Recombinant erIL-15 NK cells

Abstract

To provide a genetically modified NK cell, and to provide a pharmaceutical composition comprising the genetically modified NK cell.SOLUTION: Provided is a genetically modified NK cell comprising a recombinant nucleic acid that includes a first segment encoding erLSP-IL-15. As one aspect, the NK cells are NK-92 cells, the recombinant nucleic acid are DNA, the recombinant nucleic acid further comprises a second segment encoding CD16 or high affinity CD16, and the recombinant nucleic acid further comprises a third segment encoding a chimeric antigen receptor. A pharmaceutical composition comprising a pharmaceutically acceptable carrier in combination with the genetically modified NK cell is also provided.SELECTED DRAWING: Figure 9

Description

REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority to the inventors' U.S. Provisional Patent Application No. 62 / 819,256, filed March 15, 2019, the contents of which are incorporated herein by reference in their entirety.

[0002] Sequence Listing The contents of the sequence listing ASCII text file entitled Sequence_listing_ST25, which is 8kb in size, created on February 19, 2020, and filed electronically via EFS-Web herewith, are incorporated by reference in their entirety. [Technical Field]

[0003] The present disclosure relates to genetically modified immune cells that express IL-15, and in particular, the present disclosure relates to NK cells that express and intracellularly retain modified IL-15 and further express at least one of a high-affinity variant of CD16 and a CAR (chimeric antigen receptor). [Background technology]

[0004] The background discussion includes information that may be useful in understanding the present disclosure. No admission is made that any of the information provided herein is prior art or relevant to the invention claimed herein, or that any publication specifically or implicitly referenced is prior art.

[0005] All publications and patent applications herein are incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. In the event that a definition or use of a term in an incorporated reference is inconsistent with, or inconsistent with, or contrary to the definition of that term provided herein, the definition of the term provided herein shall apply and the definition of the term in the reference shall not apply.

[0006] NK-92 cells are desirable in various aspects of cell-based therapy because they have a relatively broad spectrum of cytotoxic activity against various tumor cells without apparent toxicity to allogeneic cells in the recipient. Furthermore, NK-92 cells can be cultured using relatively simple methods, making them an attractive option for adoptive cancer immunotherapy. Unfortunately, the growth and function of NK-92 cells are highly dependent on IL-2, which increases costs when NK-92 cells are required on a large scale. To circumvent the problems associated with the need for such cytokines, NK-92 cells have been transformed to express and retain IL-2 intracellularly (see, e.g., Exp Hematol 33:159-164). While such modified cells have indeed become independent of exogenous cytokines, various disadvantages remain. Notably, IL-2 released from such modified cells can increase IL-2-mediated effects in vivo when such cells are used in mammals, which is particularly harmful when IL-2 stimulates immunosuppression in the tumor microenvironment (typically via the growth and proliferation of myeloid-derived suppressor cells (MDSCs) and regulatory T cells (Tregs)).

[0007] In another example, NK-92 cells were transfected to express IL-15 from a cDNA cloned into the pcDNA3 expression vector (see, e.g., Haematologica, 2004;89:338-347); such transfected cells continuously produced high levels of IL-15 in the culture supernatant, which appeared to allow the cells to proliferate significantly more rapidly in response to stimulation with low doses of IL-2 or IL-15. Furthermore, the cumulative number of cells in long-term culture was also significantly greater than that of non-transfected cells. However, when such cells are used in vivo, high levels of secreted IL-15 may be a clinical problem.

[0008] Similarly, NK-92 cells were transformed using a viral transfection system to express a recombinant native form of IL-15 (see, e.g., Cancer Immunol Immunother (2012) 61:1451-1461). Although such recombinant cells were able to grow in the absence of exogenous cytokines and expressed the recombinant CAR, the transfection efficiency was relatively low, the amount of IL-15 produced intracellularly was relatively low, and the amount of IL-15 secreted into the culture medium was low. Furthermore, the cytotoxicity of the recombinant cells was reduced compared to the parental NK-92 cell line. Notably, when similar IL-15 was expressed from a plasmid, the NK-92 cells so generated were not completely independent of exogenous growth factors, thus limiting the in vivo use of such recombinant cells.

[0009] Thus, even though various modified immune cells, particularly modified NK cells, are known in the art, all or nearly all suffer from various disadvantages. Consequently, there is a need to provide improved modified NK cells that exhibit desirable growth characteristics while maintaining targeted cytotoxicity. Summary of the Invention

[0010] Disclosed herein are various recombinant cells, compositions, and methods in which NK cells are genetically modified to intracellularly express, secrete, or present IL-15 or a variant thereof, rendering the modified NK cells independent of exogenous cytokines and enabling stimulation / activation of other immune-competent cells in the vicinity of the modified NK cells.

[0011] In one aspect of the present subject matter, the inventors contemplate genetically modified NK cells and methods for producing such cells, wherein the genetically modified NK cells comprise a recombinant nucleic acid comprising a first segment encoding erLSP-IL-15. Most typically, the NK cells are NK-92 cells, and the recombinant nucleic acid is DNA (e.g., a linear plasmid). Preferably, but not necessarily, the IL-15 portion in erLSP-IL-15 comprises a codon-optimized human IL-15 sequence.

