RU2776890C2 - Cell therapy based on improved natural killer cells - Google Patents

Abstract

FIELD: cell biology; medicine.

SUBSTANCE: present invention relates to cell biology and medicine, in particular to a combination of natural killer cells (hereinafter – NK) or a line of NK cells, providing a cytotoxic effect, with an antibody-antagonist relatively to IL-6R and a method for the treatment of blood cancer, the cells of which express IL-6R, using the specified combination.

EFFECT: present invention allows for the improvement of efficiency and improvement of therapy of blood cancer, the cells of which express IL-6R.

51 cl, 70 dwg, 1 tbl, 11 ex

Description

Introduction

The present invention relates to therapies in which natural killer (NK) cells or NK cell lines are used as effector cells for the treatment of cancer. It relates to combination therapies, methods of treatment and compositions, in particular for the treatment of blood cancers.

State of the art invention

Despite significant investment in many types of physical, drug and other therapies, cancer in humans remains a significant cause of death in all age groups.

For example, 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 , Mounce 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.

Natural killer (NK) cells are cytotoxic lymphocytes with distinct phenotypes and effector functions that differ from natural killer T cells (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 several 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 (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 improved survival with AML treatment (Ruggeri, Capanni 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 (mentioned above) 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 shows 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.

Sifaldi et al. (Journal Arthritis Rheumatol. November 2015; 67(11): 3037-46) reported that IL-6 reduces NK cell cytotoxicity in mice and human patients with arthritis. In the work of Kahn et al. (Hum Reprod. October 10, 2014; 29(10): 2176-89) it has been demonstrated that NK cytotoxicity in the peritoneal fluid of patients suffering from endometriosis can be reversed by neutralizing IL-6 antibodies. The choice of IL-6/STAT3 as a target in cancer therapy was proposed by Bon et al. (PLoS One. October 7, 2013; 8(10): e75788) without substantiating data. In addition, IL-6 has previously been shown to play a role in upregulating PD-L1 expression in certain myeloma cell lines (Tamura et al., Leukemia. Feb. 2013; 27(2): 464-72). Separately, other researchers report that IL-6 antagonists provoked a decrease in tumor control (Eidorn et al., Cancer Immunol Immunother. May 2017; 66(5): 667-671).

There is a need for an alternative and preferably improved cancer therapy using such NK cells and using NK cells in general.

The purpose of the present invention is to provide a combination therapy using NK cells and NK cell lines that are more effective, eg more cytotoxic, than NK cell-only therapies. More specific embodiments are directed to providing methods for treating certain cancers, for example, blood cancers such as leukemia.

annotation

Provided herein are methods for treating cancer using IL-6 antagonists in combination with NK cells. Cells may be taken from patients, in which case the intervention may involve the administration of an antagonist, thereby relying on the patient's already available NK cells. The cells may be administered as part of a therapy, in which case they may be autologous, allogeneic, primary cells or cell lines, etc. Together, these therapies are referred to as a combination because both NK cells and antagonists are required.

The invention provides methods of treatment, including the administration of antagonists, the administration of antagonists and cells, and the administration of cells (in which antibodies are separately present, for example, as part of a coupled therapy). The invention provides a combination for use in the treatment of cancer. The invention further provides compositions comprising both cells and antagonists.

In addition, the invention provides NK cells modified to reduce or eliminate IL-6 receptor expression.

In particular, diseases treatable in accordance with the present invention using the NK cells described herein include cancers such as blood cancer (eg, leukemia) and, in particular, acute myeloid leukemia and myeloma. Thus, human tumors and cancers can be treated. References to tumors in this document include references to neoplasms.

Detailed description

Accordingly, the present invention uses natural killer (NK) cells or an NK cell line in combination therapy. As detailed below, NK cells and NK cell lines can also be genetically modified to increase their anti-cancer cytotoxic activity in such therapies. Together, primary NK cells and NK cell lines will be referred to as NK cells (unless the context requires otherwise). The NK cells described herein additionally use IL-6 signaling antagonists (alone or in combination with NK cells).

The invention thus provides, inter alia, a natural killer (NK) cell or NK cell line in combination with an IL-6 antagonist for use in the treatment of cancer. Accordingly, expression of IL-6 receptors and/or expression of one or more inhibitory checkpoint receptor ligands, eg PDL-1 and/or PDL-2, occurs in cancer.

Similarly, the invention provides methods for treating cancer comprising administering to a patient an effective amount of a combination of an NK cell and an IL-6 antagonist. Again, in cancer there is expression of IL-6 receptors and/or expression of one or more inhibitory checkpoint receptor ligands, eg PDL-1 and/or PDL-2.

The NK cell or cell line may additionally have a pre-linked IL-6 antagonist. The antagonist and cells may also be unbound as single or separate preparations.

The present combination therapy can also be used as an adjunct, separate anticancer therapy, as therapies in which endogenous NK cells are used as immune effector cells. Thus, the treatment of cancer, including antibody-dependent cell-mediated cytotoxicity (ADCC), can be supplemented by using the methods and compositions described herein.

NK cells or cell lines to be used in accordance with the present invention may be genetically modified to reduce the expression of the IL-6 receptor. Separately, the NK cell line can be selected from known lines, such as NK-92 or KHYG-1, as shown in the examples below. A cell or cell line may show a high level of expression of the E-selectin ligand on the cell surface. The expression of the E-selectin ligand is determined using the HECA-452 antibody. In a specific embodiment, the NK cell or cell line exhibits a high level of E-selectin ligand on the cell surface. Preferably, a high level of E-selectin ligand on the cell surface is at least two-, four-, six-, eight-, or ten-fold in binding of the HECA-452 antibody relative to the binding of the isotype control antibody. This manifestation can be measured, for example, by flow cytometry. The KHYG-1 cell line is the only such cell line that expresses high levels of the HECA-452 antigen compared to other NK cell lines such as NK-92 cells. Other methods of modifying a cell to express a high level of E-selectin ligand include, for example: 1) chemical treatment with GDP-fucose substrate and alpha-1,3-fucosyltransferase-VI enzyme; and 2) expression or overexpression of FUT6 or FUT7. In addition, a cell line on the cell surface may exhibit low levels of expression of a TRAIL receptor, such as DR4 or DR5. For example, compared to NK-92 cells, KHYG-1 cells show low levels of DR4 or DR5 expression. Expression of the TRAIL receptor on the cell surface can be quantified, for example, by flow cytometry, as detailed in the examples.

This document further suggests therapies in which NK cells are initially present in the patient; thus, for the treatment of cancer of the present invention, it is proposed to use an IL-6 antagonist in which cancer cells express IL-6 receptors, as well as cancer treatments comprising administering an effective amount of an IL-6 antagonist.

As detailed in the examples, the present combination has been shown to be effective in in vitro models of cancer, and in particular in the expression of IL-6 receptors in cancer. Additionally, cancers in which one or more inhibitory checkpoint receptor ligands are expressed, for example, in response to or during treatment, are amenable to the treatment of the present invention. In a specific example, expression of PDL-1 and/or PDL-2 occurred in cancers treated with the combination of the present invention.

The invention also provides compositions comprising an NK cell or cell line and an IL-6 antagonist. The cells in the present compositions may be further modified in accordance with one or more of the modifications set forth hereinafter.

Using the present invention, IL-6 antagonists act by blocking IL-6 signaling and thereby inhibit IL-6 activity.

Examples of useful IL-6 antagonists of the present invention include antibodies, respectively, as indicated below. These include, for example, IL-6 antibodies as well as IL-6R and gp130 antibodies. These antibodies bind to IL-6, IL-6R or gp130 to inhibit the association between IL-6 and IL-6R or IL-6R and gp130.

Thus, these antibodies block IL-6 signaling by inhibiting its activity and thereby reduce IL-6 signaling in NK cells and/or (cancer) target cells.

Beneficial antibodies thereby reduce or stop IL-6 signaling and also mediate this effect on NK/target cells. One feature of specific embodiments of the present invention is that, along with the first effect of blocking the direct effect of IL-6 on NK cells (otherwise IL-6 would reduce the activity of NK cells), there is a second effect of blocking the effect of IL -6 per (cancer) target cells; otherwise, IL-6 would provoke or promote a reaction in the target cell that would reduce the cytotoxic activity of NK cells. Thus, it has been demonstrated that the blocking effect of IL-6 on target cells prevents the expression of inhibitory checkpoint receptor ligands in the target cell; thus, the target cell is more vulnerable to the cytotoxic effects of the NK cell.

