KR20150041554A - Protocol and media for storage and transport of nk-92 cell line - Google Patents

Abstract

Disclosed in the present invention is a storage medium for delivery of NK-92 cell having enough density to provide NK-92 cell of treating amount and including NK-92 cell not radiated when delivering to human serum, IL-2 of about 200 IU/mL, and a treatment facility. The storage medium has the temperature maintained in ±5°C of selected temperature between about 20 to 40°C for the NK-92 cell to survive to be administrated to a patient in at least 24 hours after arrangement of NK-92 cell into the storage medium. Moreover, disclosed is a method for delivering NK-92 cell, which enables the NK-92 cell to survive tobe administrated to a patient in at least 24 hours after arrangement of NK-92 cell into the storage medium.

Description

TECHNICAL FIELD [0001] The present invention relates to a protocol and a medium for storing and transporting NK-92 cell lines.

The present invention relates to the storage and delivery of NK-92 cells, which allows cells to survive for a period of at least 24 hours after placement into, for example, a storage medium, such that they can be administered to a patient.

Certain cells of the immune system have cytotoxic activity against a particular target cell. Natural Killer (NK) cells, which generally account for about 10 to 15% of circulating lymphocytes, are capable of expressing target cells, including virus-infected cells and many malignant cells, nonspecifically with respect to the antigen, without prior immunosensitive sensitization (Herberman et al., Science 214: 24 (1981)). The death of the target cell occurs by inducing cell lysis. NK cells were found to be effective in both in vitro and in vivo treatment of patients with advanced cancer. However, endogenous NK cells (i. E., Cells harvested from a donor or from a patient) are still difficult to use and apply with immunotherapy. It is difficult to propagate NK cells in vivo while maintaining their in vivo tumor-targeting, tumoricidal and viricidal abilities, and this is the clinical use of these in adoptive cell immunotherapy (Melder et al., Cancer Research 48: 3461-3469 (1988), Stephen et al., Leuk. Lymphoma 377-399 (1992), Rosenberg et al., New Engl. J. Med 316: 889-897 (1987)). In addition, the preparation of endogenous NK cells comprises T cells and / or other immune effector cells that should be removed if the NK cells are used in a donor-independent patient treatment.

The embodiments described herein generally relate to a storage medium for delivery and / or delivery of NK-92 cells and also methods of using the cells that carry and carry the NK-92 cells.

SUMMARY OF THE INVENTION

The NK-92 cell line is a unique cell line that has been found to proliferate in the presence of interleukin 2 (IL-2) (Gong et al., Leukemia 8: 652-658 (1994)). These cells have high cytolytic activity against various cancers. The NK-92 cell line is a homogeneous population of NK cells with extensive anti-tumor cytotoxicity with predictable yield after proliferation. Phase I clinical trials confirmed its safety profile and anti-tumor responses have been observed in certain patients with advanced cancer.

One limitation of patient treatment with NK-92 cells is their ability to store and / or carry the cells for long periods of time. NK cells used in patient therapy must be kept under current good manufacturing processes (cGMP), which limits the number of facilities that can culture and manufacture NK-92 cell lines. To date, the use of NK-92 cells in patient therapy has been limited by the availability of a culture and production facility very close to the hospital where treatment is to be performed. NK-92 cells are manufactured at a facility next to the hospital and then hand-delivered to a medical team for use in patient care. Recently, frozen NK-92 cells show dramatically reduced cytotoxicity after thawing. Even after several days of recovery by incubation, the number and cytotoxicity of NK-92 cells are still suboptimal.

So far, studies on endogenous NK cells have shown that IL-2 (1000 IU / mL) during transport is very important for NK cell activation but does not need to be maintained at 37 < 0 > C and 5% (Koepsell et al., Transfusion 53: 398-403 (2013)). However, endogenous NK cells are significantly different from NK-92 cells, mainly due to their origin: NK-92 is a cancer-derived cell line whereas endogenous NK cells are harvested from a donor (or patient) Are treated for infusion into the patient. Endogenous NK cell preparations are heterogeneous cell populations whereas NK-92 cells are homogeneous clonal cell lines. NK-92 cells proliferate easily while maintaining cytotoxicity, whereas endogenous NK cells do not. In addition, a heterogeneous population of endogenous NK cells does not aggregate at high density. Thus, maximization of survival and cytotoxicity after storage and / or transport of these two cell types requires very different considerations.

