KR20210079381A - CIML NK cells and methods therefor (CIML NK CELLS AND METHODS THEREFOR) - Google Patents

Abstract

Cytokine-induced memory-like (CIML) NK cells with enhanced cytotoxicity are presented. Most typically, CIML NK cells are derived from the mononuclear cell fraction of peripheral blood or umbilical cord blood. In a further contemplated embodiment, CIML NK cells are expanded and induced in a contained and automated production environment that substantially reduces operational complexity and production cost.

Description

CIML NK cells and methods therefor (CIML NK CELLS AND METHODS THEREFOR)

The present disclosure relates to compositions, methods, and devices for generating and/or culturing activated immune competent cells, in particular to memory like NK cells generated from umbilical cord blood (CB) or peripheral blood (PB). will be.

The background description contains information that may be useful in understanding the present disclosure. It is not an admission that any information provided herein is prior art or relevant to the presently claimed invention, or 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. To the extent a definition or use of a term in an incorporated reference is inconsistent or contradictory 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, which are often characterized as cytotoxic lymphocytes that exhibit antibody-dependent cytotoxicity through the targeted release of granulesin and perforin. Most NK cells have a specific cell surface marker profile (eg, CD3 ⁻ , CD56 ⁺ , CD16 ⁺ , CD57 ⁺ , CD8 ⁺ ) in addition to a diverse set of activating and inhibitory receptors. Although NK cells have recently become an important component of certain cancer treatments, the generation of significant amounts of NK cells (especially autologous NK cells) has been a significant obstacle because the fraction of NK cells in whole blood is relatively low.

To obtain therapeutically significant amounts of NK and NK-like cells, NK cells can be generated from a variety of progenitor cells. For example, various stem cell factors (SCFs), FLT3 ligands, interleukin (IL)-2, IL-7 and IL-15 are responsible for cord blood-derived cytokine-induced killer (CIK). It has been reported in various in vitro approaches to induce and expand cells ( 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 another approach, human hemangioblasts were sequentially exposed to two different cytokine cocktails as described in WO2011/068896, and various cytokine cocktails were used with post-embryonic hematopoietic stem cells as taught in WO2012/128622. . At least some of these methods provide for a significant n-fold expansion of NK cells, but methods and reagents for such expansion are both time- and resource-intensive. Moreover, it should be noted that many of the known methods also require NK cell culture in a feeder cell layer, which is often problematic from a technical and regulatory standpoint.

In a more simplified method, acute myeloid leukemia (AML) cells can be exposed to a TpoR agonist to induce AML cells to form NK cells. However, such an approach may not be feasible as a source of therapeutic cell preparations. Alternative methods also relied on culturing peripheral blood cells in the presence of various interleukins, stem cell factors, and FLT3 ligands as disclosed in WO 2011/103882. Alternatively, US 2013/0295671 teaches a method of stimulating pre-existing NK cells with anti-CD16 and anti-CD3 antibodies along with cytokines. Although procedurally simpler, such methods still require sophisticated manipulation of cells and require specific reagents, adding significant costs.

In a further known method, US 10,125,351 discloses umbilical cord blood or peripheral as a source of cells, in which nucleated cells are isolated via density gradient separation, and then these nucleated cells are cultured in a medium containing interferon, interleukin, CD3 antibody and human albumin. The use of blood is described. Most advantageously, such a method permits perfusion culture in a bioreactor, thereby significantly reducing operational difficulties. Unfortunately, however, the yield of NK cells is relatively low.

Regardless of the particular mode of production, cultured NK cells will typically not exhibit memory-like properties particularly desirable for cancer immunotherapy. In at least some attempts to produce memory-like NK cells, cultured NK cells were exposed to IL-12, IL-15, and IL-18, and the NK cells so exposed displayed a memory-like phenotype and exhibited a memory-like phenotype and CD94, NKG2A, NKG2C. , and CD69 expression, and lack of CD57 and KIR (see Blood (2012) Vol.120, No.24; 4751-4760). Similarly, as described in WO 2018/089476, memory-like NK cells were obtained by pre-activating NK cells using various stimulatory cytokines and then contacting the pre-activated cells with PM21 particles, EX21 exosomes, or FC21 support cells. prepared. In another approach to generating memory-like NK cells, freshly isolated NK cells were exposed to IL-18/IL-12-TxM fusion protein complexes, as described in WO 2018/165208. While such methods typically produced at least some NK cells with memory-like properties, the cytotoxicity of such activated NK cells to selected target cells is presumably due to the lack or low expression of specific activating receptors and/or expression of specific inhibitory receptors. still lower than optimal.

Thus, although various methods for generating memory-like NK cells are known in the art, all or almost all of them suffer from various disadvantages. Consequently, there is a need to provide improved systems and methods for producing memory-like NK cells, particularly autologous memory-like NK cells, in significant amounts. Moreover, the improved systems and methods will also allow for automation of cell culture and NK cell activation, and will have substantially reduced reagent requirements to make such methods clinically and commercially viable.

The present 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 are expanded to a desired amount, and then the expanded NK cells are induced with a mixture of cytokines to form cytokine induced memory like (CIML) NK cells. can be produced in a two-step process. Expansion of NK cells is preferably performed in an enrichment process using N-803 and an anti-CD16 agonist antibody and optionally an anti-CD3 antibody. Activation to obtain memory-like properties is then performed with a combination of stimulatory cytokines, most preferably with IL-12/IL-15/IL-18 or IL-18/IL-12-TxM fusion protein complexes.

Unexpectedly, in addition to up-regulation of activation markers and IFN-γ secretion, so activated expanded memory-like NK cells had increased expression of CD25 and DNAM-1, an NK activation receptor, and down-regulated expression of TIGIT, an inhibitory receptor, , which probably contributed to or even caused the observed increase in toxicity of CIML NK cells. Most notably, the CIML NK cells presented herein also exhibited significant cytotoxicity against the otherwise NK resistant tumor cell line MS-1 at a relatively low effector to target ratio.

In one aspect of the subject matter of the present invention, we provide one step of isolating a mixture of mononuclear cells from a biological fluid, and another step of contacting the mixture of mononuclear cells with an anti-CD16 antibody and N-803 to expand NK cells. A method for producing CIML NK cells with enhanced cytotoxicity is contemplated, comprising the steps of: In a further step, the expanded NK cells are treated with a stimulatory cytokine composition (typically IL-18/IL-12-TxM fusion protein complex, a mixture of IL-12, N-803, and IL-18, or IL-12, IL -15, and a mixture of IL-18) to generate CIML NK cells with enhanced cytotoxicity. If desired, contemplated methods may further comprise contacting the CIML NK cells with N-803 after restimulating the CIML NK cells.

