CN113195706A

CN113195706A - CIML NK cells and methods thereof

Info

Publication number: CN113195706A
Application number: CN201980082326.4A
Authority: CN
Inventors: 罗希特·杜加尔; 拉吉特·辛哈; 李文钊; 詹森·艾萨克森; 派翠克·松吉翁
Original assignee: NantKwest Inc
Current assignee: ImmunityBio Inc
Priority date: 2019-07-08
Filing date: 2019-07-08
Publication date: 2021-07-30
Also published as: SG11202104256XA; WO2021006876A1; JP2022540267A; EP3853353A4; KR20210079381A; AU2019456438A1; EP3853353A1; CA3117134A1; IL283997A

Abstract

Cytokine-induced memory-like (CIML) NK cells with enhanced cytotoxicity are proposed. Most commonly, these CIML NK cells are derived from the monocyte fraction of peripheral blood or umbilical cord blood. In a further contemplated aspect, these CIML NK cells are expanded and induced in a closed and automated production environment that greatly reduces operational complexity and production costs.

Description

CIML NK cells and methods thereof

Technical Field

The present disclosure relates to compositions, methods and devices for generating and/or culturing activated immunoreceptive cells, particularly when it relates to memory-like NK cells produced by Cord Blood (CB) or Peripheral Blood (PB).

Background

The background description includes information that may be useful in understanding the present disclosure. This is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, nor that any publication specifically or implicitly referenced is prior art.

All publications and patent applications herein are incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. Where 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.

Natural Killer (NK) cells constitute a group of innate immune cells that are often characterized as cytotoxic lymphocytes that exhibit antibody-dependent cytotoxicity via targeted release of granulysin and perforin. Most NK cells have a specific cell surface marker profile (e.g., CD3) in addition to a collection of various activating and inhibitory receptors^-、CD56⁺、CD16⁺、CD57⁺、CD8⁺). Although NK cells have recently become an important component of certain cancer treatments, the generation of large numbers of NK cells (especially autologous NK cells) has been very difficult due to the relatively low proportion of NK cells in whole blood.

To obtain therapeutically significant numbers of NK cells and NK-like cells, NK cells can be generated from various precursor cells. For example, various Stem Cell Factors (SCF), FLT3 ligands, Interleukins (IL) -2, IL-7 and IL-15 have been introduced in various bodiesReports have been made in a method of inducing and amplifying cytokine-induced killer (CIK) cells derived from umbilical cord blood ex-situ (Anticancer Research)]30:3493-3500(2010)). Similarly, CD34⁺Hematopoietic cells can be exposed to IL-12 and other agents, as reported in US 2018/0044636. In other methods, human hemangioblasts are sequentially exposed to two different cytokine mixtures, as described in WO 2011/068896, and the different cytokine mixtures are used with post-embryonic hematopoietic stem cells, as taught in WO 2012/128622. Although at least some of these methods provide significant n-fold expansion of NK cells, the methods and reagents used for such expansion require both time and resources. Furthermore, it should be noted that many known methods also require culturing NK cells on a feeder cell layer, which is often problematic from a technical and regulatory point of view.

In a simpler approach, Acute Myeloid Leukemia (AML) cells can be exposed to TpoR agonists, thereby inducing the AML cells to form NK cells. However, this approach may not be feasible as a source of therapeutic cell preparations. Alternative methods also rely on culturing peripheral blood cells in the presence of various interleukins, stem cell factors and FLT3 ligands, as disclosed in WO 2011/103882. In another approach, US 2013/0295671 teaches methods of stimulating already existing NK cells with anti-CD 16 and anti-CD 3 antibodies and cytokines. Although simpler in procedure, such methods still require elaborate manipulation of the cells and add significantly to the cost due to the specific reagents required.

In a further known method, US 10,125,351 describes the use of umbilical cord blood or peripheral blood as a source of cells, which are subjected to density gradient separation to separate nucleated cells, which are then incubated with a medium containing interferon, interleukin, CD3 antibody and human albumin. Most advantageously, this method is suitable for perfusion culture in bioreactors and therefore significantly reduces the operational difficulties. Unfortunately, however, the yield of NK cells is relatively low.

Regardless of the particular mode of production, cultured NK cells typically do not exhibit memory-like characteristics, which are particularly desirable for cancer immunotherapy. In at least some attempts to generate memory-like NK cells, cultured NK cells are exposed to IL-12, IL-15 and IL-18, so the exposed NK cells exhibit a memory-like phenotype and are associated with expression of CD94, NKG2A, NKG2C and CD69 and lack of CD57 and KIR (see Blood [ Blood ] (2012) Vol.120, No. 24; 4751-4760). Similarly, NK cells were preactivated by using various stimulatory cytokines and then the preactivated cells were contacted with PM21 particles, EX21 exosomes or FC21 feeder cells as described in WO 2018/089476. In another method of generating memory-like NK cells, freshly isolated NK cells are exposed to an IL-18/IL-12-TxM fusion protein complex, as described in WO 2018/165208. While such methods typically produce at least some NK cells with memory-like characteristics, the cytotoxicity of such activated NK cells on selected target cells is still not optimal, possibly due to lack or low expression of specific activating receptors and/or specific inhibitory receptors.

Thus, even though various methods of generating memory-like NK cells are known in the art, all or almost all of them suffer from various drawbacks. Accordingly, there is a need to provide improved systems and methods for producing large numbers of memory-like NK cells, particularly autologous memory-like NK cells. In addition, the improved systems and methods will also allow automation of cell culture and NK cell activation and will have significantly reduced reagent requirements to make such methods clinically and commercially viable.

Disclosure of Invention

The inventors have discovered compositions, methods and devices that enable the generation and expansion of memory-like NK cells in a conceptually simple and efficient manner. Advantageously, the memory-like NK cells may be generated in a two-step process, wherein the NK cells are expanded to a desired number and wherein the expanded NK cells are then induced with a mixture of cytokines, thereby forming cytokine-induced memory-like (CIML) NK cells. The expansion of NK cells is preferably performed during enrichment with N-803 and anti-CD 16 agonist antibodies and optionally anti-CD 3 antibodies. Activation is then performed with a combination of stimulating cytokines, most preferably with IL-12/IL-15/IL-18 or IL-18/IL-12-TxM fusion protein complexes to obtain memory-like characteristics.

Unexpectedly, in addition to upregulating activation markers and IFN- γ secretion, the expanded memory-like NK cells so activated had increased expression of CD25 and the NK activation receptor DNAM-1 and downregulated expression of the inhibitory receptor TIGIT, which could lead to or even cause the high toxicity observed with CIML NK cells. Most notably, the CIML NK cells presented herein exhibited significant cytotoxicity to other NK-resistant tumor cell lines MS-1 even at relatively low effector to target ratios.

