CD16 High CD57 High NK-92MI Cells

Abstract

Cells, cell-based therapeutic compositions, and methods are presented that are CD16+CD57+ NK-92MI cells that inherently express CD16 and CD57 and exhibit IL-2-independent growth.

Description

Technical Field

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 345,275, filed May 24, 2022, by the inventors hereof, which is incorporated herein by reference.

[0002] The field of the invention is compositions and methods for cell-based therapies, and in particular, the invention relates to NK cells with enhanced cytotoxicity that inherently express CD16 and CD57.

Background Art

[0003] The background description may include information that is useful in understanding the present invention. None of the information provided herein is admitted to be prior art or related to the present invention currently claimed, nor is any publication, whether expressly or implicitly mentioned, admitted to be prior art.

[0004] All publications and patent applications herein are incorporated by reference to the same extent as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference. Where the definition or use of a term in the incorporated reference conflicts with or contradicts the definition of the term provided herein, the definition of the term provided herein applies and the definition of the term in the reference does not apply.

[0005] Natural killer (NK) cells constitute a group of innate immune cells, but are often characterized as cytotoxic lymphocytes that exhibit antibody-dependent cytotoxicity via the targeted release of granzyme and perforin. More recently, NK cells have become an important component of certain cancer therapies, but the generation of therapeutically effective amounts of NK cells, particularly autologous NK cells, remains a rather large hurdle because the proportion of NK cells in whole blood is relatively low.

[0006] To obtain therapeutically effective amounts of NK cells and NK-like cells, NK cells can be generated from various progenitor cells. For example, various stem cell factors (SCF), FLT3 ligand, interleukin (IL)-2, IL-7, and IL-15 have been reported in various in vitro approaches for inducing and expanding cytokine-induced killer (CIK) cells derived from umbilical cord blood (Anticancer Research 30:3493-3500 (2010)). Similarly, CD34 + hematopoietic cells can be exposed to IL-12, and other agents are reported in US Patent Application Publication No. 2018 / 0044636. In yet other approaches, human angioblasts are sequentially exposed to two different cytokine cocktails as described in International Publication No. WO 2011 / 068896, and the different cytokine cocktails are used with post-embryonic hematopoietic stem cells as taught in International Publication No. WO 2012 / 128622. At least some of these methods result in a significant n-fold expansion of NK cells, but the methods and reagents for such expansion require both excessive time and resources. Moreover, many of the known methods also require culturing NK cells on a feeder cell layer, which is often problematic from a technical and regulatory perspective.

[0007] To avoid issues associated with the generation of therapeutically relevant amounts of NK cells from progenitor cells or isolated NK cells, the NK-92 cell line can be used. NK-92 cells are interleukin-2 (IL-2)-dependent natural killer cells derived from peripheral blood mononuclear cells of a 50-year-old Caucasian male with rapidly progressive non-Hodgkin lymphoma and are commercially available from the ATCC as CRL-2407. Advantageously, NK-92 cells can be grown in large quantities and are made suitable for cancer cell immunotherapy by the ability of the natural killer cell line NK-92 to efficiently kill a wide range of malignant cells. However, cells of the NK-92 cell line generally do not express CD16 and thus lack the ability to bind to target antibodies for targeted cytotoxic effects (notably antibody-dependent cell cytotoxicity (ADCC)).

[0008] More recently, NK cells expressing CD16 from recombinant nucleic acids transfected into NK cells have been produced as described, for example, in US Patent Application Publication No. 2019 / 0321402. Unfortunately, such RNA-based expression is not usually stable over multiple generations during NK cell expansion to obtain therapeutically relevant amounts of NK cells. In an alternative approach, a FACS sorting strategy has been used against the NK-92 cell line to select NK cells with endogenous CD16 expression as described in International Publication No. 2020 / 150475 (see also Zih-Fei Cheng (2021) Biochemistry and Biophysics Reports Volume 26). Here, a human NK-92-derived cell line, oNK-1, was established that endogenously expresses CD16 in addition to high levels of NK activation markers and low levels of NK inhibitory markers characteristic of the parental NK-92 cell line.

[0009] Interestingly, when these cells are covalently conjugated with the monoclonal antibody, trastuzumab, the cells so modified exerted in vitro enhanced HER2 binding specificity and cytotoxicity against various types of cancer cells with grade 1 (MCF-7), grade 2 (OVCAR-3) and grade 3 (SK-OV-3) HER2 expression as compared to control oNK cells (see, for example, Proceedings of the Annual Meeting of the American Association for Cancer Research 2020;2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2169). Unfortunately, production and CD16 expression in oNK cells required very large amounts of IL-2 (500 units / mL).