Preferably, but not necessarily, the biological fluid is whole blood or umbilical cord blood, and the mixture of mononuclear cells is not further treated to enrich for NK cells. Most typically, the mixture of mononuclear cells contains about 100 to 500 x 10 ⁶ cells and/or contacting the mixture comprises a volume of about 100 to 300 ml or a cell density of ^{about 1 x 10 6 cells/ml} is performed with In a further embodiment, in the step of contacting the mixture the anti-CD16 antibody may be present at a concentration of 0.05 to 0.5 mcg/ml and/or in the step of contacting the mixture N-803 may be present at a concentration of 0.1 to 1.0 nM have. Optionally, contacting the mixture may further comprise contacting the mixture of mononuclear cells with an anti-CD3 antibody (eg, at an anti-CD3 antibody concentration of 0.1-1.0 ng/ml).

In some embodiments the stimulatory cytokine composition comprises an IL-18/IL-12-TxM fusion protein complex, while in other embodiments the stimulatory cytokine composition comprises a mixture of IL-12, N-803, and IL-18, In a further aspect the stimulatory cytokine composition comprises a mixture of IL-12, IL-15, and IL-18. Most typically, the NK cells are ^{expanded to a total cell number of about 0.5 to 5.0 x 10 9} cells, and/or contacting the expanded NK cells with the stimulatory cytokine composition is performed in the same vessel as the step of expanding the NK cells. do.

Thus, from another perspective, we also contemplate a method of activating NK cells to form CIML NK cells with enhanced cytotoxicity. Such methods include providing expanded NK cells (typically expanded from mononuclear cells of whole blood or umbilical cord blood) and converting the expanded NK cells to IL-18/IL-12-TxM fusion protein complex, IL-12, N-803. and contacting the CIML NK cells with enhanced cytotoxicity comprising the additional step of contacting with a stimulatory cytokine composition, which may comprise a mixture of IL-18, or a mixture of IL-12, IL-15, and IL-18. will create

As mentioned previously, NK cells are considered to be expanded from whole blood or umbilical cord blood. Thus, NK cells may be autologous to a subject receiving a transfusion comprising CIML NK cells. Preferably, the stimulatory cytokine composition comprises an IL-18/IL-12-TxM fusion protein complex. However, in further embodiments, the stimulatory cytokine composition may also comprise a mixture of IL-12, N-803, and IL-18, or a mixture of IL-12, IL-15, and IL-18. Typically, expanded NK cells have a total cell number of about 0.5 to 5.0 x 10 ^{9 cells.}

CIML NK cells with enhanced cytotoxicity are cytotoxic to MS-1 cells and/or CIML NK cells with enhanced cytotoxicity have reduced expression of CD16 compared to expanded NK cells contacted only with N-803 CIML NK cells with and/or with enhanced cytotoxicity have reduced expression of TIGIT compared to expanded NK cells contacted only with N-803, and/or CIML NK cells with enhanced cytotoxicity are N- It is further contemplated to have increased expression of CD25 and/or DNAM1 compared to expanded NK cells contacted only with 803.

Accordingly, we also contemplate CIML NK cells with enhanced cytotoxicity, exhibiting cytotoxicity to MS-1 cells of at least 50% killing with an effector to target cell ratio of 5 or less. In a further aspect, the CIML NK cells have reduced expression of CD16 compared to expanded NK cells contacted only with N-803 and/or reduced expression of TIGIT compared to expanded NK cells contacted only with N-803 and/or has increased expression of CD25 and/or DNAM1 compared to expanded NK cells contacted only with N-803.

Without limiting the subject matter of the present invention, the CIML NK cells are preferably autologous to a subject receiving an infusion comprising the 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 variant thereof, IL-2 or variant thereof, and/or IL-15 or variant thereof from recombinant nucleic acid.

In a further contemplated aspect, the inventors also contemplate pharmaceutical compositions comprising a pharmaceutically acceptable carrier in association with CIML NK cells as set forth herein. Consequently, the use of CIML NK cells as presented herein in medicine, particularly in the treatment of cancer, is contemplated.

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

Various objects, features, aspects, and advantages will become more apparent from the following detailed description of preferred embodiments, taken in conjunction with the accompanying drawings in which like numbers indicate like elements.

1 depicts an exemplary schematic of an IL-18/IL-12-TxM fusion protein complex.
2 depicts exemplary results from peripheral blood NK cell expansion using selected combinations of autologous PBMCs and specific antibodies.
3 depicts exemplary results from a cytotoxicity assay of cord blood-derived CIML NK cells against MS-1 target cells.
4 depicts exemplary results for expression of selected phenotypic markers on CIML NK cells derived from cord blood.
5 depicts exemplary results from a cytotoxicity assay of peripheral blood derived CIML NK cells against MS-1 target cells.
6 depicts exemplary results for expression of selected phenotypic markers on peripheral blood derived CIML NK cells.
7 depicts an exemplary activated cluster phenotype of cord blood-derived CIML NK cells after IL-18/12 TxM exposure.
8 depicts exemplary CD25 expression on CIML NK cells derived from cord blood following IL-18/12 TxM exposure.
9 depicts an exemplary activation cluster phenotype of cord blood-derived CIML NK cells upon restimulation.
10A-10C depict exemplary results for apoptotic activity of cord blood-derived CIML NK cells after 24 hours (10a), 48 hours (10b), and 72 hours (10c) of IL-18/12 TxM exposure.
11 depicts exemplary results for the apoptotic activity of CIML NK cells derived from cord blood cultured in a single-box culture environment and clustering of activations from such cells.
12 depicts exemplary results for NK marker expression on peripheral blood derived CIML NK cells after IL-18/12 TxM exposure.
13 depicts exemplary results from a cytotoxicity assay of peripheral blood derived CIML NK cells following IL-18/12 TxM exposure to K562 target cells.
14 depicts exemplary results for IFN-γ staining of peripheral blood-derived CIML NK cells after restimulation.
15 depicts exemplary results from a cytotoxicity assay of peripheral blood derived CIML NK cells following exposure to N-803.
16 depicts exemplary results for IFN-γ staining of CIML NK cells derived from cord blood after restimulation and exemplary results from a cytotoxicity assay after exposure to N-803.

