Method of generating CIK NKT cells from cord blood

Abstract

To provide a natural killer T cell (NKT cell) therapy that does not need activation with α-galactosyl ceramide (Gal-Cer).SOLUTION: Provided herein are CIK NKT cells that can be activated in the absence of Gal-Cer. In some embodiments, more than 50% of the cells in the CIK NKT cell population express CD56 and CD3, and less than 10% of the cells in the population express Va24. Also provided are compositions and kits comprising a plurality of CIK NKT cells from the population. Methods of producing the CIK NKT cells and methods of using the cells to treat cancer are also provided.SELECTED DRAWING: Figure 2

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 62 / 696,131, filed July 10, 2018, which is incorporated herein by reference. [Background technology]

[0002] background Natural killer T cells (NKT cells) are a subset of T lymphocytes that express natural killer (NK) cell surface markers. A subset of NKT cells, termed invariant NKT cells (iNKT), expresses a highly restricted T cell receptor (TCR). iNKT cells play a key role in linking innate and adaptive immune responses and have been implicated in various diseases, including infectious diseases, allergies, asthma, autoimmunity, and tumor surveillance (Juno et al. PLoS Pathog. 2012;8(8)). However, their activation typically requires the CD1d-restricted lipid ligand α-galactosylceramide (Gal-Cer). The procedures required to introduce Gal-Cer (e.g., in the form of Gal-Cer / CD1d tetramers) can significantly increase the cost of treating patients with these NKT cells and, in some cases, limit their therapeutic utility. Thus, there remains a need for safe and cost-effective NKT cell therapies that can be used to treat patients with a wide range of histologies. [Prior art documents] [Non-patent literature]

[0003] [Non-Patent Document 1] Juno et al. PLoS Pathog. 2012;8(8) Summary of the Invention

[0004] overview Provided herein is a CIK NKT cell that can be activated in the absence of Gal-Cer.In some embodiments, more than 50% of the cells in the CIK NKT cell population express CD56 and CD3, and less than 10% of the cells in the population express Va24.Also provided is a composition and kit comprising a plurality of CIK NKT cells derived from the population.Also provided is a method for generating CIK NKT cells and a method for using the cells to treat cancer.

[0005] The present disclosure provides a population of CIK NKT cells, wherein greater than 50% of the cells in the population express CD56 and CD3, and less than 10% of the cells in the population express Va24.

[0006] Optionally, the CIK NKT cell population can kill target cells in the absence of α-galactosylceramide (Gal-Cer). Optionally, the target cells are cancer cells. The cancer cell line can be selected from the group consisting of myeloid leukemia cells, medulloblastoma cells, and monocytic cells. Optionally, the cancer cells are selected from the group consisting of K562 cells, Daudi cells, DAOY cells, and THP-1 cells.

[0007] Optionally, the CIK NKT cells kill a plurality of target cells with an EC50 of 1.0 to 10.0. Optionally, the CIK NKT cells can kill target cells with an EC50 that is 90% or more and 110% or less than the IC50 at which the CIK NKT cells kill the target cells in the presence of Gal-Cer.

[0008] The present disclosure also provides a composition comprising a plurality of CIK NKT cells derived from any of the above populations of CIK NKT cells and a physiologically acceptable excipient.

[0009] Also provided is a kit for treating cancer comprising a plurality of CIK NKT cells derived from any of the above populations of CIK NKT cells and a container and / or label indicating that the kit is for treating cancer.

[0010] Also provided is a method for enriching CIK NKT cells from an umbilical cord blood sample, comprising isolating mononuclear cells from the umbilical cord blood sample; and contacting the isolated mononuclear cells with one or more agents selected from the group consisting of IL-7, ALT-803 or IL-15, FLT3 ligand, and Gal-Cer, thereby enriching CIK NKT cells. IL-7, if present, may be at a concentration ranging from 5 to 20 ng / mL. ALT-803, if present, may be at a concentration ranging from 100 to 300 ng / mL. FLT3 ligand, if present, may be at a concentration ranging from 5 to 20 ng / mL. Gal-Cer, if present, may be at a concentration ranging from 2 to 10 μg / mL.

[0011] Optionally, the method further comprises isolating enriched CIK NKT cells from the remainder of the cord blood sample. Optionally, the method further comprises contacting the isolated CIK NKT cells with anti-CD3, anti-CD28, and IL2 to expand the CIK NKT cells. Optionally, the method further comprises contacting the isolated CIK NKT cells with Gal-Cer. Optionally, Gal-Cer is used in the form of a Gal-Cer-loaded CD1d tetramer. Optionally, the anti-CD3 antibody may be present in an amount of 5 ng / mL to 60 ng / mL. Optionally, the anti-CD28 antibody is present in an amount of 0.1 μg / mL to 2 μg / mL. Optionally, IL-2 is present at a concentration of 50 ng / mL to 500 ng / mL. Optionally, the enrichment and / or expansion of CIK NKT cells does not include interferon-γ.

[0012] Populations of CIK NKT cells generated by the above-described methods of enriching, isolating, and expanding CIK NKT cells are also provided.

[0013] Also provided is a method for treating cancer or a viral infection in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of CIK NKT cells derived from any of the populations of CIK NKT cells described above, thereby treating the cancer. 2 1x10 8 ~Approx. 1×10 11 CIK NKT cells are administered to a patient. Optionally, the cancer is selected from the group consisting of leukemia, lymphoma, polycythemia vera, multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, sarcoma, and carcinoma. Optionally, the cells are administered to the patient by a route selected from the group consisting of intravenous, intraperitoneal, and subcutaneous. Optionally, the method further comprises administering an antibody.

[0014] Also provided is the population of CIK NKT cells of claim 1, wherein the CIK NKT cells express a CAR and / or a cytokine, and more than 50% of the cells in the CIK NKT cell population express CD56 and CD3, and less than 10% of the cells in the population express Va24.

[0015] The foregoing general description and the following detailed description are exemplary and explanatory and are intended to provide further explanation of the present disclosure. Other objects, advantages, and novel features will become readily apparent to those skilled in the art. [Brief explanation of the drawings]

[0016] The objects, features, and advantages will be more readily appreciated by reference to the following disclosure when considered in conjunction with the accompanying drawings. [Figure 1] FIG. 1 is a schematic diagram of the pathways used by CIK NKT cells to kill target cells. [Figure 2] Figure 2A shows the results of flow cytometry analysis of CIK NKT cells stained with CD3, CD56, and Va24: Figure 2A is a forward scatter and size scatter plot; Figure 2B is a CD3 and CD56 plot; and Figure 2C is a Va24 plot. [Figure 3] Figure 3A shows the killing of DAOY cells by cord blood CIK NKT cells in the presence (represented by circles) or absence (represented by squares), and Figure 3B shows the killing of luciferase-expressing THP1 cells by cord blood CIK NKT cells (represented by squares) or peripheral blood iNKT cells (represented by circles). [Figure 4] 1 shows the killing of K562, DAOY, and Daudi cells by cord blood CIK NKT cells. DETAILED DESCRIPTION OF THE INVENTION

[0017] Detailed Description Overview The present application provides cytokine-induced killer NKT cells (CIK NKT cells) that can kill target cells in a non-CD1d-restricted manner, i.e., independent of Gal-Cer / CD1d tetramer formation. CIK NKT cells can be used to target a wide range of target cells and do not induce graft-versus-host disease (GVHD). GVHD occurs due to the infiltrating ability of lymphocytes to infiltrate organs such as the intestine, skin, and liver, causing widespread inflammation. CIK NKT cells do not express chemokine receptors important for targeting to GVHD organs, but they have been shown to express receptors that promote tumor homing, thus preventing GVHD induction. Compared to existing techniques for generating CIK NKT cells, which typically produce heterogeneous populations consisting of CD3+ single-positive cells, CD56+ single-positive cells, and CD3+ / CD56+ double-positive cells, with double-positive cells typically accounting for 30% or less, the method of the present invention produces a cell population consisting primarily of CD3+ / CD56+ double-positive cells. In one aspect, at least 50% of the CIK NKT cells in the population express both CD56 and CD3, and less than 10% of the cells in the population express Va24. The method for generating CIK NKT cells does not require exposing the cells to interferon-γ, thereby reducing costs. Thus, the present application provides a safe and cost-effective NKT cell therapy that can be widely used to treat various diseases, such as cancer, without causing clinically adverse symptoms such as GVHD.

