CD16 high-CD57 high-NK-92MI cell

Abstract

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

Description

[0001] This application claims priority to our co-pending U.S. provisional patent application serial number 63 / 345,275 filed on May 24, 2022, and is incorporated herein by reference. Technical Field

[0002] The field of the invention is compositions and methods for cell-based therapies, and particularly as it relates to NK cells with enhanced cytotoxicity that naturally express CD16 and CD57. Background Art

[0003] The Background Description includes information that may be useful in understanding the present invention. No admission is made that any of the information provided herein is prior art or relevant to the presently claimed invention, nor is an admission made that any publication specifically or implicitly referenced is prior art.

[0004] All publications and patent applications are incorporated herein by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. If a definition or use of a term in an incorporated reference is inconsistent or contrary to the definition of that term provided herein, the definition of that term provided herein applies and the definition of that term in the reference does not apply.

[0005] Natural killer (NK) cells constitute a group of innate immune cells, which are often characterized as cytotoxic lymphocytes that exhibit antibody-dependent cellular cytotoxicity via target-directed release of granulysins and perforins. Although NK cells have recently become an important component of certain cancer treatments, it is still very difficult to generate therapeutically meaningful numbers of NK cells, especially autologous NK cells, due to the relatively low proportion of NK cells in whole blood.

[0006] In order to obtain therapeutically meaningful numbers of NK cells and NK-like cells, NK cells can be generated from various precursor cells. For example, various stem cell factors (SCFs), FLT3 ligands, interleukins (IL)-2, IL-7, and IL-15 have been reported in various methods for inducing and expanding umbilical cord blood-derived cytokine-induced killer (CIK) cells in vitro (Anticancer Research [Anticancer Research] 30:3493-3500 (2010)). Similarly, CD34 +Hematopoietic cells can be exposed to IL-12 and other agents, as reported in US2018 / 0044636. In other methods, human hemangioblasts are sequentially exposed to two different cytokine mixtures, as described in WO 2011 / 068896, and different cytokine mixtures are used together with post-embryonic hematopoietic stem cells, as taught in WO 2012 / 128622. Although at least some of these methods provide a significant n-fold expansion of NK cells, the methods and reagents for such expansion require both time and resources. Further, it should be noted that many known methods also require NK cells to be cultured on a feeder cell layer, which is often problematic from a technical and regulatory perspective.

[0007] In order to avoid the problems associated with generating therapeutic amounts of NK cells from precursor cells or separated 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 white male with rapidly progressive non-Hodgkin's lymphoma and can be commercially obtained from ATCC as CRL-2407. Advantageously, NK-92 cells can be grown on a large scale, and the ability of the natural killer cell line NK-92 to effectively kill a wide range of malignant cells makes it suitable for cellular immunotherapy for cancer. However, cells of the NK-92 cell line do not generally express CD16 and therefore lack the ability to bind to targeting antibodies to achieve target-specific cytotoxicity (and especially antibody-dependent cellular cytotoxicity (ADCC)).

[0008] Recently, NK cells expressing CD16 from recombinant nucleic acids transfected into NK cells have been produced, as described in, for example, US2019 / 0321402. Unfortunately, in the process of NK cell amplification to obtain a large number of NK cells for treatment, such RNA-based expression is generally not stable over multiple generations. In an alternative approach, as described in WO 2020 / 1504752018, a FACS sorting strategy is used on the NK-92 cell line to select NK cells with endogenous CD16 expression (see also: Zih-Fei Cheng (2021) Biochemistry and Biophysics Reports [biochemistry and biophysics report] Vol. 26). Here, a human NK-92-derived cell line oNK-1 is established, which endogenously expresses CD16, as well as high levels of NK activation markers and low levels of NK inhibition markers that are unique to the parental NK-92 cell line.

[0009] Interestingly, when these cells were covalently conjugated with the monoclonal antibody trastuzumab, the cells so modified exhibited human epidermal growth factor 2 (HER2) binding specificity and enhanced cytotoxicity against various types of cancer cells with grade 1 (MCF-7), grade 2 (OVCAR-3), and grade 3 (SK-OV-3) HER2 expression compared to control oNK cells in vitro (see, e.g., Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; April 27-28 and June 22-24, 2020. Philadelphia (PA): AACR; Cancer Res [Cancer Research] 2020; 80(16 Suppl): Abstract No. 2169). Unfortunately, very high amounts of IL-2 (500 units / mL) are required for production and CD16 expression in oNK cells.