There are other problems with NK-92 cells. For example, NK-92 is still viable and requires the presence of IL-2 to proliferate and will proliferate under IL-2 as low as 1 IU / mL (Gong et al., Supra). In addition, the cell shape and / or phenotype varies with the amount of IL-2 used in the medium (Gong et al., Supra). For example, IL-2 receptor (CD-25) expression is inversely proportional to IL-2 concentration (Gong et al., Supra).

In addition, aggregation of NK-92 cells at high concentrations has a negative impact on their use in treating patients. This results in the challenge that in the absence of IL-2, NK-92 cells lose viability while in the presence of IL-2 the cells proliferate. Thus, under normal cell culture conditions, the number of NK-92 cells will doubling after about 26 to 32 hours. These difficulties are further complicated by the fact that NK-92 cells must be able to be delivered to the hospital for at least 24 hours after they are placed into the storage medium. To prevent aggregation during delivery, the rate of NK-92 cell proliferation at any given IL-2 concentration should be considered.

For example, a time of at least about 24 hours allows for the placement of cells into the storage medium, delivery to a hospital, and subsequent administration of appropriate procedures for NK-92 cell injection into the patient. Once the NK-92 cells arrive at the hospital (or other treatment facility), they are washed and transferred to a solution suitable for injection into the patient, such as phosphate buffered saline (PBS). NK-92 cells are also irradiated to prevent in vivo proliferation. The radiation dose is designed to maintain cytotoxicity, but it has a deleterious effect on cell survival. Thus, in order to minimize degradation of the cell population during conversion of the NK-92 cell population into a form useful for treatment, it is important that the storage medium used for transport maintains the NK-92 cell viability as high as possible.

Thus, there is still a need for a storage medium to deliver the NK-92 cell line, which allows the NK-92 cells to be cultured and manufactured in a central facility and distributed to remote hospitals or other treatment facilities to be taken to the patient shortly after receipt. This need includes methods for minimizing cell proliferation to the point at which substantial aggregation occurs, while maintaining NK-92 cell viability and cytotoxicity during storage and / or delivery. The present invention provides a storage medium and method for storage and / or delivery of non-irradiated NK-92 cells, and addresses the above-mentioned need.

In some embodiments, the invention provides a method for the storage and / or delivery of NK-92 cells that maintains the survival and cytotoxicity of NK-92 cells for the treatment of patients at a remote facility from the NK- Storage medium and method. The present invention demonstrates that storing NK-92 cells at a minimum variable temperature in the presence of low levels of IL-2 is able to prevent substantial aggregation due to limited cell growth but also cell survival and cytotoxicity It was predicted based on an amazing discovery. For the therapeutic use of patients in remote treatment facilities, the cells can be delivered under conditions provided by the present invention after manufacturing NK-92 cells for therapy in a central facility, and cGMP-specific No facilities are needed. Thus, the present invention allows NK-92 products as "off-the-shelf" reproducible with minimal cell manipulation in a treatment facility. The present invention also provides a method of delivering a homogeneous NK-92 cell product having a consistent cell viability to a destination.

In one aspect, the invention described herein generally relates to cell storage media for delivery and / or delivery of cells, such as sterile isotonic NK-92 cell storage for delivery and / or delivery of NK-92 cells As the medium, the storage medium may be human serum or human serum albumin; NK-92 cells not irradiated and not substantially aggregated, having an initial density sufficient to deliver therapeutic amounts of NK-92 cells upon delivery to the treatment facility; And IL-2 at a concentration of about 200 IU / mL and maintained within ± 5 ° C. between about 20 ° C. and about 40 ° C. selected for delivery in the presence of sufficient oxygen to maintain the viability of the cells so that NK-92 cells Is capable of surviving the NK-92 cells so that the cells can be administered to the patient for at least a period of at least 24 hours after placement into the storage medium. In a preferred embodiment, the medium chiryoryang of NK-92 cells was about 2.5 × 10 ⁶ cells / mL or less at the time of delivery to the treatment facility. In some embodiments, the NK-92 cell density is maintained between about 1 x 10 ⁴ cells / mL to about 1 x 10 ⁷ cells / mL during delivery. In some embodiments, NK-92 cell density is maintained between about 0.5 × 10 ⁶ cells / mL to about 8 × 10 ⁶ cells / mL for handling. In some embodiments, the medium is maintained within +/- 2 DEG C of the temperature selected for delivery. In some embodiments, the medium comprises from about 1% to about 5% human serum or human serum albumin. In some embodiments, the temperature selected for delivery is between about 25 캜 and about 38 캜. In some embodiments, the initial cell density is between about 1 x 10 ⁶ cells / mL to about 6 x 10 ⁶ cells / mL.