In one aspect of the inventive subject matter, the inventors contemplate a method of producing CIML NK cells with enhanced cytotoxicity, the method comprising the step of separating a mixture of monocytes from a biological fluid, and another step of contacting the mixture of monocytes with an anti-CD 16 antibody and N-803 to expand the NK cells. In another step, the expanded NK cells are contacted with a stimulating cytokine composition (typically comprising an IL-18/IL-12-TxM fusion protein complex, a mixture of IL-12, N-803, and IL-18, or a mixture of IL-12, IL-15, and IL-18) to generate CIML NK cells with enhanced cytotoxicity. Where desired, contemplated methods may further comprise the step of contacting the CIML NK cells with N-803 after restimulation of the CIML NK cells.

Preferably, but not necessarily, the biological fluid is whole blood or cord blood and the mixture of mononuclear cells is not further processed to enrich for NK cells. Most typically, the mixture of monocytes contains about 100-⁶A cell, and/or in a volume of about 100-⁶The step of contacting the mixture is performed at a cell density of one cell/ml. In other embodiments, the anti-CD 16 antibody may be present at a concentration of 0.05-0.5mcg/ml during the step of contacting the mixture, and/or N-803 may be present at a concentration of 0.1-1.0nM during the step of contacting the mixture. Optionally, the step of contacting the mixture can further comprise the step of contacting the mixture of monocytes with an anti-CD 3 antibody (e.g., anti-CD 3 antibody concentration of 0.1-1.0 ng/ml).

While in some aspects the stimulating cytokine composition comprises an IL-18/IL-12-TxM fusion protein complex, in other aspects the stimulating cytokine composition comprises a mixture of IL-12, N-803, and IL-18, and in further aspects the stimulating cytokine composition comprises a mixture of IL-12, IL-15, and IL-18. Most typically, NK cells are expanded to about 0.5-5.0X 10⁹The total cell number of individual cells, and/or the step of contacting the expanded NK cells with the stimulating cytokine composition is performed in the same vessel as the step of expanding NK cells.

Thus, from a different perspective, the inventors also contemplate a method of activating NK cells to form CIML NK cells with enhanced cytotoxicity. Such methods will include the step of providing expanded NK cells (typically expanded from monocytes of whole blood or cord blood), and the further step of contacting the expanded NK cells with a stimulating cytokine composition, which may include an IL-18/IL-12-TxM fusion protein complex, a mixture of IL-12, N-803, and IL-18, or a mixture of IL-12, IL-15, and IL-18, such that CIML NK cells with enhanced cytotoxicity are generated.

As indicated previously, it is contemplated that NK cells are expanded from whole blood or cord blood. Thus, NK cells may be autologous relative to the individual receiving the infusion comprising CIML NK cells. Preferably, the stimulating cytokine composition comprises an IL-18/IL-12-TxM fusion protein complex. However, in other embodiments, the stimulating cytokine composition may also include a mixture of IL-12, N-803, and IL-18 or a mixture of IL-12, IL-15, and IL-18. Typically, the total cell number of the expanded NK cells is about 0.5-5.0X 10⁹And (4) cells.

It is further contemplated that CIML NK cells with enhanced cytotoxicity will be cytotoxic to MS-1 cells; CIML NK cells with enhanced cytotoxicity have reduced expression of CD16 compared to expanded NK cells contacted with N-803 alone; CIML NK cells with enhanced cytotoxicity have reduced TIGIT expression compared to expanded NK cells contacted with N-803 alone; and/or the CIML NK cell with enhanced cytotoxicity has increased expression of CD25 and/or DNAM1 compared to the expanded NK cell contacted with N-803 alone.

Thus, the inventors also contemplate a CIML NK cell with enhanced cytotoxicity that exhibits cytotoxicity to MS-1 cells of at least 50% killing at an effector to target cell ratio equal to or less than 5. In other aspects, the CIML NK cell has reduced expression of CD16 as compared to an expanded NK cell contacted with N-803 alone, has reduced expression of TIGIT as compared to an expanded NK cell contacted with N-803 alone, and/or has increased expression of CD25 and/or DNAM1 as compared to an expanded NK cell contacted with N-803 alone.

Although not limiting to the subject matter of the invention, the CIML NK cells are preferably autologous cells relative to the individual receiving the infusion comprising CIML NK cells. In other embodiments, the CIML NK cells may also be recombinant NK cells. For example, such recombinant cells may express CD16 or a variant thereof, IL-2 or a variant thereof, and/or IL-15 or a variant thereof from a recombinant nucleic acid.

In a further contemplated aspect, the inventors also contemplate a pharmaceutical composition comprising a pharmaceutically acceptable carrier in combination with CIML NK cells as set forth herein. Thus, the use of CIML NK cells as proposed herein in medicine and in particular in the treatment of cancer is contemplated.

Accordingly, the present inventors also contemplate a method of treating an individual in need thereof with CIML NK cells, the method comprising the step of administering to the individual a therapeutically effective amount of CIML NK cells as set forth herein. Preferably, the CIML NK cells are autologous cells of the individual, and/or the CIML NK cells are peripheral blood or cord blood derived NK cells.

Various objects, features, aspects and advantages will become more apparent from the following detailed description of preferred embodiments, along with the accompanying drawings in which like numerals represent like components.

Drawings

FIG. 1 depicts an exemplary schematic of an IL-18/IL-12-TxM fusion protein complex.

Figure 2 depicts exemplary results of peripheral blood NK cell expansion using autologous PBMC and selected specific antibody combinations.

FIG. 3 depicts exemplary results of a cytotoxicity assay of cord blood-derived CIML NK cells against MS-1 target cells.

FIG. 4 depicts exemplary results of selected phenotypic marker expression on cord blood-derived CIML NK cells.

FIG. 5 depicts exemplary results of cytotoxicity assays of CIML NK cells of peripheral blood origin on MS-1 target cells.

Fig. 6 depicts exemplary results of selected phenotypic marker expression on peripheral blood-derived CIML NK cells.

FIG. 7 depicts exemplary activated cluster phenotypes of cord blood-derived CIML NK cells following IL-18/12TxM exposure.

FIG. 8 depicts exemplary CD25 expression on cord blood-derived CIML NK cells following IL-18/12TxM exposure.

FIG. 9 depicts exemplary activated cluster phenotypes of cord blood-derived CIML NK cells after restimulation.

FIGS. 10A-10C depict exemplary results of cell killing activity of cord blood-derived CIML NK cells after 24 hours (10A), 48 hours (10B), and 72 hours (10C) IL-18/12TxM exposure.

FIG. 11 depicts exemplary results of cell killing activity of cord blood-derived CIML NK cells grown in a single-tank culture environment and clustering of activation from such cells.

FIG. 12 depicts exemplary results of NK marker expression on peripheral blood-derived CIML NK cells following IL-18/12TxM exposure.

FIG. 13 depicts exemplary results of cytotoxicity assays of peripheral blood-derived CIML NK cells against K562 target cells following IL-18/12TxM exposure.

FIG. 14 depicts exemplary results of IFN- γ staining of peripheral blood-derived CIML NK cells following restimulation.