[0012] In further embodiments, the recombinant nucleic acid may further comprise a second segment encoding CD16 or a high-affinity CD16 and / or a third segment encoding a chimeric antigen receptor and / or a fourth segment encoding a protein that confers immune stimulation, prevents checkpoint inhibition, a protein that binds to / inhibits a cytokine involved in immune suppression, and / or the IL-15 receptor alpha chain. It is also contemplated that the recombinant nucleic acid may comprise a promoter strong enough to drive expression of erLSP-IL-15 in an amount sufficient to (a) render the engineered NK cells independent of exogenous cytokines and (b) enable stimulation / activation of other immune-competent cells in the vicinity of the engineered NK cells. Optionally, the engineered NK cells may comprise an antibody tethered to the cells via CD16.

[0013] In a further aspect of the inventive subject matter, the inventors also contemplate pharmaceutical compositions comprising the genetically modified NK cells described herein in combination with a pharmaceutically acceptable carrier. Most typically, such compositions are formulated for infusion into a patient and contain at least 1 x 10 cells per dosage unit. 9 It may contain cells.

[0014] Thus, the inventors also contemplate the use of the genetically modified NK cells described herein in the treatment of cancer. While such use may be the infusion of cells as a stand-alone therapy, other contemplated uses also include the administration of drugs that penetrate the TME, reduce immunosuppression, stimulate immune-competent cells, cancer vaccine compositions, and / or help maintain the immune response and promote the development of memory cells.

[0015] Various objects, features, aspects and advantages will become more apparent from the following detailed description of preferred embodiments, taken in conjunction with the accompanying drawings, in which like numerals represent like elements and in which: [Brief explanation of the drawings]

[0016] [Figure 1] FIG. 1 shows an exemplary graph depicting the growth rates of various NK cells as indicated. [Figure 2] FIG. 2 shows exemplary results from immunophenotyping of NK cells as shown. [Figure 3] 3-5 show exemplary results for the cytotoxicity of the various NK cells indicated. [Figure 4] 3-5 show exemplary results for the cytotoxicity of the various NK cells indicated. [Figure 5] 3-5 show exemplary results for the cytotoxicity of the various NK cells indicated. [Figure 6] 6-8 show further exemplary results for the cytotoxicity of the various NK cells indicated. [Figure 7] 6-8 show further exemplary results for the cytotoxicity of the various NK cells indicated. [Figure 8] 6-8 show further exemplary results for the cytotoxicity of the various NK cells indicated. [Figure 9] FIG. 9 shows the schematic layout of an exemplary recombinant nucleic acid used herein. Specific Description of the Invention

[0017] The inventors have now discovered that modified NK cells can be generated that produce sufficient amounts of recombinant IL-15 to provide IL-2 / IL-15-independent growth and stimulation while maintaining cytotoxicity and functional expression of recombinant CD16 and / or CAR. Furthermore, in at least some embodiments, contemplated modified NK cells not only produce sufficient intracellular IL-15 (particularly erLSP-IL-15) to allow growth and proliferation in the absence of cytokines, but also secrete IL-15 in amounts that support immunostimulation (or reversal of immunosuppression) in the tumor microenvironment (TME) when such cells are present in the TME. Thus, the NK cells of the present subject matter advantageously allow for simplified culture and expansion while providing a means for recombinant targeted cytotoxicity via CD16 and / or CAR.

[0018] Previously prepared NK-92 derivatives expressing IL-2 with an endoplasmic retention sequence appeared to support the growth and proliferation of immunosuppressive cells, such as myeloid-derived suppressor cells (MDSCs) and regulatory T cells (T-regs). These negative regulatory factors may attenuate any antitumor effects of immune cells, particularly aNK, haNK, and t-haNK, as well as donor NK cells. On the other hand, recombinant IL-15 only supports the function of immune-activated cells, without the negative effects of IL-2.

[0019] However, it should be understood that expression of IL-15 and its variants may adversely affect the growth and / or function of NK cells, particularly NK-92 cells. In particular, it is not expected that biologically active forms of IL-15 and its variants can be expressed in amounts that support cell proliferation and activity without adversely interfering with further desired recombinant proteins in the target NK cells, such as CD16 and / or CAR. Furthermore, overexpression and secretion of IL-15 and its variants beyond immune-stimulatory levels may result in systemic side effects in recipients of such cells. From various perspectives, preferred engineered NK cells will produce and secrete IL-15 at a sufficiently low level to avoid systemic side effects, yet maintain the desired beneficial effects, growth, and stimulatory properties of the engineered cells and immune cells within the TME. In particular, the inventors have now discovered that NK cells can be prepared that have a desirable balance between intracellularly formed and retained IL-15 to stimulate growth and proliferation in an autocrine manner (i.e., without the need for exogenous cytokines) and secreted IL-15 that provides an immunostimulatory effect on other immune-competent cells in the TME.