[0010] NK-92MI (ATCC: CRL-2408) is an IL-2 independent variant of NK-92 generated using particle-mediated gene transfer of hIL-2 cDNA. NK-92MI cells exhibit potent cytotoxic activity against various tumor cells. Nevertheless, the Fc receptor (CD16) that normally mediates ADCC is either completely absent or present only at very low levels on NK-92MI cells, and these cells are therefore unable to induce ADCC for target-specific cell killing. Although circulating NK cells have also been shown to be more potent in CD16-mediated ADCC activity when they are also positive for CD57 (see for example Sandra Lopez-Verges (2010) Blood 2010 116(19)), the proportion of CD16+CD57+ cells in the blood is very low and these cells are representative of mature NK cells. Consequently, it has not been achieved to make a therapeutically effective amount by concentrating or increasing them. NK-92MI chimeric cells have also been generated using viral transduction to express a recombinant chimeric receptor that can bind to the Fc portion of human immunoglobulins (see for example Ying Chen (2017) Oncotarget 6;8(23)). However, the CD57 expression of these cells has not been reported and was not naturally expected either.

[0011] Accordingly, even though various compositions and methods of NK cell therapy are known in the art, most or all of them have several drawbacks, especially when such cells are required in economically significant amounts and when such cells express CD16 and CD57 for enhanced cytotoxic activity. SUMMARY OF THE INVENTION MEANS FOR SOLVING THE PROBLEM

[0012] The subject matter of the present invention therefore relates to various cells, as well as cell-based compositions and methods, which are CD16+CD57+NK-92MI cells that inherently express CD16 and CD57 and exhibit IL-2-independent growth. Among other things, such cells have significantly enhanced cytotoxic effects and can be produced simply and cost-effectively.

[0013] In one aspect of the subject matter of the present invention, the inventors contemplate CD16+CD57+NK-92MI cells that inherently express CD16 and CD57 and exhibit IL-2-independent growth. Typically, CD16 is CD16a or CD16b. Particularly contemplated cells express a greater amount of NKG2D compared to parental NK-92MI cells (ATCC: CRL-2408). Furthermore, while CD56 is typically present on the cell surface, CD3 is typically not present on the cell surface. Additionally, the cells presented herein may further comprise one or more of CD4, CD25, NKp30, NKp44, NKp46, CD27, OX40, CD107a, NKG2A, PD-1, TIGIT on the cell surface.

[0014] Phenotypically, the cells presented herein will generally have enhanced direct cytotoxic effects against Ramos cells and / or exhibit potent ADCC in the presence of antibodies against target cells and surface proteins on the target cells, compared to parental NK-92MI cells (ATCC: CRL-2408). Furthermore, the contemplated cells will also have high levels of expression of CD57, CD16, and NKG2D after thawing and expansion, compared to parental NK-92MI cells (ATCC: CRL-2408). Additionally, in at least some embodiments, the cells will have faster replenishment after degranulation and / or a faster cell doubling time compared to parental NK-92MI cells (ATCC: CRL-2408).

[0015] If desired, the CD16+CD57+NK-92MI cells may also be transfected with recombinant nucleic acids encoding chimeric antigen receptors, homing receptors, chemokine receptors, TGF-β traps and / or checkpoint inhibitors. Alternatively or additionally, the CD16+CD57+NK-92MI cells may also have antibodies or antibody fragments bound to the surface of the cells (e.g., via CD16 or a linker).

[0016] In another aspect of the subject matter of the present invention, the inventors also contemplate a composition comprising a plurality of NK-92MI cells presented herein in a medium, wherein at least 10% or at least 50% or at least 90% of the NK-92MI cells are CD16+CD57+NK-92MI cells that inherently express CD16 and CD57 and show IL-2-independent growth. As can be seen, the contemplated media include growth media, cryopreservation media, and pharmaceutically acceptable media for injection. Further, the composition may further comprise an antibody that may or may not bind to CD16 of the CD16+CD57+NK-92MI cells. In a further contemplated aspect, the plurality of cells are between 1×10 8 ~1×10 10 cells, the medium is a pharmaceutically acceptable medium for injection, and the cells and the medium are placed in a polymer bag.

[0017] In a further aspect of the subject matter of the present invention, the inventors contemplate a cell culture comprising a plurality of dividing cells in a medium, the cells being the CD16+CD57+NK-92MI cells presented herein, and the medium being substantially free of IL-2. In a typical embodiment, the plurality of dividing cells are preferably maintained in a single culture vessel from the beginning of the culture and during growth until a predetermined amount of cells is obtained. Thus, the contemplated cell culture may contain at least 1×10 7 cells per culture vessel. Usually, but not necessarily, the medium contains human AB serum or is a serum-free medium.

[0018] Furthermore, in a further aspect of the subject matter of the present invention, the inventors contemplate a method for preparing CD16+CD57+NK-92MI cells that inherently express CD16 and CD57 and exhibit IL-2-independent growth. Such a method typically includes the step of providing a plurality of NK-92MI cells and another step of enriching CD16+CD57+NK-92MI cells that inherently express CD16 and CD57 using anti-CD16 antibody and anti-CD57 antibody.

[0019] For example, each of the anti-CD16 antibody and the anti-CD57 antibody may be fluorescently labeled, and the antibodies may be used (usually in order) to enrich using fluorescence-activated cell sorting (FACS). As will be readily appreciated, FACS may be performed using single-pass sorting or using multiple repetitions of FACS. Furthermore, the contemplated method may also include the step of growing the enriched population of CD16+CD57+NK-92MI cells.

