaaattag 8220

ctggacatgg tggcgcacgc tgcaatccca gctacttggg aggcagaggc aggaggatca 8280

cttgaatgaa cccgggaggt ggaggttgaa gtgagcaaaa acaaaccccc tacaattcag 8340

cctaggatat gtttattaaa tttacatttg tctttttgct taagattgct ttggtattca 8400

tcctcttttt ggttccatat gaattttagg atttttttct aattctgtga aaaaaatgat 8460

gttgatattt tgatgggaat tgcattgaac ctaaatattg ctttgggaag tgtgatcatt 8520

ttcacaatat tgattctgcc aatccatgag catgggatat atttctattt tgctgtgtca 8580

tctacgattt ctttctgcag cattttgttg ttcttcttgt agagatcttt cacctcctca 8640

gttaggtata ttcttagata tttttaattt tttgcaactg atgtacaagg gattgagttt 8700

tgcagcaacc tggatgagct ggaggccatt attcatgaca ccacatccag ctaatttttg 8760

tatttcttgt agagatgagg ttttgccatg ttgcccaggc tggtcttgaa ctcctgggcc 8820

caagtgaccc gcccgccttg acctcccaaa gtgctgggac tgcaggcatg agccacggtg 8880

cctggcccat catagcactt ttgatcatta ggataattcc ttctccttgt catttttgga 8940

cacatgcttc ccacatgcct catcttccag agagggtttc caccagggct gtgctgggag 9000

ttaaggctgg aaaaggggag atggttccac ctgccagtgc cacatgagtc tactcaggggc 9060

tgtaaccagc agggagggtc cagtgtgagc ctcagactcg catgtgggac agacgcccat 9120

gtgtgacaac gctgcagtga atctgtttca cacacatgga ggaggcggct cagggctgac 9180

catggacctg agtcaatgag cagagatatc ccagtgccat ccacaaacac aggggagaag 9240

gagccacaac ttcccacttt catccaaaac cccgacccct ccctgtctgt gagggccctg 9300

gggttctcct ctgtctcata cagaggcaga aacctccccc ttagtgaccc ccagctttgc 9360

aagtcaccag cagcccctcg gcgctggcat cttctgcttc ttaaggtttc ctgcctatga 9420

caggaagtct catttctcat ttttcttcatt ggaccatggc tacatatttc agacacatta 9480

taagtaggtt ttcccagtgt taggagcaga tgtgggctgt tgagcacata agtcactcac 9540

<210> 2

<211> 101

<212> PRT

<213> Homo sapiens

<400> 2

Gly Ala Tyr Pro Lys Pro Thr Leu Ser Ala Gln Pro Ser Pro Val Val

1 5 10 15

Thr Ser Gly Gly Arg Val Thr Leu Gln Cys Glu Ser Gln Val Ala Phe

20 25 30

Gly Gly Phe Ile Leu Cys Lys Glu Gly Glu Asp Glu His Pro Gln Cys

35 40 45

Leu Asn Ser Gln Pro His Ala Arg Gly Ser Ser Arg Ala Ile Phe Ser

50 55 60

Val Gly Pro Val Ser Pro Asn Arg Arg Trp Ser His Arg Cys Tyr Gly

65 70 75 80

Tyr Asp Leu Asn Ser Pro Tyr Val Trp Ser Ser Pro Ser Asp Leu Leu

85 90 95

Glu Leu Leu Val Pro

100

<210> 3

<211> 23

<212> DNA

<213> Homo sapiens

<400> 3

gagtcacagg tggcatttgg cgg 23

<210> 4

<211> 23

<212> DNA

<213> Homo sapiens

<400> 4

cgaatcgcag gtggtcgcac agg 23

<210> 5

<211> 20

<212> DNA

<213> Homo sapiens

<400> 5

gggagcccca tttaacacga 20

<210> 6

<211> 20

<212> DNA

<213> Homo sapiens

<400> 6

gggaactca gggaactcca 20

<210> 7

<211> 7375

<212> DNA

<213> Homo sapiens

<400> 7

ctttggacct tcttcaactc tgttttgtct ctgttgagtt aaggctttta agaacacctg 60

aattctttcc ttctgcaaaa ccagaggcag cttcttttcc gcctattttc agtttatttc 120

ttgtgatttt agtttttttc tcttaaccaa atgctaaatg gatttaggag aaataaactt 180

atttgtaaag ctgtcaaggg accattagaa ggatggtgct tcacagatag aatacagttt 240

