Combination therapy with immunomodulatory agents

Abstract

A method of treating cancer includes administering modified natural killer (NK) cells in combination with an immunomodulatory agent. Compositions including the modified NK cells in combination with an immunomodulatory agent are also described.

Description

[Technical Field]

[0001] The present invention relates to the modification of natural killer (NK) cells and NK cell lines to produce derivatives with a more cytotoxic phenotype. Additionally, the present invention relates to methods for producing the modified NK cells and NK cell lines, compositions containing the cells and cell lines, and the use of the cells, lines, and compositions in the treatment of cancer. [Background technology]

[0002] Immune cells typically require target cells to present antigens via the major histocompatibility complex (MHC) before triggering an immune response that leads to the death of the target cell, allowing cancer cells that do not present MHC class I to largely evade the immune response.

[0003] However, NK cells can recognize cancer cells in the absence of MHC class I expression, and therefore play an important role in the body's defense against cancer.

[0004] On the other hand, in certain circumstances, cancer cells exert the ability to attenuate the cytotoxic activity of NK cells through the expression of ligands that bind to inhibitory receptors on the NK cell membrane. Resistance to cancer may involve a balance between these and other factors.

[0005] In this context, cytotoxicity refers to the ability of immune effector cells, such as NK cells, to induce cancer cell death by releasing cytolytic compounds or by binding to receptors on the cancer cell membrane to induce apoptosis of the cancer cells. Cytotoxicity is influenced not only by signals that induce the release of cytolytic compounds, but also by signals that inhibit their release. Therefore, increased cytotoxicity reduces the likelihood that cancer cells will attenuate the cytotoxic activity of NK cells, as described above, resulting in more efficient cancer cell death.

[0006] Expression of cytokine-inducible SH2-containing protein (CIS) is induced by certain growth cytokines and is a critical negative regulator of interleukin-15 (IL-15) signaling in natural killer (NK) cells. CIS is encoded by the CISH gene. CISH expression is associated with suppressed cell proliferation and reduced cytotoxic activity against several cancer cell lines when maintained at low cytokine concentrations. Therefore, knocking out CISH expression in NK cells has recently been reported to be beneficial for NK cell cytotoxicity against cancer cells (Zhu et al., 2020, "Metabolic Reprogramming via Deletion of CISH in Human iPSC-Derived NK Cells Promotes In Vivo Persistence and Enhances Anti-tumor Activity," Cell: Vol. 27(2): pp. 224-237). However, as with all genetic modifications, even if some modifications are more beneficial than others, it is essential that therapeutic approaches using genetically modified cells be supported by solid scientific evidence.

[0007] As alternatives to cell-based cancer treatments, other options are available, such as small molecule and antibody therapies.

[0008] Nevertheless, new and improved cancer therapies are needed that utilize different strategies (e.g., cell-based, antibody-based, small molecule, etc.) to maximally target and kill cancer cells. Summary of the Invention [Problem to be solved by the invention]

[0009] The present invention aims to provide modified NK cells and NK cell lines with a more cytotoxic and / or more durable phenotype that targets cancer cells. It also aims to provide methods for producing modified NK cells and NK cell lines, compositions containing the cells or cell lines, and their use in therapy, particularly in the treatment of cancer. Particular embodiments aim to provide treatments for identified cancers. Particular embodiments aim to combine two or more therapies in a synergistic manner to improve the cytotoxicity and durability of the modified cells. [Means for solving the problem]

[0010] Provided herein are modified NK cells and modified NK cell lines with a more cytotoxic phenotype, and methods for producing the cells and cell lines. Also provided are compositions of modified NK cells and modified NK cell lines, and the use of the compositions for treating cancer. In this case, cytotoxicity refers to the killing of cancer cells, as described above.

[0011] The present invention provides NK cells and NK cell lines in combination with immunomodulatory agents for use in the treatment of cancer, wherein the NK cells or NK cell lines have been modified to reduce expression of CISH.

[0012] Further provided in accordance with the present invention, both generally and specifically, are methods of treating cancer, e.g., hematological cancers, using immunomodulatory agents in combination with modified NK cells and modified NK cell lines, wherein the modified NK cells and modified NK cell lines have been engineered to have reduced CIS function. Additionally, the cells may optionally lack expression of one or more checkpoint inhibitory receptors, express one or more TRAIL ligands, express one or more chimeric antigen receptors, express one or more growth-promoting cytokines, and / or express one or more Fc receptors.

[0013] In particular, diseases treatable according to the present invention include cancers, such as solid cancers and hematological cancers, more particularly multiple myeloma. In particular, tumors and cancers in humans can be treated. Reference herein to a tumor includes reference to a neoplasm. [Brief explanation of the drawings]

[0014] [Figure 1] Figure 1 shows that CISH KO NK cells act synergistically with the IMiD iberdomine to enhance their cytotoxic properties against cancer cells. DETAILED DESCRIPTION OF THE INVENTION

[0015] Thus, the present invention provides natural killer (NK) cells or NK cell lines in combination with an immunomodulatory agent for use in the treatment of cancer, wherein the NK cells or NK cell lines have been modified to have reduced expression of CISH (compared to the same NK cells or NK cell lines that have not been modified).

[0016] The present invention also provides natural killer (NK) cells or NK cell lines for use in the treatment of cancer, wherein the NK cells or NK cell lines have been modified to have reduced expression of CISH (compared to the same NK cells or NK cell lines that have not been modified), and the NK cells or NK cell lines are administered in combination with an immunomodulatory agent.