[0010] NK-92MI (ATCC: CRL-2408) is an IL-2-independent variant of NK-92 that was prepared using particle-mediated gene transfer of hIL-2 cDNA. NK-92MI cells show potent cytotoxicity against different tumor cells. However, the Fc receptor (CD16) that normally mediates ADCC is completely absent or present only at very low levels on NK-92MI cells, and these cells will therefore be unable to induce ADCC for target-specific cell killing. Although it has been demonstrated that CD16-mediated ADCC activity is more effective if circulating NK cells are also CD57-positive (see, e.g., Sandra Lopez-Vergès (2010) Blood 2010116 (19)), the proportion of CD16+CD57+ cells in the blood is very low, as these cells represent mature NK cells. Therefore, they have not yet been enriched and proliferated to therapeutically meaningful numbers. NK-92MI chimeric cells were also generated to express a recombinant chimeric receptor that can bind to the Fc portion of human immunoglobulin using viral transduction (see, e.g., Ying Chen (2017) Oncotarget 6; 8 (23)). However, CD57 expression by these cells was neither reported nor reasonably expected.

[0011] Thus, although various compositions and methods of NK cell therapy are known in the art, all or nearly all of them have several disadvantages, particularly where large numbers of such cells are required in an economical manner, and where such cells express CD16 and CD57 to enhance cytotoxicity. Summary of the invention

[0012] The present subject matter relates to various cells and cell-based compositions and methods thereof, wherein the cells are CD16+CD57+NK-92MI cells that naturally express CD16 and CD57 and exhibit IL-2-independent growth. Most notably, such cells have significantly enhanced cytotoxicity and can be produced in a simple and cost-effective manner.

[0013] In one aspect of the subject matter of the present invention, the inventors envision a CD16+CD57+NK-92MI cell that naturally expresses CD16 and CD57 and exhibits IL-2-independent growth. Most commonly, CD16 is CD16a or CD16b. Compared to parental NK-92MI cells (ATCC: CRL-2408), the cells envisioned in particular express NKG2D in higher amounts. In addition, CD3 is generally not present on the surface of the cell, while CD56 is generally present on the surface of the cell. In addition, the cells proposed herein may further include one or more of the following on the surface of the cell: CD4, CD25, NKp30, NKp44, NKp46, CD27, OX40, CD107a, NKG2A, PD-1, TIGIT.

[0014] Phenotypically, the cells proposed herein will generally have enhanced direct cytotoxicity to Ramos cells compared to the parental NK-92MI cells (ATCC: CRL-2408), and / or the cells proposed herein will exhibit strong ADCC in the presence of target cells and antibodies against surface proteins on target cells. In addition, the envisioned cells will also have high levels of CD57, CD16, and NKG2D expression after thawing and expansion compared to the parental NK-92MI cells (ATCC: CRL-2408). Further, in at least some embodiments, these cells will have faster replenishment, and / or faster cell doubling time after degranulation compared to the parental NK-92MI cells (ATCC: CRL-2408).

[0015] Where necessary, 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 in addition, 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 inventive subject matter, the inventors also contemplate a composition comprising a plurality of NK-92MI cells as set forth herein in a culture 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 naturally express CD16 and CD57 and exhibit IL-2-independent growth. As will be appreciated, the contemplated culture medium includes growth medium, cryopreservation medium, and pharmaceutically acceptable medium for infusion. In addition, the composition may further comprise an antibody that may or may not bind to CD16 of the CD16+CD57+NK-92MI cells. In further contemplated aspects, the plurality of cells is at 1×10 8 Up to 1×10 10 The cells are placed in a polymer bag and the culture medium is a pharmaceutically acceptable medium for infusion.

[0017] In a further aspect of the inventive subject matter, the inventors contemplate a cell culture comprising a plurality of dividing cells in a culture medium, wherein the cells are CD16+CD57+NK-92MI cells as set forth herein, wherein the culture medium is substantially free of IL-2. In a typical embodiment, the plurality of dividing cells are preferably maintained in a single culture vessel from the start of the culture and during the growth period until a predetermined number of cells is obtained. Thus, the contemplated cell culture may contain at least 1×10 7 cells / culture vessel. Most commonly, but not necessarily, the culture medium contains human AB serum or is serum-free.

[0018] In still another aspect of the inventive subject matter, the inventors contemplate a method for preparing CD16+CD57+NK-92MI cells that naturally express CD16 and CD57 and exhibit IL-2-independent growth. Such a method generally includes the step of providing a plurality of NK-92MI cells, and another step of enriching CD16+CD57+NK-92MI cells that naturally express CD16 and CD57 using anti-CD16 antibodies and anti-CD57 antibodies.