Immunotherapy in cancer treatment uses a growing variety of cell-based components, and NK cells have recently become a promising modality. Although some NK cells are currently available in relatively large quantities, the production of therapeutically significant amounts of autologous and/or memory-like NK cells remains problematic at best. Unfortunately, many current methods require the use of support layers or the differentiation of isolated CD34+ hematopoietic stem cells (HSCs), which are both time consuming and resource intensive. Moreover, because of the various manipulation steps required, such methods typically require human interaction and are susceptible to contamination. In addition, conversion of NK cells to a memory-like phenotype may reduce cytotoxicity or fail to deliver sufficient amounts of such cells in at least some protocols.

We now show that once mononuclear cells are obtained from biological fluids (eg, whole blood, umbilical cord blood), they can be easily converted into memory-like NK cells in a simple and effective manner that can even be fully automated. Various systems, compositions, and methods have been discovered for generating positive NK cells (eg, at least 0.5 x 10 ^{9 NK cells).} Advantageously, such NK cells may be autologous NK cells and may be induced with a memory-like phenotype to generate cytokine-induced memory-like (CIML) NK cells with enhanced cytotoxicity. Notably, as will be explained in more detail below, the CIML NK cells so generated will have superior cytotoxicity compared to other (CIML) NK cells, such as MS-1 cells (Merkel cell carcinoma cells). , will have significant killing ability against target cells that are otherwise resistant to NK cell cytotoxicity or even inactive.

)) NK 세포의 공급원, 이전의 확장 조건, 및 아마도 확장 및 사이토카인 유도의 중단되지 않은(예를 들어, 배지, 배양 조건 등의 변화) 성질에 기인할 수 있으며, 이는 활성화 인자의 과발현 및 억제 수용체의 저발현을 초래할 수 있음을 고려한다. 본원에 제시된 CIML NK 세포의 다른 주목할 만한 특징들 중에서도, CIML NK 세포는 전형적으로 5 이하의 이펙터 대 표적 세포 비로 적어도 50% 사멸의 MS-1 세포에 대한 세포독성을 나타낼 것이고, N-803과만 접촉한 확장된 NK 세포와 비교하여 TIGIT(억제 수용체)의 감소된 발현, 및 N-803과만 접촉한 확장된 NK 세포와 비교하여 CD25 및/또는 DNAM1(활성화 보조 수용체)의 증가된 발현을 가질 것이다. 따라서, 용어 "향상된 세포독성을 갖는 NK 세포"는 5 이하의 이펙터 대 표적 세포 비로 적어도 50% 사멸의 MS-1 세포에 대한 세포독성, N-803과만 접촉한 확장된 NK 세포와 비교하여 TIGIT(억제 수용체)의 감소된 발현, 및/또는 N-803과만 접촉한 확장된 NK 세포와 비교하여 CD25 및/또는 DNAM1(활성화 보조 수용체)의 증가된 발현을 나타내는 NK 세포를 지칭한다. 더욱이, 고려되는 CIML NK 세포는 전형적으로 CD16의 감소된 발현도 나타낼 것이다. 가장 전형적으로, CIML NK 세포는 상기 3 가지 파라미터(즉, 다른 경우라면 NK 내성인 세포에 대한 세포독성, 활성화 수용체의 증가된 발현, 억제 수용체의 감소된 발현) 모두를 나타낼 것이다.Without wishing to be bound by any theory or hypothesis, the inventors found that enhanced cytotoxicity (naive

))) the source of NK cells, previous expansion conditions, and possibly due to the uninterrupted (e.g., change in medium, culture conditions, etc.) nature of expansion and cytokine induction, which overexpression and inhibition of activating factors It is contemplated that it may lead to underexpression of the receptor. Among other notable characteristics of CIML NK cells presented herein, CIML NK cells will typically exhibit cytotoxicity to MS-1 cells of at least 50% death with an effector to target cell ratio of 5 or less, and contact only with N-803. decreased expression of TIGIT (inhibiting receptor) compared to one expanded NK cell, and increased expression of CD25 and/or DNAM1 (activating co-receptor) compared to expanded NK cells contacted only with N-803. Thus, the term “NK cells with enhanced cytotoxicity” refers to a cytotoxicity to MS-1 cells of at least 50% killing with an effector to target cell ratio of 5 or less, TIGIT ( inhibitory receptor) and/or increased expression of CD25 and/or DNAM1 (activating co-receptor) compared to expanded NK cells contacted only with N-803. Moreover, contemplated CIML NK cells will typically also exhibit reduced expression of CD16. Most typically, CIML NK cells will exhibit all three parameters (ie, cytotoxicity to cells that are otherwise NK resistant, increased expression of activating receptors, decreased expression of inhibitory receptors).

In one exemplary process contemplated herein, NK cells are expanded in a first step from a fraction of a biological fluid containing mononuclear cells, preferably to a total cell number ^{of about 0.5 to 5.0 x 10 9 cells.} Notably, such expansion can be achieved with relatively small volumes to medium cell densities (e.g., 100 to 300 ml or about 0.5 to 5.0 x 10 ⁶ cells/m) without the need for support cells or other manipulations requiring alteration of the culture vessel. ml) in a single reactor. Once the desired NK cell amount is achieved, the so expanded NK cells are then contacted with a stimulatory cytokine composition in a second step to activate the NK cells to a memory-like phenotype. Preferably, but not necessarily, the stimulatory cytokine composition will comprise an IL-18/IL-12-TxM fusion protein complex as exemplarily shown in FIG . 1 . However, the stimulatory cytokine composition can also include a mixture of IL-12, N-803, and IL-18, or a mixture of IL-12, IL-15, and IL-18. Cytokine stimulation will typically be performed for a period of 4 to 24 hours, and the CIML NK cells so generated may be resting (preferably in the presence of N-803) or restimulated prior to injection.

For example, whole blood or umbilical cord blood can be used as the starting material to be treated to obtain mononuclear cells. Most typically, treatment can be performed using conventional density gradient centrifugation (e.g., using Ficoll-Paque Plus™ (hydrophilic soluble polysaccharide, density 1.077 g/mL) commercially available from GE Lifesciences). have. Once the mononuclear cells are detached from the centrifuge tube, the cells are washed and resuspended in activation medium (eg, NK MACS supplemented with 10% human AB serum). The activation medium may further comprise N-803 at a concentration of about 0.4 nM, and an anti-CD16 antibody at a concentration of about 1.0 mcg/ml.

