Production of CIK NKT cells from umbilical cord blood

Abstract

Provided herein are methods and customized culture medium compositions for culturing CIK NKT cells.

Description

[0001] This application is a divisional application of an invention patent application with an application date of July 1, 2019, application number 201980046254.8, and invention name “Generating CIK NKT cells from umbilical cord blood”.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims priority to U.S. Provisional Application No. 62 / 696,131, filed on July 10, 2018, which is incorporated herein by reference. Background Art

[0004] Natural killer T cells (NKT cells) represent a subset of T lymphocytes that express natural killer (NK) cell surface markers. A subset of NKT cells, called invariant NKT cells (iNKT), expresses a highly restricted T cell receptor (TCR). Although iNKT cells play an important role in connecting innate and adaptive immune responses and have been implicated in a variety of diseases, such as infectious diseases, allergies, asthma, autoimmunity, and tumor monitoring (Juno et al. PLoS Pathog. 2012; 8 (8)), their activation generally 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 using these NKT cells to treat patients and in some cases limit the scope of their therapeutic use. Therefore, there is still a need for safe and cost-effective NKT cell therapies that can be used to treat patients with a wide range of tissue types. Summary of the invention

[0005] Provided herein are CIK NKT cells that can be activated in the absence of Gal-Cer. In some embodiments, greater than 50% of the cells in the CIK NKT cell population express CD 56 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. Also provided are methods of producing the CIN NKT cells and using the cells to treat cancer.

[0006] 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.

[0007] Optionally, the population of CIK NKT cells can kill target cells in the absence of alpha-galactosylceramide (Gal-Cer). Optionally, the target cells are cancer cells. The cancer cell line can be selected from myeloid leukemia cells, medulloblastoma cells and monocytes. Optionally, the cancer cells are selected from K562 cells, Daudi cells, DAOY cells and THP-1 cells.

[0008] Optionally, the CIK NKT cells kill a plurality of the target cells with an EC50 between 1.0 and 10.0. Optionally, the CIK NKT cells can kill the target cells with an EC50 that is not less than 90% of the EC50 at which the CIK NKT cells kill the target cells in the presence of Gal-Cer and not more than 110% of the EC50.

[0009] The present disclosure also provides a composition comprising a plurality of CIK NKT cells from any one of the above-mentioned CIK NKT cell populations and a physiologically acceptable excipient.

[0010] Also provided is a kit for treating cancer, comprising a plurality of CIK NKT cells from any one of the above CIK NKT cell populations and a container and / or a label indicating that the kit is for treating cancer.

[0011] Also provided is a method for enriching CIK NKT cells from an umbilical cord blood sample, the method comprising: isolating monocytes from the umbilical cord blood sample; and contacting the isolated monocytes with one or more agents selected from IL-7, ALT-803 or IL-15, FLT3 ligand and Gal-Cer, thereby enriching CIK NKT cells. IL-7, if present, can be in a concentration range of 5 to 20 ng / mL. ALT-803, if present, can be in a concentration range of 100 to 300 ng / mL. FLT3 ligand, if present, can be in a concentration range of 5 to 20 ng / mL. Gal-Cer, if present, can be in a concentration range of 2 to 10 μg / mL.

[0012] Optionally, the method further comprises separating the enriched CIK NKT cells from the rest of the cord blood sample. Optionally, the method further comprises contacting the isolated CIK NKT cells with anti-CD3, anti-CD28 and IL2 to amplify the CIK NKT cells. Optionally, the method further comprises contacting the isolated CIK NKT cells with Gal-Cer. Optionally, the Gal-Cer is used in the form of a CD1d tetramer loaded with Gal-Cer. Optionally, the anti-CD3 antibody is present in an amount of 5ng / mL to 60ng / mL. Optionally, the anti-CD28 antibody is present in an amount of 0.1μg / mL to 2μg / mL. Optionally, IL-2 is present in a concentration of 50ng / mL to 500ng / mL. Optionally, the enrichment and / or amplification of CIK NKT cells does not include interferon-γ.