[0019] For example, each of the anti-CD16 antibody and the anti-CD57 antibody can be fluorescently labeled, and the antibodies can be used for enrichment (usually sequentially) using fluorescence activated cell sorting (FACS). As will be readily appreciated, FACS can be performed using a single pass sorting or using iterative rounds of FACS. In addition, the contemplated method can also include a step of amplifying the enriched population of CD16+CD57+NK-92MI cells.

[0020] In another aspect of the subject matter of the present invention, the inventors envision a method for treating cancer, the method comprising administering a therapeutically effective amount of a cell as proposed herein or a composition comprising such a cell to an individual in need thereof. Most commonly, the individual is a mammal (e.g., a human), and / or the cancer is a solid cancer. As will be readily understood, cells or compositions are administered by infusion, and treatment may also include the step of co-administering antibodies, checkpoint inhibitors, immunostimulants, and / or cancer vaccines.

[0021] Various objects, features, aspects and advantages of the present subject matter will become more apparent from the following detailed description of preferred embodiments and the accompanying drawings, in which like numerals represent like components. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Depicted are exemplary FACS results of CD16 and CD57 enrichment in sorted NK-92MI cells.

[0023] Figure 2 Exemplary results of the surface phenotype of parental and sorted NK-92MI cells are shown.

[0024] Figure 3 Depicted are graphs showing exemplary results of ADCC of Ramos target cells by sorted NK-92MI cells in the presence of Rituximab (Rituxan) antibody.

[0025] Figure 4 Depicted are graphs showing exemplary results of ADCC of HCC827 target cells by sorted NK-92MI cells in the presence of Avelumab antibody.

[0026] Figure 5 Described are exemplary FACS results demonstrating that sorted NK-92MI cells maintain high expression of CD57 and CD16 after freeze-thawing.

[0027] Figure 6 Exemplary results are shown for the surface phenotype of parental and sorted NK-92MI cells following freeze-thaw.

[0028] Figure 7 Depicted are graphs showing exemplary results of ADCC of sorted NK-92MI cells against Ramos target cells following freeze-thaw. DETAILED DESCRIPTION

[0029] The inventors have discovered that NK cells with high universal and target-specific cytotoxicity can be prepared, which not only express CD16 and CD57 in large quantities, but can also grow without the need for exogenous addition of IL-2. Unexpectedly, these cells were isolated from the NK-92MI cell line, which was previously not known to express CD16 and CD57 in large quantities. In this context, it should be understood that the NK-92MI cell line is not a collection of naturally occurring cells, but a cell line established by transforming the NK-92 cell line with human IL-2 (hIL-2) cDNA by particle-mediated gene transfer (see Hum Gene Ther [Human Gene Therapy] May 20, 1999; 10 (8): 1359-73).

[0030] Although not wishing to be bound by a particular theory or hypothesis, the inventors envision that upon expansion of the NK-92MI cell line, some cells undergo certain genetic changes and possible epigenetic changes, which can lead to natural and stable expression of CD16 and CD57. Although rare in the entire cell population of the NK-92MI cell line, the inventors note that such cells can be enriched via affinity-based separation methods. Notably, such enrichment is facilitated by the fact that NK-92MI cells express IL-2 from recombinant and genomic-integrated nucleic acid sequences, making such cells independent of exogenously added IL-2.

[0031] In a further unexpected discovery, the inventors also observed that the CD16+CD57+NK-92MI cells thus enriched also expressed NKG2D (a major regulator of cytotoxicity) in significantly higher amounts compared to the NKG2D expression of the NK-92MI cell line. The presence of such increased NKG2D even further enhanced the ubiquity and ADCC-type cytotoxicity of the isolated / enriched CD16+CD57+NK-92MIIL-2-independent cells.

[0032] As used herein, the phrase "cells that naturally express CD16 and CD57" or "natural and stable expression of CD16 and CD57" refers to cells that express CD16 and CD57 from corresponding genes in the cell genome, wherein these genes are not recombinant genes introduced into the cell. Also as used herein, the phrase "exhibits IL-2-independent growth" with respect to one or more cells refers to the ability of cells to undergo n-fold expansion (n is at least 10) in a culture medium provided without IL-2, without exogenous addition of IL-2 thereto, or without providing IL-2 thereto through a feeder cell layer. Similarly, as further used herein, the phrase "culture medium substantially free of IL-2" refers to a culture medium having an amount of IL-2 that is insufficient to support the growth of NK-92 cells (ATCC: CRL-2407). Therefore, a culture medium substantially free of IL-2 will contain an amount of IL-2 that is 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 can secrete IL-2 into the culture medium and all secreted amounts are included within the scope of this definition.