Fig. 15 depicts exemplary results of cytotoxicity assays of peripheral blood-derived CIML NK cells after exposure to N-803.

FIG. 16 depicts exemplary results of IFN- γ staining of cord blood-derived CIML NK cells after restimulation and exemplary results of cytotoxicity assays after exposure to N-803.

Detailed Description

Immunotherapy in cancer treatment increasingly utilizes various cell-based components, and NK cells have recently become a promising approach. Although some NK cells are available in relatively high numbers, the generation of therapeutically significant numbers of autologous NK cells and/or memory-like NK cells remains problematic at best. However, many of the current methods require the use of feeder layers or the differentiation of isolated CD34+ Hematopoietic Stem Cells (HSCs), which is time and resource intensive. Furthermore, such methods typically require human interaction and are susceptible to contamination due to the various operating steps required. In addition, conversion of NK cells to a memory-like phenotype may, in at least some approaches, reduce cytotoxicity or fail to deliver sufficient amounts of such cells.

The inventors have now found that a therapeutically significant number of NK cells (e.g., at least 0.5X 10) can be generated in a simple and efficient manner⁹Individual NK cells) that can be readily converted into memory-like NK cells, which can be fully automated even once mononuclear cells are obtained from biological fluids (e.g., whole blood, cord blood). Advantageously, such NK cells may be autologous NK cells and may be induced into a memory-like phenotype to give cytokine-induced memory-like (CIML) NK cells with enhanced cytotoxicity. Notably, and as described in more detail below, the CIML NK cells so generated will have superior cytotoxicity compared to other (CIML) NK cells and will have significant killing capabilities even against other target cells such as MS-1 cells (Merkel) cell carcinoma cells) that are resistant or even inert to NK cell cytotoxicity.

While not wishing to be bound by any theory or hypothesis, the inventors contemplate that the enhanced cytotoxicity may be due to the source of the (original) NK cells, previous expansion conditions, and may be due to an uninterrupted (e.g., change in culture medium, culture conditions, etc.) expansion and cytokine-induced nature, which may result in over-expression of the activating factor and under-expression of the inhibitory receptor. Among other noteworthy features of the CIML NK cells presented herein, the CIML NK cells will typically exhibit cytotoxicity to MS-1 cells of at least 50% killing at an effector cell to target cell ratio equal to or less than 5, will have reduced TIGIT (inhibitory receptor) expression compared to expanded NK cells contacted with N-803 alone, and increased CD25 and/or DNAM1 (activating co-receptor) expression compared to expanded NK cells contacted with N-803 alone. Thus, the term "NK cell with enhanced cytotoxicity" refers to a cell exhibiting cytotoxicity to MS-1 cells of at least 50% killing at an effector to target cell ratio equal to or less than 5, exhibiting reduced TIGIT (inhibitory receptor) expression as compared to an expanded NK cell contacted with N-803 alone, and/or increased expression of CD25 and/or DNAM1 (activating co-receptor) as compared to an expanded NK cell contacted with N-803 alone. Moreover, contemplated CIML NK cells will also typically exhibit reduced expression of CD 16. Most commonly, CIML NK cells will exhibit all three of the above parameters (i.e., cytotoxicity to otherwise resistant cells, increased expression of activated receptors, decreased expression of inhibitory receptors).

In one exemplary method contemplated herein, NK cells are expanded in a first step from a mononuclear cell-containing biofluid fraction, preferably up to about 0.5-5.0X 10⁹Total number of cells per cell. Notably, such amplification can be achieved in a single reactor in relatively small volumes and at moderate cell densities (e.g., 100-⁶Cell density per cell/ml) without feeder cells or other manipulations of the culture vessel. Once the desired number of NK cells is reached, the so expanded NK cells are contacted with a stimulating cytokine composition in a second step to activate the NK cells to a memory-like phenotype. Preferably, but not necessarily, the stimulating cytokine composition will include an IL-18/IL-12-TxM fusion protein complex, as exemplarily depicted in FIG. 1. However, the cytokine-stimulating composition may further compriseIncluding IL-12, N-803 and IL-18 mixture or IL-12, IL-15 and IL-18 mixture. Cytokine stimulation will typically be performed for a period of 4-24 hours, and the CIML NK cells so produced may be allowed to settle or restimulated (preferably in the presence of N-803) prior to infusion.

For example, whole blood or umbilical cord blood may be used as a raw material, which is processed to obtain monocytes. Most typically, conventional density gradient centrifugation (e.g., using Ficoll-Paque Plus) can be used^TM(hydrophilic soluble polysaccharide, density 1.077g/mL), available from general Life sciences, GE Life sciences). Once the monocytes were isolated from the centrifuge tube, the cells were washed and resuspended in activation medium (e.g., NK MACS supplemented with 10% human AB serum). The activation medium may also contain N-803 at a concentration of about 0.4nM and anti-CD 16 antibody at a concentration of about 1.0 mcg/ml.

Most typically, the monocytes have a volume of 1-2X 10 in a total volume of about 200ml⁶Density of individual cells/ml, and cells and medium in a single container. After about 3-4 days, the cells were fed with fresh medium containing N-803, and a further feeding cycle was performed about every three days by recovery, rapid expansion and culture of the apices. Notably, successful NK cell expansion in this protocol was significantly dependent on the proper selection of stimulating factors, as exemplarily shown in fig. 2. Here, over 20,000-fold dramatic amplification was observed when using anti-CD 3 and anti-CD 16 monoclonal antibodies, whereas the anti-CD 16 antibody alone failed to produce the same dramatic effect. Notably, the presence of anti-4-1 BB antibodies appears to prematurely deplete NK cell proliferation.

When the desired amount is reached, it is usually about 0.5 to 5.0X 10⁹Total cells and/or when the desired expansion (e.g., at least 100-fold expansion) is achieved, then the cell culture is terminated. Notably, despite being seemingly simple, the cell culture so obtained contained more than about 85% NK cells, less than about 8% NKT cells, less than about 2.5% T cells, and less than about 1.2% Double Negative (DN) T cells after about three weeks. Furthermore, it should be recognized that the entire culture process may be in a self-contained bioreactionIn a single container within the container, which greatly reduces the risk of contamination and eliminates the handling of reagents and cells during the incubation step.

When the desired number of cells is reached, the cells can be transferred to fresh medium for subsequent cytokine stimulation. Alternatively, cytokine stimulation to generate a memory-like phenotype can be performed in the same medium, typically by adding a medium containing a stimulating cytokine composition comprising the IL-18/IL-12-TxM fusion protein complex (or a mixture of IL-12, N-803, and IL-18, or a mixture of IL-12, IL-15, and IL-18). In most cases, cytokine stimulation will be performed for a period of about 4-24 hours, and more typically 12-16 hours. As will be readily appreciated, the cells may then be transferred into an infusion medium prior to infusion. In addition, the phenotype and/or cytotoxicity of CIML NK cells can be determined, exemplary results are shown in more detail below.