[0020] In yet another aspect of the subject matter of the present invention, the inventors contemplate a method for treating cancer, the method comprising administering to an individual in need thereof a therapeutically effective amount of the cells presented herein or a composition comprising, for example, such cells. Typically, the individual is a mammal (e.g., a human) and / or the cancer is a solid cancer. As will be readily appreciated, the cells or composition are administered by injection, and the treatment may also include the step of co-administering an antibody, a checkpoint inhibitor, an immunostimulant, and / or a cancer vaccine.

[0021] The various objects, features, aspects and advantages of the subject matter of the present invention will become more apparent from the following detailed description of the preferred embodiments in addition to the accompanying drawings in which the same numerals indicate the same components.

Brief Description of the Drawings

[0022]

Figure 1

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Figure 7

[0023] The inventors have discovered that it is possible to prepare NK cells that not only express large amounts of CD16 and CD57, but also have a high degree of general and target-specific cytotoxic effects and can grow without the need for exogenous addition of IL-2. Surprisingly, these cells were isolated from the NK-92MI cell line, which was previously not known to express significant amounts of CD16 and CD57. In this regard, it should be recognized that the NK-92MI cell line is not a collection of naturally occurring cells, but a cell line established by the transformation of the NK-92 cell line with human IL-2 (hIL-2) cDNA by particle-mediated gene transfer (see Hum Gene Ther. 1999 May 20;10(8):1359-73).

[0024] While not wishing to be bound by any particular theory or hypothesis, the inventors believe that during the expansion of the NK-92MI cell line, some of the cells undergo changes in certain genes, presumably epigenetic changes, which can lead to the stable expression of CD16 and CD57 as they are originally. Although rare in the entire population of cells in the NK-92MI cell line, the inventors noted that such cells can be enriched via affinity-based separation methods. In particular, such enrichment was driven by the fact that NK-92MI cells express the recombinant genomic integrated nucleic acid sequence IL-2, rendering such cells independent of exogenously added IL-2.

[0025] Even more unexpectedly, the inventors also observed that such enriched CD16+CD57+NK-92MI cells also express NKG2D, a major regulator of cytotoxicity, in significantly greater amounts compared to the NKG2D expression of the NK-92MI cell line. Such an increase in the presence of NKG2D further enhanced the general and ADCC-type cytotoxic effects of the isolated / enriched CD16+CD57+NK-92MI IL-2-independent cells.

[0026] As used herein, the phrases "cells that inherently express CD16 and CD57" or "the inherent stable expression of CD16 and CD57" refer to cells that express CD16 and CD57 from their respective genes in the genome of the cells, and these genes are not recombinant genes introduced into the cells. Further, as used herein, the phrase "showing IL-2-independent growth" with respect to a cell refers to the ability of the cell to cause n-fold proliferation (where n is at least 10) in a culture medium in which IL-2 is absent, not exogenously added, or not provided by a feeder cell layer when added. Similarly, as further used herein, the phrase "the medium is substantially free of IL-2" refers to a medium in which the amount of IL-2 is insufficient to support the growth of NK-92 cells (ATCC: CRL-2407). Thus, a medium that is substantially free of IL-2 will contain IL-2 in an amount less than 10 IU / mL or less than 5 IU / mL or less than 1 IU / mL. However, it should be noted that CD16+CD57+NK-92MI cells may secrete IL-2 into the medium, and all secreted amounts are included within the scope of this definition.

[0027] CD16+CD57+ NK-92MI IL-2-independent cells are isolated from the NK-92MI cell line, but it should be recognized that a number of other cell lines, including NK-92 and genetically modified NK-92 cell lines (such as NK-92Cl), are also considered suitable as parental cell lines. Usually, when the NK-92 cell line is genetically modified, the modification is generally preferably a genetically stable modification as it is passed on to descendant cells. Therefore, genetically modifications specifically contemplated include, among others, artificial chromosomes, viral genome insertion into the cell genome, CRISPR-based modifications, etc. In some embodiments, the genetic modification will include the addition of nucleic acids encoding CD16 and / or CD57, whereas in other modifications, the genetic modification will include the addition of nucleic acids encoding IL-2 (the nucleic acids may or may not include cytoplasmic or endoplasmic reticulum retention sequences). Further contemplated genetic modifications include the addition of nucleic acids encoding one or more recombinant proteins to enhance cytotoxicity, direct the cells to a specific location, etc., and particularly contemplated recombinant proteins include chimeric antigen receptors, homing receptors, chemokine receptors, (secreted or membrane-bound) TGF-β traps, and / or checkpoint inhibitors.