[0017] The present invention further provides an immunomodulatory agent for use in the treatment of cancer, wherein the immunomodulatory agent is administered in combination with natural killer (NK) cells or NK cell lines that have been modified to have reduced expression of CISH (compared to the same NK cells or NK cell lines that have not been modified).

[0018] The NK cells and NK cell lines of the present invention are also collectively referred to as NK cells (unless the context dictates otherwise). Preferably, the NK cells are human NK cells.

[0019] As described in detail below in the Examples, NK cells and NK cell lines have been genetically modified to enhance cytotoxic activity, particularly against cancer, and thus, the modification is preferably to knock down or knock out expression of CISH.

[0020] Preferably, expression of CISH is reduced by at least 50%, at least 75%, at least 90%, at least 95%, and more preferably at least 99% compared to the same unmodified NK cells or NK cell line (wild-type NK cells). Preferably, the genetic knockout of CISH is via CRISPR gene editing.

[0021] Preferably, expression of CISH is knocked out in NK cells.

[0022] A preferred immunomodulatory agent of the present invention is an Ikaros pathway modulator or a modulator of the cereblon E3 ligase complex. Preferably, the modulator is an immunomodulatory imid drug (IMiD) or cereblon E3 ligase modulator (CELMoD). In some instances, the term CELMoD is used in a manner intended to encompass the IMiD class.

[0023] Preferably, the IMiD is selected from thalidomide, lenalidomide, iberdomide, and pomalidomide. More preferably, the IMiD is selected from lenalidomide and iberdomide. Most preferably, the IMiD is lenalidomide.

[0024] Preferably, the CELMoD is selected from iverdomide (CC-220) and CC-92480. Most preferably, the CELMoD is iverdomide.

[0025] The cancer to be treated is preferably a blood cancer.The blood cancer is preferably selected from multiple myeloma, acute myeloid leukemia, and acute lymphoblastic leukemia.The blood cancer may optionally be a B-cell neoplasm.

[0026] The cancer to be treated may optionally, but preferably, be a solid cancer.

[0027] In a particularly preferred embodiment, the present invention provides NK cells in combination with lenalidomide for use in the treatment of multiple myeloma, wherein the NK cells have been modified to knock out expression of the CISH gene.

[0028] A key advantage of the present invention lies in the unexpected synergistic effects of a combined cancer therapy involving immunomodulatory drugs and NK cells with reduced CISH expression. IMiDs and CELMoDs are known to degrade Ikaros and Aiolos (hematopoietic-specific zinc finger transcription factors, key regulators of lymphocyte differentiation) through selective ubiquitination by the CRBN-CRL4 ubiquitin ligase (Kronke 2014 and Lu 2014). Research in this field to date has primarily focused on examining B and T cell development. However, Ikaros is known to antagonize DNA binding by STAT5 in pre-B cells. Because STAT5 coincidentally targets the promoter of the CISH gene and upregulates its expression, we demonstrated that IMiD / CELMoD cancer therapy unintentionally upregulates CISH expression in NK cells through Ikaros degradation. Interestingly, this also occurs in a STAT5-independent manner due to the lack of direct transcriptional repression by Ikaros. Therefore, IMiDs / CELMoDs suitable for use in the present invention (of which lenalidomide and iverdimide are mentioned examples) are those that result in upregulation of CISH expression, for example via degradation of Ikaros and / or Aiolos.

[0029] The upregulation of CISH expression in NK cells induced by IMiD / CELMoD is an uncharacterized and unintended side effect, and the present inventors have devised a cancer therapy that takes advantage of this finding. By providing NK cells with downregulated CISH in combination with IMiD / CELMoD therapy, the NK cells can maximize their cytotoxic effects without being negatively affected by upregulated CISH expression.

[0030] Therefore, the immunomodulatory agents of the present invention are Ikaros depletors.

[0031] A preferred feature of the present invention is to provide modified NK cells and modified NK cell lines that have an increased intrinsic ability to grow and proliferate rapidly in culture. This can be achieved, for example, by transfecting the cells to overexpress the growth-inducing cytokines IL-2 and / or IL-15. Preferably, the cells are modified to express exogenous soluble IL-2 (sIL-2). Furthermore, this optional modification provides a cost-effective alternative to continuously replenishing growth medium containing cytokines. These cells can be used as intermediates to prepare significant quantities of cells for further treatment (e.g., treatment to reduce division potential) prior to therapeutic application.

[0032] In another embodiment of the present invention, NK cells are provided that also express or overexpress a TRAIL ligand. The TRAIL ligand is preferably a mutant (variant) TRAIL ligand. The resulting NK cells have increased binding to the TRAIL receptor, resulting in increased cytotoxicity against cancer, particularly solid cancers, particularly ovarian, breast, and colon cancers, and blood cancers, particularly leukemia. NK cells with such combined activities may also be effective in suppressing cancer metastasis.

[0033] Preferably, the mutant / variant of TRAIL has reduced affinity (or substantially no affinity) for the "decoy" receptor compared to the binding of wild-type TRAIL to the "decoy" receptor. Such decoy receptors represent a class of TRAIL receptors that bind to TRAIL ligands but lack the ability to cause cell death, potentially acting to antagonize cell death signaling pathways. Mutant / variant TRAIL ligands may be prepared according to WO 2009 / 077857.