In yet another aspect, the invention described herein provides a method of treating NK-92 cells in which the NK-92 cells that are not irradiated and substantially not aggregated are administered to a patient for at least a 24-hour period after placement into a storage medium Lt; RTI ID = 0.0 > NK-92 < / RTI > cells while maintaining viability,

(a) providing a sterile storage container;

(b) adding to the container sterile storage medium comprising human serum or human serum albumin and IL-2 at about 200 IU / mL;

(c) adding NK-92 cells that are not irradiated and not substantially aggregated to the vessel, having an initial density sufficient to provide a therapeutic dose of NK-92 cells upon delivery to a treatment facility;

(d) sealing the vessel;

(e) maintaining said medium within ± 5 ° C between a temperature selected from about 20 ° C to about 40 ° C for transport; And

(f) delivering said NK-92 cells for up to at least 24 hours after placement into said storage medium in the presence of sufficient oxygen to maintain viability of said cells

Wherein the NK-92 cell is capable of surviving the NK-92 cell such that the NK-92 cell can be administered to the patient for a period of at least 24 hours after placement into the storage medium ≪ / RTI >

In a preferred embodiment, the medium chiryoryang of NK-92 cells was about 2.5 × 10 ⁶ cells / mL or less at the time of delivery to the treatment facility. In some embodiments, the NK-92 cell density is maintained between about 1 x 10 ⁴ cells / mL to about 1 x 10 ⁷ cells / mL during delivery. In some embodiments, NK-92 cell density is maintained between about 0.5 × 10 ⁶ cells / mL to about 8 × 10 ⁶ cells / mL for handling. In some embodiments, the medium is maintained within +/- 2 DEG C of the temperature selected for delivery. In some embodiments, a temperature controller is used to maintain the storage medium within ± 5 ° C of the temperature selected for delivery. In some embodiments, the date and time that non-irradiated NK-92 cells were added to the medium are labeled on the storage container. In some embodiments, the medium comprises from about 1% to about 5% human serum or human serum albumin. In some embodiments, the temperature selected for delivery is between about 25 캜 and about 38 캜. In some embodiments, the initial cell density is between about 1 x 10 ⁶ cells / mL to about 6 x 10 ⁶ cells / mL.

Figure 1 shows the doubling time of NK-92 cells in growth culture.
Figure 2A is a graph showing the effect of the concentration of 4 × 10 ⁵ cells / mL (green triangle) at a density of 1 × 10 ⁶ cells / mL (red circle) or 6 × 10 ⁶ cells / mL (blue square) Indicating cytotoxicity of NK-92 cells stored overnight in the incubator. Figure 2B shows cytotoxicity of cells after dilution and stabilization.
Figure 3 shows the results of flow cytometric analysis of NK-92 cells transfected overnight at a density of 1 x ¹⁰⁶ cells / mL or 6 x ¹⁰⁶ cells / mL.

Before the present compositions and methods are disclosed and described, it is to be understood that the aspects described below are not limited to the specific compositions, methods, or uses thereof, but may of course vary. It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only, and is not intended to be limiting.

In the present specification and claims, various terms are defined that have the following meanings.

It should be noted that, as used in this specification and the appended claims, the singular forms include references to plural objects, unless the context clearly indicates otherwise.

As used herein, the term " about "means that the value may vary by +/- 15% and still fall within the scope of the invention. For example, "IL-2 at a concentration of about 200 IU / mL" includes an IL-2 concentration between 170 IU / mL and 230 IU / mL.

As used in describing the present invention, "natural killer (NK) cells" are cells of the immune system that kill target cells without restriction by MHC class in the absence of specific antigen stimulation. The target cell may be a tumor cell or a cell bearing a virus. NK cells are characterized by the presence of CD56 and absence of CD3 surface markers.

The term "endogenous NK cell" is used to refer to an NK cell derived from a donor and is distinct from the NK-92 cell line. Endogenous NK cells are an inhomogeneous group of cells, generally rich in NK cells. The endogenous NK cells may be for the treatment of autologous or allogeneic origin of the patient.

The immortalized NK cell line, NK-92, was originally obtained from a patient with non-Hodgkin's lymphoma. For purposes of the present invention, unless otherwise indicated, the term "NK-92" refers to the original NK-92 cell line as well as the modified NK-92 cell line (eg, by introduction of an exogenous gene). NK-92 cells and exemplary, non-limiting variations thereof are described in U.S. Patent Nos. 7,618,817, 8,034,332, and 8,313,943, all of which are incorporated herein by reference in their entirety.