[0018] The present disclosure also provides compositions and kits comprising a plurality of CIK NKT cells derived from the population. Methods of generating CIK NKT cells and methods of using CIK NKT cells to treat cancer are also provided.

[0019] Terminology Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0020] In this specification and the claims that follow, reference will be made to a number of terms that shall be defined to have the following meanings.

[0021] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. As used herein, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. Thus, for example, reference to "a natural killer cell" includes a plurality of natural killer cells.

[0022] All numerical designations, including ranges, e.g., pH, temperature, time, concentration, amount, and molecular weight, are approximations which are varied (+) or (-) by increments of 0.1 or 1.0, as appropriate. It is to be understood that all numerical designations can be preceded by the term "about," although not always explicitly stated.

[0023] As used herein, a "+" when used to indicate the presence of a particular cell marker means that the cell marker is detectably present compared to an isotype control in fluorescence-activated cell sorting; or is detectable above background in quantitative or semi-quantitative RT-PCR.

[0024] As used herein, "-" when used to indicate the presence of a particular cell marker means that the cell marker is not detectably present compared to an isotype control in fluorescence-activated cell sorting; or is not detectable above background in quantitative or semi-quantitative RT-PCR.

[0025] As will be understood by those skilled in the art, for any and all purposes, particularly in terms of providing a written description, all ranges disclosed herein also encompass any and all possible subranges and combinations of those subranges. Any recited range can be readily recognized as fully expressing and allowing that same range to be divided into at least equal halves, thirds, fourths, fifths, tenths, etc. As a non-limiting example, each range discussed herein can be readily divided into a lower third, a middle third, an upper third, etc. As will also be understood by those skilled in the art, all language such as "up to," "at least," "greater than," "less than," and the like, refers to a range that includes the recited number and can be subsequently divided into subranges as described above. Finally, as will be understood by those skilled in the art, a range includes each individual member. Thus, for example, a group having 1 to 3 cells refers to a group having 1, 2, or 3 cells. Similarly, a group having 1 to 5 cells refers to a group having 1, 2, 3, 4, or 5 cells, and so on.

[0026] It is also to be understood, although not necessarily stated, that the reagents described herein are merely exemplary, and that equivalents of such reagents are known in the art.

[0027] "Optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and that the description includes instances when the event or circumstance occurs and instances when it does not occur.

[0028] The term "comprising" is intended to mean that the compositions and methods include the recited elements, but do not exclude other elements. When used to define compositions and methods, "consisting essentially of" is intended to mean excluding other elements that are of some essential importance to the combination. For example, a composition consisting essentially of the elements defined herein does not exclude other elements that do not materially affect the basic and novel characteristics of the claim. "Consisting of" is intended to mean excluding more than trace amounts of other components and substantial method steps. Embodiments defined by each of these transition terms are within the scope of this disclosure.

[0029] As used herein, the terms "cytotoxicity" and "cytolysis" when used to describe the activity of effector cells such as NK cells are intended to be synonymous. Generally, cytotoxic activity is associated with the killing of target cells by any of a variety of biological, biochemical, or biophysical mechanisms. Cytolysis more specifically refers to the activity of an effector cell in lysing the plasma membrane of a target cell, thereby destroying its physical integrity. This results in the death of the target cell. Without wishing to be bound by theory, it is believed that the cytotoxic effect of NK cells is due to cytolysis.

[0030] The term "killing" in relation to a cell / cell population is intended to include any type of manipulation that results in the death of that cell / cell population.

[0031] The term "cytokine" or "cytokines" refers to a general class of biological molecules that act on cells of the immune system. Exemplary cytokines include, but are not limited to, FLT3 ligand, interferons, and interleukins (IL), particularly IL-2, IL-12, IL-15, IL-18, and IL-21.

[0032] The terms "patient," "subject," "individual," and the like, are used interchangeably herein and refer to any animal, or cells thereof, whether in vitro or in situ, that are amenable to the methods described herein. In certain non-limiting embodiments, the patient, subject, or individual is a human.

[0033] The term "treating" or "treatment" encompasses the treatment of a disease or disorder described herein in a subject, such as a human, and includes (i) inhibiting the disease or disorder, i.e., halting its development; (ii) alleviating the disease or disorder, i.e., causing regression of the disorder; (iii) slowing the progression of the disorder; and / or (iv) inhibiting, alleviating, or slowing the progression of one or more symptoms of the disease or disorder. The term "administering" a monoclonal antibody or natural killer cells to a subject or "administration" of a monoclonal antibody or natural killer cells to a subject includes any route of introducing or delivering the antibody or cells to perform its intended function. Administration can be by any route suitable for delivering the cells or monoclonal antibody. Thus, delivery routes can include intravenous, intramuscular, intraperitoneal, or subcutaneous delivery. In some embodiments, CIK NKT cells are administered directly to the tumor, for example, by intratumoral injection. In some embodiments, the modified CIK NKT cells described herein are administered parenterally, for example, by injection, infusion, or implantation (subcutaneously, intravenously, intramuscularly, intravesically, or intraperitoneally).

[0034] The term "expression" refers to the production of a gene product.

[0035] As used herein, the term "cytotoxicity" when used to describe the activity of effector cells, such as NK cells, relates to the killing of target cells by any of a variety of biological, biochemical, or biophysical mechanisms.

[0036] The terms "reduce," "decreased," "reduction," and "decrease" are all used herein to refer to a decrease of at least 10% compared to a reference level, e.g., a decrease of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% compared to a reference level, or a decrease of up to and including 100% (i.e., a level that is absent compared to a reference sample), or any decrease between 10 and 100%.

[0037] The term "cancer" refers to any type of cancer, neoplasm, or malignant tumor found in mammals, including leukemia, carcinoma, and sarcoma. Exemplary cancers include brain cancer, breast cancer, cervical cancer, colon cancer, head and neck cancer, liver cancer, kidney cancer, lung cancer, non-small cell lung cancer, melanoma, mesothelioma, ovarian cancer, sarcoma, gastric cancer, uterine cancer, and medulloblastoma. Further examples include Hodgkin's disease, non-Hodgkin's lymphoma, multiple myeloma, neuroblastoma, ovarian cancer, rhabdomyosarcoma, primary thrombocytosis, primary macroglobulinemia, primary brain tumors, cancer, malignant pancreatic insulinoma, malignant carcinoid, bladder cancer, precancerous skin lesions, testicular cancer, lymphoma, thyroid cancer, neuroblastoma, esophageal cancer, genitourinary cancer, malignant hypercalcemia, endometrial cancer, adrenocortical carcinoma, neoplasms of the endocrine and exocrine pancreas, and prostate cancer.