[0028] In a further contemplated aspect, note that the parental cell line (e.g., NK-92MI) may be used for cell sorting or other types of enrichment of CD16 and CD57 immediately after thawing from cryopreservation medium or after at least 1 or at least 2 or at least 3 or at least 5 or at least 10 or at least 20 passages in culture medium. Usually, such culture medium will be serum-free and will not contain exogenous IL-2. In other embodiments, the medium contains human AB serum but does not contain exogenously added IL-2. Similarly, it is generally preferred that the culture medium does not contain an additional feeder cell layer.

[0029] For cell isolation and enrichment, it is generally preferred that cells be isolated using a selection process that includes the use of anti-CD16a and anti-CD57 antibodies. However, in alternative embodiments, the antibody may also be an anti-CD16b antibody, an anti-CD16c antibody, an anti-CD32 antibody, and / or an anti-CD64 antibody. Most commonly, the cell selection process is a FACS process, although various alternative selection processes are also considered suitable for use herein, including magnetic bead separation where the antibody is conjugated to beads, microplate separation where the antibody is conjugated to the walls of wells, microfluidic separation where the antibody is conjugated to the walls of microfluidic channels, and the like. Regardless of the method of separation, it should be recognized that cell separation can be performed in a single pass using both anti-CD16a and anti-CD57 antibodies or in multiple sequential passes where the cells are first enriched for CD16+ cells and then further enriched for CD57+ cells.

[0030] Also, as can be readily appreciated, the cells may be subjected to a growth / proliferation step, and in particular the isolation may be performed sequentially. Such growth and proliferation may start from a large number of cells (e.g., the untreated output of the isolation step) or from those with a reduced number of cells. Such a reduction in number may be obtained by subculturing under selected conditions or by serial dilution in order to ultimately produce a monoclonal cell product. Once the desired CD16+CD57+ NK-92MI IL-2-independent cells are obtained, it is contemplated that these cells can then be further characterized for one or more parameters and that the cells can be confirmed to be positive for CD16 and CD57.

[0031] If the CD16+CD57+NK-92MI IL-2-independent cells are not of monoclonal origin, it is also conceivable that the cell population so obtained can be further tested and / or selected for further desirable traits. In particular, CD16+CD57+NK-92MI IL-2-independent cells can be selected or confirmed for faster replenishment after degranulation, reduced cell doubling time, optimal growth density, etc. (all compared to the parental NK-92MI cell line). Similarly, CD16+CD57+NK-92MI IL-2-independent cells can be selected or confirmed for cytotoxicity / efficacy against various cancer cells (which may be cells of solid or blood-derived cancers), cytokine expression, in particular IFN-γ secretion, migration to target tissues, etc.

[0032] Once isolated, it should be recognized that the cells can be further genetically modified, usually by expression of one or more recombinant proteins, to contain one or more desirable properties. For example, further proteins that are particularly suitable for expression preferably include chimeric antigen receptors targeting cancer or tumor-associated antigens (such as CD19, CD20, GD2, HER-2, CD30, EGFR, FAP, CD33, CD123, PD-L1, IGF1R, CSPG4 or B7-H4) or neoantigens, homing receptors (such as G protein-coupled receptors (GPCRs), chemokine receptors such as CCR7, CXCR2 or receptors for CXCL14, cytokine receptors, cell adhesion molecules, selectins or integrins), TGF-β traps which may be membrane-bound or secreted and / or checkpoint inhibitors. Exemplary proteins, sequences and constructs for such modifications are described in WO 2020 / 028656 pamphlet which is incorporated herein by reference. In addition, it should be recognized that the cells presented herein may be further modified by one or more target-specific binders such as antibodies and fragments thereof including scFv constructs.

[0033] As a result, the cells presented herein should be recognized as being useful in a variety of therapeutic applications, particularly for injection into individuals in need thereof. Accordingly, the inventors also contemplate compositions in which the cells presented herein are placed in a growth medium for the preparation of a therapeutically effective amount of the cells, in a cryopreservation medium for the storage of such cells, or in a pharmaceutically acceptable medium for injection.

[0034] As will be readily appreciated, the cells presented herein may be administered to a subject by a variety of routes, with intravenous infusion over a period of time being particularly preferred. Typically, for a single administration of cells, the period is between 5 and 130 minutes. Optionally, the period is between 90 and 120 minutes. Optionally, the period is between 15 and 30 minutes. Preferably, the cells presented herein can be administered daily, twice or three times a week, once a week, twice a week, once a month or at longer intervals.

[0035] With regard to the number of cells to be administered, the cells presented herein can be administered in amounts from about 1000 cells / injection to about 10,000,000,000 cells / injection or less, such as about, at least about, at most about 1×10 10 、1×10 9 、1×10 8 、1×10 7 、5×10 7 、1×10 6 、5×10 6 、1×10 5 、5×10 5 、1×10 4 、5×10 4 、1×10 3 、5×10 3 cells, or any range between any two numbers, including endpoints, is contemplated to be administrable to a subject. For example, 1×10 8 ~1×10 10Individual cells are administered to a subject. As will be readily appreciated, the cells can be administered from the production process without intermediate storage in a cryopreservation medium or the cells can be administered after thawing. If desired, the cryopreservation medium may be replaced with an infusion medium prior to infusion. In a further contemplated aspect, it should be recognized that the cells presented herein may also be irradiated, particularly when the cells are administered in a therapeutic composition, to reduce or arrest proliferation. Thus, all methods or irradiations are considered suitable for use herein, particularly low energy e-beam irradiations and gamma irradiations at doses typically between 2 and 20 Gy, more typically between 6 and 10 Gy (see, for example, Front. Immunol. June 2021, Vol. 12, Article 684052).