^{Most typically, mononuclear cells have a density of 1-2 x 10 6} cells/ml in a total volume of about 200 ml, and the cells and medium are in a single container. After about 3 to 4 days, the cells are fed with fresh medium containing N-803, and an additional feeding cycle of recovery, rapid expansion, and culture culmination is performed about every 3 days. Notably, successful NK cell expansion in such a manner was highly dependent on the appropriate selection of stimulatory factors as exemplarily shown in FIG . 2 . At this time, a dramatic expansion of over 20,000 fold was observed from day 0 when using anti-CD3 and anti-CD16 monoclonal antibodies, whereas anti-CD16 antibodies alone did not produce the same dramatic effect. Notably, the presence of anti-4-1BB antibody appeared to prematurely deplete proliferation of NK cells.

Cell culture is then terminated upon reaching a desired amount, typically about 0.5 to 5.0 x 10 ⁹ total cells, and/or upon reaching a desired expansion (eg, at least 100 fold expansion). Notably, despite the apparent simplicity, the cell culture so obtained yielded greater than about 85% NK cells, less than about 8% NKT cells, and less than about 2.5% T cells, and less than about 1.2% NK cells after about 3 weeks. of double negative (DN) T cells. Moreover, it should be recognized that the entire culturing process can be performed in a single vessel within a self-contained bioreactor, which substantially reduces the risk of contamination and eliminates reagent and cell handling during the culturing step.

When the desired cell mass is reached, the cells can be transferred to fresh medium for subsequent cytokine stimulation. Alternatively, cytokine stimulation to produce a memory-like phenotype is typically an IL-18/IL-12-TxM fusion protein complex (or a mixture of IL-12, N-803, and IL-18, or IL- 12, IL-15, and a mixture of IL-18) by adding an additional medium with a stimulatory cytokine composition comprising a mixture of IL-18). In most cases, cytokine stimulation will be performed for about 4 hours to 24 hours, more typically 12 hours to 16 hours. As will be readily appreciated, the cells may then be transferred to a transfusion medium prior to injection. In addition, the phenotype and/or cytotoxicity of CIML NK cells can be determined, with exemplary results presented in more detail below.

In the context of suitable biological fluids, it is generally contemplated that the fluid may be autologous to the subject to be provided with NK cells isolated in the methods presented herein. Accordingly, particularly preferred biological fluids include fresh whole blood, umbilical cord blood (frozen or fresh), and cells isolated in a leukocyte apheresis procedure. However, it should be understood that the biological fluid may be any fluid containing NK cells (typically among other cell types). For example, suitable alternative biological fluids include whole blood from an allogeneic donor, which may or may not be matched for compatible MHC types. Therefore, freshly donated whole blood or archived cord blood by individuals other than NK cell recipients, as well as samples from blood banks with an approaching expiry date, are considered suitable for use. Moreover, it should be noted that if the biological fluid is umbilical cord blood, the umbilical cord blood can be donated after matching and a sufficient MHC match with the NK cell recipient. Likewise, it should be noted that methods for isolating or enriching mononuclear cells can vary considerably, and those skilled in the art will readily be able to identify the most suitable method for isolation and enrichment. For example, where the biological fluid is whole blood or umbilical cord blood, the fluid is preferably processed via gradient density centrifugation using any suitable medium (eg, Ficoll-Hypaque). Alternatively, mononuclear cells can be obtained directly from the patient by leukocyte apheresis, or the biological fluid can be depleted of red blood cells using antibodies. In another method, mononuclear cells can be isolated using magnetic bead separation in which the beads are coated or otherwise bound to an antibody that binds to the mononuclear cells.

Likewise, the specific properties of the medium for activation and supply need not be limited to NK MACS medium, but it should be recognized that any medium known to support the growth of NK cells is considered suitable for use herein. However, most preferably, defined media is used, which may be supplemented with human AB serum.

Proliferation of NK cells in a mixture of mononuclear cells is preferably stimulated and supported with a combination of an anti-CD16 antibody and N-803, and optionally an anti-CD3 antibody. There are various sources of anti-CD16 antibodies known/commercially available in the art, and particularly preferred anti-CD16 antibodies have agonist (activating) activity and are specific for human CD16. However, activators other than anti-CD16 antibodies are also considered suitable for use herein, including anti-CD16 antibody fragments and fusion proteins with anti-CD16 antibody fragments. Additionally or alternatively, contemplated activators are also CD314 or NKG2D, the natural cytotoxic receptors CD335 (NKp46), CD336 (NKp44) and CD337 (NKp30), CD226 (DNAM-1), CD244 (2B4), among others, among others. , a member of the CD158 or killer immunoglobulin-like receptor (KIR) family with a short cytoplasmic tail (KIR2DS and KIR3DS) and CD94/NKG2C.

The concentration of anti-CD16 antibody will typically follow what is already known in the art for activation of NK cells. Thus, suitable concentrations for an anti-CD16 antibody are about 0.01 to 5.0 mcg/ml, more typically about 0.01 to 0.3 mcg/ml, or about 0.05 to 0.5 mcg/ml, or about 0.1 to 1.0 mcg/ml, or about It will be between 1.0 and 5.0 mcg/ml. With respect to the duration of exposure to anti-CD16 antibody, a mixture of mononuclear cells is most typically produced when the mononuclear cells are dissociated and first (and/second, and/or third) contact with the activation medium, anti-CD16 antibody Exposure to only a single, two, or three doses of Those skilled in the art will readily recognize appropriate schedules and dosages to achieve NK cell activation. Most typically, exposure of mononuclear cells to anti-CD16 antibody coincides with exposure of mononuclear cells with N-803. However, in a less preferred embodiment, exposure of mononuclear cells to anti-CD16 antibody is sequentially followed by exposure of mononuclear cells to anti-CD16 antibody with N-803 (preferred sequence is exposure of mononuclear cells to anti-CD16 antibody first) .

If desired, stimulating/supporting proliferation can also typically include contacting the cells with an anti-CD3 antibody at the same time as contacting the cells with an anti-CD16 antibody. As mentioned above, the concentration of anti-CD3 antibody will typically follow what is already known in the art for activation of NK cells. Thus, suitable concentrations for an anti-CD3 antibody are from about 0.01 to 10.0 ng/ml, more typically from about 0.01 to 0.1 ng/ml, or from about 0.1 to 0.5 ng/ml, or from about 0.3 to 1.0 ng/ml, or It will be about 1.0 to 5.0 ng/ml. Likewise, with respect to the duration of exposure to the anti-CD3 antibody, a mixture of mononuclear cells is most typically when the mononuclear cells are isolated and first (and/second, and/or third) contact with the activation medium, anti- Exposure to only a single, two, or three doses of CD3 antibody is generally contemplated. Those skilled in the art will readily recognize appropriate schedules and dosages to achieve NK cell activation.