[0038] The term "therapeutically effective amount" or "effective amount" refers to the amount required to improve the symptoms of a disease compared to an untreated patient. The effective amount of an active compound used to practice the present disclosure for the therapeutic treatment of a disease will vary depending on the mode of administration, the age, weight, and general health of the subject. Ultimately, the attending physician or veterinarian will determine the appropriate amount and administration regimen. Such an amount is referred to as an "effective" amount.

[0039] Headings and subheadings may be used herein for the convenience of the reader, but they are not intended to affect the scope of the disclosure. In addition, some terms used herein are more specifically defined below.

[0040] Cytokine-induced killer natural killer T cells (CIK NKT cells) Natural killer T cells are a heterogeneous group of T cells that share properties of both T cells and natural killer cells. For example, NKT cells not only express the αβ T cell receptor but also various molecular markers typically associated with NK cells, such as NKp44, NKp46, and NKp30. NKT cells comprise only approximately 0.1% of all peripheral blood T cells. NKT cells have been implicated in suppressing autoimmunity and graft rejection, promoting resistance to pathogens, and promoting tumor immunity.

[0041] NKT cells are typically classified into type I and type II based on differences in T cell receptor (TCR) usage. Type 1 NKT cells, commonly referred to as invariant NKT cells, are NKT cells that express a highly restricted T cell receptor that includes the invariant TCR α chain, Va24.

[0042] Invariant NKT cells are typically activated by recognizing the lipid ligand α-galactosylceramide (Gal-Cer) presented by CD1d. CD1d is a member of the CD1 family of glycoproteins expressed on the surface of various antigen-presenting cells and is involved in the presentation of lipid antigens. In contrast to class I and II major histocompatibility complex (MHC) molecules, which present protein antigens to CD8+ and CD4+ T cells, respectively, CD1 molecules can capture and process both foreign and self lipid antigens for presentation to T cells. Gal-Cer is typically derived from pathogenic cells, such as bacteria.

[0043] Compared to type I NKT cells, type II NKT cells have a more diverse TCR repertoire and higher sequence diversity. Type II NKT cells are Gal-Cer insensitive, i.e., their activation does not depend on the presence of Gal-Cer.

[0044] The CIK NKT cells disclosed herein belong to the category of type II NKT cells. CIK NKT cells can be generated via cytokine induction. In some cases, they are generated via cytokine induction, for example, from umbilical cord blood. In some cases, they are generated during the process of enriching or isolating iNKT cells from umbilical cord blood samples. However, CIK NKT cells differ from typical iNKT cells in several aspects. Phenotypically, they express Va24, a marker of the α chain of the TCR receptor, and unlike iNKT cells, the percentage of cells expressing Va24 can be 70% or more, CIK NKT cells have reduced expression of Va24. For example, the percentage of CIK NKT cells expressing Va24 can be less than 10% of the total population of CIK NKT cells. In terms of cytotoxicity, unlike iNKT cells, which are activated by recognizing the glycolipid α-galactosylceramide (Gal-Cer), CIK NKT cells do not require Gal-Cer for activation and can kill cells in the absence of (Gal-Cer) (see Figures 3 and 4).

[0045] Thus, in terms of phenotype, the CIK NKT cells provided herein typically have high expression levels of CD56 and CD3 and low expression of Va24. In some cases, at least 90% of a CIK NKT population generated from cord blood cells expresses CD56 and CD3, and less than 10% of the cells in the population express Vα24.

[0046] Methods for isolating and culturing CIK NKT cells Collection of umbilical cord blood Human umbilical cord blood is rich in hematopoietic stem cells and can be used as a source for generating CIK NKT cells. To collect umbilical cord blood, a human placenta is typically collected immediately after delivery after birth. The placenta can be transported in a sterile, insulated transport device (which maintains the placenta at 20-28°C), for example, in a cord blood collection kit substantially as described in U.S. Patent No. 7,147,626. Preferably, the placenta is delivered to the laboratory 4-24 hours after delivery.

[0047] The placenta can be subjected to a conventional cord blood recovery process. Typically, the placenta is exsanguinated using a needle or cannula with the assistance of gravity (see, e.g., Anderson, U.S. Pat. No. 5,372,581; Hessel et al., U.S. Pat. No. 5,415,665). A needle or cannula is usually placed in the umbilical vein, and the placenta can be gently massaged to aid in the drainage of cord blood from the placenta. Such cord blood recovery may be performed commercially, for example, by LifeBank Inc., Cedar Knolls, NJ, ViaCord, Cord Blood Registry, and CryoCell. Preferably, the placenta is allowed to drain by gravity without further manipulation to minimize tissue disruption during cord blood recovery.

[0048] Methods for collecting cord blood cells are well known, for example, as described in US20150225697. Cord blood mononuclear cells (Comics) can be isolated from collected cord blood using methods well known in the art, for example, density gradient methods using Ficoll-Paque. Reagents suitable for isolating Comics are commercially available, for example, from Stem cell Technology Inc.

[0049] Enrichment of CIK NKT cells To enrich for CIK NKT cells, comics can be cultured for a certain period of time in the presence of various cytokines. Enrichment refers to increasing the proportion of target cells in a heterogeneous cell population (e.g., comics). The enrichment period can be 2 days to 3 weeks, e.g., 1 to 2 weeks, 5 to 10 days, or about 2 weeks. Various growth media, such as Roswell Park Memorial Institute medium (RPMI) or Dulbecco's modified Eagle's medium (DMEM), can be used. Optionally, the medium further contains human AB serum and / or Gal-Cer. Optionally, human AB serum is present at 5 to 15% v / v, e.g., about 10% v / v. Optionally, Gal-Cer is present at a concentration of 2 to 10 μg / mL, e.g., about 5 μg / mL. Suitable cytokines that can be added to the medium can include one or more cytokines selected from the group consisting of stem cell factor, FLT3 ligand, IL-7, and ALT-803 or IL-15. In some embodiments, FLT3 ligand is present at a concentration in the range of 5 to 20 ng / mL, e.g., 10 ng / mL; IL-7 is present at a concentration in the range of 5 to 20 ng / mL, e.g., 10 ng / mL; and / or ALT-803 is present at a concentration in the range of 100 to 300 ng / mL, e.g., about 175 ng / mL.

[0050] FLT3 ligand is a hematopoietic four-helix bundle cytokine that is structurally homologous to stem cell factor (SCF) and colony-stimulating factor 1 (CSF-1). In synergy with other growth factors, FLT3 ligand stimulates the proliferation and differentiation of various hematopoietic progenitor cells. It is the major growth factor that stimulates the proliferation of dendritic cells.

[0051] ALT-803 is a complex consisting of the human IL-15 mutant IL-15N72D (a substitution at residue position 72) and an IL-15Rα sushi-Fc fusion protein (see Zhu et al. J. Immunol. 2009; 183:3598-607, the relevant disclosures of which are incorporated herein by reference).