[0013] Also provided is a population of CIK NKT cells produced by the above method for enriching, isolating and expanding CIK NKT cells.

[0014] Also provided is a method for treating cancer or viral infection in a patient in need thereof, the method comprising administering to the patient a therapeutically effective amount of CIK NKT cells from any one of the CIK NKT cell populations described above, thereby treating cancer. Optionally, about 1×10 8 About 1x10 11 The method further comprises administering an antibody 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 via a route selected from intravenous, intraperitoneal, and subcutaneous. Optionally, the method further comprises administering an antibody.

[0015] Also provided is a population of CIK NKT cells as described in claim 1, wherein the CIK NKT cells express CAR and / or cytokines, and greater than 50% of the cells in the CIK NKT cell population express CD 56 and CD3 and less than 10% of the cells in the population express Va24.

[0016] 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 be apparent to those skilled in the art. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] The objects, features and advantages will be more readily understood with reference to the following disclosure when considered in conjunction with the accompanying drawings.

[0018] Figure 1 Schematic diagram of the pathway used by CIK NKT cells to kill target cells.

[0019] Figure 2A Shown are the results of flow cytometric analysis of CIK NKT cells stained with CD3, CD56, and Va24. Figure 2A are the forward scatter plot and the size scatter plot; Figure 2B CD3 and CD56 plots are shown; and Figure 2C A Va24 map is shown.

[0020] Figure 3A Killing of DAOY cells by cord blood CIK NKT cells in the presence (indicated by circles) or absence (indicated by squares) is shown. Figure 3B Killing of luciferase-expressing THP1 cells by cord blood CIK NKT cells (indicated by squares) or peripheral blood iNKT cells (indicated by circles) is shown.

[0021] Figure 4 The killing of K562 cells, DAOY cells and Daudi cells by umbilical cord blood CIK NKT cells is shown. DETAILED DESCRIPTION

[0022] Overview

[0023] The present disclosure provides cytokine-induced killer NKT cells (CIK NKT cells), which can kill target cells in a non-CD1d restricted manner (e.g., independent of the formation of Gal-Cer / CD1d tetramers). CIK NKT cells can be used to target a wide range of target cells and will not trigger graft-versus-host disease (GVHD). GVHD occurs due to the invasive ability of lymphocytes to infiltrate organs (e.g., intestines, skin, and liver) and cause a large amount of inflammation therein. It has been shown that CIK NKT does not express chemokine receptors that are important for targeting GVHD organs but expresses receptors that help to home to tumors, so they do not trigger GVHD. As compared with the prior art of generating CIK NKT cells (which generally produces a heterogeneous population consisting of CD3+, CD56+ single positive cells and CD3+ / CD56+ double positive cells, and the double positive cells usually account for 30% or less), the present method produces a cell population consisting mainly of CD3+ / CD56+ double positive cells. In one aspect, at least 50% of CIK NKT cells in the population express both CD56 and CD3 and less than 10% of cells in the population express Va24. The method for producing CIK NKT cells does not require exposing cells to interferon-γ, which saves costs. Therefore, the present application provides a safe and cost-effective NKT cell therapy that can be widely used to treat various diseases (e.g., cancer) without causing clinical adverse reactions (e.g., GVHD).

[0024] The present disclosure also provides compositions and kits comprising a plurality of CIK NKT cells from the population. Also provided are methods of producing the CIK NKT cells and using the CIK NKT cells to treat cancer.

[0025] the term

[0026] Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art.

[0027] In this specification and the claims that follow, reference will be made to a number of terms which shall be defined to have the following meanings:

[0028] 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.

[0029] All numerical designations (e.g., pH, temperature, time, concentration, amount, and molecular weight, including ranges) are approximate values ​​varying in increments of 0.1 or 1.0 ((+) or (-)) where appropriate. It should be understood that all numerical designations may be preceded by the term "about", although not always explicitly stated.

[0030] As used herein, when used to indicate the presence of a particular cell marker, "+" means that the cell marker is detectably present in fluorescence activated cell sorting relative to an isotype control; or detectable above background in quantitative or semiquantitative RT-PCR.