ttattaatga tgcctagaca aatcctgcca ttagcccaag ggctcagaaa gttagcagcc 300

360

attgaataaa attgggattt aggaggaccc ttgtactcca ggaaattctc caagtctcca 420

cttagttatc cagatcctca aagtgaacat gaagcttcag tttcaaattg aatacatttt 480

ccatccatgg attggcttgt tttgttcagt tgagtgcttg aggttgtctt ttcgacgtaa 540

cagctaaacc cacggcttcc tttctcgtaa aaccaaaaca aaaaggcttt ctattcaagt 600

gccttctgtg tgtgcacatg tgtaatacat atctgggatc aaagctatct atataaagtc 660

cttgattctg tgtgggttca aacacatttc aaagcttcag gatcctgaaa ggttttgctc 720

tacttcctga agacctgaac accgctccca taaagccatg gcttgccttg gatttcagcg 780

gcacaaggct cagctgaacc tggctaccag gacctggccc tgcactctcc tgttttttct 840

tctcttcatc cctgtcttct gcaaaggtga gtgagacttt tggagcatga agatggagga 900

ggtgtttctc ctacctggggt ttcatttgtt tcagcagtca aaggcagtga tttatagcaa 960

agccagaagt taaaggtaaa actccaatct ggcttggctg gctctgtatt ccaggggccag 1020

caggggagcag ttgggcggca gcaaataagg caaagagata gctcagaaca gagcgccagg 1080

tatttagtag gggcttcatg aatgcatgtg agttggttta gtagagagac acaggcaatt 1140

tcagaccctt ctatgagact ggaagtgatt taagagggaa aggatagcca tagtcctgaa 1200

tacatttgag ctgggtttca ggatgagctc acaagttcct ttaaaaaaaa ttgacttaag 1260

caaatcctgg gaagagtttt tttgctatac aattcaaggt tttaaggtcc tcggattcat 1320

atactttata aatgaattag ccagcttgtt taaaatgtag ggaaattgtg ggaagaatgc 1380

cttctttact taattcaagg tttttaaggtt ctcttaatca attctactag ctaattagcc 1440

1500

agaaagccac cagtctgttt ggcatacaat acttaattgt tgcctgacct acgtgtgggt 1560

ttcagatgca gatcctcagt tttcagctct tcagagactg acaccaggtt tgttacacgg 1620

cttaaaatga tgagtatatc cattgaatct caaccttatc tctctctaga ccttcttggt 1680

taagaaacca tgtagtttgt atgaagtagg tactcaaaag atatttgatg atttaatttt 1740

tactggagaa gaaatattca tatatgtttt cttattttta catgttttaa atatgtaaag 1800

attaaataaa cactcttaga agtatttaaa ttttcctaaag taaatttatc tcaaccagta 1860

acaggaccct cccaatactg gaaagttgag tgtgaccgca tttagtggtg atgagtgtga 1920

gcttgcttgg gggagagggca ggacatttag gatttcttaa gcttagagtc aatacaataa 1980

agattattga gtgctcactt gggtgggcta taatcactgc tcacaggagt tcatgaacca 2040

caagtaaaag agtgaggaga tatgattagc tcacaaataa ctttaataca gagcagaaag 2100

taatgaacta ctgcaatgga gttatcacag tgctaaggat gctcagaggg catctctgat 2160

aggcagaggt gagggttagg gaaggaagct gtagtctagc tagctagagc tgctggaata 2220

gacatgacaa tggctgctgc caaactgttt tctcttctga ggacagatgt cccgtgcaag 2280

tggcttggtg gaagggacta gtgtctctaa tatagggtga tttataagca ggaaagtgtg 2340

tcctagaaat tcagaccaga gtgatagatt ggaattggat catgggggac tcattgaatg 2400

ttatttattg tatttgtttt tgcgatcagt gttagtaaag tgtcaaaggg attgagcaga 2460

tgagtgacat catgcaacac aagttttgag tttcacttgt cagactgact ggagaggggc 2520

ctggttagtt acagggaaggt aatttggcat gcagccacta tttttgagtt gatgcaagcc 2580

tctctgtatg gagagctggt ctcctttatc ctgtgggaaa agagaacaaa ggagcatggg 2640

agtgttcaag ggaaggagaa ataaagggca gagaggcagc ggtggtgtca ggggaagccc 2700