[0033] Although CD16+CD57+NK-92MIIL-2 independent cells are isolated from NK-92MI cell lines, it should be understood that many other cell lines, including NK-92 and genetically modified NK-92 cell lines (e.g., NK-92CI) are also considered suitable as parental cell lines. Most commonly, in the case where the NK-92 cell line is genetically modified, it is generally preferred that such modifications are genetically stable modifications because the modifications are passed to daughter cells. Therefore, the genetic modifications envisioned in particular include artificial chromosomes, viral genomes inserted into cell genomes, CRISPR-based modifications, and the like. In some embodiments, the genetic modification will include the addition of nucleic acids encoding CD16 and / or CD57, while in other modifications, the genetic modification will include the addition of nucleic acids encoding IL-2 (which may or may not include cytoplasmic or endoplasmic reticulum retention sequences). Further contemplated genetic modifications will include the addition of nucleic acids encoding one or more recombinant proteins to enhance cytotoxicity, target 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.

[0034] In further contemplated aspects, it should be noted that the parental cell line (e.g., NK-92MI) can be used for cell sorting or other types of enrichment for CD16 and CD57 directly after thawing from the cryopreservation medium or after passage in the medium for at least 1 generation, or at least 2 generations, or at least 3 generations, or at least 5 generations, or at least 10 generations, or at least 20 generations. Most commonly, such a culture medium will be serum-free and will not contain exogenous IL-2. In other embodiments, the culture medium contains human AB serum, but does not contain exogenously added IL-2. Similarly, it is generally preferred that the culture medium will not include an additional feeder cell layer.

[0035] For cell separation and enrichment, it is generally preferred that cells are separated using a selection process that includes the use of anti-CD16a antibodies and anti-CD57 antibodies. However, in alternative embodiments, the antibody can also be anti-CD16b antibodies, anti-CD16c antibodies, anti-CD32 antibodies and / or anti-CD64 antibodies. Most commonly, the cell selection process will be a FACS process, however, various alternative selection processes are also considered suitable for use herein, including magnetic bead separation in which antibodies are coupled to beads, microplate separation in which antibodies are coupled to the pore wall, microfluid separation in which antibodies are coupled to the microfluidic channel wall, etc. Regardless of the separation method, it should be understood that the separation of cells can be performed using both anti-CD16a antibodies and anti-CD57 antibodies in a single pass, or in multiple continuous passes, in which cells are first enriched in CD16+ cells, and then these CD16+ cells are further enriched in CD57+ cells.

[0036] It is also easy to understand that cells can be subjected to growth / expansion steps, especially when separated in a sequential manner. Such growth and expansion can start from many cells (e.g., the original output of the separation step) or from cells reduced in number. Such a reduction in number can be obtained by subculture under selected conditions or by serial dilution to ultimately produce a monoclonal cell product. Once the desired CD16+CD57+NK-92MIIL-2 independent cells are obtained, it is envisioned that these cells can then be further characterized for one or more parameters, and it can be verified that these cells are positive for CD16 and CD57.

[0037] In the case where CD16+CD57+NK-92MIIL-2 independent cells are not monoclonal sources, it is also envisioned that the cell colonies thus obtained can be further tested and / or selected for other ideal traits. In addition, CD16+CD57+NK-92MIIL-2 independent cells can be selected or verified for rapid replenishment after degranulation, reduced cell doubling time, optimal growth density, etc. (all relative to parental NK-92MI cell line). Similarly, CD16+CD57+NK-92MIIL-2 independent cells can be selected or verified for cytotoxicity / efficacy, cytokine expression and especially IFN-γ secretion, migration to target tissues, etc. to multiple cancer cells (which can be cells of solid cancer or hematogenous cancer).

[0038] It should be understood that once separated, the cell can be further genetically modified to include one or more desirable properties, typically by expressing one or more recombinant proteins. For example, particularly preferred other proteins suitable for expression include chimeric antigen receptors, homing receptors (e.g., G protein-coupled receptors (GPCRs), chemokine receptors (such as CCR7, CXCR2 or CXCL14 receptors), cytokine receptors, cell adhesion molecules, selectins or integrins) preferably targeting cancer or tumor-associated antigens (e.g., CD19, CD20, GD2, HER-2, CD30, EGFR, FAP, CD33, CD123, PD-L1, IGF1R, CSPG4 or B7-H4) or neoantigens), membrane-bound or secreted TGF-β traps and / or checkpoint inhibitors. Exemplary proteins, sequences and constructs for such modifications are described in WO 2020 / 028656 (which is incorporated herein by reference). Additionally, it should be understood that the cells presented herein can be further modified with one or more target-specific binding agents such as antibodies and fragments thereof (including scFv constructs).