[0052] CIK NKT cells can be isolated from the enriched culture by methods well known in the art, for example, by incubating the enriched culture with magnetic beads conjugated with an antibody against the Va24-J18 chain of the TCR so that the CIK NKT cells bind to the magnetic beads, and then isolating the CIK NKT cells bound to the beads in the presence of a magnetic field. One example of a suitable antibody that can be used is the 6B11 antibody, which is commercially available from suppliers such as Biolegend (San Diego, CA). A suitable reagent for isolating CIK NKT cells is from Miltenyi Biotec, Germany. CIK NKT cells are typically isolated in PBS containing serum (e.g., human AB serum). Optionally, the isolation solution also contains EDTA.

[0053] Thus, provided herein is a method for enriching CIK NKT cells from umbilical cord blood, the method comprising isolating mononuclear cells from a cord blood sample; and contacting the isolated monocytes with one or more agents selected from the group consisting of IL-7, ALT-803, FLT3 ligand, and Gal-Cer to enrich for CIK NKT cells.

[0054] Expansion of CIK NKT cells CIK NKT cells isolated as described above can be expanded in an appropriate growth medium. Expansion refers to growing an isolated population of target cells so that the number of target cells increases. In some embodiments, the growth medium is NK Macs medium, available from Miltenyi Biotec, Germany. In some embodiments, the growth medium is supplemented with IL-2, anti-CD3 antibody, and / or anti-CD28 antibody in amounts appropriate for the expansion of NKT cells. In some embodiments, the anti-CD3 antibody is present at a concentration of 5 ng / mL to 60 ng / mL, e.g., 20 ng / mL. In some embodiments, the anti-CD28 antibody is present at a concentration of 0.1 μg / mL to 2 μg / mL, e.g., 0.5 μg / mL. In some embodiments, IL-2 is present at a concentration of 50 ng / mL to 500 ng / mL, e.g., 200 ng / mL. In some embodiments, the growth medium comprises human AB serum (e.g., about 10% v / v). In some embodiments, the growth medium further comprises a Gal-Cer-loaded CD1d tetramer. In some embodiments, the Gal-Cer-loaded CD1d tetramer is a pre-assembled tetramer commercially available, for example, from ProImmune (Oxford, UK). Methods for constructing Gal-Cer-loaded CD1d tetramers are well known. Typically, Gal-Cer lipids are co-incubated with CD1d protein oligomerized on a streptavidin surface to form a tetramer. Once Gal-Cer binds to the CD1d complex, it is column-purified and used as a reagent for expansion. Some exemplary methods for preparing Gal-Cer loaded CD1d tetramers are described at www.proimmune.com / ecommerce / pdf_files / PS_DE000-RPE_V1.1%20%28CD1d%20Tetramer%20Empty%20%28R-PE%20Labeled%29%29.pdf and proimmune.com / ecommerce / pdf_files / ST14.pdf.In some embodiments, Gal-Cer-loaded CD1d tetramers are used in an amount such that the concentration of Gal-Cer in the growth medium is about 20-200 ng / mL, e.g., 50-150 ng / mL, or 80-120 ng / mL, or about 100 ng / mL Gal-Cer. In some embodiments, CIK NKT cells are grown and expanded for several days or weeks to reach a quantity of cells suitable for various applications.

[0055] Accordingly, the present disclosure provides a method for expanding CIK NKT cells from umbilical cord blood, the method comprising isolating mononuclear cells from a cord blood sample; and contacting the isolated monocytes with one or more agents selected from the group consisting of IL-7, ALT-803, FLT3 ligand, and Gal-Cer to enrich for CIK NKT cells.

[0056] Phenotyping of CIK NKT cells In certain embodiments, the CIK NKT cell population can be assessed by detecting one or more functionally relevant markers, such as CD56 and CD3 (markers of NKT cells) and the TCR receptor Va24 (high expression of which indicates that the NKT cells are invariant NKT cells).

[0057] In some embodiments, provided herein are CIK NKT cell populations that contain a lower percentage of Va24+ cells compared to typical invariant NKT cells. The CIK NKT cell population contains about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, or about 10% Va24+ cells. In some embodiments, the CIK NKT cell population contains 0-20%, 5-10%, 1-7%, or 4-8% Va24+ cells. In some embodiments, the CIK NKT cell population contains 20% or less, 15% or less, or 10% or less Va24+ cells.

[0058] In some embodiments, provided herein are CIK NKT cell populations comprising a proportion of CD56+ cells substantially similar to that found in typical invariant NKT cells. The CIK NKT cell population comprises at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% CD56+ cells. In some embodiments, the CIK NKT cell population comprises 50-100%, 70-100%, 85-100%, 90-100%, 95-100%, or 98-100% CD56+ cells. In some embodiments, the CIK NKT cell population comprises 50% or more, 70% or more, 85% or more, 90% or more, 93% or more, or 95% or more CD56+ cells.

[0059] In some embodiments, provided herein are CIK NKT cell populations comprising a proportion of CD3+ cells substantially similar to that found in typical invariant NKT cells. The CIK NKT cell population comprises at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% CD3+ cells. In some embodiments, the CIK NKT cell population comprises 50-100%, 70-100%, 85-100%, 90-100%, 95-100%, or 98-100% CD3+ cells. In some embodiments, the CIK NKT cell population comprises 50% or more, 70% or more, 85% or more, 90% or more, 93% or more, or 95% or more CD3+ cells.

[0060] In some embodiments, provided herein are CIK NKT cell populations comprising a proportion of CD56+ CD3+ cells substantially similar to that found in typical invariant NKT cells. The CIK NKT cell population comprises at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% CD56+ CD3+ cells. In some embodiments, the CIK NKT cell population comprises 50-100%, 70-100%, 85-100%, 90-100%, 95-100%, or 98-100% CD56+ CD3+ cells. In some embodiments, the CIK NKT cell population comprises 50% or more, 70% or more, 85% or more, 90% or more, 93% or more, or 95% or more CD56+ CD3+ cells.

[0061] CIK NKT cell cytotoxicity Optionally, the cytotoxic activity of isolated or enriched natural killer cells can be evaluated in a cytotoxicity assay using, for example, tumor cells, such as cultured K562, DAOY, THP-1, LN-18, U937, WERI-RB-1, U-118MG, HT-29, HCC2218, KG-1, or U266 tumor cells, or the like, as target cells. The CIK NKT cells disclosed herein can kill target cells regardless of MHC type and regardless of the presence of αGal-Cer.

[0062] Assays for assessing cytotoxicity are well known, for example the MTT assay, which is a system based on the tetrazolium compound MTT. Briefly, after a treatment period in which target cells are contacted with CIK NKT cells, freshly diluted MTT solution (2.5 mg mL -1) was added to each well, and the plate was incubated for 4 hours at 37°C in a humidified 5% CO2 atmosphere. At the end of the incubation period, the medium was removed, and the formazan product was dissolved in 100 μL of dimethyl sulfoxide. Cell viability was assessed by measuring absorbance at 570 nm using a SUNRICE Tecan absorbance reader (Schoeller). The compound concentration resulting in 50% cell growth inhibition (IC50) was calculated from a curve constructed by plotting cell viability (%) versus drug concentration (μM). Readings were converted to a percentage of control (cell viability). Non-limiting methods for cell killing assays include the methods described in the literature, which are incorporated herein by reference in their entirety. www.rsc.org / suppdata / mt / c4 / c4mt00112e / c4mt00112e1.pdf These include the sulforhodamine B (SRB) assay, and the neutral red (NR) assay as described in

[0063] The effectiveness of CIK NKT cells in killing target cells can be assessed by EC50. As used herein, EC50 refers to the effector-to-target ratio used in the assay when 50% of the target cells are killed. In some embodiments, CIK NKT cells can kill multiple target cells with an EC50 of 1-10, e.g., 1-8, 2-6, 2-5.5, or 3-7. In some embodiments, the target cells are THP-1, and the EC50 is 4.64. In some embodiments, the target cells are DAOY, and the IC50 is 3.69. In some embodiments, the target cells are K562, and the IC50 is 2.6.