acaggagtta acagcagggt tgcctcaacc tagagaggaa gcgacctggt gccctcggct 2760

ctgtggcttc cttcatctaa caacatcttc cactctacaa caatgccagg gaaggcggag 2820

gctggtacag tgcatcaaga cacagctact cctgggtgac agaggttcag ggccagctca 2880

ctaagtaggc agaagttttt gacatatact ttgagagata aagcaagatt ctgtacctca 2940

accttcagaa tttcccctac cactcattat agttccggag ctatatagct cctatcattc 3000

3060

tgaaaaaaat gaaggacatg ggggaagtgt gacttgcccc aaatcacata tttcatggta 3120

gagccaggtc ttctgtttgt catatcagtg ttcttcctgc cacaaccatc ttgaagaatc 3180

tatttctcag taagaaaata tctttatgga gagtagctgg aaaacagttg agagatggag 3240

gggaggctgg gggtgtggag aggggaaggg gtaagtgata gattcgttga aggggggaga 3300

aaaggccgtg gggatgaagc tagaaggcag aagggcttgc ctgggcttgg ccatgaagga 3360

gcatgagttc actgagttcc ctttggcttt tccatgctag caatgcacgt ggcccagcct 3420

gctgtggtac tggccagcag ccgaggcatc gccagctttg tgtgtgagta tgcatctcca 3480

ggcaaagcca ctgaggtccg ggtgacagtg cttcggcagg ctgacagcca ggtgactgaa 3540

gtctgtgcgg caacctacat gatggggaat gagttgacct tcctagatga ttccatctgc 3600

acgggcacct ccagtggaaa tcaagtgaac ctcactatcc aaggactgag ggccatggac 3660

acgggactct acatctgcaa ggtggagctc atgtacccac cgccatacta cctgggcata 3720

ggcaacggaa cccagattta tgtaattggt gagcaaagcc atttcactga gttgacacct 3780

gttgcattgc agtcttctat gcacaaaaac agttttgttc cttaatttca ggaggtttac 3840

ttttaggact gtggacattc tctttaagag ttctgtacca catggtagcc ttgcttattg 3900

tgggtggcaa ccttaatagc attctgactg taaaataaaa tgatttgggg aagttggggc 3960

tctcgctctg gagtgctaac catcatgacg tttgatctgt acttttgata tgatatgatg 4020

ctcctgggga agtagtccca aatagccaaa cctattggtg ggctacccat gcaatttagg 4080

ggtggacctc aaggcctgga agctctaatg tccttttttc accaatgttg gggagtagag 4140

ccctagagtt taaaactgtc tcagggaggc tctgctttgt tttctgttgc agatccagaa 4200

ccgtgcccag attctgactt ccctcctctgg atccttgcag cagttagttc ggggttgttt 4260

ttttatagct ttctcctcac agctgtttct ttgagcaaaa tggtgagtgt ggtgctgatg 4320

gtgcaccatg tctgatgggg atacctttag tggtatcaac tggccaaaag atgatgttga 4380

4440

cgcactagaa ccgtaggcat tggcatttga ggtttcagaa tgactaatat tttagatgaa 4500

tttgtttgac attgaatgtt catgtgcttc tgagcagggt ttcaatttga gtaaccgttg 4560

caataacatg gggcagctgt tttgctcttt gtcttcatga caactgtact taagctaaca 4620

gccctgaaac atgagattag gctgggcaga atgctgctag agaggaccac ttggatggtc 4680

tttattctcc ttctccatgt ccctctccat cacctggaag tcacctctgg gtgccactct 4740

ggtgccttcc ttgtcgaagc tgtagctgct cacatgacac ctatccctgt tatccagttt 4800

4860

tatcacaggt gttggtattg gtggtagaag aggtagaatt atggaatcag gcctcctgtc 4920

aggatttctt tttgacagtc ccctctcagac acctctgcct aaggccagct ttgccattac 4980

aaactctccc ttctccctct ctcccttctt ctcttcctct tccttcttct cgctctttct 5040

ctctctctct ttctccctct ctgtctctta tacacataca caaagatata ctctattcca 5100

acatcctcta cccaacctga cagagatgtc ctttgctgta ggttcagcag tggggatgag 5160