[0039] Therefore, it should be understood that the cells proposed herein can be used for a variety of therapeutic purposes, and in particular for infusion into individuals in need thereof. Therefore, the inventors also contemplate a composition in which the cells proposed herein are placed in a growth medium for preparing a therapeutic amount of cells, placed in a cryopreservation medium for storing such cells, or placed in a pharmaceutically acceptable medium for infusion.

[0040] As will be readily appreciated, the cells presented herein can be administered to a subject by a variety of routes, and a particularly preferred route is intravenous infusion over a period of time. Typically, for a single dose of cells, the period of time is between 5 and 130 minutes. Optionally, the period of time is between 90 and 120 minutes. Optionally, the period of time 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.

[0041] With respect to the number of cells administered, it is contemplated that the cells presented herein may be administered to a subject at from about 1000 cells / infusion to as high as about 10 billion cells / infusion, such as about, at least about, or at most about 1×10 cells / infusion. 10 pcs, 1×10 9 pcs, 1×10 8 pcs, 1×10 7 pcs, 5×10 7 pcs, 1×10 6 pcs, 5×10 6 pcs, 1×10 5 pcs, 5×10 5 pcs, 1×10 4 pcs, 5×10 4 pcs, 1×10 3 pcs, 5×10 3 cells, or any range between any two numbers, including the endpoints. For example, 1×10 8 Up to 1×10 10 Individual cells are administered to a subject. As will be readily understood, cells can be administered from the production process without intermediate storage in a cryopreservation medium, or cells can be administered after thawing. Where necessary, cryopreservation medium can be replaced with an infusion medium before infusion. In further contemplated aspects, it should be understood that the cells proposed herein can also be irradiated to reduce or eliminate proliferation, especially when cells are administered in a therapeutic composition. Therefore, it is believed that all modes or irradiation are suitable for use herein, and particularly include low-energy electron beam irradiation and gamma irradiation, typically with a dose between 2 and 20 Gy, more typically between 6 and 10 Gy (see, e.g., Front. Immunol. [Immunology Frontier] June 2021, Vol. 12, Article 684052).

[0042] It will therefore be appreciated that the CD16+CD57+NK-92MI cells of the subject matter of the present invention can be used for a variety of therapeutic needs. However, it is particularly contemplated that the cells are used in methods for treating cancer (e.g., solid tumors or hematogenous cancers) in a subject, wherein such cells are typically administered in a pharmaceutically acceptable composition comprising a therapeutically effective amount of the cells. Advantageously, these cells will form part of a multimodal treatment strategy that includes additional therapeutic agents such as antibodies, checkpoint inhibitors, immunostimulants, cancer vaccines, and / or metronomic low-dose chemotherapy.

[0043] Examples

[0044] The inventors used a BD FACS Melody cell sorter and fluorescently labeled anti-CD16 and anti-CD57 antibodies to sort CD57+CD16+NK cells from a parental NK-92MI cell line (e.g., commercially available from ATCC as CRL-2408). After sorting, the cells were cultured for up to 126 days and high expression of CD16 and CD57 was observed by flow cytometry (1.98% CD16 in parental cells versus 85.7% CD16 in sorted cells, and 2.69% CD57 in parental cells versus 32.1% CD57 in sorted cells). Figure 1 Described are exemplary results of such CD16 and CD57 enrichment in 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 presented as dot plots. Bar graphs compare the percentage expression of the corresponding markers on parental cells and sorted cells.

[0045] Surprisingly, as Figure 2 It can be concluded that the sorted cells also showed higher expression of NKG2D (a major regulator of cytotoxicity) when compared to the parental cells. Figure 2 As can be seen, the expression of receptors such as CD56 and CXCR4 is comparable on both cell lines. Notably, the sorted cells also maintained high viability throughout the culture period. Figure 2 Described are exemplary results of the surface phenotype of parental NK-92MI and sorted CD16+CD57+NK-92MI cells. Here, surface marker expression was analyzed by flow cytometry-based staining, and the results are presented as histograms below. Light grey and dark grey indicate marker staining on unstained and stained cells, respectively.