With respect to suitable biological fluids, it is generally contemplated that the fluid may be autologous with respect to the individual that will receive the NK cells isolated in the methods presented herein. Thus, particularly preferred biological fluids include fresh whole blood, cord blood (frozen or fresh), and cells isolated in a leukapheresis procedure. However, it should be understood that the biological fluid may also be any fluid containing NK cells (typically in other cell types). For example, suitable alternative biological fluids include whole blood from allogeneic donors, which may or may not match compatible MHC types. Therefore, samples in blood banks near the expiration date are considered suitable for use, as well as whole freshly donated blood or stored cord blood from individuals other than the NK cell recipient. Also, it should be noted that where the biological fluid is cord blood, the cord blood may be matched and donated after sufficient MHC matches with the NK cell recipient. Also, it should be noted that the manner in which monocytes are isolated or enriched may vary widely, and one of ordinary skill in the art will readily appreciate the most suitable method of isolation and enrichment. For example, where the biological fluid is whole blood or umbilical cord blood, the fluid is preferably processed by gradient density centrifugation using any suitable medium (e.g., Ficoll-Hypaque). Alternatively, monocytes may be obtained directly from the patient by leukapheresis, or the antibodies may be used to remove red blood cells from the biological fluid. In still further methods, magnetic bead separation may be used to isolate monocytes, wherein the magnetic beads are coated or otherwise coupled to antibodies that bind to monocytes.

Likewise, it should be recognized that the specific nature of the medium used for activation and supply need not be limited to NK MACS medium, but that all known media that support NK cell growth are considered suitable for use herein. However, it is most preferred to use defined media and can be supplemented with human AB serum.

Preferably the combination of anti-CD 16 antibody and N-803, and optionally anti-CD 3 antibody, is used to stimulate and support the proliferation of NK cells in a cocktail of monocytes. There are various sources of anti-CD 16 antibodies known/commercially available in the art, and a particularly preferred anti-CD 16 antibody has agonist (activation) activity and is specific for human CD 16. However, activators other than anti-CD 16 antibodies are also considered suitable for use herein, including anti-CD 16 antibody fragments and fusion proteins with anti-CD 16 antibody fragments. Additionally or alternatively, contemplated activators also include CD314 or NKG2D, natural cytotoxic receptors CD335(NKp46), CD336(NKp44) and CD337(NKp30), CD226(DNAM-1), CD244(2B4), CD158 or members of the killer immunoglobulin-like receptor (KIR) family carrying a short cytoplasmic tail (KIR2DS and KIR3DS), and CD94/NKG2C, and the like.

The concentration of anti-CD 16 antibody will generally follow the concentrations known in the art for activating NK cells. Thus, a suitable concentration of anti-CD 16 antibody will be about 0.01-5.0mcg/ml, more usually about 0.01-0.3mcg/ml, or about 0.05-0.5mcg/ml, or about 0.1-1.0mcg/ml, or about 1.0-5.0 mcg/ml. With respect to the duration of exposure to the anti-CD 16 antibody, it is generally contemplated that the mixture of monocytes will be exposed to only a single, two or more doses of anti-CD 16 antibody, most typically when the monocytes are isolated and contacted with the activation medium a first (and/or second and/or third) number of times. One of ordinary skill in the art will readily recognize the appropriate schedule and dosage to achieve NK cell activation. Most commonly, exposure of monocytes to anti-CD 16 antibody is performed simultaneously with exposure of monocytes to N-803. However, in a less preferred embodiment, exposure of the monocytes to the anti-CD 16 antibody is sequential to exposure of the monocytes to N-803 (it is the preferred sequence that the monocytes are first exposed to the anti-CD 16 antibody).

Where desired, proliferation stimulation/support may also include contacting the cell with an anti-CD 3 antibody, typically at the same time as contacting the cell with an anti-CD 16 antibody. As mentioned above, the concentration of anti-CD 3 antibody will generally follow the concentrations known in the art for activating NK cells. Thus, a suitable concentration of anti-CD 3 antibody will be about 0.01-10.0ng/ml, more usually about 0.01-0.1ng/ml, or about 0.1-0.5ng/ml, or about 0.3-1.0ng/ml, or about 1.0-5.0 ng/ml. Also, with respect to the duration of exposure to the anti-CD 3 antibody, it is generally contemplated that the mixture of monocytes will be exposed to only a single, two or more doses of anti-CD 3 antibody, most typically when the monocytes are isolated and contacted with the activation medium the first (and/or second and/or third) times. One of ordinary skill in the art will readily recognize the appropriate schedule and dosage to achieve NK cell activation.

As for N-803, it is considered that N-803 (IL-15N 72D: IL-15 R.alpha.Su/IgG 1 Fc complex with human sequence; see US 2019/0023766, commercially available from ImmunityBio) is preferable as a reagent in activation and supply to the medium. However, various alternative agents having IL-15 activity are also contemplated as suitable for use herein. In this context, and without wishing to be bound by any theory or hypothesis, the inventors contemplate that N-803 enables NK cell growth and expansion through continuous signaling. In contrast, IL-15 has a very short life span as an isolated cytokine and signaling activity is usually very short. In the case of IL-15 added to the growth medium as an isolated cytokine, signaling will be pulsed or intermittent. In contrast, in the case of N-803, the stability of IL-15 is significantly extended and signaling is considered to be continuous. In addition, it will be appreciated that N-803 also provides the physiological context (i.e., the IL-15R-alpha chain) and the form of N72D as a super agonist. Thus, any stable IL-15 compound is also specifically considered suitable for use herein.

For example, all compounds and complexes that affect IL-15 signaling are considered suitable for use herein, provided that such compounds and complexes have a longer serum half-life than isolated/recombinant and purified IL-15 alone. Furthermore, it is generally preferred that the stabilized IL-15 compound will include at least a portion of the human sequence of IL-15 and/or IL-15R α. For example, suitable compounds include P22339 (a complex of IL-15 and the Sushi domain of the IL-15 Ra chain with a disulfide bond linking the IL-15/Sushi domain complex to IgG1 Fc to increase its half-life; see Nature, Scientific Reports [ Nature, science Reports ] (2018)8:7675), and XBAM 24306 (which is an IL-15/IL-15 Ra-Fc heterodimer (see, e.g., WO 2018/071919)).