With respect to N-803, N-803 (IL-15N72D:IL-15RαSu/IgG1 Fc complex with human sequence; see US 2019/0023766, commercially available from ImmunityBio) is preferred as an agent in activation and feed medium. are considered However, various alternative agents having IL-15 activity are also considered suitable for use herein. In this context, without wishing to be bound by any theory or hypothesis, we believe that N-803 enables the growth and expansion of NK cells through continuous signaling. In contrast, IL-15 as an isolated cytokine has a very short lifespan and signaling activity is typically very short. This means that when IL-15 as an isolated cytokine is added to the growth medium, the signaling will be pulsed or intermittent. In contrast, when N-803 is given, the stability of IL-15 is dramatically prolonged and signaling is considered continuous. Moreover, it should be recognized that N-803 also provides a physiological context (ie, IL-15 R-alpha chain) and a form of N72D that acts as a super agonist. Accordingly, any stabilized IL-15 compound is expressly considered suitable for use herein.

For example, all compounds and complexes that result in IL-15 signaling are considered suitable for use herein as long as such compounds and complexes have a serum half-life longer than isolated/recombined and purified IL-15 alone. do. Moreover, it is generally preferred that the stabilized IL-15 compound comprises at least a portion of the human sequence for IL-15 and/or IL-15 Ra. For example, a suitable compound is P22339 (a complex of IL-15 and the Sushi domain of the IL-15Ra chain, having a disulfide bond linking the IL-15/Sushi domain complex with an IgG1 Fc to increase its half-life, ; Nature, Scientific Reports (2018) 8: 7675), and the IL-15/IL-15Rα-Fc heterodimer XmAb24306 (see, eg, WO 2018/071919).

In a further particularly contemplated embodiment, the mixture of mononuclear cells, after separation from the biological fluid, is placed in a cell culture vessel along with a medium containing an anti-CD16 (and optionally anti-CD3) antibody and N-803. Activates NK cells. Most preferably, the vessel is a cell culture flask having at least one wall (or a portion thereof) that allows light to pass through such that cell shape, staining, and/or growth can be observed with a microscope or other optical instrument. Thus, cells can be continuously or periodically monitored in the bioreactor, and the measurements so obtained (eg, cell size, cell number, cell distribution, etc.) are used to automate the automation of the control unit logically coupled to the bioreactor. It should be noted that supply schedules can be triggered or modified. Most typically, as shown in FIG. 2 , the feeding of fresh medium with N-803 may be performed using a predefined schedule, typically every 3 days, wherein preferably each feeding is continuous. will include N-803 to maintain signaling. In the examples below, specific volumes are suitable for expanding NK cells to cell densities consistent with cell growth, however, it should be understood that volumes can be adjusted to accommodate specific growth patterns. To this end, it should also be understood that the feeding may be continuous or the predetermined volume may be changed in response to the growth kinetic observed in the vessel.

In most cases, the yield of NK cells at the end of the culture 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 are NKT cells, DN T cells, and T cells. For example, the remaining NKT cells will typically be no more than 10%, or no more than 8%, or no more than 7%, or no more than 6% of all living cells, while the remaining T cells will typically be no more than 5% of all living cells. , or 4% or less, or 3% or less, or 2% or less, and the remaining DN T cells will typically be 3% or less, or 2% or less, or 1.5% or less, or 1% or less of all living cells.

Thus, viewed from another point of view, the systems and methods contemplated herein allow for a remarkably high expansion of NK cells, a typical expansion being at least 80 times the number of NK cells originally present in the mixture of mononuclear cells, or It should be understood that at least 100 times, or at least 120 times, or at least 130 times, or at least 140 times. Such expansion is particularly noteworthy in terms of being a very simple way of activation and culturing (a one-pot process). Indeed, once the mixture of mononuclear cells is placed in a cell culture vessel, the entire process can be continued within the same vessel and only with the addition of medium. Thus, complicated handling and expensive reagents are completely avoided, and the risk of contamination is significantly reduced.

As already mentioned above, NK cells are about 0.1 to 1.0 x 10 ⁹ cells, or about 0.3 to 3.0 x 10 ⁹ cells, or about 0.5 to 5.0 x 10 ⁹ cells, or about 0.7 to 7.0 x 10 ^{9 cells.} can be expanded to a total number of canine cells, or about 1 to 10 x 10 ^{9 cells, or even more.} The exact number of expanded NK cells will typically depend on the particular purpose of the NK cells, the culture conditions, and the starting cell number, among other things. Upon reaching the desired amount of cells, cytokine stimulation can then be performed in expansion medium, typically by adding fresh medium containing the stimulatory cytokine composition.

In most cases, the stimulatory cytokine composition will include 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, as discussed in more detail below, suitable cytokines may also be derivatives of said cytokines, particularly preferred derivatives including fusion complexes. Additionally, it should be recognized that one or more of the cytokines may also be expressed in expanded NK cells following transfection with an appropriate recombinant nucleic acid (eg, transient expression from a plasmid or viral expression vector).

For example, in some embodiments, the stimulatory cytokine composition will comprise an IL-18/IL-12-TxM fusion protein complex, particularly preferred fusion protein complexes are described in WO 2018/165208, incorporated herein by reference have. In such a case, it should be understood that the fusion protein complex provides three cytokine functions (IL-12, IL-15, and IL-18) in a stabilized form through binding of human IgG to the Fc portion. Moreover, without wishing to be bound by any theory or hypothesis, the Fc portion of the fusion protein complex may provide additional stimulatory signals, presumably through interaction with CD16 on expanded NK cells. However, other fusion protein complexes based on N-808 are also expressly contemplated herein. For example, a suitable fusion protein complex may comprise a targeting scFv moiety, or a cytokine moiety other than (or in addition to) IL-12 and IL-18. Of course, in many cases IL-18/IL-12-TxM fusion protein complexes are preferred, but alternative TxM fusion protein complexes are also considered suitable and particularly contemplated fusion complexes are IL15/IL- as described in WO 2018/165208. 15Ralpha moiety, and at least one additional cytokine selected from the group consisting of IL-7, IL-18, and IL-21. Thus, among other suitable choices, contemplated TxM fusion complexes include IL-18/IL-7 TxM and/or IL-18/IL-21 TxM.