[0064] Modification of CIK NKT cells Chimeric Antigen Receptor The CIK NKT cells generated as described above can be further engineered to express chimeric antigen receptor (CAR) on the cell surface.Optionally, the CAR is specific for tumor-specific antigen.Tumor-specific antigens are described, for example and not limited to, in US 2013 / 0189268; WO 1999024566 A1; US ​​7098008; and WO 2000020460 A1, each of which is incorporated herein by reference in its entirety. Tumor-specific antigens include, but are not limited to, NKG2D, CS1, GD2, CD138, EpCAM, EBNA3C, GPA7, CD244, CA-125, ETA, MAGE, CAGE, BAGE, HAGE, LAGE, PAGE, NY-SEO-1, GAGE, CEA, CD52, CD30, MUC5AC, c-Met, EGFR, FAB, WT-1, PSMA, NY-ESO1, AFP, CEA, CTAG1B, CD19, and CD33. Additional non-limiting tumor-associated antigens and their associated malignancies can be found in Table 1.

[0065] Table 1. Tumor-specific antigens and associated malignancies TIFF2025015816000002.tif31142TIFF2025015816000003.tif242160TIFF2025015816000004.tif92160

[0066] In some embodiments, the CAR targets CD19, CD33, or CSPG-4.

[0067] In examples, variant polypeptides are made using methods known in the art, such as oligonucleotide-mediated (site-directed) mutagenesis, alanine scanning, and PCR mutagenesis. Site-directed mutagenesis (Carter, 1986; Zoller and Smith, 1987), cassette mutagenesis, restriction-selection mutagenesis (Wells et al., 1985), or other known techniques can be performed on cloned DNA to generate CD16 variants (Ausubel, 2002; Sambrook and Russell, 2001).

[0068] Optionally, the CAR targets an antigen associated with a specific cancer type. Optionally, the cancer is selected from the group consisting of leukemia (including acute leukemia (e.g., acute lymphocytic leukemia, acute myeloid leukemia (including myeloblastic, promyelocytic, myelomonocytic, monocytic, and erythroleukemia)) and chronic leukemia (e.g., chronic myeloid (granulocytic) leukemia and chronic lymphocytic leukemia)), polycythemia vera, lymphoma (e.g., Hodgkin's disease and non-Hodgkin's disease), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, including, but not limited to, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, chordoma, angiosarcoma, endothelial sarcoma, lymphangiosarcoma, and solid tumors, including sarcomas and carcinomas such as lymphangiosarcoma, synovium, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatocellular carcinoma, cholangiocarcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testicular tumor, lung cancer, small cell lung cancer, bladder cancer, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, and retinoblastoma.

[0069] In some embodiments, the polynucleotide encoding CAR is mutated to change the amino acid sequence encoding CAR without changing the function of CAR.For example, polynucleotide substitutions that result in amino acid substitutions at "non-essential" amino acid residues can be carried out in the CAR disclosed above.CAR can be engineered as described in, for example, patent publication numbers WO 2014039523; US 20140242701; US ​​20140274909; US 20130280285; and WO 2014099671, each of which is incorporated herein by reference in its entirety.Optionally, CAR is CD19 CAR, CD33 CAR, or CSPG-4 CAR.

[0070] Further modifications - cytokines In some embodiments, the CAR-expressing CIK NKT cells are further modified to express at least one cytokine. In certain embodiments, the at least one cytokine is IL-2, IL-12, IL-15, IL-18, IL-21, or a variant thereof. In preferred embodiments, the cytokine is IL-12. A representative polypeptide of IL-12 is available under Accession No. IF45_A ( https: / / www.ncbi.nlm.nih.gov / protein / 1F45_A ) and the amino acid sequence set forth in accession number IF45_B (https: / / www.ncbi.nlm.nih.gov / protein / 1F45_B).

[0071] therapeutic application The present disclosure also provides a method for treating any type of cancer in a subject at any stage. Non-limiting examples of suitable cancers include carcinoma, melanoma, or sarcoma. In some embodiments, the present invention is used to treat cancers of hematopoietic origin, such as leukemia or lymphoma. In some embodiments, the cancer is a solid tumor.

[0072] In some embodiments, the method for treating any type of cancer in a subject comprises administering a therapeutically effective amount of CIK NKT cells to the patient, thereby treating the cancer. The CIK NKT cells are derived from a population of CIK NKT cells, wherein more than 90% of the cells in the population express CD56 and CD3, and less than 10% of the cells in the population express Va24.

[0073] The present disclosure also provides a method for treating any type of viral infection, comprising administering to a patient a therapeutically effective amount of CIK NKT cells, thereby treating cancer. The CIK NKT cells are derived from a population of CIK NKT cells, wherein more than 90% of the cells in the population express CD56 and CD3, and less than 10% of the cells in the population express Va24.

[0074] Also provided is a method for treating a subject in need thereof using the CIK NKT cells described herein. In some embodiments, the subject or patient is suffering from cancer or an infectious disease, such as a viral infection.

[0075] CIK NKT cells can be administered to an individual in absolute numbers of cells, e.g., from about 1000 cells / injection up to about 10 billion cells / injection, e.g., about, at least about, or at most about 1 x 10 cells / injection. 8 , 1×10 7 , 5×10 7 , 1×10 6 , 5×10 6 , 1×10 5 , 5×10 5 , 1×10 4 , 5×10 4 , 1×10 3 , 5×10 3 (and so forth) CIK NKT cells, or any range including the endpoints between any two numbers. Thus, the present disclosure also provides for administering a cell count of 1×10 8 , 1×10 7 , 5×10 7 , 1×10 6 , 5×10 6 , 1×105 , 5×10 5 , 1×10 4 , 5×10 4 , 1×10 3 , or 5 × 10 3 The present invention provides a composition comprising a plurality of CIK NKT cells, such as 10 CIK NKT cells.

[0076] In other embodiments, the individual receives about 1000 cells / injection / ml 2 up to approximately 10 billion cells / injection / m 2 up to, e.g., about, at least about, or at most about 1 x 10 per injection 8 pieces / m 2 , 1×10 7 pieces / m 2 , 5×10 7 pieces / m 2 , 1×10 6 pieces / m 2 , 5×10 6 pieces / m 2 , 1×10 5 pieces / m 2 , 5×10 5 pieces / m 2 , 1×10 4 pieces / m 2 , 5×10 4 pieces / m 2 , 1×10 3 pieces / m 2 , 5×10 3 pieces / m 2 (etc.) CIK NKT cells, or any range including the endpoints between any two numbers, can be administered.

[0077] In other embodiments, CIK NKT cells can be administered to such individuals in relative numbers of cells, e.g., from about 1000 cells per kilogram of the individual up to about 10 billion cells per kilogram of the individual, e.g., about, at least about, or at most about 1 x 10 cells per kilogram of the individual. 8 , 1×10 7 , 5×10 7 , 1×10 6 , 5×10 6 , 1×10 5 , 5×10 5 , 1×104 , 5×10 4 , 1×10 3 , or 5 x 10 3 (and so on) CIK NKT cells, or any range between any two numbers, including the endpoints, can be administered.