In a further particularly contemplated embodiment, the mixture of monocytes is placed in a cell culture vessel after separation from the biological fluid, together with a medium containing anti-CD 16 (and optionally anti-CD 3) antibodies and N-803, to activate NK cells. Most preferably, the container is a cell culture flask having at least one wall (or portion thereof) that is transparent to light so that the cells can be observed for shape, staining and/or growth using a microscope or other optical instrument. It should therefore be noted that the cells may be monitored continuously or periodically in the bioreactor and the measurements thus obtained (e.g. cell size, cell number, cell distribution, etc.) may be used to trigger or modify an automatic feeding schedule in a control unit logically coupled to the bioreactor. Most commonly, as shown in fig. 2, the supply of N-803 to fresh medium may be performed using a predefined schedule, typically once every three days, wherein preferably each supply will include N-803 to maintain continuous signaling. Although the specific volume in the examples below is suitable for expanding NK cells to a cell density consistent with cell growth, it should be understood that the volume may be adjusted to accommodate a particular growth pattern. To this end, it should also be understood that the supply may be continuous, or the predetermined volume may be altered in response to growth kinetics observed in the vessel.

In most cases, the yield of NK cells at the end of incubation is typically at least 80%, or at least 82%, or at least 85%, or at least 88%, or at least 90%, or at least 92%, or at least 94% of all living cells, the remainder being NKT cells, DN T cells and T cells. For example, the remaining NKT cells will typically be equal to or less than 10%, or equal to or less than 8%, or equal to or less than 7%, or equal to or less than 6% of all viable cells, while the remaining T cells will typically be equal to or less than 5%, or equal to or less than 4%, or equal to or less than 3%, or equal to or less than 2% of all viable cells, and the remaining DN T cells will typically be equal to or less than 3%, or equal to or less than 2%, or equal to or less than 1.5%, or equal to or less than 1% of all viable cells.

Thus, and from a different perspective, it is understood that the systems and methods contemplated herein are capable of significantly high expansion of NK cells, and typically expansion is at least 80-fold, or at least 100-fold, or at least 120-fold, or at least 130-fold, or at least 140-fold relative to the number of NK cells originally present in the mixture of monocytes. This amplification is particularly notable in view of the very simple manner of activation and incubation (one-pot method). Indeed, once the mixture of monocytes is placed in the cell culture vessel, the whole process can be continued in the same vessel and can be maintained only by the addition of culture medium. Thus, complicated handling and expensive reagents are completely avoided and the risk of contamination is greatly reduced.

As already mentioned above, NK cells can be expanded to about 0.1-1.0X 10⁹Individual cell, or about 0.3-3.0X 10⁹Individual cell, or about 0.5-5.0X 10⁹About 0.7 to 7.0X 10 per cell⁹Individual cell, or about 1-10X 10⁹Individual cells, or even higher total cell numbers. The exact number of NK cells expanded will generally depend on, among other things, the other objectives of the NK cells, the culture conditions and the starting cell number. When the desired number of cells is reached, cytokine stimulation may then be performed in the expansion medium, usually by adding fresh medium containing a stimulating cytokine composition.

In most cases, the stimulating cytokine composition will comprise one or more activating cytokines, such as IL-2, IL-12, IL-15, IL-21, and to a lesser extent IL-4 and IL-7. Of course, and as discussed in more detail below, suitable cytokines may also be derivatives of the above cytokines, with particularly preferred derivatives including fusion complexes. Still further, it will be appreciated that cytokines may also be expressed in the amplified NK cells following transfection with the appropriate recombinant nucleic acid (e.g., from transient expression of a plasmid or viral expression vector).

For example, in some embodiments, the stimulating cytokine composition will comprise an IL-18/IL-12-TxM fusion protein complex, with particularly preferred fusion protein complexes described in WO2018/165208, incorporated herein by reference. In this case, it will be appreciated that the fusion protein complex provides three cytokine functions (IL-12, IL-15 and IL-18) in a stable form via its coupling to the Fc portion of human IgG. Furthermore, while not wishing to be bound by any theory or hypothesis, the Fc portion of the fusion protein complex may provide a further stimulatory signal, perhaps through interaction with CD16 on the expanded NK cells. However, other fusion protein complexes based on N-808 are also specifically contemplated herein. For example, a suitable fusion protein complex may include a targeting scFv moiety, or a cytokine moiety different from (or complementary to) IL-12 and IL-18. It should of course be noted that although the IL-18/IL-12-TxM fusion protein complex is preferred in many cases, it is also contemplated that an alternative TxM fusion protein complex will be suitable and that a specifically contemplated fusion complex will include an IL15/IL-15 α moiety as described in WO2018/165208, and at least one additional cytokine selected from the group consisting of IL-7, IL-18 and IL-21. Thus, in other suitable alternatives, contemplated TxM fusion complexes include IL-18/IL-7TxM and/or IL-18/IL-21 TxM.

Thus, in other examples, the stimulating cytokine composition may further comprise a derivative of IL-15, and particularly preferred derivatives are N-803-based derivatives. Such derivatives have an increased signalling effect compared to IL-15 itself due to the presence of the IL-15R α chain, and exemplary suitable derivatives are described in WO 2016/004060 and WO 2018/075989. Most often, where N-803 or similar fusion proteins are used, additional cytokine function will be provided by the individual cytokines, and in particular IL-7, IL-12, IL-21 and IL-18. Thus, in yet another aspect of the inventive subject matter, the stimulating cytokine composition may further comprise IL-7, IL12, IL-15, IL-21, and IL-18 as individual cytokines. Thus, in other options, such individual cytokines may be added alone or in combination with other individual cytokines or the TxM construct, each of which may be recombinant (or even recombinantly expressed in a cell).

Thus, it will be appreciated that stimulation of one or more of the cytokines may also (temporarily) be expressed from the recombinant nucleic acid transfected into the amplified NK cells. For example, suitable transfection methods include viral transfection, wherein the recombinant nucleic acid is a viral expression vector. Alternatively, the recombinant nucleic acid can also be transfected into cells using electroporation or lipofection using methods well known in the art. Furthermore, where electroporation or lipofection is employed, it is generally preferred that the nucleic acid is RNA (however, DNA is also considered suitable for use herein).

Regardless of the particular type of stimulating cytokine composition, it is generally contemplated that the cytokine or cytokines are present in the culture medium at a concentration effective to produce an NK cell memory-like phenotype. Thus, suitable total cytokine concentrations will be between 0.1nM and 1.0nM, or between 0.5nM and 5.0nM, or between 1.0nM and 10nM, or between 10nM and 50nM, and in some cases even higher. Where multiple cytokines are used, it is generally preferred that the cytokines are present at substantially equimolar concentrations (+/-50% deviation). On the other hand, where the stimulating cytokine composition comprises an IL-18/IL-12-TxM fusion protein complex, the complex may be present at 0.5nM to 5.0nM, or 1.0nM to 10nM, or 10nM to 50nM or even higher.

With respect to the timing of the stimulation of the cytokine composition, it is generally preferred to first expand the NK cells to the desired (usually final) number prior to exposure to the stimulation cytokine composition. However, in alternative aspects, the stimulating cytokine composition may be added to the expanding population of NK cells, starting at about 70% of the final desired cell number, or starting at about 80% of the final desired cell number, or starting at about 90% of the final desired cell number. In most aspects of the inventive subject matter, exposure to the stimulating composition will last from about 2 hours to 48 hours, or from 4 hours to 8 hours, or from 8 hours to 12 hours, or from 12 hours to 24 hours, and in some cases even longer.