Examples of IL-6 antibodies suitable for use in the combination therapy of the present invention include siltuximab (FDA approved antibody), olokizumab (CDP6038), elsilimomab, BMS-945429 (clazakizumab/ALD518 ) MH-166 and sirukumab (CNTO 136). Examples of IL-6R antibodies include tocilizumab (an antibody approved by the US Food and Drug Administration), sarilumab, PM-1, and AUK12-20. An example of a gp130 antibody is AM64.

Additional IL-6 antagonists useful in the combination therapy of the present invention include mAb 1339 (OP-R003), PF-04236921, MEDI 5117, C326 (AMG-220), 6a, sgp130Fc (FE301), ALX-0061 , NRI, SANT-7, ERBF, ERBA, MDL-A, SC144, raloxifene (keoxifene/LY156758/evista), bazedoxifene (viviant), and LMT-28.

Monoclonal bodies are prepared by standard techniques using one position from, for example, IL-6, IL-6R or gp130 as the sensitizing antigen for immunization.

Antibodies specific for IL-6R can bind one or both types of existing IL-6R, i.e. membrane-bound IL-6R; and soluble IL-6R (sIL-6R) separated from the cell membrane. SIL-6R mainly consists of the extracellular domain of IL-6R attached to the cell membrane and differs from membrane-bound IL-6R in that it lacks a transmembrane domain and/or an intracellular domain.

Antibodies specific for IL-6, IL-6R or gp130 can be administered separately or simultaneously with NK cells or NK cell lines of the present invention. Since the optimal pharmacokinetics of NK cells and NK cell lines may differ from the optimal pharmacokinetics of antibodies, NK cells and NK cell lines can be administered on a different schedule than for IL-6, IL-6R or gp130 antibodies. For example, an antibody of the present invention may be administered weekly, while NK cells and cell lines may be administered twice a week. Another example is that the antibody of the present invention may be administered biweekly, while NK cells and cell lines may be administered weekly.

Unless otherwise indicated, the term "antibody" as used herein includes antigen-binding fragments of antibodies, i.e. fragments of antibodies that retain the ability to bind specifically to the antigen associated with a full-length antibody, for example, fragments that retain one or more CDRs (complementary detectable regions). Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments; diabody; linear antibodies; single chain antibody molecules, such as single chain variable region (scFv) fragments, nanobodies, and multispecific antibodies formed from antibody fragments with separate specificities, such as bispecific antibodies. Preferably, the antibodies are humanized such that the individual's immune response to the antibody is reduced. For example, antibodies can be chimeric, eg, a non-human variable region with a human constant region, or with a CDR grafted; for example, non-human CDR regions with human constant region and variable region framework sequences.

As stated above, current observations indicate that, along with NK cell activity, IL-6 signaling in cancer cells indirectly suppresses NK cell cytotoxicity by increasing the amount of inhibitory checkpoint receptor (cIR) ligands on the cancer cell membrane, and this other effect IL-6 signaling can also be prevented or attenuated. These cIR ligands bind cIR to NK cells and reduce the cytotoxicity of these cells.

Inhibitory checkpoint receptor ligands mediated by cancer-expressing IL-6R include ligands for cIR CD96 (TACTILE), CD152 (CTLA4), CD223 (LAG-3), CD279 (PD-1), CD328 (SIGLEC7) , SIGLEC9, TIGIT and TIM-3.

In accordance with the present invention, only an effective dose of IL-6 antagonists can be administered to a patient. IL-6 antagonists are able to prevent IL-6 signaling in cancer cells and endogenous NK cells. The inhibitory effect of IL-6 on NK cell cytotoxicity - both directly, through signaling in NK cells, and indirectly, through signaling in cancer cells - is prevented by IL-6 antagonists.

Thus, the present invention provides IL-6 antagonists for use in the treatment of cancers, for example, in which IL-6R is expressed. The cancer to be treated may be a solid tissue tumor such as a liver tumor including hepatocellular carcinoma; lung tumor; non-small cell lung cancer; a tumor of the pancreas, including adenocarcinoma or acinar cell carcinoma of the pancreas; bowel cancer; stomach cancer; kidney cancer, including renal cell carcinoma (RCC) and transitional cell carcinoma (TCC, also known as urothelial cell carcinoma); ovarian cancer; prostate cancer; mammary cancer; or cervical cancer. The types of cancers to be treated should preferably be hematological malignancies, also referred to as blood cancers, which include leukemia, myeloma and lymphoma. The preferred choice of cancer for treatment is from acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), hairy cell leukemia, T-cell prolymphocytic leukemia, large granular leukemia lymphocytes, Hodgkin's lymphoma, non-Hodgkin's lymphoma, including T-cell and B-cell lymphomas, asymptomatic myeloma, indolent multiple myeloma (SMM), multiple myeloma (MM), or light chain myeloma.

The combination therapy of the present invention may be administered with NK cells in general, including but not limited to autologous or allogeneic cells or specific lines such as NK92, KHYG-1 or others. In the specific examples below, selected NK cells are used for illustrative purposes only. Therapy can be used modified NK cells, as described in the description.

In a first example of such modified cells, NK cells with or without reduced inhibitory checkpoint receptor (cIR) function are provided/used. Thus, in the examples below, NK cells are formed by knocking out one or more cIR genes. Preferably, these receptors are specific cIRs. 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, TIGIT and /or TIM-3.

Also contemplated/used are NK cells in which one or more inhibitory receptor signaling pathways are knocked out or show 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 enhanced 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 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, a 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.

As used herein, 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 in both the "resting" and "active" states 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.

Thus, a NK cell without an inhibitory checkpoint receptor gene selected from CD96 (TACTILE), CD152 (CTLA4), CD223 (LAG-3), CD279 (PD-1), CD328 ( SIGLEC7), SIGLEC9, TIGIT and TIM-3. In a particular embodiment, the NK cell or cell line lacks two or more CD96 (TACTILE), CD152 (CTLA4), CD223 (LAG-3), CD279 (PD-1), CD328 (SIGLEC7), SIGLEC9, TIGIT, and TIM- 3. In a specific embodiment, the NK cell or cell line lacks two or more CD96 (TACTILE), CD152 (CTLA4), CD279 (PD-1), or CD328 (SIGLEC7). In a certain embodiment, the NK cell or cell line lacks three or more CD96 (TACTILE), CD152 (CTLA4), CD279 (PD-1), or CD328 (SIGLEC7) In a specific embodiment, the NK cell or cell line lacks CD96 ( TACTILE) and CD328 (SIGLEC7).

Alternatively, the NK cell may show reduced expression of an inhibitory checkpoint receptor selected from CD96 (TACTILE), CD152 (CTLA4), CD223 (LAG-3), CD279 (PD-1), CD328 (SIGLEC7), SIGLEC9, TIGIT and TIM-3. In a certain embodiment, the NK cell or cell line may show down-expression of two or more of CD96 (TACTILE), CD152 (CTLA4), CD223 (LAG-3), CD279 (PD-1), CD328 (SIGLEC7), SIGLEC9, TIGIT, and TIM-3. In a particular embodiment, the NK cell or cell line exhibits reduced expression of two or more CD96 (TACTILE), CD152 (CTLA4), CD279 (PD-1) or CD328 (SIGLEC7). In a certain embodiment, the NK cell or cell line shows reduced expression of three or more CD96 (TACTILE), CD152 (CTLA4), CD279 (PD-1), or CD328 (SIGLEC7). In a certain embodiment, the NK cell or cell line shows reduced expression of CD96 (TACTILE) and CD328 (SIGLEC7). NK cells can be modified to reduce inhibitory checkpoint receptor expression by using, for example, inhibitory small RNA constructs, short hairpin RNA (plasmid or viral vector) constructs, or desensitizing technology.

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). Specific embodiments, described in more detail below, 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, NK cells expressing a TRAIL ligand or preferably a mutated (variant) TRAIL ligand are provided/used. 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. Reduced affinity for the bait 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 exhibited by the TRAIL variant/mutation refers to the baseline affinity that is exhibited by wild-type TRAIL. Preferably, the affinity is increased by at least 10%, more preferably by at least 25%, 50%, or 100%, compared to wild-type TRAIL development.

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, 100 or even 1000 times or more greater than the affinity for one or more DR4, DcR1 and DcR2. More preferably, the affinity for DR5 is at least 1.5, 2, 5, 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.

Combination therapy provides the opportunity to further improve the treatment of cancer.

Further, specific embodiments include/use a 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 a 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 a particular embodiment, the TRAIL receptor variant has two mutations in the human TRAIL amino acids, D269H and E195R. In another embodiment, the TRAIL receptor variant has three mutations in the human amino acids TRAIL, G131R, N199R, and K201H.