aaatacagct ctcaaacagg ataactaaag cttattatct tatcaagctt gttcccttgc 5220

agacaagatt gatcaattat cataggcttt ctgggtgttc tttctgaagc tttctcaaag 5280

tctctttctc ctatcttcca ttcaaggcaa atgattgcca tttaacatca aaatcacagt 5340

tatttatcta aaataaattt taatagctga atcaagaaaa tctcctgagg tttataattc 5400

tgtatgctgt gaacattcat ttttaaccag ctagggaccc aatatgtgtt gagttctatt 5460

atggttagaa gtggcttccg tattcctcag tagtaattac tgtttctttt tgtgtttgac 5520

agctaaagaa aagaagccct cttacaacag gggtctatgt gaaaatgccc ccaacagagc 5580

cagaatgtga aaagcaattt cagcctttatt ttattcccat caattgagaa accattatga 5640

agagagagt ccatatttca atttccaaga gctgaggcaa ttctaacttt tttgctatcc 5700

agctattttt atttgtttgt gcatttgggg ggaattcatc tctctttaat ataaagttgg 5760

atgcggaacc caaattacgt gtactacaat ttaaagcaaa ggagtagaaa gacagagctg 5820

ggatgtttct gtcacatcag ctccactttc agtgaaagca tcacttggga ttaatatggg 5880

gatgcagcat tatgatgtgg gtcaaggaat taagttaggg aatggcacag cccaaagaag 5940

gaaaaggcag ggagcgagg agaagactat attgtacaca ccttatattt acgtatgaga 6000

6060

tgtatgatta catcaaggct tcaaaaatac tcacatggct atgttttagc cagtgatgct 6120

6180

atatatatat atatatattt taatttgata gtattgtgca tagagccacg tatgtttttg 6240

tgtatttgtt aatggtttga atataaacac tatatggcag tgtctttcca ccttggggtcc 6300

6360

tgctaactag cttggaaact ggatgaggtc atagcagtgc ttgattgcgt ggaattgtgc 6420

tgagttggtg ttgacatgtg ctttggggct tttacaccag ttcctttcaa tggtttgcaa 6480

ggaagccaca gctggtggta tctgagttga cttgacagaa cactgtcttg aagacaatgg 6540

cttactccag gagacccaca ggtatgacct tctaggaagc tccagttcga tgggcccaat 6600

6660

6720

atgcaacaac atgtataata tttttaatta aataaaaatc tgtggtggtc gttttccgga 6780

gttgtcttta tcatccttgc atttgaatat tgtgttcaaa tttttgattg attcattcag 6840

tatctggtgg agtctccaat attagaaata ctggaaacaa actgaaaaac cacaaaagga 6900

6960

gtatccatcc tctttccttt tgatttcttc accactattt gggatataac gtgggttaac 7020

acagacatag cagtccttta taaatcaatt ggcatgctgt ttaacacagg ttcttcacct 7080

cccctttctt accgcctgct ttctcagctc aactatcaca ggcattacag ttgtcatggc 7140

aaccccaatg ttggcaacca cgtcccttgc agccattttg atctgccttc ctgaaatata 7200

gagcttttcc ctgtggcttc caaatgaact attttgcaaa tgtggggaaa acacacacct 7260

gtggtcctat gttgctatca gctggcacac ctaggcctgg cacactaagc cctctgtgat 7320

tcttgcttaa ccaatgtata gtctcagcac atttggtttc cacttaaggt ttcct 7375

<210> 8

<211> 36

<212> PRT

<213> Homo sapiens

<400> 8

Met Ala Cys Leu Gly Phe Gln Arg His Lys Ala Gln Leu Asn Leu Ala

1 5 10 15

Thr Arg Thr Trp Pro Cys Thr Leu Leu Phe Phe Leu Leu Phe Ile Pro

20 25 30

Val Phe Cys Lys

35

<210> 9

<211> 23

<212> DNA

<213> Homo sapiens

<400> 9

cactcacctt tgcagaagac agg 23

<210> 10

<211> 23

<212> DNA

<213> Homo sapiens

<400> 10

ccttgtgccg ctgaaatcca agg 23

<210> 11

<211> 29

<212> DNA

<213> Homo sapiens

<400> 11

aggacccttg tactccagga aattctcca 29

<210> 12

<211> 26

<212> DNA

<213> Homo sapiens