Thus, in another example, the stimulatory cytokine composition may also include a derivative of IL-15, with a particularly preferred derivative based on N-803. Such derivatives will advantageously have an increased signaling effect compared to IL-15 itself due to the presence of the IL-15Rα chain, exemplary suitable derivatives are described in WO 2016/004060 and WO 2018/075989. Most typically, when N-803 or similar fusion proteins are used, additional cytokine function will be provided by individual cytokines, particularly IL-7, IL-12, IL-21, and IL-18. Accordingly, in another aspect of the subject matter of the present invention, the stimulatory cytokine composition may also include IL-7, IL12, IL-15, IL-21, and IL-18 as individual cytokines. Thus, among other options, such individual cytokines may be added alone or in combination with other individual cytokines or TxM constructs, each of which may be recombinant (or even recombinantly expressed in cells). .

Accordingly, it should be understood that one or more of the stimulatory cytokines may also be expressed (transiently) from recombinant nucleic acids transfected with expanded NK cells. For example, suitable transfection methods include viral transfection wherein the recombinant nucleic acid is a viral expression vector. On the other hand, recombinant nucleic acids can also be transfected into cells using electroporation or lipofection using methods well known in the art. Also, when electroporation or lipofection is used, it is typically preferred that the nucleic acid is RNA (however, DNA is also considered suitable for use herein).

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

With regard to the timing of the stimulatory cytokine composition, it is generally preferred that the NK cells first expand to a desired (typically final) amount prior to exposure to the stimulatory cytokine composition. However, in alternative embodiments, the stimulatory cytokine composition comprises expanding NK starting at about 70% of the desired final cell mass, starting at about 80% of the desired final cell mass, or starting at about 90% of the desired final cell mass. can be added to the cell population. In most embodiments of the present subject matter, the exposure to the irritant composition lasts 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. will last a long time

Exposure to the stimulatory cytokine composition can typically be terminated by replacing the medium with fresh medium or medium suitable for infusion. On the other hand, it is also shown that the CIML NK cells so produced may undergo a resting phase which may last from 0 to 4 hours, 4 hours to 12 hours, 12 hours to 24 hours, or 1 day to 4 days, and even longer before subsequent use. are considered As will also be readily understood, CIML NK cells can also be restimulated to further increase cytotoxicity, which will typically be accomplished using at least one stimulatory cytokine such as IL2 or IL-15. . Most preferably, as shown in more detail below, restimulation provided an unexpectedly high cytotoxicity (compared to IL-15 itself) when N-803 was used. Moreover, it should be noted that restimulation will typically follow standard protocols well known in the art.

Regardless of the final treatment of the CIML NK cells, the CIML NK cells will be used for infusion into a subject in need thereof, most typically the subject is considered to be diagnosed with cancer. As will also be readily understood, CIML NK cells can be isolated from cancer vaccines (eg, recombinant (adeno)virus vaccines, recombinant yeast vaccines, recombinant bacterial vaccines), chemotherapeutic agents, checkpoint inhibitors, N-803 or TxM based A treatment regimen may be formed in which a therapeutic agent, and/or a targeted interleukin (eg, NHS-IL12) is provided.

Without limiting the subject matter of the present invention, CIML NK cells enable continuous monitoring, _{continuous management of CO 2} and O ₂ levels, and continuous monitoring to detect cell density (confluence). It is further contemplated as being expanded and/or activated in a culture environment where Among other options for such an environment, a particularly preferred environment is an automated cell culture and harvesting apparatus, for example as described in WO 2015/165700. Such a 'GMB-in-a-box' system advantageously allows control over feeding schedules, gas control, and enables real-time detection of cell density, growth (dynamics) and cell health as well as significantly reducing Dramatically reduce the possibility of contamination due to the specified handling requirements.

It should be noted that, in a further contemplated aspect, advantageously the systems and methods presented herein also enable the generation of ^{CD56 dim} and CD56 ^{bright NK cells, particularly when the NK cells are generated from peripheral blood.} Depending on additional culture conditions, CD56 ^bright NK cells can then differentiate into ^{CD56 dim cells.} Such distinct NK cell populations can then be used for distinct treatment options due to their distinct maturation and cytotoxicity profiles. In addition, it is to be understood that the compositions, systems and methods will also be suitable for generating NKT cells upon appropriate stimulation and culture.

Example

In view of the above, and as provided in more detail below, one exemplary method is to isolate CBMC or PBMC by a single Ficoll centrifugation step, and then, cells are cultured in NK MACS medium with 10% human AB serum. 0.4 nM N-803 and about 0.1 mcg/ml of anti-CD16 antibody (eg, clone B73.1 commercially available from BD Biosciences), and optionally about 0.5 ng/ml of anti-CD3 antibody it entails Typically, 100-150 mL (typically 135 mL) of CBMC at 1 million cells/ml was used as starting material with the above reagents. For dilution with N-803 twice a week (3 to 5 days apart) at the regimens of 1:2 and 1:10 compared to the existing volume with the corresponding concentration of N-803 for a final concentration of 0.4 nM. Medium was used. Expansion cultures are typically terminated when expanded NK cells constitute about 90% to 99% (eg, 98%) of all cells. Upon termination, cytokine induction can be performed as described in more detail below.

MNCs were freshly isolated from either umbilical cord blood or peripheral blood. It was washed twice with complete NKMACS medium (NKMACS + supplement + 10% hu-AB-serum). MNCs were suspended in 150 mL of medium at a density of 1x10^6 cells/mL in a GMP box (500 mL volume). 150 mL cell suspension was supplemented with anti-CD16 antibody (1 mcg/mL) and N-803 (0.4 nM). Imaging of the GMP box was started and cells were propagated according to pre-programmed steps. Cells in GMP boxes were alternately supplemented with either 10X cytokine medium or 2X cytokine medium. NK enrichment (phenotype for CD3, CD56, and CD16 expression) and cell health (cell number, viability, and cell density) were regularly monitored and plotted.

Cytokine induction to generate CIML NK cells from expanded NK cells started when they reached the point where 98% of all cells were NK cells. For this purpose, a box of 500 mL and a density of 2.3x10^6 cells/mL was divided equally into two separate boxes. Thus, the 500 mL cell suspension became 250 mL in each of the two boxes, and the cells were diluted 1:1 with fresh medium. Subsequently, IL18/12 TxM was added to a final concentration of 10 nM (for controls and comparisons, N-803 was used at a final concentration of 0.07 nM) and cells were transformed into IL-18/IL-12-TxM fusion protein CIML NK cells were obtained by incubation with the complex for 16 h. 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-CD16 antibody (BD bioscience, San Diego, CA); NK MACS medium with NK supplements, antibodies for staining for phenotypic analysis (aCD3, aCD16, aCD56, aNKp30, aNKp44, aNKp46, aNKG2A, aNKG2D, aTIGIT, aCD34, aTRAIL, aCD57, aCXCR3, and aCCR5) (California, USA) Miltenyi Biotec, San Diego); human AB serum (Access Biologicals, San Diego, CA); N-803, GMP in box kit (Nantbio Inc., Culver City, CA). The IL-18/IL-12-TxM fusion protein complex was obtained from ImmunityBio.