[0020] With regard to suitable NK cells, it is generally contemplated that the NK cells may be autologous NK cells from the subject who will receive the genetically modified NK cells. Such autologous NK cells may be isolated from whole blood or cultured from progenitor or stem cells using methods well known in the art. However, it should also be understood that the NK cells need not be autologous, but may be allogeneic or xenogeneic NK cells. In particularly preferred embodiments of the present subject matter, the genetically engineered NK cells are NK-92 cells or derivatives thereof. For example, in one particularly preferred embodiment of the present subject matter, the genetically engineered NK cells are NK-92 derivatives modified to have reduced or abolished expression of at least one killer cell immunoglobulin-like receptor (KIR), which typically renders such cells constitutively activated (through the absence or reduction of inhibition).

[0021] NK-92 cells exhibit an unusual receptor expression profile, expressing a relatively large number of activating receptors (e.g., NKp30, NKp46, 2B4, NKGD, E, and CD28). Conversely, NK-92 cells also express few inhibitory receptors (e.g., NKGA / B, low levels of KIR2DL4, and ILT-2) and lack most killer cell inhibitory receptors (KIRs) clonally expressed on conventional NK cells. In addition, NK-92 cells express relatively high levels of molecules involved in the perforin-granzyme cytolytic pathway and additional cytotoxic effector molecules, including tumor necrosis factor (TNF)-superfamily members FasL, TRAIL, TWEAK, and TNF-alpha, demonstrating their ability to kill via alternative mechanisms. Furthermore, NK-92 cells also express other molecules involved in immune effector cell regulation (e.g., CD80, CD86, CD40L, and TRANCE), although their relevance in NK killing is unclear. Notably, however, these particularly desirable traits will not be adversely affected by the modifications described herein. Indeed, in at least some embodiments, one or more of the above-described activator and / or effector proteins may be overexpressed in response to intracellular expression of IL-15, as described in more detail below.

[0022] Additionally, suitable NK cells may have one or more modified KIRs mutated, such as to reduce or abolish interaction with MHC class I molecules. Of course, it should be noted that one or more KIRs may also be deleted or silenced (e.g., via miRNA, siRNA, etc.). Most typically, more than one KIR will be mutated, deleted, or silenced, and particularly contemplated KIRs include those with two or three domains and short or long cytoplasmic tails. From various perspectives, modified, silenced, or deleted KIRs may include KIR2DL1, KIR2DL2, KIR2DL3, KIR2DL4, KIR2DL5A, KIR2DL5B, KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS4, KIR2DS5, KIR3DL1, KIR3DL2, KIR3DL3, and KIR3DS1. Such modified cells may be prepared using protocols well known in the art. Alternatively, such cells may also be commercially obtained as aNK cells (activated natural killer cells) from NantKwest (see URL www.nantkwest.com). Such cells may then be additionally genetically modified to express IL-15 or a variant thereof, as discussed further below.

[0023] In another preferred embodiment of the present subject matter, the genetically engineered NK cells may also be NK-92 derivatives modified to express a high-affinity Fcγ receptor (CD16). Sequences of high-affinity variants of Fcγ receptors are well known in the art (see, e.g., Blood 2009 113:3716-3725), and all methods of production and expression are considered suitable for use herein. Expression of such receptors may allow for specific targeting of tumor cells in a patient using antibodies specific for tumor cells (e.g., neoepitopes), specific tumor types (e.g., her2neu, PSA, PSMA, etc.), or associated with cancer (e.g., CEA-CAM). Advantageously, such antibodies are commercially available and can be used in conjunction with the cells (e.g., Fcγ receptor-bound). Alternatively, such cells may also be obtained commercially from NantKwest as haNK cells ("high affinity natural killer cells"). Such cells may then be further genetically modified to express IL-15 or a variant thereof, as discussed further below.

[0024] Alternatively or additionally, genetically engineered NK cells may also be genetically engineered to express a chimeric antigen receptor (CAR). In particularly preferred embodiments, the CAR has an scFv portion or other ectodomain with binding specificity for tumor-associated antigens, tumor-specific antigens, and cancer neoepitopes. Accordingly, as will be readily understood, suitable CARs include first-, second-, and third-generation CARs (see, e.g., Immunotherapy (2015) 7(5):487-497). As previously mentioned, there are numerous methods for genetically engineering NK cells to express such chimeric T cell receptors, and all methods are considered suitable for use herein. Alternatively, such cells may also be commercially obtained as taNK cells ("targeted activated natural killer cells") from NantKwest. Such cells may then be further genetically modified to express IL-15 or a variant thereof, as further discussed below.

[0025] When cells are engineered to have an affinity for a cancer-associated antigen or an antibody with specificity for a cancer-associated antigen, it is contemplated that any known cancer-associated antigen would be considered suitable for use. For example, cancer-associated antigens include CEA, MUC-1, CYPB1, etc. Similarly, when cells are engineered to have an affinity for a cancer-specific antigen or an antibody with specificity for a cancer-specific antigen, it is contemplated that any known cancer-specific antigen would be considered suitable for use. For example, cancer-specific antigens include PSA, Her-2, PSA, Brachyury, etc. When cells are engineered to have an affinity for a cancer neoepitope or an antibody with specificity for a cancer neoepitope, it is contemplated that any known method for identifying neoepitopes would lead to suitable targets. For example, neoepitopes may be identified in a first step from a patient tumor by genome-wide analysis of a tumor biopsy (or lymphatic biopsy or metastatic biopsy) and a matched normal tissue (i.e., a non-diseased tissue from the same patient) through a synchronous comparison of the omics information thus obtained. The neoepitopes thus identified can then be further filtered for matching to the patient's HLA type to increase the likelihood of antigen presentation of the neoepitopes. Most preferably, such matching can be performed in silico.