[0034] The mutants / variants may have increased affinity for individual TRAIL receptors, such as DR4 and DR5. Wild-type TRAIL is known to typically have a KD of greater than 2 nM for DR4, greater than 5 nM for DR5, and greater than 20 nM for the decoy receptor DcR1 (WO 2009 / 077857; measured by surface plasmon resonance), or approximately 50 nM-100 nM for DR4, 1 nM-10 nM for DR5, and 175 nM-225 nM for DcR1 (Truneh, A. et al. 2000; measured by isothermal titration calorimetry and ELISA). Thus, an appropriate definition of increased affinity for DR4 is a KD of less than 2 nM or less than 50 nM, respectively, and an appropriate definition of increased affinity for DR5 is a KD of less than 5 nM or less than 1 nM, respectively. A suitable definition of reduced affinity for the decoy receptor DcR1 is a KD of greater than 50 nM or greater than 225 nM, respectively. In either case, the increased or decreased affinity of the TRAIL mutant / variant is relative to the baseline affinity of wild-type TRAIL. Preferably, the affinity is increased by at least 10%, at least 25%, at least 50%, at least 100%, and more preferably at least 1000% compared to that of wild-type TRAIL.

[0035] Preferably, the TRAIL variant has increased affinity for DR5 relative to its affinity for DR4, DcR1, and DcR2. Preferably, the affinity for DR5 is at least 1.5-fold, 2-fold, 5-fold, 10-fold, 100-fold, or even 1,000-fold greater than its affinity for one or more of DR4, DcR1, and DcR2. More preferably, the affinity for DR5 is at least 1.5-fold, 2-fold, 5-fold, 10-fold, 100-fold, or even 1,000-fold greater than its affinity for at least two, preferably all, of DR4, DcR1, and DcR2.

[0036] Preferably, the TRAIL variant has increased affinity for one or both of DR4 and DR5 compared to the affinity for wild-type TRAIL, and the affinity for DR4 and / or DR5 is preferably at least 1.5-fold, 2-fold, 5-fold, 10-fold, 100-fold, or even 1,000-fold or more than the affinity for wild-type TRAIL.

[0037] Further specific embodiments include NK cells expressing mutant TRAIL ligands with reduced or no affinity for the TRAIL decoy receptor. Further specific embodiments include NK cells expressing mutant TRAIL ligands with reduced or no affinity for the TRAIL decoy receptor and increased affinity for DR4 and / or DR5.

[0038] Binding affinity may be measured according to any suitable method known in the art. Preferably, binding affinity is measured using surface plasmon resonance, isothermal titration calorimetry, or ELISA.

[0039] In certain embodiments, the TRAIL variant comprises at least one amino acid substitution at a position selected from the group consisting of: 131, 149, 159, 160, 189, 191, 193, 195, 199, 200, 201, 203, 204, 212, 213, 214, 215, 218, 240, 251, 261, 264, 266, 267, 269, and 270.

[0040] In certain embodiments, the TRAIL variants are G131R, G131K, R149I, R149M, R149N, R149K, S159R, G160E, Y189A, Y189Q, R191K, Q193H, Q193K, Q193S, Q193R, E195R, N199V, N199R, N199H, T200H, K201R, K201H, D203A, K204E, and at least one substitution selected from the group consisting of K204D, K204Y, K212R, Y213W, T214R, S215D, S215E, S215H, S215K, S215D, D218H, D218A, Y240A, K251D, K251E, K251Q, T261L, H264R, I266L, D267Q, D269A, D269H, and H270D.

[0041] In certain embodiments, the TRAIL variants are G131R, G131K, R149I, R149M, R149N, R149K, S159R, G160E, Y189A, Y189Q, R191K, Q193H, Q193K, Q193S, Q193R, E195R, N199V, N199R, N199H, T200H, K201R, K201H, D203A, K204E, and at least two substitutions selected from the group consisting of K204D, K204Y, K212R, Y213W, T214R, S215D, S215E, S215H, S215K, S215D, D218H, D218A, Y240A, K251D, K251E, K251Q, T261L, H264R, I266L, D267Q, D269A, D269H, and H270D.

[0042] In certain embodiments, the TRAIL variants are G131R, G131K, R149I, R149M, R149N, R149K, S159R, G160E, Y189A, Y189Q, R191K, Q193H, Q193K, Q193S, Q193R, E195R, N199V, N199R, N199H, T200H, K201R, K201H, D203A, K204E, and at least three substitutions selected from the group consisting of K204D, K204Y, K212R, Y213W, T214R, S215D, S215E, S215H, S215K, S215D, D218H, D218A, Y240A, K251D, K251E, K251Q, T261L, H264R, I266L, D267Q, D269A, D269H, and H270D.

[0043] In certain embodiments, the amino acid substitutions of the TRAIL variant are G131R, G131K, R149I, R149M, R149N, R149K, S159R, G160E, Y189A, Y189Q, R191K, Q193H, Q193K, Q193S, Q193R, E195R, N199V, N199R, N199H, T200H, K201R, K201H, D203A, K 204E, K204D, K204Y, K212R, Y213W, T214R, S215D, S215E, S215H, S215K, S215D, D218H, D218A, Y240A, K 251D, K251E, K251Q, T261L, H264R, I266L, D267Q, D269A, D269H, H270D, T214R / E195R, T214R / D269H, Y 189A / Q193S / N199V / K201R / Y213W / S215D, Y213W / S215D, N199R / K201H, N199H / K201R, G131R / N199R / K 201H, G131R / N199R / K201H / R149I / S159R / S215D, G131R / R149I / S159R / S215D, G131R / N199R / K201H / R 149I / S159R / S215D, G131R / D218H, Y189Q / R191K / Q193R / H264R / I266L / D267Q, T261L / G160E, T261L / H270D, T261L / G160E / H270D, and T261L / G160E / H270D / T200H (the use of " / " indicates multiple amino acid substitutions).