[0046] To examine whether the sorted cells are cytotoxic to different cancer cell lines, parental or sorted NK-92MI were co-cultured with target cells such as Ramos and HCC827 cells at different effector to target ratios in a Calcein AM based assay. Figure 3 As can be seen, sorted but not parental cells showed enhanced direct cytotoxicity towards Ramos cells (non-specific β-gal treatment was comparable to direct cytotoxicity; 7% Ramos lysis for parental cells versus 55% Ramos lysis for sorted cells). Figure 3 Exemplary results of ADCC of Ramos target cells by sorted NK-92MI cells are shown. More specifically, ADCC of Ramos target cells by parental (unsorted) and sorted NK-92MI cells in the presence of rituximab was measured in a calcein release assay. Samples were tested in triplicate. For different effector to target ratios, the percentage (%) of calcein release as an indicator of target cell lysis is shown.

[0047] ADCC of parental and sorted NK-92MI cells was also tested against two tumor cell lines, including HCC827 and Ramos cells, in the presence of Avelumab and Rituximab, respectively. As expected, parental cells lacking CD16 failed to induce ADCC-mediated lysis of Ramos cells. In contrast, the sorted cells induced effective ADCC against Ramos cells (3% ADCC of parental cells against Ramos vs. 94% ADCC of sorted cells against Ramos). Similar effective ADCC activity was observed when the sorted cells were mixed with HCC827 cells in the presence of Avelumab, as shown from Figure 4 It can be seen from the results that Figure 4 Described are exemplary results of ADCC of HCC827 target cells by sorted NK-92MI cells. Here, ADCC of HCC827 target cells by sorted NK-92MI cells in the presence of Avelumab was determined in a calcein release assay. Samples were tested in triplicate. For different effector to target ratios, the percentage (%) of calcein release as an indicator of target cell lysis is shown.

[0048] The expression stability after freeze-thaw cycles was also evaluated. The sorted cells were frozen in a LN2 freezer in a proprietary culture medium from ImmunityBio. After thawing and expansion, the sorted cells maintained high expression of CD57, CD16, and NKG2D, and exemplary results are shown in Figure 5 and Figure 6 middle. Figure 5Describe exemplary results, these results show that the sorted NK-92mi cells maintain high expression of CD57 and CD16 after freezing and thawing. Here, the expression of surface markers on parental (unsorted) and sorted NK-92MI cells was analyzed by flow cytometry-based staining, and the results are presented as dot plots below. The bar graph compares the expression percentage of the corresponding markers on parental cells and sorted cells. Figure 6 Describe the exemplary results of the surface phenotype of parental and sorted NK-92MI cells after freezing and thawing. Here, surface marker expression was analyzed by flow cytometry-based staining, and the results are presented as histograms below. Light gray and dark gray indicate marker staining on unstained and stained cells, respectively.

[0049] Figure 7 Exemplary results of ADCC after freezing and thawing of sorted NK-92MI cells are shown. Here, ADCC of NK-92MI cells thawed in the presence of rituximab against Ramos target cells was determined in a calcein release assay. Samples were tested in triplicate. For different effector-to-target ratios, the percentage (%) of calcein release as an indicator of target cell lysis is shown. As can be seen, freeze-thaw cycles had no effect on CD16-mediated ADCC activity against CD20-expressing Ramos cells.

[0050] Based on the above, it will be appreciated that the sorted cell line unexpectedly provides CD16+CD57+NK-92MI cells, which naturally express CD16 and CD57 and exhibit IL-2-independent growth in long-term culture and after freeze-thaw, with a significant increase in NKG2D.

[0051] For the above experiment, the inventors sorted CD57+CD16+NK cells from parental NK-92MI (IL-2 independent cell line). About 60 million living cells with greater than 90% viability were mixed with fluorescently labeled anti-CD16 and anti-CD57 antibodies for cell sorting using BD FACS Melody cell sorter. The NK cells sorted out by CD57+CD16+ were cultured in immunobiology company specific culture medium for up to 126 days. On different days, flow cytometry analysis was performed using Miltenyi MACSQuant analyzer 10, in which cells were stained at room temperature for 10-30min with fluorescent dye-conjugated antibodies. 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, in which the sorted cells were mixed with tumor cells such as Ramos cells loaded with calcein in the presence / absence of rituximab antibodies. NK cells induce lysis of calcein-loaded target cells, resulting in the release of intracellular fluorescent calcein into the assay medium. The fluorescence level of released calcein is proportional to the number of lysed cells and is therefore a measure of NK effector cell cytotoxicity.