[0078] In other embodiments, the total dose is expressed as m of body surface area. 2 It can be calculated as follows: 1 m 2 Approximately 1 x 10 11 , 1×10 10 , 1×10 9 , 1×10 8 , 1×10 7 The average person is about 1.6 to 1.8 meters tall. 2 In a preferred embodiment, about 1 billion to about 3 billion CIK NKT cells are administered to a patient. In another embodiment, the amount of CIK NKT cells injected per dose is determined based on the number of cells per m² of body surface area. 2 It can be calculated as follows: 1 m 2 1x10 11 , 1×10 10 , 1×10 9 , 1×10 8 , 1×10 7 The average human body surface area is 1.6-1.8 m 2 is.

[0079] In other embodiments, CIK NKT cells can be administered to such individuals in relative numbers of cells, e.g., from about 1000 cells per kilogram of the individual up to about 10 billion cells per kilogram of the individual, e.g., about, at least about, or at most about 1 x 10 cells per kilogram of the individual. 8 , 1×10 7 , 5×10 7 , 1×10 6 , 5×10 6 , 1×10 5 , 5×10 5 , 1×10 4 , 5×10 4 , 1×10 3 , or 5 x 103 (and so on) CIK NKT cells, or any range between any two numbers, including the endpoints, can be administered.

[0080] The CIK NKT cells can be administered once to a cancer patient, or multiple times, for example, once every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 hours, or once every 1, 2, 3, 4, 5, 6, or 7 days, or once every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more weeks, or any range including the endpoints between any two numbers, throughout the treatment period.

[0081] In some embodiments, the CIK NKT cells are administered in a composition comprising the CIK NKT cells and a vehicle, such as human serum or its equivalent. In some embodiments, the vehicle comprises human serum albumin. In some embodiments, the vehicle comprises human plasma. In some embodiments, the vehicle comprises about 1% to about 15% human serum or a human serum equivalent. In some embodiments, the vehicle comprises about 1% to about 10% human serum or a human serum equivalent. In some embodiments, the vehicle comprises about 1% to about 5% human serum or a human serum equivalent. In preferred embodiments, the vehicle comprises about 2.5% human serum or a human serum equivalent. In some embodiments, the serum is human AB serum. In some embodiments, a serum substitute acceptable for use in human therapy is used in place of human serum. Such serum substitutes are known in the art or may be developed in the future. It is contemplated that concentrations greater than 15% human serum can be used, but concentrations greater than about 5% are cost-prohibitive. In some embodiments, the CIK NKT cells are administered in a composition comprising the CIK NKT cells and an isotonic solution that supports cell viability. In some embodiments, the CIK NKT cells are administered in a composition reconstituted from a cryopreserved sample.

[0082] Pharmaceutically acceptable compositions containing CIK NKT cells can contain various carriers and excipients. Various aqueous carriers, such as buffered saline and the like, can be used. These solutions are sterile and generally free of undesirable substances. Suitable carriers and excipients and their formulations are described in Remington: The Science and Practice of Pharmacy, 21st Edition, David B. Troy, ed., Lippicott Williams & Wilkins (2005). A pharmaceutically acceptable carrier refers to a material that is not biologically or otherwise undesirable; that is, the material can be administered to a subject without causing undesirable biological effects or interacting in a deleterious manner with other components of the pharmaceutical composition in which it is contained. When administered to a subject, the carrier is optionally selected to minimize degradation of the active ingredient and minimize adverse side effects in the subject. As used herein, the term "pharmaceutically acceptable" is used synonymously with "physiologically acceptable" and "pharmacologically acceptable." Pharmaceutical compositions generally include agents for buffering and preservation during storage, and depending on the route of administration, may include buffers and carriers for appropriate delivery.

[0083] These compositions for in vivo or in vitro use can be sterilized by the sterilization technique used for cells.The composition can contain acceptable auxiliary substances required to approach physiological conditions, such as pH adjusting and buffering agents, toxicity adjusting agents, and the like, for example, sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, and the like.The concentration of cells and / or other active substances in these formulations can vary and is selected mainly based on fluid volume, viscosity, body weight, and the like, according to the specific administration method selected and the needs of the subject.

[0084] In one embodiment, the CIK NKT cells are administered to a patient in combination with one or more other treatments or agents for the cancer being treated. In some embodiments, the one or more other treatments for the cancer being treated include, for example, antibodies, radiation, chemotherapy, stem cell transplantation, or hormone therapy.

[0085] In some embodiments, the CIK NKT cells and the other cancer agent / treatment are administered simultaneously or nearly simultaneously (e.g., within about 1, 5, 10, 15, 20, or 30 minutes of each other). In some embodiments, the CIK NKT cells and the other cancer agent / treatment are administered sequentially. In some embodiments, the other cancer treatment / agent is administered 1 day, 2 days, or 3 days after administration of the CIK NKT cells.

[0086] In one embodiment, the other cancer drug is an antibody. In one embodiment, CIK NKT cells are administered in combination with an antibody that targets diseased cells. In one embodiment, CIK NKT cells and antibodies can be administered to patients together, for example, in the same formulation; separately, for example, in separate formulations, simultaneously; or separately, for example, with different administration schedules or at different times of the day. When administered separately, the antibody can be administered via any suitable route, such as intravenous or intratumoral injection.

[0087] In some embodiments, the CIK NKT cells of the present disclosure are used in combination with therapeutic antibodies and / or other anti-cancer drugs. Therapeutic antibodies can be used to target cells expressing cancer- or tumor-associated markers. Examples of monoclonal antibodies for cancer therapy are listed in Table 2.

[0088] Table 2: Exemplary therapeutic monoclonal antibodies TIFF2025015816000005.tif100160TIFF2025015816000006.tif234160TIFF2025015816000007.tif57160

[0089] Such administration of CIK NKT cells can be performed simultaneously with or sequentially to administration of the monoclonal antibody. In some embodiments, the CIK NKT cells are administered to a subject after the subject has been treated with the monoclonal antibody. Alternatively, the CIK NKT cells can be administered simultaneously with the monoclonal antibody, e.g., within 24 hours.

[0090] In some embodiments, the CIK NKT cells are administered intravenously. In some embodiments, the CIK NKT cells are infused directly into the bone marrow.

[0091] Accordingly, the present disclosure provides a method of treating cancer or a viral infection in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of CIK NKT cells derived from a population of CIK NKT cells using the methods disclosed herein, thereby treating the cancer.

[0092] kit Also disclosed is a kit for treating cancer or infectious diseases using a composition comprising an amount of CIK NKT cells as described herein. In some embodiments, the kit of the present disclosure may also include at least one monoclonal antibody.

[0093] In certain embodiments, the kit may include additional compounds, such as therapeutically active compounds or drugs, to be administered prior to, concurrently with, or after administration of the CIK NKT cells. Examples of such compounds include antibodies, vitamins, minerals, fludrocortisone, ibuprofen, lidocaine, quinidine, chemotherapeutic agents, etc.

[0094] In various embodiments, the kit's instruction manual includes instructions for using the kit's components in the treatment of cancer or infectious diseases. The instruction manual may further include information on how to handle (e.g., thaw and / or culture) CIK NKT cells. The instruction manual may further include guidance on dosage and frequency of administration.