[0044] In certain embodiments, the amino acid substitutions in the TRAIL variant are selected based on the variant having increased affinity for DR5, and such substitutions may be selected from the group consisting of D269H, E195R, T214R, D269H / E195R, T214R / E195R, T214R / D269H, N199V, Y189A / Q193S / N199V / K201R / Y213W / S215D, Y213W / S215D, D269A, and Y240A.

[0045] In certain embodiments, the amino acid substitutions in the TRAIL variants are selected based on variants having increased affinity for DR4, and these substitutions include G131R, G131K, R149I, R149M, R149N, R149K, S159R, Q193H, W193K, N199R, N199R / K201H, N199H / K201R, G131R / N199R / K201H ... 01H / R149I / S159R / S215D, G131R / R149I / S159R / S215D, G131R / D218H, K201R, K201H, K204E, K204D, K204L, K204Y, K212R, S215E, S215H, S215K, S215D, D218H, K251D, K251E, K251Q, and Y189Q / R191K / Q193R / H264R / I266L / D267Q.

[0046] In certain embodiments, the amino acid substitutions in the TRAIL variants are selected based on the variants having reduced affinity for the TRAIL decoy receptor, and such substitutions may be selected from the group consisting of T261L, H270D, T200H, T261L / G160E, T261L / H270D, T261L / G160E / H270D, T261L / G160E / H270D / T200H, D203A, and D218A.

[0047] Treatment of cancer using engineered NK cells expressing TRAIL or TRAIL variants can optionally be enhanced by administering to the patient an agent capable of upregulating the expression of TRAIL death receptors on cancer cells. This agent can be administered before, in combination with, or following the administration of engineered NK cells. However, it is preferable that the agent be administered before the administration of engineered NK cells. The agent upregulates the expression of DR5 on cancer cells. The agent can optionally be a chemotherapeutic agent, such as a proteasome inhibitor, such as bortezomib, and can be administered at a low dose that can upregulate the expression of DR5 in cancer. Other examples of DR5 inducers include gefitinib, piperlongumine, doxorubicin, alpha-tocopherol succinate, and HDAC inhibitors.

[0048] In certain embodiments of the present invention, NK cells are provided that have been further modified to reduce or eliminate the function of checkpoint inhibitory receptors. NK cells may be produced in which one or more checkpoint inhibitory receptor genes have been knocked down / knocked out. Preferably, these receptors are specific checkpoint inhibitory receptors. More preferably, these checkpoint inhibitory receptors are one or more, or all, of TIGIT, CD96 (TACTILE), CD152 (CTLA4), CD223 (LAG-3), CD279 (PD-1), CD328 (SIGLEC7), SIGLEC9, and / or TIM-3. In other embodiments, NK cells are provided in which one or more inhibitory receptor signaling pathways have been knocked out or have their function reduced, again resulting in reduced or eliminated inhibitory receptor function. For example, SHP-1, SHP-2, and / or SHIP-mediated signaling pathways are knocked out by genetic modification of the cells.

[0049] Reducing the function of checkpoint inhibitory receptors is preferred over other inhibitory receptors, as their expression occurs subsequent to NK cell activation. Canonical or "classical" inhibitory receptors, such as the majority of the KIR family, NKG2A, and LIR-2, bind to MHC class I and are therefore primarily involved in reducing the problem of self-targeting. Therefore, knockout of checkpoint inhibitory receptors is preferred. The reduced or absent function of these receptors according to the present invention prevents cancer cells from suppressing immune effector functions that might otherwise occur if the receptors were fully functional. Thus, these embodiments of the present invention offer an important advantage over NK cells, which are less susceptible to suppression of cytotoxic activity by cancer cells, and are therefore useful in the treatment of cancer.

[0050] As used herein, reference to an inhibitory receptor generally refers to a receptor expressed on the plasma membrane of an immune effector cell, such as a NK cell, which upon binding to its complementary ligand generates an intracellular signal that reduces the cytotoxic activity of the immune effector cell. These inhibitory receptors are expressed in both the "resting" and "activated" states of immune effector cells and are often involved in providing the immune system with a "self-tolerance" mechanism that inhibits cytotoxic responses against the body's cells and tissues. One example is the inhibitory receptor family "KIR," which is expressed on NK cells and recognizes MHC class I expressed on healthy cells of the body.

[0051] Also, in this specification, checkpoint inhibitory receptors are generally considered to be a subset of the above-mentioned inhibitory receptors.However, unlike other inhibitory receptors, checkpoint inhibitory receptors are expressed at high levels during the long-term activation and cytotoxicity of immune effector cells, such as NK cells.This phenomenon is useful for attenuating chronic cytotoxicity, for example, at inflammatory sites.Examples include checkpoint inhibitory receptors PD-1, CTLA-4, and CD96, all of which are expressed on NK cells.

[0052] In the present invention, it is preferable not to reduce the function of inhibitory receptors that bind to MHC class I.