[0036] Accordingly, it should be recognized that the CD16+CD57+NK-92MI cells that are the subject of the present invention can be used in a variety of therapeutic needs. However, the cells are used in methods for treating cancer (e.g., solid tumors or blood-derived cancers) in a subject, and a therapeutically effective amount of the cells is particularly contemplated to be administered in a pharmaceutically acceptable composition containing such cells. Advantageously, these cells form part of a combinatorial therapeutic strategy that includes additional therapeutic agents such as antibodies, checkpoint inhibitors, immunostimulants, cancer vaccines, and / or metronomic low-dose chemotherapy.

Examples

[0037] The inventors sorted CD57+CD16+ NK cells from the parental NK-92MI cell line (commercially available from ATCC as, for example, CRL-2408) using a BD FACS Melody cell sorter and fluorescently labeled anti-CD16 and anti-CD57 antibodies. After sorting, the cells were cultured for up to 126 days, and high CD16 and CD57 expression was observed by flow cytometry (1.98% CD16 in parental cells vs. 85.7% CD16 in sorted cells and 2.69% CD57 in parental cells vs. 32.1% CD57 in sorted cells). Figure 1 shows exemplary results for such enrichment of CD16 and CD57 in the sorted NK-92MI cells. Here, surface marker expression on parental (unsorted) and sorted NK-92MI cells was analyzed by flow cytometry-based staining, and the results are shown as dot plots. The bar graph compares the percent expression of each marker on parental and sorted cells.

[0038] Unexpectedly, the sorted cells also showed higher expression of NKG2D, a major regulator of cytotoxicity, when compared to parental cells, as can be seen from Figure 2. On the other hand, the expression of receptors such as CD56 and CXCR4 was equivalent in both cell lines, as can be seen from Figure 2. In particular, the sorted cells also maintained a high viability throughout the culture period. Figure 2 shows exemplary results for surface phenotyping of parental NK-92MI cells and sorted CD16+CD57+ NK-92MI cells. Here, surface marker expression was analyzed by flow cytometry-based staining, and the results are shown as histograms below. Light gray and dark gray indicate marker staining of unstained and stained cells, respectively.

[0039] To examine whether the sorted cells exert a cytotoxic effect on various cancer cell lines, parental or sorted NK-92MI was co-cultured with target cells such as Ramos cells and HCC827 cells at various effector-to-target ratios in a calcein-based assay. As can be seen from Figure 3, the sorted cells rather than the parental cells showed enhanced direct cytotoxic effects on Ramos cells (nonspecific beta-gal treatment corresponds to direct cytotoxic effects; 7% Ramos lysis by parental cells vs. 55% Ramos cell lysis by sorted cells). Figure 3 shows exemplary results for the ADCC of sorted NK-92MI cells against Ramos target cells. More specifically, the ADCC of parental (unsorted) and sorted NK-92MI cells against Ramos target cells in the presence of rituxan was determined in a calcein release assay. Samples were tested in triplicate. The percentage of calcein release (%) as an indicator of target cell lysis is shown for various effector-to-target ratios.

[0040] The ADCC of parental and sorted NK-92MI cells was also tested against two tumor cell lines including HCC827 cells and Ramos cells in the presence of abemaciclib and rituximab, respectively. As expected, the parental cells lacking CD16 did not induce lysis mediated by the ADCC of Ramos cells. In contrast, the sorted cells induced potent ADCC of Ramos cells (3% ADCC against Ramos by parental cells vs. 94% ADCC against Ramos by sorted cells). Similar potent ADCC activity was observed when sorted cells were mixed with HCC827 cells in the presence of abemaciclib, as can be seen from the results in Figure 4 showing exemplary results for the ADCC of sorted NK-92MI cells against HCC827 target cells. Here, the ADCC of sorted NK-92MI cells against HCC827 target cells in the presence of abemaciclib was determined in a calcein release assay. Samples were tested in triplicate. The percentage of calcein release (%) as an indicator of target cell lysis is shown for various effector-to-target ratios.

[0041] Expression stability was also evaluated after freeze-thaw cycles. The sorted cells were frozen in ImmunityBio appropriate medium in an LN2 freezer. After thawing and expansion, the sorted cells maintained high expression of CD57, CD16, and NKG2D, and exemplary results are shown in FIGS. 5 and 6. FIG. 5 shows exemplary results establishing that sorted NK-92mi cells maintained high expression of CD57 and CD16 after freeze-thaw. Here, surface marker expression on parental (unsorted) and sorted NK-92MI cells was analyzed by flow cytometry-based staining, and the results are shown below as dot blots. The bar graph compares the percentage of expression of each marker on parental and sorted cells. FIG. 6 shows exemplary results of surface phenotyping of parental and sorted NK-92MI cells after freeze-thaw. Here, surface marker expression was analyzed by flow cytometry-based staining, and the results are shown below as histograms. Light gray and dark gray indicate marker staining of unstained and stained cells, respectively.