[0095] In certain embodiments, the kit further comprises one or more containers filled with one or more compositions described herein, for example, compositions comprising CIK NKT cells as described herein. Optionally, such containers can be accompanied by a label indicating that the kit is for treating cancer as described herein. Optionally, the label also includes a notice in the form prescribed by a government agency regulating the manufacture, use, or sale of pharmaceuticals or biological products, which notice reflects the approval by the government agency for manufacture, use, or sale for human administration.

[0096] Although the present disclosure and examples illustrate the generation and use of CIK NKT cells derived from umbilical cord blood samples, those skilled in the art will recognize that CIK NKT cells can also be generated from other hematopoietic progenitor cell samples using approaches similar to those described herein.

[0097] Disclosed are materials, compositions, and components that can be used in, in conjunction with, used in preparation for, or are products of the disclosed methods and compositions. These and other materials are disclosed herein, and when combinations, subsets, interactions, groups, etc. of these materials are disclosed, it is understood that each is specifically contemplated and described herein, even though specific reference to various individual and collective combinations and permutations of these compounds may not be explicitly disclosed. For example, if a method is disclosed and discussed, and several modifications that can be made to several molecules comprising that method are discussed, any and all combinations and permutations of the method and possible modifications are specifically contemplated unless specifically indicated otherwise. Similarly, any subset or combination of these is also specifically contemplated and disclosed. This concept applies to all aspects of the present disclosure, including, but not limited to, steps in methods using the disclosed compositions. Thus, if there are various additional steps that can be performed, it is understood that each of these additional steps can be performed with any particular method step or combination of method steps of the disclosed methods, and that each such combination or subset of combinations is to be considered specifically contemplated and disclosed.

[0098] Example The present disclosure includes the following non-limiting aspects. Aspect 1. A population of CIK NKT cells, wherein greater than 50% of the cells in the population express CD56 and CD3, and less than 10% of the cells in the population express Va24. Aspect 2. The population of CIK NKT cells of embodiment 1, wherein the CIK NKT cells are capable of killing target cells in the absence of α-galactosylceramide (Gal-Cer). Aspect 3. The population of CIK NKT cells of embodiment 1, wherein the target cells are cancer cells. Aspect 4. The population of CIK NKT cells of embodiment 1, wherein the cancer cell line is selected from the group consisting of myeloid leukemia cells, medulloblastoma cells, and monocytic cells. Aspect 5. The population of CIK NKT cells of embodiment 3, wherein the cancer cells are selected from the group consisting of K562 cells, Daudi cells, DAOY cells, and THP-1 cells. Aspect 6. 6. The population of CIK NKT cells of embodiments 2 to 5, wherein the CIK NKT cells kill a plurality of target cells with an EC50 of 1.0 to 10.0. Aspect 7. 7. The population of CIK NKT cells of embodiment 6, wherein the CIK NKT cells are capable of killing target cells with an EC50 that is at least 90% and not more than 110% of the EC50 at which the CIK NKT cells kill the target cells in the presence of Gal-Cer. Aspect 8. A composition comprising a plurality of CIK NKT cells derived from the population of CIK NKT cells of any of aspects 1 to 7, and a physiologically acceptable excipient. Aspect 9. A kit for treating cancer, comprising a plurality of CIK NKT cells derived from the population of CIK NKT cells of any of aspects 1 to 7, the kit further comprising a container and / or a label indicating that the kit is for treating cancer. Aspect 10. 1. A method for enriching CIK NKT cells from an umbilical cord blood sample, comprising the steps of: isolating mononuclear cells from the cord blood sample; and Contacting the isolated mononuclear cells with one or more agents selected from the group consisting of IL-7, ALT-803 or IL-15, FLT3 ligand, and Gal-Cer, thereby enriching for CIK NKT cells. Aspect 11. The method of embodiment 10, wherein IL-7, if present, is at a concentration in the range of 5 to 20 ng / mL. Aspect 12. 12. The method of embodiment 10 or 11, wherein ALT-803, if present, is at a concentration in the range of 100 to 300 ng / mL. Aspect 13. The method of any of embodiments 10 to 12, wherein the FLT3 ligand, if present, is at a concentration in the range of 5 to 20 ng / mL. Aspect 14. The method of any of embodiments 10 to 13, wherein Gal-Cer is present at a concentration in the range of 2 to 10 μg / mL. Aspect 15. Isolating enriched CIK NKT cells from the remainder of the cord blood sample. 11. The method of embodiment 10, further comprising: Aspect 16. contacting the isolated CIK NKT cells with anti-CD3, anti-CD28, and IL2 to expand the CIK NKT cells. 16. The method of embodiment 15, further comprising: Aspect 17. contacting the isolated CIK NKT cells with Gal-Cer 17. The method of embodiment 16, further comprising: Aspect 18. The method of embodiment 17, wherein the Gal-Cer is present in the form of a Gal-Cer-loaded CD1d tetramer. Aspect 19. The method of embodiment 16, wherein the anti-CD3 antibody is present in an amount of 5 ng / mL to 60 ng / mL. Aspect 20. 20. The method of any of embodiments 16-19, wherein the anti-CD28 antibody is present in an amount of 0.1 μg / mL to 2 μg / mL. Aspect 21. The method of any of embodiments 16-20, wherein IL-2 is present at a concentration of 50 ng / mL to 500 ng / mL. Aspect 22. The method of any of aspects 16 to 21, wherein the generation of CIK NKT cells does not involve interferon-γ. Aspect 23. 1. A method of treating cancer or a viral infection in a patient in need thereof, comprising: Administering a therapeutically effective amount of CIK NKT cells derived from the population of CIK NKT cells of any of embodiments 1 to 7 to a patient, thereby treating cancer. Aspect 24. Patient's body surface area 1 m 2 Approximately 1 x 10 8 ~Approx. 1×10 11 The method of embodiment 23, wherein the cells are administered to the patient. Aspect 25. 24. The method of embodiment 23, wherein the cancer is selected from the group consisting of leukemia, lymphoma, polycythemia vera, multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, sarcoma, and carcinoma. Aspect 26. 26. The method of embodiment 25, wherein said cells are administered to the patient by a route selected from the group consisting of intravenous, intraperitoneal, and subcutaneous. Aspect 27. 27. The method of any of embodiments 23 to 26, further comprising administering an antibody. Aspect 28. A population of CIK NKT cells generated by the method of any one of aspects 10 to 27. Aspect 29. The population of CIK NKT cells of embodiment 1, wherein the CIK NKT cells express a CAR and / or a cytokine. [Example]

[0099] The following examples are for illustrative purposes only and should not be construed as limiting. There are a variety of alternative techniques and procedures available to those skilled in the art that could similarly successfully perform the following examples.

[0100] Example 1: Phenotype of CIK-NKT cells Umbilical cord blood mononuclear cells (Comics) were isolated from cord blood samples by density gradient analysis using Ficoll-Paque and Sepmate™ (Stem cell Technology Inc.). Comics were incubated in RPMI medium containing 10% human AB serum for 2 weeks with Gal-Cer (5 μg / mL), FLT3-L (10 ng / mL), IL-7 (10 ng / mL), and ALT-803 (175 ng / mL) to enrich for NKT cells. NKT cells were isolated by affinity chromatography using reagents from Miltenyi Biotec, Germany. Isolated cells were then expanded in NK Macs medium containing 10% human AB serum in the presence of anti-CD3 antibody (20 ng / mL), anti-CD28 antibody (0.5 μg / mL), and IL-2 (200 ng / mL) and activated overnight with Gal-Cer-loaded CD1d tetramer. Gal-Cer-loaded CD1d tetramers were from ProImmune, Oxford, UK, and were used at a concentration of 100 ng / mL Gal-Cer. After 1 week of expansion, the cells were stained with antibodies recognizing CD3, CD56, or Va24. Results showed that the majority of cells (97.1%) were positive for both CD56 and CD3 (Figure 2B), and a small percentage of cells (6.45%) were positive for Va24 (Figure 2C). This indicates that cord blood CIK NKT cells express low levels of Va24, but retain intact CD3 and CD56 expression.