[0053] The engineered cells of the invention may express chimeric antigen receptors (CARs). Membrane-bound CARs typically contain a targeting sequence (commonly a single-chain fragment (scFv) derived from an antibody), usually a hinge (to overcome steric hindrance issues), a spacer, a transmembrane element, and an internal signaling domain.

[0054] Preferably, the CAR comprises a targeting region that binds to an antigen selected from PD-L1, VEGF, VEGFR2, PD-1, MUC-1, CLL-1, TIM-3, CD19, EGFR, CD38, GD2, SLAMF7, CTLA4, CCR4, CD20, PDGFRα, HER2, CD33, CD30, CD22, CD79B, Nectin-4, and TROP2.

[0055] Preferably, the CAR comprises a targeting region that binds to an antigen selected from HER2, TIM-3, MUC-1, CD38, CD96, CLL-1, SLAMF7, and CD 19. Most preferably, the CAR binds to an antigen selected from HER2, CD38, and MUC-1.

[0056] Preferably, the CAR targets CD38, and the NK cells expressing the CAR are further modified to reduce CD38 expression. Preferably, expression of the CD38 gene is knocked out.

[0057] Examples of sequences known to bind to aberrantly glycosylated MUC-1 (such sequences include 5E5, SM3, and HMFG2) can be used as targeting sequences and are appropriately incorporated into the CARs of the present invention. Preferably, the CAR comprises an HMFG2 sequence. Nevertheless, additional sequences for targeting aberrantly glycosylated MUC-1 may be identified by screening methods known in the prior art, and high-affinity sequences can be used to produce CAR-NK cells that target MUC-1.

[0058] Examples of sequences known to bind to CD38 (such sequences include daratumumab, isatuximab, and those disclosed in WO 2018 / 104562) can be used as targeting sequences and are suitably incorporated into the CARs of the present invention. The CD38CAR preferably has reduced affinity for CD38 compared to the affinity of daratumumab for CD38. This affinity reduction is preferably at least 10%, preferably at least 25%, and more preferably at least 45% compared to the affinity of daratumumab. Furthermore, this affinity reduction is preferably 90% or less, preferably 75% or less, and more preferably 55% or less compared to the affinity of daratumumab.

[0059] The CAR used in the NK cells of the present invention may comprise or be linked to one or more NK cell costimulatory domains, such as CD28, CD134 / OX40, 4-1BB / CD137, CD3 zeta / CD247, DAP12, or DAP10, such that binding of the CAR to an antigen on a target cell promotes a cytotoxic signal in the engineered NK cell.

[0060] In any embodiment, the combination therapy of the present invention may further be used or administered in combination with an antibody specific for an antigen on a cancer cell.

[0061] The combination therapy of the invention is preferably administered in combination with an antibody that targets an antigen selected from PD-L1, VEGF, VEGFR2, PD-1, MUC-1, CLL-1, TIM-3, CD19, EGFR, CD38, GD2, SLAMF7, CTLA4, CCR4, CD20, PDGFRα, HER2, CD33, CD30, CD22, CD79B, Nectin-4, and TROP2.

[0062] Preferably, the antibody used in combination with the therapeutic methods of the invention is selected from atezolizumab, avelumab, bevacizumab, brentuximab, blinatumomab, cemiplimab, cetuximab, daratumumab, dinutuximab, durvalumab, elotuzumab, enfortumab, gemtuzumab, ibritumomab, ipilimumab, inotuzumab, isatuximab, mogamulizumab, necitumumab, nivolumab, obinutuzumab, ofatumumab, olaratumumab, panitumumab, pembrolizumab, pertuzumab, polatuzumab, ramucirumab, rituximab, sacituzumab, tositumomab, and trastuzumab.

[0063] More preferably, the antibody used in combination with the therapeutic methods of the present invention is trastuzumab or daratumumab.

[0064] The NK cells of the present invention may be treated or pre-treated to render them incapable of division, thereby further reducing the risks described above and reducing or eliminating the tendency to form tumors in the patient, as compared to, for example, T cells.

[0065] The NK cells of the present invention are for use in therapy, particularly in treating cancer in patients. The cancer is suitably a CD38-expressing cancer. The cancer is preferably myeloma, leukemia, or solid cancer. The cancer is particularly preferably multiple myeloma.

[0066] In general, NK cell-resistant cancers are well known in the art (Pardoll, D M Immunity (2015) 42:605-606). The sensitivity of cancer cells to NK cell-mediated killing is determined by several factors. There is a balance between positive and negative signals, delivered primarily by the interaction of membrane receptors on NK cells with ligands on cancer cells. It is often the balance of the expression of ligands for these receptors that determines whether a cancer is sensitive or resistant to killing by NK cells (Yokoyama, W M Immunol Res (2005) 32:317-325).

[0067] The susceptibility of cancers to NK cell-mediated cytotoxicity is generally understood to fall into one of the following categories: highly resistant, resistant, sensitive, and highly sensitive. In the laboratory, cancer cells can be screened for their susceptibility to NK cell-mediated cytotoxicity using cytotoxicity assays. Each category is understood to correspond to the proportion of cancer cells killed during exposure to NK cells at a particular effector:target (E:T) ratio for a particular period of time.

[0068] In an example of the present invention, a cancer is said to be highly resistant to NK cell-mediated killing if 25% or less of the cancer cells are killed after incubation with NK cells for up to 15 hours at an E:T ratio of up to 5:1. The NK cells are preferably KHYG-1 cells.