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 does not adversely affect the 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, which is administered at a low dose capable of causing an increase in DR5 expression on cancer cells, can be used as an agent.

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, gefinitib, 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 and/or uses 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 and/or uses NK cells and NK cell lines, preferably KHYG-1 cells, as well as derivatives thereof, modified to express one or more CARs. In general, CARs that bind to cancer antigen, e.g., CD38, CD319/SLAMF-7, TNFRSF17/BCMA, SYND1/CD138, CD229, CD47, Her2/Neu, epidermal growth factor receptor (EGFR), CD123/IL3-RA, CD19, CD20, CD22, mesothelin, EpCAM, MUC1, MUC16, Tn antigen, NEU5GC, NeuGcGM3, GD2, CLL-1, HERV-K. Also contemplated are CARs that bind to blood cancer-specific antigens, e.g. CLL-1, HERV-K.

For use in cancer therapy, CARs specifically bind 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, a bispecific CAR can 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 myeloma), 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 may have 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/use 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 the growth medium with cytokines on an ongoing basis.

The present invention also provides/uses 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 technique, such as CRISPR, is used to generate a novel NK cell line with enhanced cytotoxicity, such as a KHYG-1 cell derivative.

In some embodiments, the invention provides a method for obtaining an NK cell or cell line that 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/uses 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 thereof 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 purposes 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 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.

The present invention also provides NK cells per se with reduced or absent IL-6R function, eg genetically engineered NK cells without IL-6 receptor function. The NK cells of the present invention can also be modified, as described herein, with reduced or absent function of one or more cIRs to express mutated TRAIL or all three modifications.

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 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.

Modified NK cells can be administered in amounts greater than about 1×10 ⁶ cells/kg, 1×10 ⁷ cells/kg, 1×10 ⁸ cells/kg, 1×10 ⁹ cells/kg and 1×10 ¹⁰ cells/kg . In certain embodiments, the modified NK cells are administered in an amount from about 1×10 ⁶ to 1×10 ¹¹ cells/kg. In certain embodiments, the modified NK cells are administered in an amount from about 1×10 ⁷ to 1×10 ¹⁰ cells/kg. In certain embodiments, the modified NK cells are administered in an amount from about 1×10 ⁸ to 1×10 ¹⁰ cells/kg. In certain embodiments, the modified NK cells are administered in an amount from about 1×10 ⁹ to 1×10 ¹⁰ cells/kg. In certain embodiments, the modified NK cells are administered in an amount from about 1×10 ⁷ to 1×10 ⁹ cells/kg. In certain embodiments, the modified NK cells are administered in an amount from about 1×10 ⁷ to 1×10 ⁸ cells/kg. In certain embodiments, the modified NK cells are administered in an amount from about 1×10 ⁸ to 1×10 ⁹ cells/kg. In the case of a hematological malignancy, the cells may be administered intravenously. In the case of a solid tissue tumor, intratumoral or intraperitoneal methods can be used to introduce cells.

IL-6 antagonist antibodies, as well as combinations thereof, can be administered to a patient at a concentration of from about 0.1 to 30 mg/kg, for example, 0.4 mg/kg, 0.8 mg/kg, 1.6 mg/kg, or 4 mg/kg of body weight. In a preferred embodiment, the IL-6 antagonist antibodies described herein, as well as combinations thereof, are administered to the patient at a frequency of once every twenty-six weeks or less, for example, once every sixteen weeks or less, once every eight weeks or less. , or once every four weeks or less. In another preferred embodiment, the IL-6 antagonist antibodies are administered to the patient at a frequency of about once in a period of one week, once in a period of two weeks, once in a period of three weeks, or once times over a period of four weeks.

It is contemplated that the effective dose may depend upon the characteristics of the patient, e.g., age, sex, pregnancy status, body mass index, lean body mass, the condition or conditions for which the formulation is intended, other health conditions of the patient that may affect metabolism or tolerability of the formulation, IL-6 levels in the patient, and resistance to exposure to the formulation (eg, in a patient who develops antibodies against the formulation).

Modified NK cells that are administered using an IL-6 antagonist can also be administered with certain ancillary drugs including interleukin-2 (IL-2), interleukin 8 (IL-8), interleukin-12 (IL-12), interleukin-15 (IL-15) or a proteasome inhibitor such as Bortezomib, Carfilzomib, Ixazomib, or a combination thereof. In certain embodiments, any of IL-2, IL-8, IL-12, IL-15, or a proteasome inhibitor may be administered to a patient prior to administration of the modified NK cell. In certain embodiments, any of IL-2, IL-8, IL-12, IL-15, or a proteasome inhibitor may be administered to a patient during administration of the modified NK cell. In certain embodiments, any of IL-2, IL-8, IL-12, IL-15, or a proteasome inhibitor may be administered to a patient following administration of the modified NK cell. In certain embodiments, IL-2, IL-8, IL-12, IL-15 activity can be provided by a non-interleukin agonist for IL-2, IL8, IL-12, and IL-15 receptors. For example, an interleukin-12 agonist may be ALT-803 or ALT-801; the interleukin-15 agonist may be NIZ985.

This document also contemplates certain adjuvants in treatment, preferably intermittent administration of cyclophosphamide or a tetracycline antibiotic. Any of these adjuvants may be administered before or during treatment with the modified NK cell. They can also be administered simultaneously during a course of treatment using a modified NK cell and an IL-6 antagonist, such as an IL-6 antibody.

An anti-tetracycline antibody such as doxycycline may be administered at a concentration of about 50 to 300 mg per day, or at a concentration of about 100 to 200 mg per day orally or intravenously. Other equivalent tetracycline antibiotics may also be used, such as tetracycline, doxycycline, minocycline, tigecycline, demeclocycline, metacycline, chlortetracycline, oxytetracycline, lymecycline, meclocycline, or rolitetracycline.

Cyclophosphamide can be administered orally and intravenously. In certain embodiments, the implementation of the introduction of cyclophosphamide is intermittent, for example, in successive small doses. In certain embodiments, the implementation of cyclophosphamide is administered orally at a dose of about 100 mg to 250 mg per day or every other day for one, two, three, four or more weeks. In certain embodiments, cyclophosphamide is administered orally at a dose of about 50 mg per day for one, two, three, four or more weeks. In certain embodiments, the implementation of cyclophosphamide is administered intravenously at a dose of about 1000 mg to 250 mg per week for one, two, three, four or more weeks. In certain embodiments, the implementation of cyclophosphamide is administered intravenously at a dose of about 750 mg, 500 mg and 250 mg or less per week for one, two, three, four or more weeks.

The present invention provides a method of treating cancer comprising administering to a patient an effective amount of a combination of an NK cell and an IL-6 antagonist. The methods include preferred and additional features of other aspects of the invention as described herein.

The present invention described herein provides a method of treating cancer comprising administering to a patient an effective amount of (a) an NK cell and (b) an IL-6 antagonist. The NK cell and IL-6 antagonist are additionally administered separately. In addition, the patient is pretreated with an IL-6 antagonist prior to administration of the NK cell.

The invention also provides a pharmaceutical composition comprising an NK cell or cell line and an IL-6 antagonist. Pharmaceutical compositions include preferred and additional features of other aspects of the invention, as described herein.

Unless the context clearly indicates otherwise, the singular form used herein refers to plural references.

The term "about" as used herein means a volume close to that specified, such as 10%, 5%, or 1%.

Unless otherwise indicated, the term "antibody" as used herein includes antigen-binding fragments of antibodies, i.e. fragments of antibodies that retain the ability to bind specifically to the antigen associated with a full-length antibody, for example, fragments that retain one or more CDRs (complementary detectable regions). Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments; diabody; linear antibodies; single chain antibody molecules, such as single chain variable region (scFv) fragments, nanobodies, and multispecific antibodies formed from antibody fragments with separate specificities, such as bispecific antibodies. Preferably, the antibodies are humanized such that the individual's immune response to the antibody is reduced. For example, antibodies can be chimeric, eg, a non-human variable region with a human constant region, or with a CDR grafted; for example, non-human CDR regions with human constant region and variable region framework sequences.

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

1. Natural killer (NK) cell or NK cell line in combination with an IL-6 antagonist for use in cancer treatment.

2. An NK cell or cell line for use in Embodiment 1, wherein the cancer expresses IL-6 receptors.

3. An NK cell or cell line for use in embodiment 1 or 2, wherein the cancer expresses PDL-1 and/or PDL-2.

4. An NK cell or cell line for use in any of the preceding embodiments, wherein the IL-6 antagonist is an antibody that binds one of IL-6, IL-6R, or gp130.