[0031] As used herein, when used to indicate the presence of a particular cell marker, "-" means that the cell marker is not detectably present in fluorescence activated cell sorting relative to an isotype control; or cannot be detected above background in quantitative or semi-quantitative RT-PCR.

[0032] As will be understood by those skilled in the art, for any and all purposes, particularly in providing written descriptions, all ranges disclosed herein also encompass any and all possible sub-ranges and combinations of sub-ranges thereof. Any listed range can be easily considered to fully describe the same range that is decomposed into at least equal half, one-third, one-quarter, one-fifth, one-tenth, etc., and make it possible. As a non-limiting example, each range discussed herein can be easily decomposed into lower third, middle third, and upper third, etc. As will be understood by those skilled in the art, all languages ​​(such as "up to", "at least", "greater than", "less than", etc.) include the enumerated numbers, and refer to the ranges that can be subsequently decomposed into the sub-ranges discussed above. Finally, as will be understood by those skilled in the art, ranges include each individual member. Therefore, for example, a group with 1-3 cells refers to a group with 1, 2, or 3 cells. Similarly, a group with 1-5 cells refers to a group with 1, 2, 3, 4, or 5 cells, and so on.

[0033] It is also understood, although not always explicitly stated, that the reagents described herein are exemplary only and that equivalents thereof are known in the art.

[0034] "Optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0035] The term "comprising" is intended to mean that the compositions and methods include the recited elements, but not excluding others. When used to define compositions and methods, "consisting essentially of" shall mean excluding other elements that have any substantial significance to the composition. For example, a composition consisting essentially of the elements defined herein will not exclude other elements that would not substantially affect the basic and novel features of the claims. "Consisting of" shall mean excluding more than trace amounts of other ingredients and substantial method steps. Embodiments defined by each of these transition terms are within the scope of the present disclosure.

[0036] As used herein, the terms "cytotoxicity" and "cytolytic" are synonymous when used to describe the activity of effector cells (e.g., NK cells). Typically, cytotoxic activity involves killing of target cells by any of a variety of biological, biochemical, or biophysical mechanisms. Cytolysis more specifically refers to the activity of effectors cleaving the cytoplasmic membrane of target cells to destroy their physical integrity. This results in killing of target cells. Without wishing to be bound by theory, it is believed that the cytotoxic effect of NK cells is due to cytolysis.

[0037] The term "killing" with respect to a cell / cell population is intended to include any type of manipulation that results in the death of that cell / cell population.

[0038] The term "cytokine" or "cytokines" refers to a general class of biomolecules that affect cells of the immune system. Exemplary cytokines include, but are not limited to, FLT2 ligand, interferons, and interleukins (IL), particularly IL-2, IL-12, IL-15, IL-18, and IL-21.

[0039] The terms "patient," "subject," "individual," and the like are used interchangeably herein and refer to any animal or cell thereof (whether in vitro or in situ) suitable for the methods described herein. In certain non-limiting embodiments, the patient, subject, or individual is a human.

[0040] The term "treating / treatment" encompasses treatment of a disease or condition described herein in a subject (e.g., a human), and includes: (i) inhibiting a disease or condition, i.e., preventing its development; (ii) alleviating a disease or condition, i.e., causing regression of the disease; (iii) alleviating the development of a condition; and / or (iv) inhibiting, alleviating, or slowing the development of one or more symptoms of a disease or condition. "Administering" a monoclonal antibody or natural killer cell (term) to a subject includes any route of introducing or delivering the antibody or cell to perform the intended function. Administration can be achieved by any route suitable for cell or monoclonal antibody delivery. Thus, the delivery route can include intravenous, intramuscular, intraperitoneal, or subcutaneous delivery. In some embodiments, CIK NKT cells are directly administered to a tumor, for example, by injection into the tumor. In some embodiments, the modified CIK NKT cells described herein are administered parenterally (e.g., by injection, infusion, or implantation (subcutaneous, intravenous, intramuscular, intracapsular, or intraperitoneal).