As used herein, "non-irradiated NK-92 cells" are NK-92 cells that have not been irradiated. Irradiation prevents cells from growing and multiplying. NK-92 cells are believed to be irradiated at some other point in the treatment facility or patient prior to treatment, since the time between irradiation and injection should not exceed 4 hours to preserve optimal activity. Alternatively, NK-92 cells may be inactivated by another mechanism.

As used in describing the present invention, "inactivation" of NK-92 cells makes them unable to grow and / or their normal function, especially their cytotoxic activity, is not possible. Inactivation may also be associated with the death of NK-92 cells. The NK-92 cells are useful for the treatment of cancer, such as, for example, after therapeutic applications, after they have purged pathologically relevant cells in the in vitro sample, or for a period sufficient to effectively kill many or all target cells in the mammalian body It can be deactivated after being present in the body. By way of non-limiting example, inactivation may be induced by administering a sensitive inactivating agent to NK-92 cells.

As used herein, the term "substantially non-aggregated" means that the NK-92 cell density is below the cell density which has a detrimental effect on aggregation. That is, the density of NK-92 cells is such that any coagulation does not significantly change their in vivo efficacy. A cell density of about 1 x 10 ⁷ cells / mL is considered the upper limit of the NK-92 cell density to maintain proper viability and unaggregated state.

The materiality of aggregation is measured by cell densities above about 1 x 10 ⁷ cells / mL. NK-92 cell densities of less than about 1 x 10 ⁷ cells / mL will not significantly alter their in vivo efficacy during transport. Since NK-92 cells will proliferate in the storage medium, the amount of cells initially added to the storage medium should reflect the extent of growth and the time at which storage is to be maintained. Such calculations are within the skill of the art.

As used in describing the present invention, the terms "cytotoxic" and "cytolytic" are intended to be synonymous with each other when used to describe the activity of effector cells such as NK cells. In general, cytotoxic activity is associated with the death of target cells by any of a variety of biological, biochemical, or biophysical mechanisms. More specifically, cell lysis refers to an activity in which an effector dissolves the cytoplasmic membrane of a target cell and destroys its physical integrity. This causes the death of the target cells. Without wishing to be bound by theory, the cytotoxic effect of NK cells is believed to be due to cell lysis.

As used in describing the present invention, a "target cell" is a cell that is killed by the cytotoxic activity of the NK cells of the present invention. In particular, they include cells that are malignant or otherwise derived from cancer, and those infected by pathogenic viruses such as HIV, EBV, CMV, or herpes.

As used in describing the present invention, "removal" is associated with the in vitro death of target cells by effector cells such as NK cells. The target cell may be included in a biological sample obtained from a mammal that is believed to suffer from a pathology associated with the presence of a target cell in the sample. The pathology can be cancer or malignant tumors due to tumor cells in the sample and can be treated by removing the tumor cells from the sample and returning the sample to the mammalian body.

As used in describing the present invention, "cancer "," tumor ", and "malignant tumor" all relate to the hyperplasia of tissue or organ. If the tissue is part of the lymphatic system or the immune system, the malignant cells may comprise non-solid tumors of circulating cells. Malignant tumors of other tissues or organs can produce solid tumors. In general, the methods of the invention can be used for the treatment of lymphocytes, circulating immune cells, and solid tumors.

As used in describing the present invention, "pathogenic virus" is a virus that causes a disease in a host. The pathogenic viruses infect cells of the host animal and the result of such infection is the deterioration of the host's health.

As used herein, the term "10 ^∧ y" is equal to 10 ^y. Therefore, 2.5 × 10 ^∧ 6 is equivalent to 2.5 × 10 ^6, or 2,500,000.

As used herein, the term "treatment facility" refers to any facility, hospital, clinic or other laboratory to which a cell is to be transported. In a preferred embodiment, the cells are delivered to a facility for treating the patient.

As used herein, a "method of transporting" refers to a method of transporting NK-92 cells, including but not limited to road transport, air transport, rail transport, and, where appropriate, Or from any other facility to the treatment facility.

The title or subheading may be used herein for the convenience of the reader, and is not intended to affect the scope of the present invention. In addition, some of the terms used herein are more specifically defined below.