[0026] Regarding IL-15 sequences suitable for expression, it is generally preferred that the IL-15 be a mammalian IL-15 sequence, most preferably a human IL-15 sequence (see, e.g., UniProt identifier P40933). Furthermore, it should be noted that suitable IL-15 sequences include various variants, and particularly contemplated variants include those with long signal peptides (LSP) and short signal peptides (SSP). LSP variants typically have a 48-amino acid signal peptide, and transcripts typically include a 316-base 5'-untranslated region (UTR), a 486-base coding sequence, and a C-terminal 3'-UTR region that is approximately 400 bases in length. SSP variants typically have a short 21-amino acid signal peptide (encoded by exons 4A and 5) that is based on alternative splicing. Notably, both isoforms produce the same mature protein but have distinct cellular transport pathways. More specifically, the IL-15 LSP isoform is detected in the Golgi apparatus, early endosomes, and endoplasmic reticulum and can exist in secreted and membrane-bound forms. On the other hand, the IL-15 SSP isoform is not secreted and appears to be restricted to the cytoplasm and nucleus, where it is thought to be involved in cell cycle regulation. Further contemplated IL-15 variants include superagonist variants, particularly the IL-15 N72D mutant, which may or may not contain additional signal peptides and / or trafficking signals, as discussed below.

[0027] Based on the clear signaling differences, the inventors therefore contemplate the use of various modifications to recombinant IL-15 to achieve appropriate expression levels, intracellular distribution, and secreted amounts. To this end, the inventors contemplate the use of various signaling moieties that can be fused to recombinant IL-15. For example, if IL-15 or an IL-15 variant is to be exported to endosomal and lysosomal compartments, a leader peptide, such as the CD1b leader peptide, may be used to sequester the (nascent) protein from the cytoplasm. Additionally or alternatively, a targeting presequence and / or targeting peptide can be used. The targeting peptide presequence can be added to the N-terminus and / or C-terminus and typically comprises 6 to 136 basic and hydrophobic amino acids. In the case of peroxisomal targeting, the targeting sequence can be at the C-terminus. Other signals (e.g., signal patches) can be used, including sequence elements that are detached from the peptide sequence and become functional upon proper peptide folding. Additionally, protein modifications, such as glycosylation, can induce targeting. Among other suitable targeting signals, the inventors contemplate peroxisomal targeting signal 1 (PTS1), a C-terminal tripeptide, and peroxisomal targeting signal 2 (PTS2), a nonapeptide located near the N-terminus.

[0028] In addition, protein sorting to endosomes and lysosomes can also be mediated by signals within the cytosolic domain of the protein, typically containing short linear sequences. Some signals, called tyrosine-based sorting signals, fit the NPXY or YXXφ consensus motif. Other signals, known as dileucine-based signals, fit the [DE]XXXL[LI] or DXXLL consensus motif. All of these signals are recognized by components of protein coats associated with the cytoplasmic face and periphery of the membrane. The YXXφ and [DE]XXXL[LI] signals are recognized with characteristic fine specificity by the adaptor protein (AP) complexes AP-1, AP-2, AP-3, and AP-4, whereas the DXXLL signal is recognized by another family of adaptors known as GGAs. Additionally, a FYVE domain, associated with vacuolar protein sorting and endosomal function, can be added. In a further embodiment, the endosomal compartment can also be targeted using the human CD1 tail sequence (see, e.g., Immunology, 122, 522-531). For example, targeting of lysosomes can be achieved using the LAMP1-TM (transmembrane) sequence, while recycling endosomes can be targeted via the CD1a tail targeting sequence and sorting endosomes can be targeted via the CD1c tail targeting sequence.

[0029] Transport to or retention within the cytosolic compartment does not necessarily require one or more specific sequence elements. However, in at least some embodiments, an N- or C-terminal cytoplasmic retention signal, such as a membrane-anchoring protein or a membrane-anchoring domain of a membrane-anchoring protein, can be added to retain the protein within the cell facing the cytosol. For example, membrane-anchoring proteins include SNAP-25, syntaxin, synaptoprevin, synaptotagmin, vesicle-associated membrane protein (VAMP), synaptic vesicle glycoprotein (SV2), high-affinity choline transporter, neurexin, voltage-gated calcium channel, acetylcholinesterase, and NOTCH.