5. NK cell or cell line for use in embodiment 4, characterized in that the IL-6 antibody is selected from siltuximab, olokizumab (CDP6038), elsilimomab, BMS-945429 (ALD518), MH-166, and sirukumab (CNTO 136) .

6. An NK cell or cell line for use in embodiment 5 or 6, wherein the IL-6R antibody is selected from tocilizumab, sarilumab, PM-1, and AUK12-20.

7. An NK cell or cell line for use in embodiment 4, wherein the gp130 antibody is AM64.

8. NK cell or cell line for use according to any of the previous embodiments in combination with a separate anticancer therapy

9. An NK cell or cell line for use in embodiment 8, characterized in that endogenous NK cells are used as immune effector cells in a separate antitumor therapy.

10. An NK cell or cell line for use according to any one of embodiments 8 or 9, characterized in that the particular antitumor therapy is antibody-dependent cell-mediated cytotoxicity (ADCC).

11. An NK cell or cell line for use according to any of the preceding embodiments, wherein the cancer is a blood cancer.

12 NK cell or cell line for use in embodiment 11, wherein the type of blood cancer is acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), Hodgkins' lymphoma, non-Hodgkin's lymphoma, including T-cell and B-cell lymphomas, asymptomatic myeloma, indolent multiple myeloma (SMM), multiple myeloma (MM), or light chain myeloma.

13. An NK cell or cell line for use in any of the preceding embodiments, wherein the NK cell or cell line has been genetically modified to reduce the expression of one or more inhibitory checkpoint receptors.

14. NK cell or cell line for use according to embodiment 13, characterized in that the inhibitory checkpoint receptors are selected from CD96 (TACTILE), CD152 (CTLA4), CD223 (LAG-3), CD279 (PD-1), CD328 (SIGLEC7), SIGLEC9, TIGIT and TIM-3.

15. An NK cell or cell line for use in any of the preceding embodiments, wherein the NK cell or cell line has been genetically modified to express a mutated TRAIL ligand.

16. NK cell or cell line for use in embodiment 15, characterized in that the mutated TRAIL ligand has increased affinity for TRAIL receptors, e.g., DR4 and/or DR5

17. An NK cell or cell line for use in embodiment 15 or 16 wherein the mutated TRAIL ligand has reduced affinity for TRAIL decoy receptors.

18. An NK cell or cell line for use in any of the previous embodiments expressing a chimeric antigen receptor (CAR).

19. An NK cell or cell line for use in embodiment 18, wherein the CAR is a bispecific CAR.

20. An NK cell or cell line for use in embodiment 19, wherein the bispecific CAR binds two ligands in one cell type.

21. An NK cell or cell line for use in embodiment 19, wherein the bispecific CAR binds one ligand on either of two separate cell types.

22. An NK cell or cell line for use in embodiments 11 and 22, wherein the CAR ligand(s) or bispecific CAR are expressed on the cancer cell.

23. An NK cell or cell line for use in embodiment 22, wherein both ligands for the bispecific CAR are expressed on the cancer cell.

24. An NK cell or cell line for use in embodiment 22, wherein the ligands for the bispecific CAR are expressed on the cancer and immune effector cell.

25. An NK cell or cell line for use in any of the previous embodiments, wherein the NK cell or cell line has been genetically modified to reduce expression of the IL-6 receptor.

26. An NK cell or cell line for use according to any of the previous embodiments, characterized in that the NK cell line is KHYG-1.

27. An IL-6 antagonist for use in the treatment of cancer, characterized in that cancer cells express IL-6 receptors.

28. An IL-6 antagonist for use in embodiment 27, wherein the cancer expresses PDL-1 and/or PDL-2.

29 An IL-6 antagonist for use in embodiments 27-28, wherein the IL-6 antagonist is an antibody that binds one of IL-6, IL-6R, or gp130.

30. An IL-6 antagonist for use in embodiment 29, wherein the IL-6 antibody is selected from siltuximab, olokizumab (CDP6038), elsilimomab, BMS-945429 (ALD518), MH-166, and sirukumab (CNTO 136).

31. An IL-6 antagonist for use in embodiment 29, wherein the IL-6R antibody is selected from tocilizumab, sarilumab, PM-1 nAUK12-20.

32. An IL-6 antagonist for use in embodiment 29, wherein the gp130 antibody is AM64.

33. An IL-6 antagonist for use according to any one of embodiments 27-32, wherein the IL-6 antagonist is used in combination with a separate anticancer therapy.

34. An IL-6 antagonist for use according to embodiment 33, characterized in that endogenous NK cells are used as immune effector cells in a separate antitumor therapy.

35. An IL-6 antagonist for use according to any one of embodiments 33 or 34, wherein the particular antitumor therapy is antibody-dependent cell-mediated cytotoxicity (ADCC).

36. An IL-6 antagonist for use in any one of embodiments 27-36, wherein the cancer is blood cancer.

37. An IL-6 antagonist for use in embodiment 36, wherein the type of blood cancer is acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), lymphoma Hodgkins, non-Hodgkin's lymphoma, including T-cell and B-cell lymphomas, asymptomatic myeloma, indolent multiple myeloma (SMM), multiple myeloma (MM), or light chain myeloma.

38. A method of treating cancer comprising administering to a patient an effective amount of a combination of an NK cell and an IL-6 antagonist.

39. The method of embodiment 38 wherein the cancer expresses IL-6 receptors.

40. The method of any one of embodiments 38-39, wherein the cancer expresses PDL-1 and/or PDL-2.

41. The method of any one of embodiments 38-40, wherein the NK cell or cell line is expected to be pre-linked with an IL-6 antagonist.

42. The method of embodiments 38-41, wherein the IL-6 antagonist is an antibody that binds one of IL-6, IL-6R, or gp130.

43. The method of embodiment 42, wherein the IL-6 antibody is selected from siltuximab, olokizumab (CDP6038), elsilimomab, BMS-945429 (ALD518), MH-166, and sirukumab (CNTO 136).

44. The method of embodiment 42, wherein the IL-6R antibody is selected from tocilizumab, sarilumab, PM-1, and AUK12-20.

45. The method of embodiment 42 wherein the gp130 antibody is AM64.

46. The method of any one of embodiments 38-45 used in combination with a separate anticancer therapy.

47. The method of embodiment 46, wherein endogenous NK cells are used as immune effector cells in a separate antitumor therapy.

48. The method of any one of embodiments 46 or 47, wherein the particular anticancer therapy is antibody-dependent cell-mediated cytotoxicity (ADCC).

49. The method of any one of embodiments 38-48, wherein the cancer is blood cancer.

50. The method of embodiment 49, wherein the type of blood cancer is acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), Hodgkins' lymphoma, non-Hodgkin's lymphoma, including T-cell and B-cell lymphomas, asymptomatic myeloma, indolent multiple myeloma (SMM), multiple myeloma (MM), or light chain myeloma.

51. The method of any one of embodiments 38-50, wherein the NK cell or cell line has been genetically modified to reduce the expression of one or more inhibitory checkpoint receptors.

52. The method of Embodiment 51 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.

53. The method of any one of embodiments 38-52, wherein the NK cell or cell line has been genetically modified to express the mutated TRAIL ligand.

54. The method of embodiment 53 wherein the mutated TRAIL ligand has increased affinity for TRAIL receptors, eg DR4 and/or DR5.

55. The method of any one of embodiments 53 or 54, wherein the mutated TRAIL ligand has reduced affinity for TRAIL decoy receptors.

56. The method of any one of embodiments 38-55, wherein the NK cell or cell line expresses a chimeric antigen receptor (CAR).

57. The method of embodiment 56 wherein the CAR is a bispecific CAR.

58. The method of embodiment 57 wherein the bispecific CAR binds two ligands on the same cell type.

59. The method of embodiment 58 wherein the bispecific CAR binds one ligand on either of two separate cell types.

60. The method of embodiment 58 or 59, wherein the ligand(s) for the CAR or the bispecific CAR are expressed on the cancer cell.

61. The method of embodiment 60 wherein the ligands for the bispecific CAR are expressed on the cancer cell.

62. The method of embodiment 60 wherein the ligands for the bispecific CAR are expressed on the cancer and immune effector cell.

63. The method of any one of embodiments 38-62, wherein the NK cell or cell line has been genetically modified to reduce expression of the IL-6 receptor.

64. The method of any one of embodiments 38-63, wherein the NK cell line is KHYG-1.

65. A composition comprising an NK cell or cell line and an IL-6 antagonist, wherein the NK cell is further modified as described herein.

66. NK cell or cell line modified to reduce or abolish IL-6 receptor function.

67. An NK cell or cell line of embodiment 66 genetically modified to reduce or eliminate the expression of IL-6R receptors.