NK -92 cell line

The NK-92 cell line has been described by Gong et al. (1994). This was found to exhibit the CD56 ^{probe light,} CD2, CD7, CD11a, CD28 , CD45 and CD54 surface markers. In addition, it does not show CD1, CD3, CD4, CD5, CD8, CD10, CD14, CD16, CD19, CD20, CD23 and CD34 markers. The growth of NK-92 cells upon incubation is dependent on the presence of recombinant interleukin-2 (rIL-2) at a low dose of 1 IU / mL sufficient to maintain proliferation. IL-7 and IL-12 do not support prolonged growth, and so are other tested cytokines including IL-1 alpha, IL-6, tumor necrosis factor alpha, interferon alpha, and interferon gamma. NK-92 is highly effective at killing certain tumor cells such as K562 (red leukemia) and Daudi (bucket lymphoma) cells, and even at a low effector: target (E: T) ratio of 1: Have high cytotoxicity (Gong et al., Supra). In addition, NK-92 cells have high cytotoxic activity against 8E5 cells that are infected with HIV and produce HIV virion. NK-92 cells are deposited with the American Type Culture Collection (ATCC) designated CRL-2407.

NK-92 cells show lytic activity against a wide range of malignant target cells. These include acute and chronic lymphocytic and circulating target cells such as myeloid leukemia, lymphoma, myeloma, melanoma, and also cell lines derived from cells from solid tumors such as prostate cancer, neuroblasts, and breast cancer cell lines. These effects are even observed at very low effector: target ratios. This dissolution is superior to cytotoxicity obtained from normal peripheral blood mononuclear cells stimulated with IL-2 for 4 days. Early-stage clinical studies performed on patients with irradiated NK-92 cells showed good tolerability, indicating beneficial effects. These studies have been performed in patients with various cancer types, including kidney and myeloma (Arai et al., Cytotherapy 10: 625-632, 2008) and lung cancer (Tonn et al., J. Hematother. & Stem Cell Res. 10: 535-544, 2001).

Composition and method

Some embodiments generally relate to storage media having one or more parameters as described herein, including media for delivery and / or delivery of NK92 cells. In one aspect, the invention described herein is a sterile isotonic NK-92 cell storage medium for the delivery and delivery of NK-92 cells, wherein the storage medium comprises human serum or human serum albumin; NK-92 cells not irradiated and not substantially aggregated, having an initial density sufficient to deliver therapeutic amounts of NK-92 cells upon delivery to the treatment facility; And IL-2 at a concentration of about 200 IU / mL and maintained within ± 5 ° C. between about 20 ° C. and about 40 ° C. selected for delivery in the presence of sufficient oxygen to maintain the viability of the cells so that NK-92 cells To allow the NK-92 cells to survive for a period of at least 24 hours after placement into the storage medium.

In yet another aspect, the invention described herein provides a method of treating NK-92 cells in which the NK-92 cells that are not irradiated and substantially not aggregated are administered to a patient for at least a 24-hour period after placement into a storage medium Lt; RTI ID = 0.0 > NK-92 < / RTI > cells while maintaining viability,

(a) providing a sterile storage container;

(b) adding to the container sterile storage medium comprising human serum or human serum albumin and IL-2 at about 200 IU / mL;

(c) adding NK-92 cells that are not irradiated and not substantially aggregated to the vessel, having an initial density sufficient to provide a therapeutic dose of NK-92 cells upon delivery to a treatment facility;

(d) sealing the vessel;

(e) maintaining said medium within ± 5 ° C between a temperature selected from about 20 ° C to about 40 ° C for transport; And

(f) delivering said NK-92 cells for up to at least 24 hours after placement into said storage medium in the presence of sufficient oxygen to maintain viability of said cells

Wherein the NK-92 cell is capable of surviving the NK-92 cell such that the NK-92 cell can be administered to the patient for a period of at least 24 hours after placement into the storage medium ≪ / RTI >

In some embodiments, the density of NK-92 cells is maintained between about 1 x 10 ⁴ cells / mL to about 1 x 10 ⁷ cells / mL during delivery. In some embodiments, the NK-92 cell density is maintained between about 0.5 x 10 ⁵ cells / mL to about 1 x 10 ⁷ cells / mL during delivery. In some embodiments, the NK-92 cell density is maintained between about 1 x 10 ⁵ cells / mL to about 1 x 10 ⁷ cells / mL during delivery. In some embodiments, the NK-92 cell density is maintained between about 1 x 10 ⁶ cells / mL to about 1 x 10 ⁷ cells / mL during delivery. In some embodiments, the NK-92 cell density is maintained between about 2 x 10 ⁵ cells / mL to about 1 x 10 ⁷ cells / mL during delivery. In some embodiments, the NK-92 cell density is maintained between about 1 x 10 ⁴ cells / mL to about 9 x 10 ⁶ cells / mL during delivery. In some embodiments, the NK-92 cell density is maintained between about 1 x 10 ⁴ cells / mL to about 8 x 10 ⁶ cells / mL during delivery. In some embodiments, the NK-92 cell density is maintained between about 1 x 10 ⁴ cells / mL to about 6 x 10 ⁶ cells / mL during delivery. NK-92 cells can be maintained at any density within any of these ranges, including endpoints.