[0041] The term "expression" refers to the production of a gene product.

[0042] As used herein, the term "cytotoxicity" when used to describe the activity of effector cells (eg, NK cells) involves the killing of target cells by any of a variety of biological, biochemical, or biophysical mechanisms.

[0043] The terms "decrease / reduce / reduction / decrease" are used throughout this document to mean a decrease of at least 10% compared to a reference level, such as 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% or up to and including 100% (i.e., no level compared to a reference sample), or any range between 10-100% compared to a reference level.

[0044] The term "cancer" refers to all types of cancers, neoplasms or malignant tumors 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, soft ovarian cancer, sarcoma, gastric cancer, uterine cancer and medulloblastoma. Other examples include Hodgkin's disease, non-Hodgkin's lymphoma, multiple myeloma, neuroblastoma, ovarian cancer, rhabdomyosarcoma, essential thrombocythemia, primary macroglobulinemia, primary brain tumors, cancer, malignant insulinoma, malignant carcinoid, bladder cancer, precancerous skin lesions, testicular cancer, lymphoma, thyroid cancer, neuroblastoma, esophageal cancer, genitourinary tract cancer, malignant hypercalcemia, endometrial cancer, adrenocortical carcinoma, pancreatic endocrine and exocrine tumors and prostate cancer.

[0045] The term "therapeutically effective amount" or "effective amount" refers to the amount required to reduce the symptoms of a disease relative to an untreated patient. The effective amount of active compound used to implement the present disclosure to treat a disease depends 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 dosage regimen. Such an amount is referred to as an "effective" amount.

[0046] For the convenience of the reader, titles or subtitles may be used in the specification, which are not intended to affect the scope of the present disclosure. In addition, certain terms used in this specification are more specifically defined below.

[0047] Cytokine-induced killer natural killer T cells (CIK NKT cells)

[0048] Natural killer T cells are a heterogeneous group of T cells that share properties of T cells and natural killer cells. For example, NKT cells express the αβ T cell receptor, but also express various molecular markers commonly associated with NK cells, such as NKp44, NKp46, and NKp30. NKT cells constitute only about 0.1% of all peripheral blood T cells. NKT cells have been implicated in the suppression of autoimmunity and transplant rejection, the promotion of pathogen resistance, and the promotion of tumor immunity.

[0049] NKT cells are generally divided into type I and type II based on differences in T cell receptor (TCR) usage. Type I NKT cells (also often referred to as invariant NKT cells) are NKT cells that express a highly restricted T cell receptor that contains a constant TCR alpha chain, Va24.

[0050] 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 class II major histocompatibility complex (MHC) molecules, which present protein antigens to CD8+ and CD4+ T cells, respectively, CD1 molecules can capture and process foreign and self lipid antigens for display to T cells. Gal-Cer is typically derived from pathogenic cells (e.g., bacteria).

[0051] Type II NKT cells have a more diverse TCR repertoire and higher sequence diversity than type I NKT cells. Type II NKT cells do not respond to Gal-Cer, i.e., their activation is not dependent on the presence of Gal-Cer.

[0052] CIK NKT cells disclosed herein belong to the category of type II NKT cells. CIK NKT cells can be produced by cytokine induction. In some cases, they are produced from umbilical cord blood by, for example, cytokine induction. In some cases, they are produced in the process of enriching or separating iNKT cells from umbilical cord blood samples. However, CIK NKT cells are different from typical iNKT cells in many aspects. Phenotypically, unlike iNKT cells expressing Va24 (a marker of the α chain of the TCR receptor) (and the percentage of iNKT cells expressing Va24 can be 70% or higher), 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 entire population of CIK NKT cells. In terms of cytotoxicity, unlike iNKT cells activated by recognizing α-galactosylceramide (Gal-Cer), a glycolipid, Gal-Cer is not required to activate CIK NKT cells and the CIK NKT cells can kill cells in the absence of (Gal-Cer). See Figures 3 and Figure 4 .