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 the human body, and in relation to IL-6 antagonists, to increase NK cytotoxicity -cells and reduce the rejection response provoked by the (cancer) target cell in response to NK cell cytotoxicity.

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 increased NK cell cytotoxicity via 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 co-cultured with KHYG-1 cells with CD96 small interfering RNA and/or variant TRAIL expression;

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

In FIG. 25 shows FACS plots of apoptosis in RPMI8226 cells co-cultured with KHYG-1 cells or KHYG-1 cells pre-exposed to IL-6 for 12 hours;

In FIG. 26 shows FACS plots of apoptosis in RPMI8226 cells with or without 12 hour pre-exposure to IL-6 co-cultured with KHYG-1 cells;

In FIG. 27 shows FACS plots of apoptosis in MM1.S cells co-cultured with KHYG-1 cells or KHYG-1 cells pre-exposed to IL-6 for 12 hours;

In FIG. 28 shows FACS plots of apoptosis in MM1.S cells with or without pre-exposure to IL-6 for 12 hours co-cultured with KHYG-1 cells;

In FIG. 29 shows FACS plots of apoptosis in K562 cells co-cultured with KHYG-1 cells or KHYG-1 cells pre-exposed to IL-6 for 12 hours;

In FIG. 30 shows FACS plots of apoptosis in K562 cells with or without 12 hours pre-exposure to IL-6 co-cultured with KHYG-1 cells;

In FIG. 31 and 32 show FACS plots of IL-6R (CD126) expression on KHYG-1 cells;

In FIG. 33 and 34 show FACS plots of gp130 (CD130) expression on KHYG-1 cells;

In FIG. 35 shows a FACS plot of IL-6R (CD126) expression on NK-2 cells;

In FIG. 36 shows a FACS plot of gp130 (CD130) expression on NK-92 cells;

In FIG. 37 shows a FACS plot of IL-6R (CD126) and gp130 (CD130) expression on U266 cells;

In FIG. 38 shows a FACS plot of IL-6R (CD126) and gp130 (CD130) expression on RPMI8226 cells;

In FIG. 39 shows FACS plots of IL-6R (CD126) and gp130 (CD130) expression on NCI-H929 cells;

In FIG. 40 shows a FACS plot of IL-6R (CD126) and gp130 (CD130) expression on KMS11 cells;

In FIG. 41 shows a FACS plot of IL-6R (CD126) and gp130 (CD130) expression on MM1.S cells;

In FIG. 42 and 43 show FACS plots of IL-6R (CD126) expression on K562 cells;

In FIG. 44 shows a FACS plot of gp130 (CD130) expression on K562 cells;

In FIG. 45 shows FACS plots of PD-L1 expression on RPMI8226 cells with or without IL-6 over 48 hours;

In FIG. 46 shows FACS plots of PD-L2 expression on RPMI8226 cells with or without IL-6 over 48 hours;

In FIG. 47 shows FACS plots of PD-L1 expression on NCI-H929 cells with or without IL-6 over 48 hours;

In FIG. 48 shows FACS plots of PD-L2 expression on NCI-H929 cells with or without IL-6 over 48 hours;

In FIG. 49 and 50 show FACS plots of PD-L1 expression on MM1.S cells with or without IL-6 over 48 hours;

In FIG. 51 and 52 show FACS plots of PD-L2 expression on MM1.S cells with or without IL-6 over 48 hours;

In FIG. 53 and 54 show FACS plots of PD-L1 expression on U266 cells with or without IL-6 over 48 hours;

In FIG. 55 and 56 show FACS plots of PD-L2 expression on U266 cells with or without IL-6 over 48 hours;

In FIG. 57-60 show FACS plots of PD-L1 expression on U266 cells with or without IL-6 over 48 hours;

In FIG. 61-64 show FACS plots of PD-L2 expression on U266 cells with or without IL-6 over 48 hours;

In FIG. 65 shows gel electrophoresis of STAT3-S727, STAT3-Tyr705, total STAT3, SHP-1, SHP-2 and P44/42 after exposure to IL-2 on a KHYG-1 cell;

In FIG. 66 shows electrophoresis of STAT3-S727, STAT3-Tyr705, total STAT3, SHP-1, SHP-2, p44/42, and total p44/42 in gel after exposure to IL-6 on a KHYG-1 cell;

In FIG. 67 shows electrophoresis of P44/42, total p44/42, and actin in gel after exposure to IL-2 and IL-6 on a KHYG-1 cell;

In FIG. 68 shows FACS plots of PD-1 expression on KHYG-1 cells cultured alone and after co-culture with K562, U937, HL60, Raji, RPMI8226, U266, or MM1.S cells for 24 hours;

In FIG. 69 shows an increase in cytotoxicity with neutralizing IL-6 against U266 cells; and

In FIG. 70 shows that IL-6 directly inhibits the cytotoxicity of the KHYG-1 cell by reducing the expression of NKG2D (70A) and increasing the expression of NKG2A (70B).

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 RNAg18 LIR2 "guideline" sequence;

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 "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 genes encoding the "classic" 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.

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 was confirmed. T7E1 endonuclease I recognizes and cleaves non-ideally matched DNAs, thereby allowing comparison of potential 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 end 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 knockout of the CTLA4 gene 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 crRNA (20 µM) and 4 µl tRNA (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: 1 450 V

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).

Stage 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 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 RNAi

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 5% humidity CO ₂ incubator 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 various effector:target (E:M) ratios.

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 (P/N: 83.3926) were purchased from SARSTEDT. A 5810R 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. The ester bonds in the cells undergo hydrolysis and form a hydrophilic ligand that no longer extends beyond 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 (5,000 cells) were transferred to the wells of the V-bottom plate and 100 µl of effector cells (KHYG-1 cells) were added at various cell concentrations to obtain an effector for targets from 1:1 to 20:1 . The tablet was centrifuged at 100 x g for 1 minute, and kept for 4 hours in an atmosphere of CO ₂ 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×g 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 tablet was kept at room temperature for 15 minutes using a tablet shaker. After K562 cells are dissolved by KHYG-1 cells, they release a 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 at all ratios 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 results in an increase in their ability to kill K562 cancer target cells.

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

Knockdown of CD328 in NK-92 Cells Using 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 interfering RNA stock solution

• Reconstitute lyophilized siRNA duplex in 66 µl RNase-free water (SIRNA dilution buffer: sc-29527) for FITC control/Small interfering RNA control, in 165 µl RNase-free water for the target gene's small interfering RNA (small interfering RNA). RNA CD328).

• Heat the tube to 90°C for 1 minute.

• Hold at 37°C for 60 minutes.

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

• One Nucleofection sample contains (per 100 µl standard cuvettes)

• Number of cells: 2×10 ⁶ cells.

• Small interfering RNA: 4 ml from 10 µM stock solution

• Nucleofector solution: 100 µl

Nucleofection

• Culture the required number of cells (passage one or two days before nucleofection, as the cells must be in the logarithmic growth phase).

• Prepare small interfering RNA for each sample.

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

• 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°C.

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

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

• Re-dissolve the cell pellet in Nucleofector's solution at room temperature to obtain a final concentration of 2×10 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.

• Mix 10 µl of cell suspension with small interfering RNA.

• 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.

• 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.

• To avoid damaging the cells, 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.

• 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, which confirms the success of the knockdown, are shown in Fig. eight.

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 (P/N: 83.3926) were purchased from SARSTEDT. A 5810R 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

• Load K562 cells with BATDA DELFIA ligand to increase fluorescence

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

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

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

Sampling for cytotoxicity

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

• Pipette 100 µl of loaded cells (5,000 cells) into a V-bottom plate.

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

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

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

• Precipitate the plate at 500 x g for 5 minutes.

• 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.

• Measure fluorescence 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

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 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 achieved by various methods. 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 using the methods described herein, an aliquot of the modified NK cells may be thawed and cultured prior to 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 resolution 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 μl L-glutamine, penicillin (100 units/ml)/streptomycin (100 mg/ml) and IL-2 (10 ng/ml). 0.5-1.0 x 10 ⁶ cells/samples were collected by centrifugation (1500 rpm x 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 BioTechnologies, 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). Nucleofector™ device (Nucleofector II, Lonza) and Nucleofector™ T kit from Lonza were used to achieve transfection efficiency up to 90% with high cell viability in KHYG-1 cells. 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 culture medium containing serum and supplements was added to 6-well plates and pre-incubated in a 5% humidity CO ₂ incubator 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 cuvette 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 5% humidity 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).