[0030] In further contemplated aspects of the present subject matter, IL-15 or IL-15 variants may also comprise one or more transmembrane segments that, after processing, present neoepitopes to the outside of the cell membrane and make them recognizable to immune-competent cells. There are numerous transmembrane domains known in the art, all of which are believed to be suitable for use herein, including those with a single alpha helix, multiple alpha helices, alpha / beta barrels, etc. For example, contemplated transmembrane domains include those derived from T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8 (e.g., CD8 alpha, CD8 beta), CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, KIRDS2, OX40, CD2, CD27, LFA-1 (CD11a, CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD160, CD19, IL2R beta, IL2R gamma, IL7R alpha, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VL A-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​or the transmembrane region of the alpha, beta, or zeta chain of PAG / Cbp.

[0031] Additionally, it is also contemplated that IL-15 or an IL-15 variant may be co-expressed with the IL-15 receptor alpha subunit. Such co-expression is believed to allow binding or other association of the IL-15 or IL-15 variant with the receptor alpha chain, stabilizing the IL-15 or IL-15 variant and / or supporting the secretion or transport / presentation of the IL-15 or IL-15 variant on the surface of the engineered NK cell. Similarly, various other proteins (other than CD16 and / or CAR) may be co-expressed with the IL-15 or IL-15 variant; suitable co-expressed proteins include various immunomodulatory compounds, particularly compounds that interfere with checkpoint inhibition (e.g., scFvs against PD1, PD-L1, CTLA4, etc.), immune stimulators (e.g., IFN-γ, IL-12, IL-21, etc.), and / or compounds that bind to / inhibit cytokines involved in immune suppression (e.g., TGF-β, IL-8, etc.).

[0032] As will be readily understood, the IL-15 or IL-15 variant and other co-expressed proteins are encoded on a recombinant nucleic acid, which may be one or more recombinant RNA or DNA molecules. Most typically, however, the recombinant nucleic acid is a polycistronic DNA construct, preferably a plasmid. However, a variety of other constructs are also contemplated as suitable for use herein, including viral vectors (which may be transfected virally or via other methods), linear DNA, DNA bound to a carrier (e.g., for ballistic transfection), and the like.

[0033] Regardless of the particular form and type of nucleic acid, it is generally preferred that the modified NK-92 cells express a sufficient amount of IL-15 or IL-15 variant to (a) render the so-transfected cells independent of exogenous cytokines and (b) enable stimulation / activation of other immune-competent cells in the vicinity of the transfected cells (typically within the TME). For example, it is contemplated that secreted or extracellular (or extracellularly displayed) recombinant IL-15 or IL-15 variant will account for between 5-10%, or between 10-20%, or between 20-30%, or between 30-50% of the total IL-15 or IL-15 variant produced in the cells. Thus, and from various perspectives, secreted or extracellular (or extracellularly displayed) recombinant IL-15 or IL-15 variant may be present in an amount of between about 20-60 pg / ml, or between about 60-80 pg / ml, or between about 80-100 pg / ml, or between about 100-150 pg / ml, or between about 150-200 pg / ml or more, whereas it is contemplated that intracellularly retained IL-15 or IL-15 variant will be present in an amount of between about 100-150 pg / ml, or between about 150-250 pg / ml, or between about 250-500 pg / ml, or between about 500-750 pg / ml or more. Thus, particularly preferred modified NK-92 cells will produce recombinant IL-15 or IL-15 variants in amounts sufficient to support autonomous growth and stimulate immune-competent cells in the TME, as well as to stimulate the establishment and maintenance of CD8+ T cell memory, although it should be noted that such amounts are insufficient to cause systemic adverse events in patients receiving such cells.

[0034] Thus, from a functional standpoint, recombinant IL-15 or IL-15 variants will be present to stimulate or enhance the effector function and / or proliferation of other NK cells (e.g., autologous NK cells in the TME), various T cells, etc., as well as to enhance or trigger Jak / STAT signaling in cells in the TME. Furthermore, by virtue of the fraction of IL-15 or IL-15 variants retained intracellularly, such cells will also be able to proliferate in the complete absence of exogenous IL-2 and / or IL-15, as described in further detail below. In further contemplated embodiments, such modified NK-92 cells also have increased sensitivity to IL-12 signaling compared to unmodified NK-92 cells, which may reduce IL-4-mediated suppression of IFN-γ, which in turn may reduce suppression of Th1 T cells in the TME.

[0035] The contemplated use of the modified NK cells is preferably in the treatment of diseases that respond to the administration of NK cells, particularly in the treatment of various cancers. As will be readily understood, the modified NK cells may be administered as the sole therapeutic agent or as a drug in a more complex regimen. For example, the modified NK cells may be part of an immunotherapy strategy in which a tumor may be initially treated with a drug that penetrates the TME (e.g., Abraxane), a drug that reduces immunosuppression (e.g., Cytoxan), a drug that stimulates various immune-competent cells (e.g., ALT-803), a cancer vaccine composition (e.g., a recombinant AdV encoding a tumor neoantigen), and / or a drug that helps maintain the immune response and promote the development of memory cells (e.g., tumor-targeted IL-12). Exemplary suitable treatment regimens are discussed in WO 2018 / 005973, which is incorporated herein by reference.