[0042] FIG. 7 shows exemplary results for ADCC of sorted NK-92MI cells after freeze-thaw. Here, ADCC of thawed NK-92MI cells against Ramos target cells in the presence of rituximab was determined in a calcein release assay. Samples were tested in triplicate. The percentage of calcein release (%) as an indicator of target cell lysis is shown for various effector-to-target ratios. As can be seen, the freeze-thaw cycle had no effect on CD16-mediated ADCC activity against CD20-expressing Ramos cells.

[0043] Based on the above, it should be recognized that the sorted cell line unexpectedly yielded CD16+CD57+NK-92MI cells that inherently expressed CD16 and CD57 and showed IL-2-independent growth along with significantly increased NKG2D during long-term culture and after freeze-thaw.

[0044] For the above experiment, the inventors sorted CD57+CD16+ NK cells from the parental NK-92MI, an IL-2-independent cell line. Approximately 60,000,000 viable cells with a survival rate of over 90% were mixed with fluorescently labeled anti-CD16 and anti-CD57 antibodies for cell sorting using a BD FACS Melody cell sorter. The CD57+CD16+ sorted NK cells were cultured in ImmunityBio-defined medium for up to 126 days. On different days, flow cytometry analysis was performed using a Miltenyi MACSQuant Analyzer 10, where the cells were stained with fluorescent dye-conjugated antibodies at room temperature for 10 - 30 minutes. The stained cells were washed and loaded for flow cytometry analysis. The functional activity of the sorted cells was evaluated using a calcein-based method, where the sorted cells were mixed with tumor cells loaded with calcein, such as Ramos cells, in the presence or absence of rituximab antibody. NK cells induced lysis of the calcein-loaded target cells, leading to the release of intracellular fluorescent calcein into the assay medium. The level of fluorescence from the released calcein was directly proportional to the number of lysed cells and thus served as a criterion for the cytotoxic action of NK effector cells.

[0045] In further experiments, the inventors performed gene expression analysis using mRNAseq and monitored the gene expression of genes over the cell sorting process. Among other findings, analysis of the mRNA seq of sorted NK-92 MI CD16 cells revealed that there were approximately 50 genes (coding and non-coding) that showed significant continuous upregulation (over 4-fold) over the two-step sorting, and exemplary results are shown in Table 1. As expected, CD16a was one of the upregulated genes. Most of the remaining upregulated genes had functions in signal transduction, metabolism, and gene expression. Unexpectedly, both Wnt6 and TRAIL DR1 were highly upregulated, with the role of Wnt6 being unknown, while the role of TRAIL DR1 would have suggested a potential (indirect) mechanism for enhanced cytotoxic action.

[0046]

Table 1

[0047]

Table 2

[0048] In addition, the inventors also identified 27 additional genes (coding and non-coding) that showed significant continuous down-regulation (more than 4-fold) over two-step fractionation, most of these genes being generally involved in cell metabolism, and exemplary results are shown in Table 2. Unexpectedly, PDGFD (platelet-derived growth factor D) is one of the genes that was significantly down-regulated, and it is a "survival factor" known in NK cells. Such a finding was particularly notable because PDGFD is known as a potent stimulator of cell growth and motility that activates NK cells by autocrine and contributes to interleukin-15-mediated NK cell survival. Its overexpression may contribute to the ability to proliferate cells sorted in the absence of exogenous IL-2 and / or IL-15.

[0049]

Table 3

[0050] The inventor further identified an additional 406 genes (coding and non-coding) that showed a tendency for upregulation during sorting. However, the upregulation data is much more complex, and some genes showed upregulation after the first sorting step but were not as upregulated after the second sorting, while others showed the opposite, showing a gradual increase after the first sorting but strong upregulation after the second sorting. Finally, other genes showed only a gradual increase after each sorting step. Exemplary data is shown in Table 3. In particular, some of the upregulated genes include CD86, CD74, CD7, CCR5, CCR10, TIM3, KLRG1, SYK, IL21R, CXCR6, CD25, 4-1BB, GZMH, CD30, LAIR1, and NKG2D, which are thought to be associated with increased cytotoxic activity or chemotaxis. On the other hand, somewhat unexpectedly, TIM3 and LAIR1, which are normally associated with a decrease in NK cells, were upregulated.