Effects of TRAIL/TRAIL variant on cytotoxicity of KHYG-1 cells

The mouse antibody CD2-APC (BD Pharmingen catalog number: 560642) was used. The Annexin V-FITC antibody was used (ImmunoTools 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 µl 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 AnnexinV 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 AnnexinV-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 these cells are involved, the development of protective strategies of cancer cells to prevent their death by a certain method is excluded. Thus, cancers cannot effectively prevent TRAIL-induced cell death by increasing the number of TRAIL decoy receptors because NK cells are modified in such a way that they remain cytotoxic under the circumstances.

Example 7 Protocol for NK Cell Therapy in Blood Cancer with 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 that expresses IL-6, IL-6R, or gp130, a DR5-containing agent, such as Bortezomib, is administered prior to the administration of modified NK cells and is therefore used at low doses to increase DR5 expression. on cancer cells, making therapy using modified NK cells more effective.

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

Since 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 apoptosis of cancer cells.

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 agent) 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% CO2 at 37°C.

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

(3) The 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 pre-treatment, 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 cancer therapy.

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 that showed a more cytotoxic phenotype and thus destroyed 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.

(1) 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 FlowJo 7.6.2 software.

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 RPMI1640 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 reaction for CD2. 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 the cytotoxicity of the NK cell to cancer cells through a mechanism less prone to perforin-associated depletion 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 are 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. .

(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).

Example 11 Direct and Indirect Effects of IL-6 on NK Cell Cytotoxicity

Materials and methods

Recombinant human IL-2 (p/n: 200-02) and IL-6 (p/n: 200-06) were purchased from PEPROTECH. IL-2 was reconstituted in a 100 mM acetic acid solution, aliquoted and stored at -80°C. IL-6 was reconstituted in PBS containing 0.1% BSA, aliquoted and stored at -80°C.

RPMI1640 media (part number: R8758) was purchased from SIGMA-ALDRICH. Fetal calf serum was purchased from SIGMA-ALDRICH (catalogue number: F7524). A solution of Penicillin-Streptomycin 100X stabilized with 10,000 units of penicillin and 10 mg streptomycin/ml (Catalog number: P4333) was purchased from SIGMA-ALDRICH. Horse serum for cell culture (catalog number: H1138-500ML) was purchased from SIGMA-ALDRICH. Alpha-MEM (part number: 12561056) was purchased from Thermo Fisher Scientific.

Phycoerythrin-labeled mouse CD126 (IL-6 receptor alpha chain) (cat. no.: 551850), phycoerythrin-labeled mouse CD130 (gp130, IL-6 receptor-associated signal transducer) (cat. no. 555757), mouse phycoerythrin-labeled isotype control IgG1k antibody (cat. no.: 555749), mouse allophycocyanin-labeled CD2 antibody (cat. no.: 560642), mouse FITC-labeled CD2 antibody (cat. no.: 555326), mouse PD-L1 antibody , labeled with allophycocyanin (catalogue number: 563741) and mouse antibody PD-L2 labeled with allophycocyanin (catalogue number: 557926) were purchased from BD Pharmingen. Allophycocyanin-labeled mouse PD1 antibody (catalog number: 329907) was purchased from Biolegend. Mouse monoclonal antibody phospho-Stat3 (Ser727) (cat. no.: 9136), rabbit monoclonal antibody phospho-Shp1 (Tyr564) (cat. no.: 8849), rabbit monoclonal antibody phospho-Shp-2 (Tyr580) (cat. no.: 5431), rabbit monoclonal antibody phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (catalog number: 4370), and rabbit monoclonal antibody p44/42 MAPK (Erk1/2) were purchased from Cell Signaling Technology. Mouse monoclonal antibody phospho-Stat3 (Tyr705) (catalog number: sc-81523) and rabbit polyclonal antibody Stat3 (catalog number: sc-7179) were purchased from Santa Cruz Biotechnology. Mouse anti-beta actin (catalog number: A5441) was purchased from SIGMA-ALDRICH. Functional IL-6 blocking antibody (p/n: 501110) and corresponding isotype control antibody (p/n: 400414) were purchased from Biolegend.

SYTOX® Green DNA Stain (P/N: S34860) for fixed cell flow cytometric analysis was purchased from Life Technologies. Annexin V-FITC antibody (catalog number: 556419) for apoptotic cell flow cytometric analysis was purchased from BD Pharmingen.

The KHYG-1 NK cell line was cultured and maintained in RPMI1640 medium containing 10% FBS, 100 U/ml penicillin/100 mg/ml streptomycin and 10 ng/ml IL-2. The NK cell line NK-92 was cultured in alpha-MEM medium containing 10% horse serum, 10% FBS, 100 U/ml penicillin/100 mg/ml streptomycin and 10 ng/ml IL-2. Every 2-3 days, the KHYG-1 and NK-92 cell culture medium was changed or the cells were divided at a 1:2 or 1:3 dilution.

K562, U937, HL60, Raji, RPMI8226, U266, MM1.S, NCI-H929, and KMS11 cells were cultured in RPMI-1640 medium supplemented with 10% FBS and 100 U/ml penicillin/100 mg/ml streptomycin.

IL-6 receptor expression

Flow cytometry was used to quantify IL-6 receptor and gp130 expression levels on various effector and target cells.

Cells (in logarithmic phase) were collected by centrifugation (1500 rpm for 5 min) and a density of 1 million cells/sample was used. Cells were washed with chilled PBS containing 0.1% BSA and 0.1% sodium azide. Finally, cells were exposed to phycoerythrin-labeled CD126, CD130 antibody, or isotype control antibody (2.5 µl/sample) in 50 µl PBS containing 0.1% BSA and 0.1% sodium azide on ice for 30 min. After washing cells twice with chilled PBS, samples were obtained on FACS Canto II (BD Biosciences). The results were analyzed using FlowJo 7.6.1 software.

Figures 31-44 show expression of IL-6 receptor (CD126) and gp130 (CD130) on KHYG-1, NK-92, RPMI8226, MM1.S, NCI-H929, U266, KMS11 and K562 cells.

KHYG-1 cells expressed low levels of CD126 (FIGS. 31 and 32) and CD130 (FIGS. 33 and 34).

NK-92 cells expressed low levels of CD126 (FIG. 35) and CD130 (FIG. 36).

MM cells (U266, RPMI8226, NCI-H929, KMS11 and MM1.S) expressed relatively high levels of CD126 and CD130 (FIGS. 37-41, respectively).

However, K562 leukemic cells were CD126 negative (FIGS. 42 and 43) but expressed relatively high levels of CD130 (FIGS. 44).

Direct inhibition of NK cell cytotoxicity by IL-6

KHYG-1, RPMI8226, MM1.S and K562 cells were cultured and maintained in the medium as described above. Cells were sampled in logarithmic phase, washed and resuspended in prewarmed appropriate media before adjusting the cell concentration to 1 million/mL. The concentration of target cells was regulated by the E:M ratio (effector:target). 0.5 ml of KHYG-1 cells and 0.5 ml of target cells were added to one well of a 24-well plate. IL-6 was added to the wells at a final concentration of 100 ng/ml. The plates were then placed in a 5% humidity CO ₂ incubator at 37° C. for 12 hours (6 or 4 hours for K562 cells). The same mixture of KHYG-1 cells and target cells was used as a control at the time point of 0 hours.

Flow cytometry was used to measure the cytotoxicity of KHYG-1 cells. Cells cultured together were harvested (at different time points), washed, first stained with CD2-APC antibody (2.5 µl/sample) and then with AnnexinV-FITC (2.5 µl/sample) and SYTOX-Green (0 .5 µl/sample) using AnnexinV buffer solution. The data were further analyzed using FlowJo 7.6.1 software. CD2 positive and negative gates were provided, which represent KHYG-1 cells and target cells, respectively. KHYG-1 cells were 100% positive for CD2, but K562, RPMI8226 and MM1.S cells were CD2 negative. Finally, the percentage of AnnexinV-FITC and SYTOX-Green positive cells (fixed cells) in the CD2 negative population was analyzed.

Figures 25-30 show that IL-6 suppressed the cytotoxicity of KHYG-1 cells against RPMI8226, MM1.S and K562 target cells at an E:M ratio of 1:1.

In the presence of IL-6 (100 ng/ml), the percentage of fixed target cells decreased when co-cultured with KHYG-1 cells. This occurred for RPMI8226 cells after 12 h incubation (Figs. 25 and 26), MM1.S cells after 12 h incubation (Figs. 27 and 28), and K562 cells after 6 h incubation (Fig. 29) or 4 h incubation ( Fig. 30).

Since K562 cells are CD126 negative, the results show that the inhibitory effect on cytotoxicity of KHYG-1 cells was directly mediated through the IL-6 receptor on KHYG-1 cells.