[0036] Regarding suitable dosages and modes of administration, it is generally preferred that the amount of cells and infusion schedule typically follow known established protocols for NK cell infusion. Thus, a typical amount of modified NK-92 cells is 5×10 7 cells / dose IV~5×10 8 or 5 x 10 cells / dose IV 8 cells / dose IV~5×10 9 or 5 x 10 cells / dose IV 9 cells / dose IV~5×10 10 Between 7 x 10 cells / dose IV and most typically 7 x 10 8 cells / dose IV~7×10 9 Between cells / dose IV (e.g., 2 x 10 9 Of course, it will be understood that if the modified NK cells express CD16 or a high affinity variant of CD16 (e.g., 158V), the cells may be co-administered with one or more antibodies that will favorably bind to the modified NK cells (either prior to infusion in vitro or sequentially, e.g., administering the antibodies prior to cell infusion). [Example]

[0037] The following examples provide representative guidance only and should not be understood as limiting the subject matter of the present invention. Unless otherwise noted, all recombinant expression constructs used the codon-optimized version of IL-15 shown as SEQ ID NO: 1, which, at least in some embodiments, resulted in high yields of recombinant protein (data not shown).

[0038] In a first set of experiments, three variants of IL-15 were created using SEQ ID NO: 1: a long signal peptide variant corresponding to wild-type IL-15 (LSP-IL-15), a long signal peptide variant with an ER-retention signal (erLSP-IL15), and a short signal peptide variant that is an alternative splice variant (SSP-IL-15). More specifically, in one embodiment, the IL-15 LSP had the nucleotide sequence shown in SEQ ID NO: 2; the IL-15 LSP had the peptide sequence of SEQ ID NO: 3; the erIL-15 LSP had the nucleotide sequence of SEQ ID NO: 4; the erIL-15 LSP had the peptide sequence of SEQ ID NO: 5; the IL-15 SSP had the nucleotide sequence of SEQ ID NO: 6; and the IL-15 had the peptide sequence of SEQ ID NO: 7.

[0039] Plasmid design and transfection: Three gBlocks flanked by KpnI / NotI restriction enzyme sites were synthesized by Integrated DNA Technologies and used to subclone the respective sequences into the similarly digested pNEUkv1-CD16-erIL2 plasmid backbone, generating the following plasmids: pNEUkv1-CD16(158V)-IRES-(KpnI)-[IL-15 LSP]-(NotI) pNEUkv1-CD16(158V)-IRES--(KpnI)-[erIL-15 LSP]-(NotI) pNEUkv1-CD16(158V)-IRES--(KpnI)-[IL-15 SSP]-(NotI)

[0040] All plasmids were verified by Sanger sequencing, linearized by SalI restriction enzyme digestion, and isolated by column purification. Cell lines were electroporated using a MaxCyte GT electroporator with 1 μg of linear DNA / 10 6The cells were electroporated (program NK-92-2-OC). pNEUkv1-CD16-IRES-[erIL-2] was prepared and electroporated in the same manner as a positive control. This experiment was repeated twice to confirm the results. NK-92 cell lines co-expressing CD16 and erIL-15 were generated by electroporating the following linearized plasmids into aNK (NK-92 "wild type"): pNEUkv1-CD16(158V)-IRES-[LSP IL-15]; pNEUkv1-CD16(158V)-IRES-[erIL-15]; and pNEUkv1-CD16(158V)-IRES-[SSP IL-15]. The positive control was transfected with pNeukv1-CD16-IRES-[erIL-2]. All samples were incubated in X-vivo-10 / 5% HS for 2 weeks in the absence of any cytokines.

[0041] To determine successful transfection of the plasmids, electroporated cells were grown in X-vivo-10 media with 5% HS for 2 weeks in the absence of any cytokines. Notably, only one of the three tested IL-15 clones was successfully renatured: the long signal peptide with an ER-retention signal (erIL-15). Furthermore, as shown in more detail below, NK-92 cells expressing erIL-15 (erLSP-IL15) maintained all relevant NK surface markers and cytotoxicity. Furthermore, these cells also produced desirable amounts of extracellular erLSP-IL15, which likely retained its biological function for immune stimulation after removal of the er / LSP segment from mature IL-15.

[0042] As can be seen in Figure 1, expression of erLSP-IL-15 did not alter the growth rate of erLSP-IL-15-expressing cells in the absence of exogenous cytokines compared to aNK or haNK (CD16+, erIL-2) cells. Thus, recombinant expression of erLSP-IL-15 advantageously provided sufficient autocrine signaling to support cell proliferation. This result was unexpected, as the ER retention sequence likely was not removed intracellularly but still maintained proper function. Furthermore, recombinant expression of erLSP-IL-15 also did not adversely affect numerous phenotypic markers of the engineered N-92 cells. Exemplary results from immunophenotyping of the newly generated cell lines are shown in Figure 2. Here, the flow cytometry surface receptor profile tested was identical to that of haNK cells and the CD16-expressing NK-92 cell line coexpressing erIL2.