The CIML NK cells so generated were tested for cytotoxicity and expression of selected surface markers. More specifically, in one set of experiments, cord blood-derived CIML NK cells were tested for Merkel cell carcinoma cells (here MS-1 cells) that are typically resistant to NK cytotoxicity. Notably, as can be seen in FIG. 3 , CIML NK cells showed significant cytotoxicity after expansion and control exposure to the IL-18/IL-12-TxM fusion protein complex, whereas cord blood cells only showed N-803. Some cytotoxicity was also observed upon exposure. 4 depicts exemplary results for surface marker expression in cord blood derived cells exposed to IL-18/IL-12-TxM fusion protein complex and N-803. As can be seen, CIML NK cells had expression of CD16, but substantially increased expression of CD25, DNAM1, and potent secretion of IFN-γ.

As can be seen in FIG. 5 , when CIML NK cells were derived from peripheral blood, similar results were obtained. At this time, CIML NK cells had substantial cytotoxicity against the MS-1 cell line, and N-803 control cells from peripheral blood also showed some cytotoxicity. Likewise, surface markers for peripheral blood-derived CIML NK cells showed decreased expression of CD16 and TIGIT, while having significant increases in CD25, DNAM1, and IFN-γ secretion as can be seen from FIG. 6 . Notably, no significant cytotoxicity was observed for MS-1 cells when NK cells were cultured using standard culture protocols or when fresh NK cells were used, and IL-12, to trigger a memory-like phenotype, This was also the case when cells were induced with IL-15, and IL-18.

Cord blood-derived CIML NK cells were also tested for the activation cluster phenotype, and FIG. 7 depicts exemplary results comparing control exposure with N-803 versus exposure to the IL-18/IL-12-TxM fusion protein complex. . As can be seen from the images, there is a significant difference in the culture morphology after overnight exposure to the IL-18/IL-12-TxM fusion protein complex versus exposure to N-803. Given the selected surface markers of these CIML NK cells, exposure to the IL-18/IL-12-TxM fusion protein complex resulted in a significant increase in CD25, a known activation-associated receptor, as shown in FIG . 8 . Once again it became clear. Clearly, cytokine stimulation with IL-12, IL-15, IL-18 function substantially increased CD25 presentation, something not typically (at least not so much) observed with conventional new NK cells.

Such an increase in activating receptors and a decrease in inhibitory receptors were also readily noticeable when observing the culture morphology in the killing assay for K562 cells as shown in FIG . 9 . At this time, upon restimulation, cord blood-derived CIML cells showed substantially increased activation clustering compared to incubation with N-803.

In a further experiment, we also investigated the time course of cytotoxicity against K562 cells as exemplarily shown in Figures 10a , 10b , and 10c showing results after 24 hours, 48 hours, and 72 hours, respectively. did. After the first 24 h time point ( FIG. 10A ), we can see the onset of increased killing ability on K562 cells _{as both TxM concentrations tested have lower EC 50 than the N-803 control.} At 48 hours ( FIG. 10B ), increased killing by the TxM treated samples is seen, but less for the N-803 control. At this point, the increase in killing of K562 is about 3-fold. At 72 hours ( FIG. 10c ), all conditions began to lose activity against K562 killing, but TxM-treated cells maintained about 3-fold enhanced killing compared to controls.

11 shows expanded cord blood-derived NK cells, expanded cord blood-derived NK cells following N-803 stimulation, and expanded cord blood-derived NK cells stimulated with IL-18/IL-12-TxM fusion protein complex for 24 hours. It provides a direct comparison. As can be seen, cytotoxicity to all cells is readily noticeable, and expanded NK cells have a slight advantage for maximal % killed but require a substantially higher E;T ratio compared to CIML cells. 12 depicts expression of selected markers of expanded peripheral blood derived NK cells following N-803 stimulation versus expanded peripheral blood derived NK cells stimulated with IL-18/IL-12-TxM fusion protein complex. As can be seen from Figure 12, there is a significant down-regulation of TIGIT (and CD16), and a significant up-regulation of CD25 indicating activation. It should be noted that down-regulation of CD16 may be accompanied by a decrease in ADCC. However, the potential reduction in ADCC is overwhelmed by the higher activation and cytotoxicity to cell lines that would otherwise be resistant to NK cell cytotoxicity. As shown in FIG. 13 , similar cytotoxicity results are found for CIML NK cells derived from peripheral blood after 24 h stimulation with IL-18/IL-12-TxM fusion protein complex. Clearly, exposure to 10 nM of the IL-18/IL-12-TxM fusion protein complex produced better cell death in the K562 assay.

The secretion of IFN-γ was tested against peripheral blood-derived CIML NK cells, and FIG. 14 shows exemplary results using various conditions. The same cells were also used in the cytotoxicity assay, and FIG. 15 shows exemplary results. As can be seen from FIG. 16 , similar results are provided for CIML NK cells derived from cord blood. Notably, cytokine induction with N-803 outperformed induction with IL-15 itself. Therefore, it should be noted that while multi-cytokine induction is preferred as indicated above, induction with N-803 is also explicitly 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 the pharmaceutical composition or drug is typically performed by a healthcare professional (e.g., For example, by a physician, nurse, etc.), indirect administration is to provide or make available a pharmaceutical composition or drug to a healthcare professional for direct administration (eg, via injection, infusion, oral delivery, topical delivery, etc.) It includes the steps of making Most preferably, the cells or exosomes are administered via subcutaneous or subdermal injection. However, in other contemplated embodiments, administration may also be intravenous injection. Alternatively or additionally, antigen presenting cells can be isolated or grown from the patient's cells, infected in vitro, and then injected into the patient. Accordingly, it should be understood that the contemplated systems and methods may be considered complete drug discovery systems (eg, drug discovery, treatment protocols, validation, etc.) for highly personalized cancer treatment.

The recitation of a range of values herein is only intended to serve as an abbreviation method for individually reciting each individual value falling within that 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 illustrative language (eg, "such as") provided in connection with particular embodiments herein is merely intended to better illuminate the full scope of the present disclosure, and However, it does not impose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the claimed invention.