To further explore the mechanism underlying the observed IL-6-induced inhibition of KHYG-1 cytotoxicity, KHYG1 cells were stimulated with 50 ng/mL IL-6 in the presence of IL-2 for 24 hours, followed by NKG2D (activating receptor) and NKG2A expression levels. (inhibitory receptor) was analyzed by flow cytometry. Negative control means FMO. Allophycocyanin-labeled NKG2D antibody (catalog number: 320807) was purchased from Biolegend. Allophycocyanin-labeled NKG2A antibody (catalogue number: FAB1059A) was purchased from R&D systems.

As can be seen in FIG. 70, IL-6 directly inhibits the cytotoxicity of the KHYG-1 cell by reducing the expression levels of the activating NKG2D receptor (70A) and increasing the expression of the inhibitory NKG2A receptor (70B).

Indirect inhibition of NK cell cytotoxicity by IL-6

MM cells in logarithmic phase were seeded at a volume of 0.5 million/ml onto RPMI1640 medium containing 10% FBS. A final concentration of 100 ng/ml IL-6 or a similar volume of vehicle (PBS) was used to treat MM cells for 48 hours. The MM cells were then harvested, washed and stained with PD-L1 and PD-L2 antibodies (2.5 µl/sample). Flow cytometric data were collected using FACS Canto II, after which the results were analyzed using FACSDIVA 8.0.1 software.

Figures 45-56 show that IL-6 increased PD-L1 and PD-L2 expression on RPMI8226, NCI-H929, MM1.S and U266 cells.

IL-6 induced upregulation of PD-L1 was observed in RPMI8226 cells (Fig. 45), NCI-H929 cells (Fig. 47), MM1.S cells (Fig. 49 and 50) and U266 cells (Fig. 53 and 54) .

IL-6-induced upregulation of PD-L2 was observed in RPMI8226 cells (Fig. 46), NCI-H929 cells (Fig. 48), MM1.S cells (Fig. 51 and 52) and U266 cells (Fig. 55 and 56) .

Since PD-L1 and PD-L2 are known to bind the inhibitory PD-1 checkpoint receptor (cIR) on NK cells, these data clearly demonstrate a second (indirect) mechanism by which IL-6 suppresses NK cell cytotoxicity by acting also on cancer cells.

These two mechanisms described indicate that IL-6 is a key cytokine involved in cancer cell viability.

Effect of antagonism of IL-6

U266 cells in logarithmic phase were seeded at 0.5 million/ml on RPMI1640 medium containing 10% FBS. A final concentration of 10 μg/ml rat IL-6 monoclonal antibody or an isotype control antibody was added to block IL-6 secreted by U266 cells. After 48 hours, U266 cells were harvested, washed and stained with PD-L1 and PD-L2 antibodies (2.5 µl/sample). Flow cytometric data were collected using FACS Canto II, after which the results were analyzed using FACSDIVA 8.0.1 software.

Figures 57-60 show that PD-L1 expression on U266 cells was significantly reduced in the presence of an IL-6 blocking antibody.

Figures 61-64 show that PD-L2 expression on U266 cells was significantly reduced in the presence of an IL-6 blocking antibody.

Thus, it can be seen that IL-6 signaling antagonism can be achieved with an IL-6 blocking antibody.

These data confirm that IL-6 signaling can regulate the expression of PD-L1 and PD-L2 on cancer cells.

The blocking of IL-6 signaling according to the present invention can thus be used in the treatment of cancer since both direct and indirect inhibition of NK cell cytotoxicity induced by IL-6 is prevented. In addition, any direct proliferative or anti-apoptotic effect of IL-6 on the cancer cell is also prevented by blocking IL-6 signaling.

To further demonstrate this, KHYG-1 cells were stimulated with U266 cells as described above in the presence of 2 μg/ml of an IL-6 blocking monoclonal antibody (purified IL-6 LEAF™ antibody, Biolegend, catalog number #501110) or a similar dose of an isotype antibody. control (Biolegend, catalog number #400413).

As can be seen in FIG. 69, blocking IL-6 with the monoclonal antibody resulted in restoration of KHYG-1 cytotoxicity to U266 cells (E:M ratio 1:1; incubation 12 hours). Thus, in addition to the above data for RPMI8226, MM1.S and K562 target cells, it can be seen that inhibition of IL-6 signaling increases the ability of KHYG-1 cells to kill target cancer cells in another cancer cell line.

IL-6 signaling in NK cells

KHYG-1 cells were cultured and kept in the medium as described above. When testing the effect of IL-6 alone, KHYG-1 cells were depleted in RPMI160 medium supplemented with 10% FBS (without IL-2) for 6 hours, after which they were stimulated with 10 ng/ml IL-2 and/or 100 ng/ml. ml IL-6.

Developing under normal conditions, KHYG-1 cells require IL-2 to promote cell proliferation and maintain cytotoxicity.

As shown in figure 65, it was found that the presence of only IL-2 caused the activation of p-STAT3 and p-P44/42, but also a drop in the expression of p-SHP1 and P-SHP2.

As shown in Figure 66, the presence of IL-6 alone caused activation of p-STAT3, p-SHP1 and p-SHP2, but also a drop in p-P44/42 expression.

As shown in figure 67, in the presence of IL-2, IL-6 retained the ability to reduce the expression of p-P44/42 and increase the expression of p-SHP1 and p-SHP2.

In models (including NK cells), p-STAT3 has been shown to be an important downstream effector for the IL-6 signaling pathway, and p-P44/42 levels are positively associated with NK cell cytotoxicity.

From the above data, it can be seen that p-SHP1 and p-SHP2 significantly influence the regulation of p-P44/42 levels. In addition, it can be seen that IL-6 anti-cytotoxic signaling dominates IL-2 pro-cytotoxic signaling, which implies an overall decrease in NK cell cytotoxicity and can be easily eliminated. Thus, antagonism of IL-6 can be proposed as a way to treat cancer.

Upregulation of NK cell PD-1 expression induced by cancer cells

KHYG-1 cells were cultured and maintained in RPMI1640 medium containing 10% FBS, 100 U/ml penicillin/100 mg/ml streptomycin and 10 ng/ml IL-2. K562, U937, HL60, Raji, RPMI8226, U266 and MM1.S cells were cultured in RPMI1640 medium supplemented with 10% FBS and 100 U/ml penicillin/100 mg/ml streptomycin. Cells were collected in logarithmic phase, washed and resuspended in prewarmed RPMI1640 medium containing IL-2 (KHYG-1 cell culture medium) before adjusting the cell concentration to 1 million/ml. The concentration of target cells was regulated by the E:M ratio (effector:target). 0.5 ml of KHYG-1 cells and 0.5 ml of target cells were added to one well of a 24-well plate and cultured for 24 hours. Cells were harvested, washed and stained with CD2-FITC (2.5 µl/sample) and PD1-APC (2.5 µl/sample) antibodies. Samples were collected using FACS Canto II and analyzed using FACSDiva 8.0.1 software. Positive and negative CD2 gates represent KHYG-1 cells and target cells, respectively. KHYG-1 cells are 100% CD2 positive, but MM cells and other blood cancer cell lines are CD2 negative. Thus, PD-1 expression in a CD2 positive population (ie, KHYG-1 cells) was analyzed.

As can be seen in Figure 68, flow cytometric analysis of PD-1 expression in KHYG-1 cells co-cultured with various blood cancer cell lines (E:M ratio = 1:1) showed that PD-1 expression was significantly induced by all tested blood cancer cell lines.

These data highlight the inhibitory effect of cancer cells on NK cell cytotoxicity, since higher expression of PD-1 on NK cells implies a greater degree of those cancers that express PD-L1 and/or PD-L2.

Thus, it can be seen that cancer cells upregulate PD-1 cIR expression on NK cells, while IL-6 directly reduces NK cell cytotoxicity and upregulates PD-L1 and PD-L2 expression on cancer cells. The increased expression of PD-1 on NK cells and the increased expression of PD-L1 and PD-L2 on cancer cells work together to significantly suppress NK cytotoxicity. In addition, IL-6 directly promotes the proliferation of cancer cells.

Blocking IL-6 signaling, as shown above, has therapeutic utility in reducing the survival of cancer cells, in particular those cancer cells that express IL-6 receptors.

Treatment protocol using an IL-6 antagonist

The following protocol has been developed for use in the treatment of patients with multiple myeloma. However, it is clear that the invention is suitable for the treatment of patients with many different types of cancer, including those in which the expression of IL-6R is observed.