In some embodiments, the initial density of the NK-92 cells is between about 0.5 × 10 ⁴ cells / mL to about 9 × 10 ⁶ cells / mL. In some embodiments, the initial density of NK-92 cells is between about 1 x 10 ⁵ cells / mL to about 9 x 10 ⁶ cells / mL. In some embodiments, the initial density of NK-92 cells is between about 1 x 10 ⁶ cells / mL to about 9 x 10 ⁶ cells / mL. In some embodiments, the initial density of NK-92 cells is between about 1 x 10 ⁶ cells / mL and about 8 x 10 ⁶ cells / mL. In some embodiments, the initial density of NK-92 cells is between about 1 x 10 ⁶ cells / mL to about 7 x 10 ⁶ cells / mL. In some embodiments, the initial density of NK-92 cells is between about 1 x 10 ⁶ cells / mL and about 6 x 10 ⁶ cells / mL. The initial density of MK-92 cells can be any density within any of these ranges, including the endpoint.

In some embodiments, the temperature selected for delivery is between about 20 캜 and about 40 캜. In a preferred embodiment, the temperature selected for delivery is between about 25 캜 and about 38 캜. In some embodiments, the temperature selected for delivery is between about 20 [deg.] C and about 25 [deg.] C. In some embodiments, the temperature selected for delivery is about room temperature. In some embodiments, the temperature selected for delivery is about 37 ° C.

 In some embodiments, the medium is maintained within +/- 5 ° C of the temperature selected for delivery. In some embodiments, the medium is maintained within +/- 4 DEG C of the temperature selected for delivery. In some embodiments, the medium is maintained within +/- 3 DEG C of the temperature selected for delivery. In some embodiments, the medium is maintained within +/- 2 DEG C of the temperature selected for delivery. In some embodiments, the medium is maintained within +/- 1 DEG C of the temperature selected for delivery.

In some embodiments, a temperature controller is used to maintain the storage medium within ± 5 ° C of the temperature selected for delivery. Such thermostats are known in the art. Exemplary temperature control devices include a gel pack heated to the desired temperature, a portable transport incubator, a bioreactor, or other suitable device known in the art.

In some embodiments, the NK-92 cells remain viable so that they can be administered to the patient for up to a period of at least 24 hours after placement into the storage medium. In some embodiments, the NK-92 cells remain viable so that they can be administered to the patient up to a period of about 14 hours after placement into the storage medium. In some embodiments, the NK-92 cells are administered to a patient for at least about 20, 16, 12, 10, 9, 8, 7, 6, 5, 4, 3, 2, And remains viable to be administered. In some embodiments, the NK-92 cells remain viable so that they can be administered to the patient over a period of more than 24 hours after placement into the storage medium. In some embodiments, the NK-92 cells remain viable so that they can be administered to the patient for up to a period of about 36, 48, 60, or 72 hours after placement into the storage medium.

In some embodiments, the NK-92 cells may be stored for a period of time after arrival at the treatment facility. For example, NK-92 cells may be stored and / or grown in the facility prior to the procedure of injecting NK-92 cells into the patient. In one embodiment, the storage medium is replaced with a growth medium (e.g., containing a higher concentration of IL-2) after delivery. In one embodiment, additional medium is added to the NK-92 cells for storage-that is, the density of the NK-92 cells is reduced. In one embodiment, the NK-92 cells are stored for 12, 24, 36, or 48 hours after delivery to the facility. In one embodiment, the storage conditions of the NK-92 cells depend on cell density, cell growth, cell viability, level of IL-2, or any combination thereof.

In some embodiments, the medium comprises human serum or its equivalent. In some embodiments, the medium comprises human serum albumin. In some embodiments, the medium comprises human plasma. In some embodiments, the medium comprises from about 1% to about 15% human serum or human serum equivalents. In some embodiments, the medium comprises about 1% to about 10% human serum or human serum equivalents. In some embodiments, the medium comprises from about 1% to about 5% human serum or human serum equivalents. In some embodiments, the medium comprises about 2.5% human serum or human serum equivalents. In some embodiments, the serum is human AB serum. In some embodiments, acceptable serum substitutes for use in human therapeutic agents are used in place of human serum. Such serum substitutes are known in the art or may be developed in the future. Although concentrations of human serum can be used in excess of 15%, concentrations above about 5% are considered to be prohibitive in terms of cost.