[0069] In an example of the present invention, a cancer is said to be resistant to NK cell-mediated killing if 50% or less of the cancer cells are killed after incubation with NK cells for up to 15 hours at an E:T ratio of up to 5:1. The NK cells are preferably KHYG-1 cells.

[0070] In the present example, a cancer is said to be susceptible to NK cell-mediated killing if more than 50% of the cancer cells are killed after incubation with NK cells for up to 15 hours at an E:T ratio of up to 5:1. The NK cells are preferably KHYG-1 cells.

[0071] In the present example, a cancer is said to be sensitive to NK cell-mediated killing if 75% or more of the cancer cells are killed after incubation with NK cells for up to 15 hours at an E:T ratio of up to 5:1. The NK cells are preferably KHYG-1 cells.

[0072] When preparing genetically modified NK cells, the modification may be performed before the cells are differentiated into NK cells. For example, pluripotent stem cells (e.g., iPSCs) can be genetically modified and then differentiated to produce genetically modified NK cells with increased cytotoxicity.

[0073] An optional aspect of the invention involves further modifying the NK cells and NK cell lines described above, for example, to express Fc receptors (which may be CD16, CD32, or CD64, including subtypes and derivatives) on the cell surface. In use, these cells may be more capable of recognizing antibody-coated cancer cells and may enhance activation of the cytotoxic response.

[0074] A further optional feature of the present invention includes adapting engineered NK cells and engineered NK cell lines to home to specific target regions of the body. NK cells of the present invention may target specific cancer cell sites. In a preferred embodiment for treating hematologic cancers, NK effectors of the present invention are adapted to home to the bone marrow. Certain NK cells are modified by fucosylation and / or sialylation to home to the bone marrow. This may be achieved by genetically modifying NK cells to express appropriate fucosyltransferases and / or sialyltransferases, respectively. Homedification of NK effector cells to tumor sites may be enhanced by disrupting tumor vasculature, for example, with metronomic chemotherapy, or by normalizing NK cell infiltration through cancer vasculature using angiogenesis-targeting agents (Melero et al., 2014).

[0075] The present invention provides a method of treating cancer in a patient, comprising: a) administering to a patient a therapeutically effective dose of an immunomodulatory drug, e.g., an IMiD; b) administering a therapeutically effective dose of NK cells to the patient; Further provided is a method wherein the NK cells are modified to reduce expression of CISH.

[0076] In a related method of the invention, there is provided a method of treating cancer in a patient, comprising: administering to the patient an immunomodulatory agent, e.g., an IMiD, in a therapeutically effective dose; Methods are provided in which the patient has already received, is receiving, or will receive a therapeutically effective dose of NK cells, and the NK cells have been modified to reduce expression of CISH.

[0077] In another related method of the invention, there is provided a method of treating cancer in a patient, comprising: administering to the patient a therapeutically effective dose of NK cells, wherein the NK cells have been modified to reduce expression of CISH; Methods are provided wherein the patient has already been administered, is being administered, or is to be administered a therapeutically effective dose of an immunomodulatory agent, such as an IMiD.

[0078] Preferably, the CISH gene is knocked out in NK cells.

[0079] Administration of the IMiD / CELMoD may occur simultaneously with, before, or after administration of the NK cells, although in a preferred method of treatment, the administration is substantially simultaneous.

[0080] Further provided is a composition (e.g., a pharmaceutical composition) comprising NK cells and an immunomodulatory agent, e.g., an IMiD, wherein the NK cells have been modified to have reduced expression of CISH.

[0081] Preferably, the CISH gene is knocked out in NK cells.

[0082] The modified NK cells, NK cell lines, and compositions thereof described hereinabove and below are suitable for the treatment of cancer, particularly cancer in humans, such as hematological cancer or solid cancer. NK cells and derivatives thereof are preferably human NK cells. For human therapy, human NK cells are preferably used. The present invention also provides a method for treating cancer in a human, comprising administering an effective amount of NK cells or a composition comprising same.

[0083] Those skilled in the art will recognize various routes of administration for delivering active agents and combinations thereof to patients in need thereof. Modified NK cells can be administered systemically or locally, for example, via the intraperitoneal route. In other embodiments, the active agent is administered more directly. Thus, administration can be direct intratumoral administration, which is particularly suitable for solid tumors.

[0084] NK cells are generally considered to be suitable for the methods, uses, and compositions of the present invention.According to the cells used in the specific examples herein, NK cells can be NK cells obtained from cancer cell lines.Advantageously, NK cells, which are preferably treated to reduce tumorigenicity, for example, by dying and / or becoming unable to divide, can be obtained from blood cancer cell lines and used in the methods of the present invention to treat blood cancer.

[0085] To make cancer-derived NK cells more acceptable for therapeutic use, they are generally treated or pretreated in some way to reduce or eliminate their tendency to form tumors in patients. The specific modified NK cell lines used in the examples are rendered nondividing, making them safe. The cell lines can be irradiated and, while retaining their killing ability, die within approximately three to four days. Therefore, certain cells and cell lines are unable to proliferate, for example, as a result of irradiation. Treatments of potential NK cells for use in the methods herein include irradiation, which prevents them from dividing and forming tumors in vivo, and genetic modifications that reduce tumorigenicity, such as inserting a sequence encoding a suicide gene that, when activated, prevents the cells from dividing and forming tumors in vivo. The suicide gene can be turned on, for example, by an exogenous agent that is circulating, subsequently causing cell death in cells expressing the gene. A further alternative is the use of monoclonal antibodies that target specific NK cells for therapeutic use. CD52, for example, is expressed on KHYG-1 cells, and binding of monoclonal antibodies to this marker can induce antibody-dependent cell-mediated cytotoxicity (ADCC) and KHYG-1 cell death.