[0052] In further experiments, the inventors used mRNAseq to carry out gene expression analysis, wherein the gene expression of genes was monitored in the whole cell sorting process. It is noteworthy that, and in other findings, the analysis of the mRNAseq of the NK-92MICD16 cells sorted reveals that there are about 50 genes (coding and non-coding) that show significant continuous upregulation (more than 4 times) in 2 step sortings, and exemplary results are described in Table 1. As expected, CD16a is one of the genes upregulated. Most of the remaining upregulated genes have the functions of signal transduction, metabolism and gene expression. Unexpectedly, both Wnt6 and TRAILDR1 are highly enriched, wherein the effect of Wnt6 is unclear, and the effect of TRAILDR1 will prompt the other potential (indirect) mechanism of enhanced cytotoxicity.

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[0056] In addition, the inventors have also identified 27 other genes (coding and non-coding) that show significant continuous downregulation (more than 4 times) in 2 step sorting, most of which are generally involved in cell metabolism, and exemplary results are described in Table 2. Unexpectedly, PDGFD (platelet-derived growth factor D) is one of the genes significantly downregulated, which is a known "survival factor" in NK cells. Such a discovery is particularly noteworthy because PDGFD is known as an effective stimulator of cell growth and movement, which activates NK cells in an autocrine manner and contributes to interleukin-15-mediated NK cell survival. Such overexpression can contribute to the ability to amplify the sorted cells in the absence of exogenous IL-2 and / or IL-15.

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[0059] The inventors further identified 406 other genes (coding and non-coding) showing an upward trend during sorting. However, the data of upregulation are much more complicated, some of which show upregulation after the 1st sorting step, but upregulation after the 2nd sorting is not so much, and other genes show the opposite situation, showing slight enrichment after the 1st sorting, but showing strong upregulation after the 2nd sorting. Finally, after each sorting step, other genes only show slight enrichment. Exemplary data are shown in Table 3. It is worth noting that some of the genes upregulated include CD86, CD74, CD7, CCR5, CCR10, TIM3, KLRG1, SYK, IL21R, CXCR6, CD25, 4-1BB, GZMH, CD30, LAIR1 and NKG2D, which are considered to be related to increased cytotoxic activity or chemotaxis. On the other hand, and somewhat unexpectedly, TIM3 and LAIR1 are upregulated, which is generally related to the reduction of NK cells.

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[0074] Finally, the inventors also identified about 350 genes (coding and non-coding) that showed a down-regulation trend during sorting, as can be seen from the data in Table 4. As above, the situation is complicated. Most notably, the down-regulated genes that are usually associated with cytotoxicity in NK cells are CD11c, CD11d, IL25, NKp44 and CD28. However, most unexpectedly, by serial sequencing, the expression of the IL-2 gene was significantly down-regulated (almost 10 times).

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[0086]

[0087] In some embodiments, the digits of the amount of the expression composition, characteristic (such as concentration), reaction conditions, etc., used to describe and claim certain embodiments of the present invention should be understood to be modified by the term "about" in some cases. Therefore, in some embodiments, the numerical parameters set forth in the written description and the appended claims are approximate values, which can vary according to the desired properties sought to be obtained in a particular embodiment. The description of the value range herein is intended only to be used as a shorthand method for individually referring 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 described herein.

[0088] As used herein, the term "administering" a pharmaceutical composition or drug refers to both direct and indirect administration of a pharmaceutical composition or drug, wherein direct administration of a pharmaceutical composition or drug is typically performed by a health care professional (e.g., a physician, nurse, etc.), and wherein indirect administration includes the step of providing the pharmaceutical composition or drug to or making available to a health care professional for direct administration (e.g., via injection, infusion, oral delivery, topical delivery, etc.). It should also be noted that the term "prognosis" or "prediction" of a condition, susceptibility to disease development, or response to a desired treatment is intended to encompass the act of predicting or predicting a condition, susceptibility, and / or response (including the rate of progression, improvement, and / or duration of a condition in a subject) (but not the act of treating or diagnosing).

[0089] All methods described herein can be performed in any suitable order, unless otherwise specified herein or otherwise clearly contradictory to the context. The use of any and all examples or exemplary language (e.g., "such as") provided with respect to certain embodiments herein is intended only to better illustrate the present invention, without limiting the scope of the present invention as originally claimed. Any language in this specification sheet should not be interpreted as indicating that any unclaimed element is necessary for practicing the present invention.

[0090] As used in the specification herein and throughout the claims, the meaning of "a, an" and "the" includes plural referents unless the context clearly indicates otherwise. Similarly, as used in the specification herein, the meaning of "in" includes "in" and "on" unless the context clearly indicates otherwise. Also as used herein, and unless the context indicates otherwise, the term "coupled to" is intended to include direct coupling (where two elements coupled to each other are in contact with each other) and indirect coupling (where at least one additional element is located between the two elements). Therefore, the terms "coupled to" and "coupled with" are used synonymously.