<400> 12

agccctact aaatacctgg cgctct 26

<---

Claims (14)

1. A human natural killer (NK) cell for cancer treatment, genetically modified to express a TRAIL variant comprising the D269H/E195R mutation. 2. A human NK cell according to claim 1 for the treatment of cancer, genetically modified by transfection or transduction with a vector containing the TRAIL D269H/E195R gene variant. 3. Human NK cell according to any one of paragraphs. 1, 2 for the treatment of cancer, further genetically modified with a wild-type NK cell to reduce the expression of one or more inhibitory checkpoint receptors selected from CD96 (TACTILE), CD152 (CTLA4), CD223 (LAG-3), CD279 (PD-1), CD328 (SIGLEC7), SIGLEC9, TIGIT or TIM-3. 4. Human NK cell according to any one of paragraphs. 1-3 for the treatment of cancer, characterized in that the NK cell targets the bone marrow. 5. Human NK cell according to any one of paragraphs. 1-4 for the treatment of cancer, characterized in that the NK cell is derived from the KHYG-1 cell line. 6. Human NK cell according to any one of paragraphs. 1-5, intended for the treatment of cancer, characterized in that the TRAIL variant is associated with one or more co-stimulatory domains of the NK cell. 7. A human NK cell according to claim 6 for the treatment of cancer, characterized in that the costimulatory domain of the NK cell is 41BB/CD137, CD3zeta/CD247, DAP12 or DAP10. 8. Human NK cell according to any one of paragraphs. 1-7, intended for the treatment of cancer, which is further genetically modified to overexpress IL-2 or IL-15, for example, by transfection or transduction. 9. Human NK cell according to any one of paragraphs. 1-8 for the treatment of cancer, characterized in that the type of cancer is solid cancer. 10. Human NK cell according to any one of paragraphs. 1-9, characterized in that the type of cancer is blood cancer. 11. Human NK cell for the treatment of blood cancer according to claim 10, characterized in that the type of blood cancer is acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia ( CML), Hodgkin's lymphoma, non-Hodgkin's lymphoma, including T-cell and B-cell lymphomas, asymptomatic myeloma, indolent multiple myeloma (SMM), active myeloma, or light chain myeloma. 12. Human NK cell according to any one of paragraphs. 1-11, intended for the treatment of cancer, characterized in that the treatment includes the introduction of an agent capable of increasing the expression of TRAIL death receptors on cancer cells. 13. A human NK cell according to claim 12 for the treatment of cancer, characterized in that the agent is capable of increasing DR5. 14. A human NK cell according to claim 13 for the treatment of cancer, characterized in that Bortezomib is used as the agent.

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