Exposure to the stimulating cytokine composition may be terminated by replacement of the medium, typically with fresh medium or a medium suitable for infusion. On the other hand, it is also contemplated that CIML NK cells so produced may be subjected to a resting time prior to subsequent use, which may last from 0-4 hours, 4-12 hours, 12-24 hours or 1-4 days, or even longer. As can also be readily appreciated, CIML NK cells can also be subjected to restimulation to further increase cytotoxicity, and are typically restimulated with at least one stimulating cytokine such as IL2 or IL-15. Most preferably, as shown in more detail below, restimulation with N-803 provides unexpectedly high cytotoxicity (compared to IL-15 itself). Furthermore, it should be noted that re-stimulation will generally follow standard protocols well known in the art.

Regardless of the final processing of CIML NK cells, it is contemplated that CIML NK cells will be used for infusion into an individual in need thereof, and most commonly, that individual will be diagnosed with cancer. As will also be readily appreciated, CIML NK cells may form a treatment regimen in which an individual receives a cancer vaccine (e.g., a recombinant (adeno) virus vaccine, a recombinant yeast vaccine, a recombinant bacterial vaccine), a chemotherapeutic agent, a checkpoint inhibitor, N-803, or TxM-based therapeutic agent, and/or targets an interleukin (e.g., NHS-IL 12).

While not limiting to the subject matter of the invention, it is further contemplated that CIML NK cells are expanded and/or activated in a culture environment that allows for continuous monitoring, continuous management of CO₂And O₂Level, and continuous monitoring to detect cell density (e.g., confluency). Among other options for such environments, a particularly preferred environment is automaticA chemocell culture and harvesting device as described in e.g. WO 2015/165700. Such "GMB in cartridge" systems advantageously allow control of the supply schedule, gas control, allow real-time detection of cell density, growth (kinetics) and cell health, and significantly reduce the potential for contamination due to significantly reduced processing requirements.

In further contemplated aspects, it should be noted that the systems and methods described herein advantageously also allow for the generation of CDs 56^DarknessAnd CD56^{Bright Light (LIGHT)}NK cells, particularly in the case where NK cells are produced from peripheral blood. According to further culture conditions, CD56^{Bright Light (LIGHT)}NK cells can subsequently differentiate into CD56^DarknessA cell. Such different NK cell populations may be used for different treatment options due to their different maturation and cytotoxicity profiles. Furthermore, it is to be understood that these compositions, systems and methods will also be suitable for the production of NKT cells under appropriate stimulation and culture.

Examples

In view of the above, and as provided in more detail below, one exemplary method entails isolating CBMC or PBMCs by a single Ficoll centrifugation step, followed by incubation of the cells with about 0.4nM N-803 and about 0.1mcg/ml anti-CD 16 antibodies (e.g., clone B73.1, available from BD Biosciences) and optionally about 0.5ng/ml anti-CD 3 antibodies in NK MACS medium with 10% human AB serum. Typically, 100-150mL (typically 135mL) of CBMC in million cells/mL is used as the starting material with the reagents described above. Dilution with medium was performed twice weekly (3-5 days apart) in the case of N-803, at 1:2 and 1:10 compared to the existing volume, corresponding to a concentration of 0.4nM N-803 for the final concentration. The expansion culture is typically terminated when the expanded NK cells constitute 90% to 99% (e.g., 98%) of all the cells. After termination, cytokine induction can be performed as described in more detail below.

MNC is freshly isolated from cord blood or peripheral blood. Washed twice with complete NKMACS medium (NKMACS + supplement + 10% hu-AB-serum). MNC is added at 1 x 10⁶Density of individual cells/mL suspended in GMP cassette (500mL volume)150mL of medium. 150mL of cell suspension was supplemented with anti-CD 16 antibody (1mcg/mL) and N-803(0.4 nM). The GMP cassette starts imaging and cells are propagated according to pre-programmed steps. The cells in the GMP cassette are supplemented with either 10X cytokine medium or 2X cytokine medium in an alternating fashion. NK enrichment (phenotype of CD3, CD56, and CD16 expression) and cell health (cell number, viability, and cell density) were monitored periodically and plotted.

Upon reaching the point where 98% of all cells are NK cells, cytokine induction was initiated to generate CIML NK cells from the expanded NK cells. For this purpose, will have 500mL and 2.3X 10⁶The boxes at individual cell/mL density were equally divided into two separate boxes. Thus, 500mL of the cell suspension was changed to 250mL in the two corresponding cassettes and the cells were diluted 1:1 with fresh medium. Subsequently, IL18/12TxM was added to a final concentration of 10nM (N-803 was used at a final concentration of 0.07nM for control and comparison) and the cells were incubated with IL-18/IL-12-TxM fusion protein complex for 16 hours, thus obtaining CIML NK cells. For further testing, cells were washed and then subjected to expression analysis and cytotoxicity assays.

Materials: MNC from umbilical cord blood and peripheral blood, anti-CD 16 antibody, BD biosciences, San Diego CA, california; NK MACS medium with NK supplements, staining antibodies for phenotypic analysis (aCD3, aCD16, aCD56, ankrp 30, ankrp 44, ankrp 46, ankrg 2A, ankrg 2D, agigi it, aCD34, acltrail, aCD57, aCXCR3, and acacr 5), santita biotech inc (Miltenyi biotech San Diego, CA); human AB serum, San Diego CA, Access Biologicals, San Diego, San Biologicals, california; n-803, GMP in box kit, south America biosciences Inc. of Calverd, Calif. (Nantbio Inc. silver City CA). IL-18/IL-12-TxM fusion protein complexes are obtained from immunobiology (ImmunityBio).

The CIML NK cells so generated were tested for cytotoxicity and selected surface marker expression. More specifically, in one set of experiments, cord blood-derived CIML NK cells were tested against merkel cell cancer cells (here MS-1 cells) that are generally resistant to NK cytotoxicity. Notably, as can be seen in fig. 3, CIML NK cells were significantly cytotoxic after expansion and control exposure to the IL-18/IL-12-TxM fusion protein complex, while some cytotoxicity was observed even when cord blood was exposed to N-803 alone. FIG. 4 depicts exemplary results of expression of surface markers in cord blood-derived cells exposed to the IL-18/IL-12-TxM fusion protein complex and N-803. As can be seen, CIML NK cells had reduced CD16 expression, but greatly increased CD25, DNAM1 expression and strong IFN- γ secretion.

As can be seen in fig. 5, similar results were obtained when CIML NK cells were derived from peripheral blood. Here, CIML NK cells are largely cytotoxic to the MS-1 cell line, and N-803 control cells from peripheral blood also show some cytotoxicity. Also, the surface markers of CIML NK cells from peripheral blood sources showed reduced expression of CD16 and TIGIT, while CD25, DNAM1 and IFN- γ secretion were significantly increased, as can be seen from fig. 6. Notably, no significant cytotoxicity was observed against MS-1 when NK cells were incubated using standard incubation protocols or in the case of fresh NK cells, even in the case of cells induced with IL-12, IL-15 and IL-18 to trigger a memory-like phenotype.