[0101] Example 2: Cytotoxicity of CIK NKT cells is independent of Gal-Cer NKT-CIK cells prepared as described in Example 2 were evaluated for cytotoxicity against the cancer cell line DAOY. DAOY cells were incubated overnight with 1 μg / mL Gal-Cer. Gal-Cer-treated DAOY cells were then incubated with the fluorescent dye, Calcien AM, for 30 minutes. The cells were washed and then incubated with CB-NKT CIK for 4 hours at various effector-to-target ratios (the highest ratio was 32:1) as indicated. DAOY cell killing was measured by cell lysis, expressed as the amount of Calcien AM dye released from the cells. Figure 3A shows the amount of Calcien AM dye released from DAOY cells induced by CB-NKT CIK. Data were expressed as the percent lysis of target cells. The results showed a significant difference in killing between the group in which target cells were loaded with Gal-Cer and the group in which they were not loaded with Gal-Cer, suggesting that Gal-Cer treatment did not confer killing specificity.

[0102] Figure 3B compares the cytotoxicity of cord blood-derived NKT-CIK cells (CB-CIK NKT cells) and PBiNKT cells (iNKT cells isolated from peripheral blood) against luciferase-expressing THP1 cells. PBiNKT cells were isolated in a manner similar to that of CB-CIK NKT cells (see Example 1), except that the source was peripheral blood instead of cord blood. THP1 cells were cocultured with CB-CIK NKT cells or PBiNKT cells at various effector-to-target ratios, as indicated, with the highest ratio being 32:1. Gal-Cer was not used in this experiment. The results show that CB-CIK NKT cells are more potent at killing THP-1 cells than PBiNKT cells.

[0103] Example 3: Cytotoxicity of CIK NKT cells against K562 cells and Daudi cells The CB-NKT CIK cells obtained as described above were assayed for their ability to kill luciferase-expressing cancer cell lines, DAOY, Daudi, and K562 (represented by triangles, squares, and circles, respectively, in Figure 4). iNKT cells isolated from peripheral blood were used as a control (inverted triangles in Figure 4). Cell killing assays were performed after 4 hours of coculture of cancer cells with effector cells (CB CIK NKT or PBiNKT). Cancer cell killing was measured by the percentage of cell lysis, as represented by the loss of luciferase in these cancer cell lines. Various effector-to-target ratios were used as indicated, with the highest being 32:1. The results demonstrate that CB-CIK NKT cells kill target cells in a non-CD1d / MHC-restricted manner.

Claims

1. A population of CIK NKT cells, wherein greater than 50% of the cells in the population express CD56 and CD3, and less than 10% of the cells in the population express Va24.

2. The population of CIK NKT cells of claim 1, wherein the CIK NKT cells are capable of killing target cells in the absence of α-galactosylceramide (Gal-Cer).

3. The population of CIK NKT cells of claim 1, wherein the target cells are cancer cells.

4. 2. The population of CIK NKT cells of claim 1, wherein the cancer cell line is selected from the group consisting of myeloid leukemia cells, medulloblastoma cells, and monocytic cells.

5. The population of CIK NKT cells of claim 3, wherein the cancer cells are selected from the group consisting of K562 cells, Daudi cells, DAOY cells, and THP-1 cells.

6. 3. The population of CIK NKT cells of claim 2, wherein the CIK NKT cells kill multiple target cells with an EC50 of 1.0 to 10.

0.

7. The population of CIK NKT cells described in claim 6, wherein the CIK NKT cells are capable of killing target cells with an EC50 that is 90% or more and 110% or less of the EC50 when the CIK NKT cells kill the target cells in the presence of Gal-Cer.

8. A composition comprising a plurality of CIK NKT cells derived from the population of CIK NKT cells of any one of claims 1 and a physiologically acceptable excipient.

9. 11. A kit for treating cancer comprising a plurality of CIK NKT cells derived from the population of CIK NKT cells of any one of claims 1, the kit further comprising a container and / or a label indicating that the kit is for treating cancer.

10. 1. A method for enriching CIK NKT cells from an umbilical cord blood sample, comprising the steps of: isolating mononuclear cells from the cord blood sample; and Contacting the isolated mononuclear cells with one or more agents selected from the group consisting of IL-7, ALT-803 or IL-15, FLT3 ligand, and Gal-Cer, thereby enriching for CIK NKT cells.

11. 11. The method of claim 10, wherein IL-7, if present, is at a concentration in the range of 5 to 20 ng / mL.

12. 11. The method of claim 10, wherein ALT-803, if present, is at a concentration ranging from 100 to 300 ng / mL.

13. The method of claim 10, wherein the FLT3 ligand, if present, is at a concentration in the range of 5 to 20 ng / mL.

14. The method of claim 10, wherein Gal-Cer is present at a concentration ranging from 2 to 10 μg / mL.

15. Isolating enriched CIK NKT cells from the remainder of the cord blood sample.

11. The method of claim 10, further comprising:

16. contacting the isolated CIK NKT cells with anti-CD3, anti-CD28, and IL2 to expand the CIK NKT cells.

16. The method of claim 15, further comprising:

17. Contacting the isolated CIK NKT cells with Gal-Cer 17. The method of claim 16, further comprising:

18. The method of claim 17, wherein Gal-Cer is present in the form of a Gal-Cer-loaded CD1d tetramer.

19. 17. The method of claim 16, wherein the anti-CD3 antibody is present in an amount of 5 ng / mL to 60 ng / mL.

20. 17. The method of claim 16, wherein the anti-CD28 antibody is present in an amount of 0.1 μg / mL to 2 μg / mL.

21. 17. The method of claim 16, wherein IL-2 is present at a concentration of 50 ng / mL to 500 ng / mL.

22. The method of claim 16, wherein the generation of CIK NKT cells does not involve interferon-γ.

23. 1. A method of treating cancer or a viral infection in a patient in need thereof, comprising the steps of:

10. Administering to a patient a therapeutically effective amount of CIK NKT cells derived from the population of CIK NKT cells according to any one of claims 1 to 7, thereby treating cancer.

24. Patient's body surface area 1 m 2 Approximately 1 x 10 8 ~Approx. 1×10 11 24. The method of claim 23, wherein the cells are administered to the patient.

25. 24. The method of claim 23, wherein the cancer is selected from the group consisting of leukemia, lymphoma, polycythemia vera, multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, sarcoma, and carcinoma.

26. 26. The method of claim 25, wherein the cells are administered to the patient by a route selected from the group consisting of intravenous, intraperitoneal, and subcutaneous.

27. 24. The method of claim 23, further comprising administering an antibody.

28. A population of CIK NKT cells generated by the method of claim 10.

29. The population of CIK NKT cells of claim 1, wherein the CIK NKT cells express a CAR and / or a cytokine.

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