[0053] Therefore, in terms of phenotype, CIK NKT cells provided herein generally have high expression levels of CD56 and CD3 and low expression of Va24. In some cases, at least 90% of the CIK NKT population generated by umbilical cord blood cells express CD56 and CD2, and less than 10% of the cells in the population express Vα24.

[0054] Methods for isolating and culturing CIK NKT cells

[0055] Collecting cord blood

[0056] Human umbilical cord blood has a high hematopoietic stem cell component and can be used as a source for producing CIK NKT cells. In order to collect umbilical cord blood, usually, the human placenta is harvested shortly after delivery. The placenta can be transported in a sterile, insulated transport device (for example, in a cord blood collection kit as described in U.S. Patent No. 7,147,626) (keeping the temperature of the placenta between 20-28°C). Preferably, the placenta is transported to the laboratory four to twenty-four hours after delivery.

[0057] The placenta can be subjected to a conventional cord blood collection process. The placenta is usually exsanguinated using a needle or cannula under gravity (see, e.g., Anderson, U.S. Pat. No. 5,372,581; Hessel et al., U.S. Pat. No. 5,415,665). The needle or cannula is usually placed into the umbilical vein and the placenta can be gently massaged to help drain the cord blood from the placenta. Such cord blood collection can be performed commercially, e.g., LifeBank Inc., Cedar Knolls, NJ, ViaCord, Cord Blood Registry, and CryoCell. Preferably, the placenta is drained by gravity without further manipulation to minimize tissue damage during the cord blood collection process.

[0058] Methods for collecting cord blood (e.g., as described in US20150225697) are well known. Cord blood mononuclear cells (COMICS) can be isolated from collected cord blood using methods known in the art (e.g., density gradients using Ficoll-Paque). Reagents suitable for isolating COMICS can be commercially available, for example, from Stem cell Technology Inc.

[0059] Enrichment of CIK NKT cells

[0060] Comics can be cultured in the presence of various cytokines for a period of time to enrich CIK NKT cells. Enrichment refers to increasing the percentage of the number of target cells in a heterogeneous cell population (e.g., Comics). The enrichment phase can be 2 days to 3 weeks, such as 1-2 weeks, 5-10 days, or about 2 weeks. Various growth media can be used, such as Roswell Park Memorial Institute (Roswell Park Memorial Institute) culture medium (RPMI) or Dulbecco's modified eagle medium (DMEM). Optionally, the culture medium further comprises human AB serum and / or Gal-Cer. Optionally, the human AB serum is present at 5-15% v / v (e.g., about 10% v / v). Optionally, the Gal-Cer is present at a concentration of 2-10 μg / mL (e.g., about 5 μg / mL). Suitable cytokines that can be added to the culture medium can include one or more cytokines selected from stem cell factor, FLT3 ligand, IL-7, and ALT-803 or IL-15. In some embodiments, FLT3 ligand is present at a concentration ranging from 5-20 ng / mL (e.g., 10 ng / mL); IL-7 is present at a concentration ranging from 5-20 ng / mL (e.g., 10 ng / mL); and / or ALT-803 is present at a concentration ranging from 100-300 ng / mL (e.g., about 175 ng / mL).

[0061] FLT3 ligand is a hematopoietic four-helix bundle cytokine and it 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 blood cell progenitor cells. It is the main growth factor that stimulates the growth of dendritic cells.

[0062] ALT-803 is a complex composed of human IL-15 mutant IL-15N72D (residue substitution at position 72) and IL-15Rαsushi-Fc fusion protein (see Zhu et al. J. Immunol. 2009; 183: 3598-607, the relevant disclosure of which is incorporated herein by reference).

[0063] CIK NKT cells can be separated from the above-mentioned enriched culture by methods well known in the art, such as incubating the enriched culture with magnetic beads coupled to antibodies against the Va24-J18 chain of TCR, so that the CIK NKT cells will be bound to the magnetic beads, and then separating the CIK NKT cells bound to the beads in the presence of a magnetic field. An example of a suitable antibody that can be used is the 6B11 antibody, which can be commercially available from a supplier (e.g., Biolegend (San Diego, CA)). Suitable reagents for separating CIK NKT cells are from Miltenyi Biotec, Germany. CIK NKT cells are usually separated in serum containing PBC (e.g., human AB serum). Optionally, the separation solution also includes EDTA.