The storage container used in the invention described herein may be any cell storage container. In some embodiments, the storage vessel may be any storage vessel known in the art. In some embodiments, the storage container is an oxygen-permeable bag. In some embodiments, the storage vessel is a tissue culture flask. In some embodiments, the storage vessel is an oxygen-permeable flask. An example of such a flask is a G-Rex flask manufactured by Wilson Wolf Manufacturing. In some embodiments, the storage vessel is an oxygen-impermeable bag having sufficient oxygen to maintain cell viability in the upper space during delivery.

In some implementations, information is labeled on the storage container. In a preferred embodiment, information on the date and time that the NK-92 cells were placed in the storage medium is labeled on the storage container. Methods of labeling containers are well known in the art. In some implementations, the label is a bar code. In some implementations, the label is a radio frequency identification (RFID) tag. In some implementations, the label is a two-dimensional barcode. In some implementations, the label is a quick response (QR) code. In some implementations, the labels are recorded, typed, or stamped. In some implementations, multiple labeling methods are used.

The following abbreviations are used herein:

Storage medium (SM) for delivery of NK-92 cells is prepared in suitably sterile containers as indicated in the table below. The medium is filtered using a 0.2 micrometer PES filter unit. IL-2 (200 IU / mL) is added to the medium for SM preparation. In a preferred embodiment, IL-2 is added just prior to use.

IL-2 is added to the above at a concentration of about 200 IU / mL, but it should be understood that other concentrations, including those described elsewhere herein, may be used.

The following examples are intended to illustrate the invention but not to limit it. All publications or references cited herein are incorporated herein by reference.

Example

Example  One: NK -92 times the cell doubling time

The NK-92 cells a view life (VueLife) ^® culture bags (American Physical fluoro (American Fluoroseal Corp.)) 2.5% human AB serum, 500 IU / mL IL-2 , asparagine, glutamine in, and supplemented with serine And cultured in X-VIVO 10 cell culture medium (Lonza, Inc.) for 2 weeks. Supplemented cell culture medium was added every 3 days as indicated in Figure 1 (2x: 2x volume of added media; 4x: 4x volume of added media; 3-4x: 3 to 4 times volume). Cell density (black circle) and total cell number (white circle) were determined by cell count. Doubling times were measured to be between 26 and 32 hours.

Example  2: NK -92 Cell cytotoxicity - Storage / transport cell density

NK-92 cells were transported at different cell concentrations in a Rat-Rex 10 flask containing SM with 450 IU IL-2 on a pre-warmed temperature control pack at 37 占 폚. The cytotoxic activity of NK-92 cells versus K562 cells was determined. K562 (red leukemia) cell line was obtained from ATCC and maintained in a continuous suspension culture in RPMI 1640 medium supplemented with 10% fetal bovine serum (FCS). Gong et al. (1994), supra], [Klingemann et al. (Effector, E) cells versus K562 (target, T) cells, as described in Cancer Immunol. Immunother. 33: 395-397 (1991) and U.S. Patent Application Serial No. 10 / 456,237. cytotoxic activity of the various E:. was evaluated by ⁵¹ Cr release assay with T ratio, the above three documents all are incorporated herein by reference in their entirety cytotoxicity is 1 × 10 ⁶ dogs transport immediately, or cells Cells / mL and allowed to stand overnight before measurement.

Figure 2A is a graph showing the effect of the concentration of 4 × 10 ⁵ cells / mL (green triangle) at a density of 1 × 10 ⁶ cells / mL (red circle) or 6 × 10 ⁶ cells / mL (blue square) Indicating cytotoxicity of NK-92 cells stored overnight in the incubator. Figure 2B shows cytotoxicity of the cells after dilution and stabilization. Cells transfected with higher density (6 × 10 ⁶ cells / mL) lost cytotoxicity immediately after transfusion but could be recovered if cells were diluted to 1 × 10 ⁶ cells / mL and stood overnight there was.

Example  3: NK -92 cell culture

NK-92 cells were delivered at different cell concentrations in vessels containing SM with 200 IU IL-2 on pre-warmed temperature control packs (e.g., 37 ° C). Cytotoxicity of NK-92 cells versus target cells was assessed, for example, by the assay described in Example 2. [ Cytotoxicity was measured either immediately after transit or after cells were diluted to 1 x 10 ⁶ cells / mL and allowed to stabilize overnight.