[0043] In this context, it should be noted that the newly created cell lines maintained full expression of functionally important surface proteins: CD16 (Fc receptor) and the activating receptor NKG2D. To test the functionality of the erIL-15-expressing cell lines and for comparison with erIL-2-expressing cell lines (haNK), cytotoxicity assays were performed using various target cell lines: K562, Raji, and HL-60. The results, shown in Figures 3–5, clearly demonstrate that the introduction of erIL-15 did not alter the spontaneous cytotoxic properties compared to haNK cells. This is an unexpected finding, as the integration of a novel / different gene may affect the cytotoxic function of NK-92 cells. Notably, as can be seen in Figures 3–5, erIL-15-expressing cells outperformed erIL-2-expressing cells at the same effector-to-target cell ratio.

[0044] To further test whether the new erIL-15-based plasmids could be incorporated into tricistronic CAR-expressing plasmids without affecting the activity of the CAR, NK-92 cell lines were generated by electroporation with tricistronic CAR_CD16_erIL-15 plasmids encoding CD19CAR, CD20CAR, or CD33CAR. The cytotoxic function of the erIL-15-expressing lines was tested against several target cell lines: K562 (CD19neg, CD20neg, CD33+), SUPB15 CD20 The cytotoxicity function of the corresponding t-haNK cells (erIL-2) against THP-1 (CD20+) and THP-1 (CD33+) was compared. Figures 6-8 show exemplary results. Here, for the top panel, killing of the transfected (tricistronic) cell line against K562 shows superior killing at all effector:target ratios tested. This result is significant because the new construct does not affect killing of the standard K562 line (top graph). Importantly, the newly created CAR kills CD20- and CD33-expressing target cell lines that are otherwise resistant to killing by wild-type NK-92.

[0045] In further experiments, the resilience and functionality of erLSP-IL-15 cells will be evaluated. For example, previously frozen erIL-2 CD19 t-haNK and erIL-15 CD19 t-haNK will be thawed and then grown in X-Vivo10 5% without cytokines. Both cell lines are expected to have comparable viability and growth characteristics in the absence of exogenously added cytokines.

[0046] In further experiments, IL-15 and IL-2 secretion is measured, where it is contemplated that IL-15 secretion is equal to or, particularly when the cells also co-express the IL-15 receptor alpha chain, at least 5%, or at least 10%, or at least 20%, or at least 40% or more superior to IL-2 secretion. Quantitatively, it is expected that the modified NK=92 cells will secrete at least 100 pg / ml, or at least 150 pg / ml, or at least 200 pg / ml or more of IL-15, and that the intracellular amount (determined by lysate) will be at least 150 pg / ml, or at least 250 pg / ml, or at least 500 pg / ml, or at least 750 pg / ml or more.

[0047] Furthermore, the modified NK-92 cells described herein are expected not to exhibit clumping or other aggregation during expansion and culture. Similarly, the cells provided herein (especially when the cells express CD16 (preferably a high-affinity variant) and / or a CAR) will have substantially identical doubling times compared to those of aNK cells or haNK cells using standard growth assays (i.e., no more than 15%, or no more than 10%, or no more than 5% deviation). Similarly, cells expressing erLSP-IL-15 will typically express functionally active recombinant CAR and / or CD16 in substantially similar amounts (i.e., no more than 15%, or no more than 10%, or no more than 5% deviation) compared to haNK cells and t-haNK cells.

[0048] Similarly, the nonspecific cytotoxicity of cells expressing erLSP-IL-15 and recombinant CAR and / or CD16 against K562 is expected to be substantially the same as aNK and haNK cells. As already shown above, CAR-mediated cell killing of SUP-B15 by NK-92 cells expressing erLSP-IL-15 and recombinant CAR is expected to be ADCC-mediated cell killing of SUPB15 CD20+.

[0049] From a functional standpoint, contemplated modified NK-92 cells presented herein will have equivalent or enhanced IFNγ secretion (or cytokine panel IFNγ / IL-8 / IL-10 / chemokines) with and without target stimulation. Furthermore, it is expected that contemplated modified NK-92 cells presented herein will have equivalent or enhanced expression of flow markers (NKp30, NKp44, NKp46, DNAM-1, NKG2D, NKG2A, NKG2C, CD94, CD96, TIGIT, PD-1, PD-L1, CTLA-4, TIM-3, LAG-3, FasL, TRAIL, T-bet, Eomes, Granzyme B, Perforin).

[0050] In further contemplated methods, RNA-seq analysis is performed to identify expression patterns that can be used to investigate the activity and / or presence of engineered NK-92 cells within a tumor, among the presence and / or activity of other immune-competent cells. RNA-seq analysis is expected to provide an RNA expression pattern that is substantially representative of activated NK cells.

[0051] As used herein, the term "administration" of a pharmaceutical composition or drug refers to both direct and indirect administration of the pharmaceutical composition or drug, where direct administration of the pharmaceutical composition or drug is typically performed by a medical professional (e.g., a doctor, a nurse, etc.), and indirect administration includes providing or making the pharmaceutical composition or drug available to a medical professional for direct administration (e.g., by injection, infusion, oral delivery, topical delivery, etc.). Most preferably, the cells or exosomes are administered by subcutaneous or subdermal injection. However, in other contemplated aspects, administration may be intravenous injection. Alternatively or additionally, antigen-presenting cells may be isolated or expanded from a patient's cells, infected in vitro, and then injected into the patient. It should be understood, therefore, that the contemplated system and method may be considered a complete drug delivery system (e.g., drug delivery, treatment protocol, validation, etc.) for highly personalized cancer treatment. It should also be understood that the contemplated treatment may be repeated over time, particularly if new neoepitopes are developed (e.g., as a result of clonal expansion).