[0051]

Table 4

[0052]

Table 5

[0053]

Table 6

[0054]

Table 7

[0055]

Table 8

[0056]

Table 9

[0057]

Table 10

[0058]

Table 11

[0059]

Table 12

[0060]

Table 13

[0061] Finally, as can be seen from the data in Table 4, the inventors also identified approximately 350 genes (coding and non-coding) that showed a tendency for down-regulation during fractionation. As described above, the situation is complex. Among them, the down-regulated genes associated with the cytotoxic effect in normal NK cells were CD11c, CD11d, IL25, NKp44, and CD28. However, most unexpectedly, the expression of the IL-2 gene was substantially down-regulated (almost one-tenth) in a series of sequencing.

[0062]

Table 14

[0063]

Table 15

[0064]

Table 16

[0065]

Table 17

[0066]

Table 18

[0067]

Table 19

[0068]

Table 20

[0069]

Table 21

[0070]

Table 22

[0071] In some embodiments, the numbers representing characteristics such as the amounts and concentrations of components, reaction conditions, etc. used to describe and claim certain embodiments of the present invention are to be understood as being optionally modified by the term "about". Accordingly, in some embodiments, the numerical parameters recited in the specification and the appended claims are approximations that may vary depending upon the desired characteristics sought to be obtained by a particular embodiment. The detailed description of the ranges of values herein is intended merely as a shorthand notation for referring individually to each separate value within the range. Unless otherwise indicated herein, each separate value is incorporated herein as if it were individually recited herein.

[0072] As used herein, the term "administering" 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 healthcare provider (e.g., a physician, nurse, etc.), and indirect administration includes the step of providing or making available the pharmaceutical composition or drug to the healthcare provider for direct administration (e.g., via injection, infusion, oral delivery, topical delivery, etc.). It should be further noted that the term "prospecting" or "predicting" a condition, susceptibility to the onset of a disease, or response to a planned treatment means encompassing the act of predicting or the prediction (not a treatment or diagnosis) of the condition, susceptibility, and / or response, including the rate of progression, improvement, and / or duration of the condition in a subject.

[0073] All methods described herein can be performed in any suitable order, unless otherwise indicated herein or clearly contradicted by the context. The use of any and all examples or exemplary representations (e.g., "such as") provided with respect to a particular embodiment herein is merely intended to more clearly illustrate the invention and does not result in a limitation on the scope of the invention, absent a separate claim. No representation herein should be construed as indicating any non-claimed element essential to the practice of the invention.

[0074] As used throughout the description herein and in the claims that follow, the meanings of "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Further, as used in the description herein, the meaning of "in" includes "in" and "on" unless the context clearly dictates otherwise. Further still, as used herein, unless the context dictates otherwise, the term "coupled to" is intended to include both direct coupling (where two elements that are coupled together are in contact with each other) and indirect coupling (where at least one additional element is located between the two elements). Thus, the terms "coupled to" and "couples with" are used synonymously.

[0075] It should be apparent to those skilled in the art that many more modifications other than those already described are possible without departing from the inventive concepts herein. The subject matter of this invention, therefore, should not be limited except as by the appended claims. Further, in interpreting both this specification and the claims, all terms should be interpreted as broadly as possible in a manner consistent with the context. In particular, the terms "comprises" and "comprising" should be interpreted as referring to elements, components, or steps in a non-exclusive manner, indicating that the referenced elements, components, or steps may be present, utilized, or combined with other elements, components, or steps not expressly referenced. Where this specification or the claims refer to at least one of something selected from the group consisting of A, B, C... and N, the text should be interpreted as requiring only one element from the group, rather than A and N or B and N, etc.

Claims

1. CD16+CD57+NK-92MI cells that inherently express CD16 and CD57 and exhibit IL-2-independent growth.

2. The cell according to claim 1, wherein the CD16 is CD16a or CD16b.

3. The cell according to claim 1 or 2, wherein NKG2D is expressed in a larger amount on the cell surface of the cell compared to NK-92MI cells.

4. The cell according to any one of claims 1 to 3, wherein CD3 is not present on the surface of the cell and CD56 is present on the surface of the cell.

5. The cell according to any one of claims 1 to 4, wherein CD4+, CD25+, NKp30+, NKp44+, NKp46+, CD27+, OX40+, CD107a+, NKG2A+, PD-1+, TIGIT+ and / or CD158+ are present on the surface of the cell.

6. The cell according to any one of claims 1 to 5, wherein the cell has enhanced direct cytotoxic effect on Ramos cells compared to NK-92MI cells.

7. The cell according to any one of claims 1 to 6, wherein the cell performs ADCC in the presence of a target cell and an antibody against a surface protein on the target cell.

8. The cell according to any one of claims 1 to 7, wherein the cell maintains high expression of CD57, CD16 and NKG2D after thawing and proliferation compared to NK-92MI cells.

9. The cell according to any one of claims 1 to 8, wherein the cell has faster replenishment after degranulation and / or a faster cell doubling time compared to NK-92MI cells.

10. The cell according to any one of claims 1 to 9, wherein the cell is transfected with a recombinant nucleic acid encoding a chimeric antigen receptor, a homing receptor, a chemokine receptor, a TGF-β trap and / or a checkpoint inhibitor.

11. The cell according to any one of claims 1 to 10, wherein the cell has an antibody or antibody fragment bound to the surface of the cell.