It will be apparent to those skilled in the art that many more modifications than those already described are possible without departing from the full scope of the concepts disclosed herein. Accordingly, the disclosed subject matter should not be limited except by the appended claims. Moreover, in interpreting both the specification and the claims, all terms are to be interpreted in the broadest possible manner consistent with the context. In particular, the terms “comprises” and “comprising” are to be construed in a non-exclusive manner to refer to an element, component, or step, in which the referenced element, component, or step may be present. indicates that it may be present, may be used, or may be combined with other elements, components, or steps not explicitly indicated. Where a claim in this specification refers to at least one selected from the group consisting of A, B, C... and N, the text is not A + N, or B + N, etc., but only one element from the group should be interpreted as requiring

Claims (40)

A method for producing cytokine induced memory like (CIML) NK cells with enhanced cytotoxicity, comprising:
isolating the mixture of mononuclear cells from the biological fluid, and contacting the mixture of mononuclear cells with an anti-CD16 antibody and N-803 to expand NK cells, wherein the NK cells at the end of the expansion are free of all living cells. constituting at least 80%; and
The expanded NK cells were treated with 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. A method comprising the step of generating CIML NK cells with enhanced cytotoxicity by contacting with a stimulatory cytokine composition comprising a. The method of claim 1 , further comprising contacting the CIML NK cells with N-803 after restimulating the CIML NK cells. 3. The method of claim 1 or 2, wherein the biological fluid is whole blood or umbilical cord blood. 3. The method of claim 1 or 2, wherein the mixture of mononuclear cells is not further treated to enrich for NK cells. 3. The method according to claim 1 or 2, wherein the mixture of mononuclear cells contains between 100 and 500 x 10 ⁶ cells. 3 . The method according to claim 1 , wherein the contacting of the mixture is carried out in ^{a volume of 100 to 300 ml or a cell density of 1×10 6 cells/ml.} 3. The method of claim 1 or 2, wherein in the step of contacting the mixture, the anti-CD16 antibody is present at a concentration of 0.05 to 0.5 mcg/ml. The method according to claim 1 or 2, wherein in the step of contacting the mixture, the N-803 is present in a concentration of 0.1 to 1.0 nM. 3. The method of claim 1 or 2, wherein contacting the mixture further comprises contacting the mixture of mononuclear cells with an anti-CD3 antibody. The method of claim 9 , wherein the anti-CD3 antibody is present at a concentration of 0.1 to 1.0 ng/ml. 3. The method of claim 1 or 2, wherein the stimulatory cytokine composition comprises the IL-18/IL-12-TxM fusion protein complex. 3. The method of claim 1 or 2, wherein the stimulatory cytokine composition comprises a mixture of IL-12, N-803, and IL-18. 3. The method of claim 1 or 2, wherein the stimulatory cytokine composition comprises a mixture of IL-12, IL-15, and IL-18. The method according to claim 1 or 2, wherein the NK cells are expanded to a total cell number ^{of 0.5 to 5.0 x 10 9 cells.} 3. The method of claim 1 or 2, wherein the step of contacting the expanded NK cells with a stimulatory cytokine composition is performed in the same vessel as the step of expanding the NK cells. 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 have been expanded from mononuclear cells of whole blood or umbilical cord blood, wherein the expanded NK cells constitute at least 80% of all living cells; and
The expanded NK cells were treated with 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. A method comprising the step of generating CIML NK cells with enhanced cytotoxicity by contacting with a stimulatory cytokine composition comprising a. The method of claim 16 , wherein the NK cells are expanded from whole blood. 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 to a subject receiving a transfusion comprising the CIML NK cells. 19. The method of any one of claims 16-18, wherein the stimulatory cytokine composition comprises the IL-18/IL-12-TxM fusion protein complex. 19. The method of any one of claims 16-18, wherein the stimulatory cytokine composition comprises a mixture of IL-12, N-803, and IL-18. 19. The method of any one of claims 16-18, wherein the stimulatory cytokine composition comprises a mixture of IL-12, IL-15, and IL-18. 19. The method according to any one of claims 16 to 18, wherein the expanded NK cells have a total cell number ^{of 0.5 to 5.0 x 10 9 cells.} 19. The method of any one of claims 16-18, wherein the CIML NK cells with enhanced cytotoxicity are cytotoxic to MS-1 cells. 19. The method of any one of claims 16-18, wherein the CIML NK cells with enhanced cytotoxicity have reduced expression of CD16 compared to expanded NK cells contacted only with N-803. 19. The method of any one of claims 16-18, wherein the CIML NK cells with enhanced cytotoxicity have reduced expression of TIGIT compared to expanded NK cells contacted only with N-803. 19. The method of any one of claims 16-18, wherein the CIML NK cells with enhanced cytotoxicity have increased expression of at least one of CD25 or DNAM1 compared to expanded NK cells contacted only with N-803. Way. Cytokine-induced memory-like (CIML) NK cells with enhanced cytotoxicity, exhibiting cytotoxicity to MS-1 cells of at least 50% death with an effector to target cell ratio of 5 or less. 29. The CIML NK cell of claim 28, wherein the CIML NK cell has reduced expression of CD16 compared to an expanded NK cell contacted only with N-803. 29. The CIML NK cell of claim 28, wherein the CIML NK cell has reduced expression of TIGIT compared to expanded NK cells contacted only with N-803. 29. The CIML NK cell of claim 28, wherein the CIML NK cell has increased expression of at least one of CD25 or DNAM1 compared to expanded NK cells contacted only with N-803. 29. The CIML NK cell of claim 28, wherein the CIML NK cells are autologous to a subject receiving an infusion comprising the CIML NK cells. 29. 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 at least one of CD16 or a variant thereof, IL-2 or a variant thereof, or IL-15 or a variant thereof from a recombinant nucleic acid. 35. A pharmaceutical composition comprising the cytokine-induced memory-like (CIML) NK cell of any one of claims 28-34 together with a pharmaceutically acceptable carrier. 35. The cytokine-induced memory-like (CIML) NK cell of any one of claims 28-34, having use in medicine. 35. The cytokine induced memory-like (CIML) NK cell of any one of claims 28-34 for use in the treatment of cancer. 35. A composition comprising cytokine-induced memory-like (CIML) NK cells for administration to a subject in a therapeutically effective amount of the cytokine-induced memory-like (CIML) NK cells of any one of claims 28-34. 39. The composition of claim 38, wherein the CIML NK cells are autologous cells of the subject. 39. The composition of claim 38, wherein the CIML NK cells are peripheral blood or umbilical cord blood-derived NK cells.

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