[0052] The recitation of ranges of values ​​herein is merely intended to serve as a shorthand method of referring individually to each individual value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually listed herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or clearly contradicted by context. The use of any and all examples or exemplary language (e.g., "etc.") provided with respect to specific embodiments herein is intended merely to better illustrate the full scope of the disclosure and does not limit the scope of the otherwise claimed invention. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the claimed invention.

[0053] It should be apparent to those skilled in the art that many further modifications beyond those already described are possible without departing from the overall scope of the concepts of the present disclosure. Accordingly, the disclosed subject matter is not limited except as by the appended claims. Moreover, in interpreting both the specification and the claims, all terms should be interpreted in the broadest possible manner consistent with the context. In particular, the terms "comprise" and "comprising" should be construed as referring to elements, components, or steps in a non-exclusive manner, indicating that the referenced element, component, or step may be present, utilized, or in combination with other elements, components, or steps not expressly referenced. When a claim of the specification refers to at least one selected from the group consisting of A, B, C... and N, it should be construed as requiring only one element of that group, rather than A+N, B+N, etc.

Claims

1. A recombinant nucleic acid comprising a first segment encoding erLSP-IL-15, said nucleic acid comprising SEQ ID NO:

4.

2. The recombinant nucleic acid of claim 1 , wherein the recombinant nucleic acid is DNA.

3. The recombinant nucleic acid of claim 2, wherein the recombinant nucleic acid is a polycistronic DNA construct.

4. The recombinant nucleic acid of claim 1 , wherein the recombinant nucleic acid is a plasmid.

5. The recombinant nucleic acid of claim 1 , wherein the recombinant nucleic acid is a viral vector.

6. The recombinant nucleic acid of claim 1 , wherein the recombinant nucleic acid is RNA.

7. The recombinant nucleic acid according to any one of claims 1 to 6, wherein the recombinant nucleic acid is a linearized plasmid.

8. The recombinant nucleic acid of any one of claims 1 to 7, wherein the recombinant nucleic acid further comprises a second segment encoding CD16 or a high-affinity CD16.

9. The recombinant nucleic acid of any one of claims 1 to 8, wherein the recombinant nucleic acid further comprises a third segment encoding a chimeric antigen receptor.

10. 10. The recombinant nucleic acid of claim 1, further comprising a fourth segment encoding a protein that confers immune stimulation, prevents checkpoint inhibition, a protein that binds to / inhibits a cytokine involved in immune suppression, and / or an IL-15 receptor alpha chain.

11. 1. A method for genetically modifying immune cells in vitro, comprising: introducing a recombinant nucleic acid into the immune cell, wherein the recombinant nucleic acid comprises a first segment encoding erLSP-IL-15, the first segment comprising SEQ ID NO: 4; Thereby producing genetically modified immune cells.

12. The method of claim 11 , wherein the immune cell is an antigen-presenting cell.

13. The method of claim 11 , wherein the recombinant nucleic acid is a viral vector.

14. The method of claim 11 , wherein the recombinant nucleic acid is an RNA molecule.

15. 13. The method of claim 11 or 12, wherein the recombinant nucleic acid is a linearized plasmid.

16. The method of claim 11 or 12, wherein the recombinant nucleic acid further comprises a second segment encoding CD16 or high-affinity CD16.

17. 17. The method of any one of claims 11 to 16, wherein the recombinant nucleic acid further comprises a third segment encoding a chimeric antigen receptor.

18. 18. The method of any one of claims 11 to 17, wherein the recombinant nucleic acid further comprises a fourth segment encoding a protein that confers immune stimulation, a protein that prevents checkpoint inhibition, a protein that binds to / inhibits a cytokine involved in immune suppression, and / or an IL-15 receptor alpha chain.

19. 19. The method of any one of claims 11 to 18, wherein the recombinant nucleic acid comprises a promoter strong enough to drive expression of erLSP-IL-15 in an amount sufficient to allow stimulation / activation of other immune competent cells in the vicinity of the genetically modified immune cells.

20. A pharmaceutical composition comprising a pharmaceutically acceptable carrier in combination with the genetically modified immune cells of any one of claims 11 to 19.

21. 21. The pharmaceutical composition of claim 20 formulated for injection into a patient.

22. At least 1 x 10 per dosage unit ９ 21. The pharmaceutical composition of claim 20, comprising cells.

23. A pharmaceutical composition comprising the genetically modified immune cells of any one of claims 11 to 19 for the treatment of cancer.

24. 24. The pharmaceutical composition of claim 23, wherein the treatment further comprises administration of a drug that enters the TME, administration of a drug that reduces immunosuppression, administration of a drug that stimulates immune-competent cells, administration of a cancer vaccine composition, and / or administration of a drug that helps maintain the immune response and promotes the development of memory cells.

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