[0086] As discussed in a paper published in Suck et al., 2006, cancer-derived NK cells and cell lines are readily irradiated using a radiation irradiator such as the GammaCell 3000 Elan. A cesium-137 source is used to control the dose of radiant energy, and a dose-response curve between, for example, 1 Gy and 50 Gy can be used to determine the optimal dose to eliminate the cells' proliferative potential while maintaining the benefits of increased cytotoxicity. This is accomplished by assaying the cells for cytotoxicity after each dose of radiation.

[0087] The use of irradiated NK cell lines for adoptive cellular immunotherapy offers significant advantages over established approaches using autologous or MHC-matched T cells. First, the use of NK cell lines with highly proliferative properties means that the expansion of modified NK cell lines can be more easily and commercially achieved. The modified NK cell lines can then be irradiated prior to administering the cells to patients. While these irradiated cells retain useful cytotoxic activity, they have a limited lifespan and, unlike modified T cells, do not circulate for long periods of time and do not cause persistent side effects.

[0088] Furthermore, the use of allogeneic engineered NK cells and NK cell lines means that they cannot inhibit NK cytotoxic responses to the same extent that MHC class I-expressing cells in the patient can inhibit the cytotoxic response of autologous NK cells. The use of allogeneic NK cells and NK cell lines to kill cancer cells benefits from the GVL effect described above, and unlike T cells, allogeneic NK cells and NK cell lines do not stimulate the development of GVHD, making them a preferable option for treating cancer via adoptive cellular immunotherapy. [Example]

[0089] The present invention will now be described in more detail and specifically with reference to the accompanying drawings, in which:

[0090] Figure 1 shows that CISH KO NK cells act synergistically with the IMiD iberdomine to enhance their cytotoxic properties against cancer cells.

[0091] For relevant examples of knockout / knockdown of inhibitory receptor function and knockin of TRAIL / mutant TRAIL, the inventors refer to WO 2017 / 017184, the contents of which are incorporated herein by reference.

[0092] For relevant examples of CD38CAR expression, the inventors refer to WO 2018 / 104562, the contents of which are incorporated herein by reference.

[0093] For relevant examples of MUC-1CAR expression, the inventors refer to WO 2019 / 101998, the contents of which are incorporated herein by reference.

[0094] Example 1 - Designed Protocol for Knockout of CISH in NK Cells NK cells were prepared to eliminate CISH function as follows: a gRNA construct was designed and prepared to target the CISH gene in NK cells, and then CRISPR / Cas9 genome editing was used to knock out CISH expression.

[0095] A total of three gRNA candidates were selected for the CISH gene, and their cleavage efficiency was determined in primary expanded NK cells. Cells were electroporated with the gRNA:Cas9 ribonucleoprotein (RNP) complex using a Maxcyte® GT, and then the knockout of CISH was analyzed by flow cytometry. The cleavage activity of the gRNA was also determined using an in vitro mismatch detection assay. T7E1 endonuclease recognizes and cleaves imperfectly matched DNA, allowing the parent CISH gene to be compared with the mutant gene after CRISPR / Cas9 transfection and non-homologous end joining (NHEJ).

[0096] The gRNA with the highest knockout efficiency is selected for further experiments to knockout CISH in primary NK cells, NK cell lines, or CD34+ progenitor cells (e.g., iPSCs that subsequently differentiate and proliferate into NK cells). Knockout of CISH is confirmed by a flow cytometry-based assay.

[0097] Example 2 - Knockout of CISH in NK cells (material) 1.Enriched cord blood-derived NK cells. 2. NK-MACS medium, premium grade IL-15, anti-CD3 VioBlue, anti-CD56 VB515, Inside staining kit, MACSquant16 (Miltenyi Biosciences). 3. Human serum albumin (Sigma). 4. Gene Knockout Kit V2 for CISH and Cas9 Recombinant Protein (Synthego). 5. Cloudz™ Cell Activation Kit (R&D Systems). 6. Anti-CISH antibody (D4D9) (Cell Signaling Technology). 7. Electroporation buffer (EB buffer), OC-100X2 Processing Assembly (PA), MaxCyte ATx Electroporation System (MaxCyte). 8. Solu-Cortef (hydrocortisone) (Pfizer). 9.Ficoll (Sytiva).

[0098] (protocol) Cord blood mononuclear cells (CBMCs) were isolated by the Ficoll method and enriched for 15 days in NK-MACS medium containing 10% human serum albumin, CD2 / NKp46 microspheres (Cloudz™), anti-CD3, anti-CD16, IL-15, and hydrocortisone.

[0099] After staining with anti-CD3 and anti-CD56 antibodies, NK cell enrichment was confirmed to be >90% by FACS (MACSquant 16) analysis. CBNK cells were washed twice with Maxcyte electroporation buffer (EB buffer) at 300 × g for 10 min to obtain 5 million cells in 75 μl of EB buffer.

[0100] sgRNA for CISH was dissolved in nuclease-free TE buffer to obtain a 200 μM stock. 1200 pmol of sgRNA was mixed with 160 pmol of Cas9 at a 7.5:1 ratio. The volume was adjusted to 25 μl with EB buffer and incubated at room temperature for 10 minutes to allow RNP complex formation.