Cord blood-derived CIML NK cell activation cluster phenotype was also tested and FIG. 7 depicts exemplary results comparing control exposure to N-803 and IL-18/IL-12-TxM fusion protein complex exposure. As can be seen from the images, there was a dramatic difference in culture morphology after overnight exposure to the IL-18/IL-12-TxM fusion protein complex versus N-803. When examining selected surface markers for these CIML NK cells, it is also evident that exposure to the IL-18/IL-12-TxM fusion protein complex causes a significant increase in CD25 (a known activation-related receptor), as shown in figure 8. Apparently, cytokine stimulation with IL-12, IL-15, IL-18 function increased CD25 presentation substantially, which is not usually observed with conventional fresh NK cells (at least to this extent).

As shown in fig. 9, this increase in activated receptors and this decrease in inhibitory receptors is also evident when culture morphology is observed in the killing assay for K562 cells. Here, cord blood-derived CIML cells showed a greatly increased clustering of activation after restimulation compared to incubation with N-803.

In further experiments, the inventors also investigated the time course of cytotoxicity against K562 cells, exemplarily depicted in fig. 10A, 10B and 10C, which show the results after 24 hours, 48 hours and 72 hours, respectively. After the first 24 hour time point (fig. 10A), EC can be seen when two TxM concentrations were tested₅₀Below the N-803 control, the killing capacity on K562 cells began to increase. At 48 hours (FIG. 10B), increased killing was seen for the TxM treated sample, but less for the N-803 control. The increase in K562 killing at this time was about 3 fold. At 72 hours (fig. 10C) all conditions had begun to lose activity on K562 killing, but TxM-treated cells maintained their killing about 3-fold enhanced compared to controls.

FIG. 11 provides a direct comparison of expanded cord blood-derived NK cells, expanded cord blood-derived NK cells stimulated with N-803, and expanded cord blood-derived NK cells stimulated with IL-18/IL-12-TxM fusion protein complex for 24 hours. As can be seen, cytotoxicity was evident for all cells, expanded NK cells had a slight advantage over% maximal killing, but required a significantly higher E compared to CIML cells; and (3) a T ratio. FIG. 12 depicts expression of selected markers for expanded peripheral blood-derived NK cells stimulated with N-803 versus expanded peripheral blood-derived NK cells stimulated with IL-18/IL-12-TxM fusion protein complex. As can be seen from fig. 12, TIGIT (and CD16) was significantly down-regulated while CD25 was significantly up-regulated, indicating activation. It should be noted that down-regulation of CD16 may be accompanied by a reduction in ADCC. However, the potential reduction in ADCC is outweighed by the higher activation and cytotoxicity against cell lines that would otherwise be resistant to NK cell cytotoxicity. As shown in FIG. 13, similar cytotoxic results were found with CIML NK cells from peripheral blood after 24 hours of stimulation with IL-18/IL-12-TxM fusion protein complex. Clearly, exposure to 10nM of the IL-18/IL-12-TxM fusion protein complex produced better cell killing in the K562 assay.

IFN- γ secretion was tested against CIML NK cells of peripheral blood origin, and figure 14 shows exemplary results using different conditions. The same cells were also used in the cytotoxicity assay and figure 15 shows exemplary results. As can be seen from FIG. 16, similar results were provided for cord blood-derived CIML NK cells. Notably, cytokine induction with N-803 outperformed induction with IL-15 itself. Thus, it should be noted that although multi-cytokine induction is preferred as shown above, induction with N-803 is also expressly contemplated.

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 by a healthcare professional (e.g., physician, nurse, etc.), and wherein indirect administration includes the step of providing the pharmaceutical composition or drug to the healthcare professional or making the pharmaceutical composition or drug available to the healthcare professional for direct administration (e.g., via injection, infusion, oral delivery, topical delivery, etc.). Most preferably, the cells or exosomes are administered via subcutaneous or intradermal injection. However, in other contemplated aspects, administration may also be intravenous injection. Alternatively or additionally, antigen presenting cells may be isolated from or grown in cells of a patient, infected in vitro, and then delivered to the patient. Thus, it should be understood that contemplated systems and methods may be considered complete drug discovery systems (e.g., drug discovery, treatment protocols, validation, etc.) for highly personalized cancer treatment.

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

It will be apparent to those skilled in the art that many more modifications besides those already described are possible without departing from the full scope of the concepts herein disclosed. Accordingly, the disclosed subject matter is to be limited only by the scope of the following claims. Moreover, in interpreting both the specification and the claims, all terms should be interpreted in the broadest possible manner consistent with the context. In particular, the terms "comprises/comprising" 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, or utilized, or combined with other elements, components, or steps that are not expressly referenced. Where the claims recite at least one of something selected from the group consisting of A, B, C … … and N, the word should be construed to require only one element of the group, rather than A plus N, or B plus N, etc.

The claims (modification according to treaty clause 19)

1. A method of producing cytokine-induced memory-like (CIML) NK cells with enhanced cytotoxicity, the method comprising:

separating a mixture of monocytes from the biological fluid and contacting the mixture of monocytes with an anti-CD 16 antibody and N-803 to expand NK cells, wherein the NK cells comprise at least 80% of all living cells at the end of the expansion; and

contacting the expanded NK cells with a stimulating cytokine composition comprising an IL-18/IL-12-TxM fusion protein complex, a mixture of IL-12, N-803, and IL-18, or a mixture of IL-12, IL-15, and IL-18, thereby generating the CIML NK cells with enhanced cytotoxicity.

2. The method of claim 1, further comprising: after restimulating the CIML NK cells, the step of contacting the CIML NK cells with N-803.

3. The method of any one of the preceding claims, wherein the biological fluid is whole blood or umbilical cord blood.

4. The method of any one of the preceding claims, wherein the mixture of monocytes is not further processed to enrich for NK cells.

5. The method of any one of the preceding claims, wherein the mixture of monocytes comprises about 100-⁶And (4) cells.

6. The method of any one of the preceding claims, wherein the step of contacting the mixture is in a volume of about 100 and 300ml or at about 1 x 10⁶Cell density of individual cells/ml.

7. The method of any one of the preceding claims, wherein the anti-CD 16 antibody is present at a concentration of 0.05-0.5mcg/ml in the step of contacting the mixture.

8. The method of any one of the preceding claims, wherein the N-803 is present at a concentration of 0.1-1.0nM in the step of contacting the mixture.

9. The method of any one of the preceding claims, wherein the step of contacting the mixture further comprises contacting the mixture of monocytes with an anti-CD 3 antibody.