Example  4: NK -92 Transport of cells

NK-92 cells were transported from Texas to Pittsburgh. Cells were formulated in one rat-lex 10 (40 x 10 ⁶ whole cells in a 40 mL volume) at 1x10 ⁶ cells / mL in GM-1 medium with IL-2. The cells were shipped using a temperature-controlled pack pre-warmed to < RTI ID = 0.0 > 37 C. < / RTI > During transport, the cells survived 94.9%.

Using a flow cytometry assay, were compared to ⁶ × 10 6 cells for dogs transport cells / mL of NK-92 cells carried as a 1 × 10 ⁶ cells / mL with respect to viability and phenotype. Figure 3 shows the results of a flow cell assay.

Claims (16)

A sterile isotonic NK-92 cell storage medium for the transport and delivery of NK-92 cells,
(i) human serum or human serum albumin;
(ii) NK-92 cells that have not been irradiated, substantially unaggregated, having an initial cell density sufficient to provide therapeutic doses of NK-92 cells upon delivery to the treatment facility; And
(iii) IL-2 at a concentration of about 200 IU / mL
And maintained within ± 5 ° C between about 20 ° C and about 40 ° C of temperature selected for delivery in the presence of sufficient oxygen to maintain the viability of the cells so that the NK-92 cells are maintained for at least 24 hours Gt; NK-92 < / RTI > cell storage medium capable of surviving said NK-92 cells such that said NK-92 cells are capable of survival until such time as the patient is able to administer said patient. 2. The storage medium of claim 1, wherein the NK-92 cell density is maintained between about 1 x 10 ⁴ cells / mL to about 1 x 10 ⁷ cells / mL during delivery. The method of claim 2, wherein the storage medium to the NK-92 cell density that is maintained between about 0.5 × 10 ⁶ cells / mL to about 8 × 10 ⁶ cells / mL for handling. The storage medium of claim 1, wherein the initial cell density is between about 1 x 10 ⁶ cells / mL to about 6 x 10 ⁶ cells / mL. 2. The storage medium of claim 1, wherein the storage medium is maintained within < RTI ID = 0.0 > 2 C < / RTI > The storage medium of claim 1, comprising from about 1% to about 5% human serum or human serum albumin. The storage medium of claim 1, wherein the temperature selected for delivery is between about 25 ° C and about 38 ° C. The NK-92 cells are maintained in the presence of the NK-92 cells so that the NK-92 cells not irradiated and not substantially aggregated can be administered to the patient for at least a 24-hour period after placement into the storage medium / RTI >
(a) providing a sterile storage container;
(b) adding to the container sterile storage medium comprising human serum or human serum albumin and IL-2 at about 200 IU / mL;
(c) adding NK-92 cells that are not irradiated and not substantially aggregated to the vessel, having an initial density sufficient to provide a therapeutic dose of NK-92 cells upon delivery to a treatment facility;
(d) sealing the vessel;
(e) maintaining said medium within ± 5 ° C between a temperature selected from about 20 ° C to about 40 ° C for transport; And
(f) delivering said NK-92 cells for up to at least 24 hours after placement into said storage medium in the presence of sufficient oxygen to maintain viability of said cells
Wherein the NK-92 cell is capable of surviving the NK-92 cell so that the NK-92 cell can be administered to the patient for a period of at least 24 hours after placement into the storage medium Way. 9. The method of claim 8, wherein the NK-92 cell density is maintained between about 1 x 10 ⁴ cells / mL to about 1 x 10 ⁷ cells / mL during delivery. 10. The method of claim 9, wherein said NK-92 cells to a density of holding between about 0.5 × 10 ⁶ cells / mL to about 8 × 10 ⁶ cells / mL for handling. 9. The method of claim 8, wherein the initial density is between about 1 x 10 ⁶ cells / mL to about 6 x 10 ⁶ cells / mL. 9. The method of claim 8, wherein the medium is maintained within +/- 2 DEG C of the temperature selected for delivery. 9. The method of claim 8, wherein a temperature regulator is used to maintain the storage medium within < RTI ID = 0.0 > 5 C < / RTI > 9. The method of claim 8, wherein the date and time that the NK-92 cells were added to the storage medium is labeled on the storage container. 9. The method of claim 8, wherein the medium comprises from about 1% to about 5% human serum or human serum albumin. 9. The method of claim 8, wherein the temperature selected for delivery is between about 25 [deg.] C and about 38 [deg.] C.

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