Once a patient has been diagnosed with an IL-6R-positive cancer, such as multiple myeloma, an aliquot of NK cells is thawed and cultured prior to administration to the patient at an effective dose. Aliquoted cells can be modified as described herein. In an alternative embodiment, transient transfection can be obtained by using, for example, viral agents, electropulse opening of cell pores, etc. For electropulse opening of cell pores, the MaxCyte Flow Electroporation platform provides the appropriate solution to enable rapid large-scale transfections in the clinic. After transfection of NK cells, they are cultured to ensure the expression of the modification and then injected into the patient's body intravenously.

Prior to this, simultaneously or after the administration of NK cells, an effective dose of the IL-6 antagonist is administered to the patient. As used herein, an IL-6 antagonist may refer to one unit or a combination of several. The IL-6 antagonist(s) may bind to IL-6, IL-6R, gp130, etc., provided that the result of binding reduces (counteracts) IL-6 signaling.

Thus, the invention provides IL-6 signaling antagonism in NK cell based cancer treatment.

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SEQUENCE LIST

<110> ONKIMMUNE LIMITED

<120> CELL THERAPY BASED ON IMPROVED NATURAL

KILLER CELLS

<130> P40309WO

<160> 12

<170> patent version 3.5

<210> 1

<211> 9540

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<213> Homo sapiens

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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 cctcacctcc 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 ccttgggtcc 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

caaataatgc ttcatgagtc agctttgcac cagccattac ctgcaagtca ttcttggaag 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 (51)

1. Combination of a natural killer (NK) cell or NK cell line providing a cytotoxic effect with an IL-6R antagonist antibody for use in the treatment of blood cancer whose cells express IL-6R receptors. 2. The combination of NK cells or NK cell lines according to claim 1, characterized in that blood cancer cells express IL-6 receptors. 3. The combination of NK cells or NK cell lines according to claim 1 or 2, characterized in that the blood cancer cells express PDL-1 and/or PDL-2. 4. The combination of NK cells or NK cell lines according to any one of the preceding claims, characterized in that the antagonist antibody comprises an antibody that binds to IL-6. 5. Combination of NK cells or NK cell line according to claim 4, characterized in that the IL-6 antibody is selected from siltuximab, olokizumab (CDP6038), elsilimomab, BMS-945429 (ALD518), MH-166 and sirukumab (CNTO 136 ). 6. The combination of NK cells or NK cell lines according to any one of paragraphs. 1-3, characterized in that the IL-6R antibody is selected from tocilizumab, sarilumab, PM-1 and AUK12-20. 7. The combination of NK cells or NK cell lines according to any one of paragraphs. 1-3, characterized in that the gp130 antibody is AM64. 8. Combination of NK cells or NK cell line according to any of the preceding paragraphs in combination with a separate anti-cancer therapy. 9. The combination of NK cells or NK cell lines according to claim 8, characterized in that endogenous NK cells are used as immune effector cells in a separate anti-cancer therapy. 10. The combination of NK cells or NK cell lines according to any one of paragraphs. 8 or 9, characterized in that a separate anti-cancer therapy is represented by antibody-dependent cell-mediated cytotoxicity (ADCC). 11. Combination of NK cells or NK cell lines according to any of the preceding claims, 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), multiple myeloma (MM), or light chain myeloma. 12. The combination of an NK cell or NK cell line according to any of the preceding claims, wherein the NK cell or NK cell line is genetically modified to reduce the expression of one or more inhibitory checkpoint receptors. 13. The combination of NK cells or NK cell lines according to claim 12, characterized in that the inhibitory checkpoint receptors are selected from CD96 (TACTILE), CD152 (CTLA4), CD223 (LAG-3), CD279 (PD-1), CD328 (SIGLEC7), SIGLEC9, TIGIT and TIM-3. 14. The combination of an NK cell or NK cell line according to any one of the preceding claims, characterized in that the NK cell or NK cell line is genetically modified to express a mutant TRAIL ligand. 15. The combination of NK cells or NK cell lines according to claim 14, characterized in that the mutant TRAIL ligand has increased affinity for TRAIL receptors, for example DR4 and/or DR5. 16. The combination of NK cells or NK cell lines according to claim 14 or 15, characterized in that the mutant TRAIL ligand has reduced affinity for TRAIL decoy receptors. 17. The combination of NK cells or NK cell line according to any one of the preceding claims, characterized in that the NK cell or NK cell line expresses a chimeric antigen receptor (CAR). 18. The combination of NK cells or NK cell lines according to claim 17, characterized in that CAR is a bispecific CAR. 19. The combination of NK cells or NK cell lines according to claim 18, characterized in that the bispecific CAR binds two ligands on the same cell type. 20. The combination of NK cells or NK cell lines according to claim 18, wherein the bispecific CAR binds one ligand on each of two separate cell types. 21. The combination of NK cells or NK cell lines according to claim 17 or 18, characterized in that the CAR ligand(s) or the bispecific CAR are expressed on the cancer cell. 22. The combination of NK cells or NK cell lines according to claim 21, characterized in that both ligands for the bispecific CAR are expressed on the cancer cell. 23. The combination of NK cells or NK cell lines for use according to claim 21, characterized in that the ligands for the bispecific CAR are expressed on the cancer and immune effector cell. 24. The combination of an NK cell or NK cell line according to any one of the preceding claims, characterized in that the NK cell or NK cell line is genetically modified to reduce expression of the IL-6 receptor. 25. The combination of NK cells or NK cell line according to any one of the preceding claims, characterized in that the NK cell line is KHYG-1. 26. A method of treating blood cancer whose cells express IL-6R, comprising administering to a patient an effective amount of a combination of an NK cell or NK cell line and an IL-6R antagonist antibody. 27. The method according to claim 25, characterized in that blood cancer cells express IL-6 receptors. 28. The method according to any one of paragraphs. 26 or 27, characterized in that blood cancer cells express PDL-1 and/or PDL-2. 29. The method according to any one of paragraphs. 26-28, characterized in that the combination of NK cells or NK cell lines is provided with a pre-linked IL-6 antagonist. 30. The method according to any one of paragraphs. 26-29, characterized in that the antagonist antibody includes an antibody that binds to IL-6. 31. The method of claim 30 wherein the IL-6 antibody is selected from siltuximab, olokizumab (CDP6038), elsilimomab, BMS-945429 (ALD518), MH-166, and sirukumab (CNTO 136). 32. The method according to any one of paragraphs. 26-29, characterized in that the IL-6R antibody is selected from tocilizumab, sarilumab, PM-1 and AUK12-20. 33. The method according to any one of paragraphs. 26-29, characterized in that the gp130 antibody is AM64. 34. The method according to any one of paragraphs. 26-33 used in combination with a separate anti-cancer therapy. 35. The method according to p. 34, characterized in that in a separate anti-cancer therapy, endogenous NK cells are used as immune effector cells. 36. The method according to any one of paragraphs. 34 or 35, characterized in that a separate anti-cancer therapy is represented by antibody-dependent cell-mediated cytotoxicity (ADCC). 37. The method according to any one of paragraphs. 26-36, 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), multiple myeloma (MM), or light chain myeloma. 38. The method according to any one of paragraphs. 26-37, characterized in that the NK cell or NK cell line is genetically modified to reduce the expression of one or more inhibitory checkpoint receptors. 39. The method of claim 38, 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. 40. The method according to any one of paragraphs. 26-39, characterized in that the NK cell or NK cell line is genetically modified to express the mutant TRAIL ligand. 41. The method of claim 40 wherein the mutant TRAIL ligand has increased affinity for TRAIL receptors, for example DR4 and/or DR5. 42. The method according to any one of paragraphs. 40 or 41, characterized in that the mutant TRAIL ligand has reduced affinity for TRAIL decoy receptors. 43. The method according to any one of paragraphs. 26-42, characterized in that the NK cells or NK cell line express a chimeric antigen receptor (CAR). 44. The method of claim 43, wherein the CAR is a bispecific CAR. 45. The method of claim 44, wherein the bispecific CAR binds two ligands on the same cell type. 46. The method of claim 44 wherein the bispecific CAR binds one ligand on each of two separate cell types. 47. The method of claim 43 or 44, wherein the ligand(s) for the CAR or the bispecific CAR are expressed on the cancer cell. 48. The method of claim 47, wherein both ligands for the bispecific CAR are expressed on the cancer cell. 49. The method of claim 47, wherein the ligands for the bispecific CAR are expressed on the cancer and immune effector cell. 50. The method according to any one of paragraphs. 26-49, characterized in that the NK cells or NK cell line are genetically modified to reduce the expression of the IL-6 receptor. 51. The method according to any one of paragraphs. 26-50, characterized in that the NK cell line is KHYG-1.

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