12. An isolated composition comprising a plurality of NK-92MI-derived cells in a medium, wherein at least 10% of the NK-92MI-derived cells are CD16+CD57+NK-92MI cells that inherently express CD16 and CD57 and show IL-2-independent growth.

13. The composition according to claim 12, comprising at least 50% of the CD16+CD57+NK-92MI cells.

14. The composition according to claim 12, comprising at least 90% of the CD16+CD57+NK-92MI cells.

15. The composition according to any one of claims 12 to 14, wherein the CD16+CD57+NK-92MI cells are the cells according to any one of claims 2 to 11.

16. The composition according to any one of claims 12 to 15, wherein the medium is a growth medium, a cryopreservation medium or a pharmaceutically acceptable medium for injection.

17. The composition according to any one of claims 12 to 16, further comprising an antibody.

18. The composition according to claim 17, wherein the antibody binds to CD16 of the CD16+CD57+NK-92MI cells.

19. The plurality of cells is 1×10 ８ ~1×10 １０among individual cells, the medium is a pharmaceutically acceptable medium for injection, and the cells and the medium are placed in a polymer bag, the composition according to any one of claims 12 to 18.

20. A cell culture containing a plurality of dividing cells in a culture medium, wherein the cells are CD16+CD57+NK-92MI cells that originally express CD16 and CD57 and show IL-2-independent growth, and the medium is a cell culture substantially free of IL-2.

21. The cell culture according to claim 20, wherein the CD16+CD57+NK-92MI cells are the cells according to any one of claims 2 to 11.

22. The cell culture according to claim 20 or 21, wherein the plurality of dividing cells are maintained in a single culture vessel from the beginning of the culture and during growth until a predetermined amount of cells is obtained.

23. The culture contains at least 1×10 ７ cells per culture vessel, the cell culture according to any one of claims 20 to 22.

24. The cell culture according to any one of claims 20 to 23, wherein the medium contains AB serum.

25. A method for preparing CD16+CD57+NK-92MI cells that originally express CD16 and CD57 and show IL-2-independent growth, comprising providing a plurality of NK-92MI cells and using anti-CD16 antibody and anti-CD57 antibody to enrich the CD16+CD57+NK-92MI cells that originally express CD16 and CD57.

26. Each of the anti-CD16 antibody and the anti-CD57 antibody is fluorescently labeled, and the step of using the antibody includes fluorescence-activated cell sorting, the method according to claim 25.

27. The method according to claim 26, wherein the fluorescence-labeled cell sorting is performed in order with respect to the use of the anti-CD16 antibody and the anti-CD57 antibody. **Claim 28** The method according to claim 26, wherein the fluorescence-labeled cell sorting includes multiple repetitions of fluorescence-labeled cell sorting. **Claim 29** The method according to any one of claims 25 to 29, further comprising the step of expanding a concentrated population of the CD16+CD57+NK-92MI cells. **Claim 30** A composition comprising a plurality of NK-92MI-derived cells in a medium for use in the treatment of cancer, wherein at least 10% of the NK-92MI-derived cells are CD16+CD57+NK-92MI cells that inherently express CD16 and CD57 and show IL-2-independent growth. **Claim 31** The composition according to claim 30, wherein the CD16 is CD16a or CD16b. **Claim 32** The composition according to claim 30, wherein the medium is a growth medium, a cryopreservation medium, or a pharmaceutically acceptable medium for injection, and the NK-92MI-derived cells are optionally irradiated. **Claim 33** The composition according to claim 30, further comprising an antibody. **Claim 34** The composition according to claim 33, wherein the antibody binds to CD16 of the CD16+CD57+NK-92MI cells. **Claim 35** The composition according to claim 30, wherein the CD16+CD57+NK-92MI cells are transfected with a recombinant nucleic acid encoding a chimeric antigen receptor, a homing receptor, a chemokine receptor, a TGF-β trap, and / or a checkpoint inhibitor. **Claim 36** The composition according to claim 30, wherein the CD16+CD57+NK-92MI cells maintain high expression of CD57, CD16 and NKG2D after thawing and proliferation as compared with NK-92MI cells.

37. The composition according to claim 30, wherein the CD16+CD57+NK-92MI cells have enhanced direct cytotoxic effect on Ramos cells as compared with NK-92MI cells.

38. The composition according to claim 30, wherein the cancer is a solid tumor.

39. The composition according to claim 30, wherein the cancer is in a human.

40. A method for treating cancer, comprising: administering to an individual in need thereof a therapeutically effective amount of the cell according to any one of claims 1 to 11 or the composition according to any one of claims 12 to 19.

41. The method according to claim 30, wherein the individual is a mammal.

42. The method according to claim 30, wherein the cancer is a solid tumor.

43. The method according to claim 30, wherein the cell or the composition is administered by injection.

44. The method according to any one of claims 30 to 34, further comprising co-administering an antibody, a checkpoint inhibitor, an immunostimulant, a cancer vaccine and / or metronomic low-dose chemotherapy.

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