10. The method of claim 9, wherein the anti-CD 3 antibody is present at a concentration of 0.1-1.0 ng/ml.

11. The method of any one of claims 1-10, wherein the stimulating cytokine composition comprises the IL-18/IL-12-TxM fusion protein complex.

12. The method of any one of claims 1-10, wherein the stimulating cytokine composition comprises a mixture of the IL-12, N-803, and IL-18.

13. The method of any one of claims 1-10, wherein the stimulating cytokine composition comprises a mixture of the IL-12, IL-15, and IL-18.

14. The method of any one of the preceding claims, wherein the NK cells are expanded to about 0.5-5.0×10⁹Total number of cells per cell.

15. The method of any one of the preceding claims, wherein the step of contacting the expanded NK cells with the stimulating cytokine composition is performed in the same vessel as the step of expanding the NK cells.

16. A method of activating NK cells to form cytokine-induced memory-like (CIML) NK cells with enhanced cytotoxicity, the method comprising:

providing expanded NK cells, wherein the NK cells are expanded from monocytes of whole blood or umbilical cord blood, wherein the expanded NK cells comprise at least 80% of all living cells; and

17. The method of claim 16, wherein the NK cells are expanded from whole blood.

18. The method of claim 16, wherein the NK cells are expanded from umbilical cord blood.

19. The method of any one of claims 16-18, wherein the NK cells are autologous relative to the individual receiving the infusion comprising the CIML NK cells.

20. The method of any one of claims 16-19, wherein the stimulating cytokine composition comprises the IL-18/IL-12-TxM fusion protein complex.

21. The method of any one of claims 16-19, wherein the stimulating cytokine composition comprises a mixture of the IL-12, N-803, and IL-18.

22. The method of any one of claims 16-19, wherein the stimulating cytokine composition comprises a mixture of the IL-12, IL-15, and IL-18.

23. The method of any one of claims 16-22, wherein the total cell number of the expanded NK cells is about 0.5-5.0 x 10⁹And (4) cells.

24. The method of any one of claims 16-23, wherein the CIML NK cells with enhanced cytotoxicity are cytotoxic to MS-1 cells.

25. The method of any one of claims 16-24, wherein the CIML NK cells with enhanced cytotoxicity have reduced expression of CD16 as compared to expanded NK cells contacted with N-803 alone.

26. The method of any one of claims 16-25, wherein the CIML NK cells with enhanced cytotoxicity have reduced TIGIT expression compared to expanded NK cells contacted with N-803 alone.

27. The method of any one of claims 16-26, wherein the CIML NK cells with enhanced cytotoxicity have increased expression of CD25 and/or DNAM1 as compared to expanded NK cells contacted with N-803 alone.

28. A cytokine-induced memory-like (CIML) NK cell with enhanced cytotoxicity, the cell exhibiting cytotoxicity to MS-1 cells of at least 50% killing at an effector-to-target cell ratio equal to or less than 5.

29. The CIML NK cell of claim 28 wherein the CIML NK cell has reduced expression of CD16 as compared to an expanded NK cell contacted with N-803 alone.

30. The CIML NK cell of claim 28 wherein the CIML NK cell has reduced TIGIT expression compared to an expanded NK cell contacted with N-803 alone.

31. The CIML NK cell of claim 28, wherein the CIML NK cell has increased expression of CD25 and/or DNAM1 as compared to an expanded NK cell contacted with N-803 alone.

32. The CIML NK cell of claim 28, wherein the CIML NK cell is autologous to the individual receiving the infusion comprising the CIML NK cell.

33. The CIML NK cell of claim 28, wherein the CIML NK cell is a recombinant NK cell.

34. The CIML NK cell of claim 33, wherein the CIML NK cell expresses CD16 or a variant thereof, IL-2 or a variant thereof, and/or IL-15 or a variant thereof from a recombinant nucleic acid.

35. A pharmaceutical composition comprising a pharmaceutically acceptable carrier in combination with the cytokine-induced memory-like (CIML) NK cell of any one of claims 28-34.

36. Use of a cytokine-induced memory-like (CIML) NK cell of any one of claims 28-34 in medicine.

37. Use of a cytokine-induced memory-like (CIML) NK cell of any one of claims 28-34 in the treatment of cancer.

38. A method of treating an individual in need with cytokine-induced memory-like (CIML) NK cells, comprising administering a therapeutically effective amount of the CIML NK cells of any one of claims 28-34.

39. The method of claim 38, wherein the CIML NK cells are autologous cells of the individual.

40. The method of claim 38, wherein the CIML NK cells are peripheral blood or cord blood-derived NK cells.

Statement or declaration (modification according to treaty clause 19)

Statement according to article 19(1)

Debate of debate

The patent office considers independent claims 1 and 16 to be obvious with respect to D1 and D2 and notes that while D1 does not teach contacting a mixture of monocytes with anti-CD 16 antibodies, D2 will provide such teaching as D2 discloses the use of irradiated autologous peripheral blood mononuclear cells and anti-CD 16 cells to expand cytotoxic natural killer cells.

Applicants respectfully disagree and note that D2 specifically teaches that a variety of cytokines, such as Interleukins (IL) -2, IL-12, IL-15, IL-18, IL-21, or combinations thereof, have been used to expand NK cells 22-24, but these cytokines are not very potent … … ". in other words, D2 forgoes the use of cytokines and instead uses anti-CD 16. Thus, no successful amplification using such cytokines is expected, as the patent office refers to. Furthermore, there is no reason why one of ordinary skill in the art would be advised to bind the anti-CD 16 antibody and N-803.

Still further, it must be understood that D1 isolated NK cells and did not amplify NK cells from PBMC, whereas D2 amplified NK cells, but in a cytokine-independent manner. Clearly, the combination of D1 and D2 failed to produce the claimed subject matter.

The patent office considers that independent claims 28-37 are obvious with respect to D1, and that the patent office considers that the cell type of claim 28 is known from the characterization of D1 considering ovarian cancer cells (see abstract), and that the percentages of claim 28 are within the routine practice adopted by a person of ordinary skill in the art, especially since such advantages can be easily foreseen. Again, the applicant respectfully expressed disagreement.

It is first noted that the cells of D1 differ from the cells claimed in the present invention for at least the reasons set forth above. Furthermore, D1 is completely silent about any cytotoxicity issues at a given E: T ratio as explicitly claimed. A simple control, known from D1, is that the cells fall within the claimed percentage without evidence support, and do not meet the threshold criterion for legibility.

Conclusion

Claims 1-40 are pending in this application. The applicant requests to grant all pending claims.

Claims

separating a mixture of monocytes from the biological fluid and contacting the mixture of monocytes with an anti-CD 16 antibody and N-803 to expand NK cells; and

14. The method of any one of the preceding claims, wherein the NK cells are expanded to about 0.5-5.0 x 10⁹Total number of cells per cell.

providing expanded NK cells, wherein the NK cells are expanded from monocytes of whole blood or umbilical cord blood; and

17. The method of claim 16, wherein the NK cells are expanded from whole blood.