[0091] It will be apparent to those skilled in the art that, without departing from the inventive concepts herein, more modifications are possible in addition to those modifications that have been described. Therefore, the subject matter of the present invention should not be limited except within the scope of the appended claims. In addition, when 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 "comprises" and "comprising" should be interpreted as referring to elements, parts or steps in a non-exclusive manner, indicating that the referenced elements, parts or steps may exist or be utilized, or be combined with other elements, parts or steps that are not explicitly referenced. When the specification or claim refers to at least one selected from the group consisting of A, B, C... to N, the text should be interpreted as requiring only one element in the group, rather than A plus N, or B plus N, etc.

Claims

1. A CD16+CD57+NK-92MI cell, which naturally expresses CD16 and CD57 and exhibits IL-2-independent growth.

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

3. The cell according to any one of the preceding claims, wherein NKG2D is expressed at a higher amount on the cell surface of the cell compared to NK-92MI cells.

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

5. A cell according to any of the preceding claims, 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 the preceding claims, wherein the cell has enhanced direct cytotoxicity towards Ramos cells compared to NK-92MI cells.

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

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

10. The cell according to any one of the preceding claims, 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 the preceding claims, 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 culture medium, wherein at least 10% of the NK-92MI-derived cells are CD16+CD57+NK-92MI cells that naturally express CD16 and CD57 and exhibit 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 of claim 12, comprising at least 90% of the CD16+CD57+NK-92MI cells.

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

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

17. The composition according to any one of claims 12-16, wherein the composition further comprises an antibody.

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

19. The composition according to any one of claims 12-18, wherein the plurality of cells is 1×10 8 Up to 1×10 10 The invention relates to a method for injecting a plurality of cells into a polysaccharide-containing pharmaceutical composition, wherein the culture medium is a pharmaceutically acceptable culture medium for infusion, and wherein the cells and the culture medium are placed in a polymer bag.

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

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

22. The cell culture according to any one of claims 20-21, wherein the plurality of dividing cells are maintained in a single culture vessel from the start of the culture and during growth until a predetermined amount of cells is obtained.

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

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

25. A method for preparing CD16+CD57+NK-92MI cells that naturally express CD16 and CD57 and exhibit IL-2-independent growth, the method comprising: Provide multiple NK-92MI cells, and These CD16+CD57+NK-92MI cells that naturally express CD16 and CD57 were enriched using anti-CD16 and anti-CD57 antibodies.

26. The method of claim 25, wherein each of the anti-CD16 antibodies and anti-CD57 antibodies are fluorescently labeled, and wherein the step of using the antibodies comprises fluorescence activated cell sorting.

27. The method according to claim 26, wherein the fluorescence activated cell sorting is performed sequentially for the use of the anti-CD16 antibodies and anti-CD57 antibodies.

28. The method of claim 26, wherein the fluorescence activated cell sorting comprises iterative rounds of fluorescence activated cell sorting.

29. The method according to any one of claims 25-29, further comprising the step of expanding the enriched population of the CD16+CD57+NK-92MI cells.

30. A composition comprising a plurality of NK-92MI-derived cells in culture for use in cancer treatment, wherein at least 10% of the NK-92MI-derived cells are CD16+CD57+NK-92MI cells that naturally express CD16 and CD57 and exhibit IL-2-independent growth.

31. The composition of claim 30, wherein the CD16 is CD16a or CD16b.

32. The composition of claim 30, wherein the culture medium is a growth medium, a cryopreservation medium, or a pharmaceutically acceptable medium for infusion, and wherein the NK-92MI-derived cells are optionally irradiated.

33. The composition of claim 30, wherein the composition further comprises an antibody.

34. The composition of claim 33, wherein the antibody binds to the CD16 of the CD16+CD57+NK-92MI cell.

35. The composition of 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.

36. The composition of claim 30, wherein the CD16+CD57+NK-92MI cells maintain high expression of CD57, CD16 and NKG2D after thawing and expansion compared to NK-92MI cells.

37. The composition of claim 30, wherein the CD16+CD57+NK-92MI cells have enhanced direct cytotoxicity against Ramos cells compared to NK-92MI cells.

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

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

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

41. The method of claim 30, wherein the subject is a mammal.

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

43. The method of claim 30, wherein the cells or the composition are administered by infusion.

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

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