[0101] 25 μl of the RNP complex was mixed with 75 μl of CBNK cells and transferred to an OC-100X2 processing assembly. Cells were electroporated using the NK5 protocol and incubated at room temperature for 15 minutes. As a control, no RNP was used.

[0102] After electroporation, cells were transferred to Grex 6-well plates in NK-MACS medium containing 10% AB and 20 ng / mL IL-15 and incubated at 37°C in a 5% CO2 incubator for 13 days. Cells were then analyzed for CISH knockout every 3–4 days. Intracellular expression of CISH was analyzed using the Inside staining kit and an anti-CISH antibody.

[0103] It was observed that the expression of CISH was successfully knocked out in NK cells.

[0104] These NK cells exhibited superior proliferation and cytokine production compared to unmodified control NK cells, and had an overall enhanced cytotoxic phenotype.

[0105] Example 3 - CISH KO NK Cells Combined with IMiDs for Myeloma Therapy The following protocol has been developed for use in treating patients with multiple myeloma: Nevertheless, it will be apparent that the present invention is suitable for treating patients with many different cancers. After a patient is diagnosed with multiple myeloma, an aliquot of CISH KO NK cells is thawed, cultured, and administered to the patient at an effective dose. The sorted cells may be further modified as described elsewhere herein. Alternatively, transient transfections can be prepared, for example, using viral means or electroporation. For electroporation, the MaxCyteFlow electroporation platform provides a suitable solution for achieving rapid, large-scale transfection in the clinic. After the NK cells are effectively modified, they can be administered intravenously to the patient.

[0106] An effective dose of lenalidomide is administered to the patient prior to, concurrently with, or after administration of the CISH KO NK cells.

[0107] Example 4 - Enhanced Cytotoxicity of CISH KO NK Cells in Combination with Ikaros Pathway Modulators NK cells from umbilical cord blood were isolated for use in wild type or CISH gene knockout (as in Example 2).

[0108] The cytotoxicity of wild-type (WT) or CISH KO NK cells alone or in the presence of 10 nM iverdomide against SKOV-3GFP ovarian cancer target cells was assessed using the following experimental setup. Human ovarian cancer target cells were plated in 96-well eSight plates and allowed to adhere. After this, fresh NK cells were added at an effector-to-target ratio (E:T) of 1:1 for 10 hours. Percent killing was calculated based on cell impedance using the RTCA eSight system. Experiments were performed using 1) WT control NK cells, 2) WT NK cells + 10 nM iverdomide, 3) CISH KO NK cells alone, and 4) CISH KO NK cells + 10 nM iverdomide.

[0109] As can be seen in Figure 1, iberdomide treatment of WT NK cells did not improve the death rate compared to WT NK cells alone. In both groups, 15% to 20% of cells died at 32 hours. Approximately 27% of CISH KO NK cells died at 32 hours, but the CISH KO NK cell composition with iberdomide significantly improved the death rate to approximately 40%.

[0110] These data demonstrate that genetic knockout of CISH in NK cells functions synergistically with iverdomid to result in an NK cell phenotype with significantly enhanced cytotoxicity.

[0111] Thus, the present invention provides NK cells in combination with an immunomodulatory agent and the use of this combination in cancer therapy.

Claims

1. A natural killer (NK) cell or NK cell line in combination with a cereblon E3 ligase modulator (CELMoD) or an immunomodulatory imid drug (IMiD) for use in the treatment of cancer, wherein the NK cell or NK cell line has been modified to have reduced expression of CISH compared to the same NK cell or NK cell line not modified.

2. 2. The NK cell or NK cell line for use according to claim 1, wherein the IMiD is selected from lenalidomide and iverdimide.

3. 3. The NK cell or NK cell line for use according to claim 1 or claim 2, wherein the IMiD is iverdomid.

4. NK cells or NK cell lines for use according to any of the preceding claims, wherein said modification is genetic knockdown of expression of CISH.

5. NK cells or NK cell lines for use according to any of the preceding claims, wherein said modification is a genetic knockout of expression of CISH.

6. 10. The NK cell or NK cell line for use according to any of the preceding claims, wherein the expression of CISH is reduced by at least 50% compared to the same NK cell or NK cell line not modified.

7. 10. The NK cell or NK cell line for use according to any of the preceding claims, wherein the expression of CISH is reduced by at least 90% compared to the same NK cell or NK cell line not modified.

8. NK cells or NK cell lines for use according to any of the preceding claims, which have been further modified to express IL-15, preferably in a soluble form (sIL-15).

9. NK cells or NK cell lines for use according to any of the preceding claims, which have been further modified to express a TRAIL variant with improved affinity for a TRAIL death receptor.

10. 10. The NK cell or NK cell line for use according to any of the preceding claims, further modified to express a chimeric antigen receptor (CAR) that binds HER2, TIM-3, CD19, CD38, MUC-1, CLL-1, SLAMF7, and / or CD96.

11. 10. The NK cell or NK cell line for use according to any of the preceding claims, wherein said cancer is a hematological cancer.

12. 13. The NK cell or NK cell line for use according to claim 12, wherein the hematological cancer is selected from multiple myeloma, acute myeloid leukemia, and acute lymphoblastic leukemia.

13. 12. The NK cell or NK cell line for use according to any one of claims 1 to 11, wherein the cancer is a solid cancer.

14. A pharmaceutical composition comprising IMiD or CELMoD and NK cells, wherein expression of CISH has been knocked out in the NK cells.

Images