[0064] Therefore, the present invention provides a method for enriching CIK NKT cells from umbilical cord blood, the method comprising: isolating monocytes from the umbilical cord blood sample; and contacting the isolated monocytes with one or more agents selected from IL-7, ALT-803, FLT3 ligand and Gal-Cer to enrich CIK NKT cells.

[0065] Expansion of CIK NKT cells

[0066] CIK NKT cells separated as above can be expanded in a suitable growth medium. Amplification refers to growing a separate population of target cells to increase the number of target cells. In some embodiments, the growth medium is NK Macs medium, which can be obtained from Miltenyi Biotec, Germany. In some embodiments, the growth medium is supplemented with IL-2, anti-CD3 and / or anti-CD28 in an amount suitable for NKT cell growth. In some embodiments, the anti-CD3 antibody is present at a concentration of 5ng / mL to 60ng / mL (e.g., 20ng / mL). In some embodiments, the anti-CD28 antibody is present at a concentration of 0.1μg / mL to 2μg / mL (e.g., 0.5μg / mL). In some embodiments, IL-2 is present at a concentration of 50ng / mL to 500ng / mL (e.g., 200ng / 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 CD1d tetramer loaded with Gal-Cer. In some embodiments, the CD1d tetramer loaded with Gal-Cer is a preassembled tetramer commercially available from, for example, ProImmune (Oxford, UK). Methods for assembling the CD1d tetramer loaded with Gal-Cer are well known. Typically, Gal-Cer lipids are co-incubated with CD1d proteins, which oligomerize on the surface of streptavidin to form tetramers. After Gal-Cer binds to the CD1d complex, it is purified and used as a reagent for amplification. Some exemplary methods for preparing the CD1d tetramer loaded with Gal-Cer are described in 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, the amount of the CD1d tetramer loaded with Gal-Cer is such that the concentration of the Gal-Cer in the growth medium is about 20-200 ng / mL, such as 50-150 ng / mL or 80-120 ng / mL or about 100 ng / mL of Gal-Cer. In some embodiments, the CIK NKT cells are grown and expanded for several days or weeks to reach a suitable number of cells for various applications.

[0067] Therefore, the present disclosure provides a method for growing CIK NKT cells from umbilical cord blood, the method comprising: isolating monocytes from the umbilical cord blood sample; and contacting the isolated monocytes with one or more agents selected from IL-7, ALT-803, FLT3 ligand and Gal-Cer to enrich CIK NKT cells.

[0068] Phenotyping of the CIK NKT cells

[0069] In certain embodiments, the CIKNKT 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).

[0070] In some embodiments, a CIK NKT cell colony is provided herein, which comprises a lower percentage of Va24+ cells compared to typical constant NKT cells. The CIK NKT cell colony comprises about 4%, about 5%, about 6%, about 7%, about 8%, about 9% or about 10% Va24+ cells. In some embodiments, the CIK NKT cell colony comprises 0-20%, 5-10%, 1-7% or 4-8% Va24+ cells. In some embodiments, the CIK NKT cell colony comprises no more than 20%, no more than 15%, no more than 10% Va24+ cells.

[0071] In some embodiments, a CIK NKT cell colony is provided herein, which comprises CD56+ cells with a percentage substantially similar to the percentage of CD56+ cells in a typical constant NKT cell colony. The CIK NKT cell colony 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 about 99% of CD56+ cells. In some embodiments, the CIK NKT cell colony comprises 50-100%, 70-100%, 85-100%, 90-100%, 95-100% or 98-100% of CD56+ cells. In some embodiments, the CIK NKT cell colony comprises no less than 50%, no less than 70%, no less than 85%, no less than 90%, no less than 93% or no less than 95% of the CD56+ cells.

[0072] In some embodiments, a CIK NKT cell colony is provided herein, which comprises CD3+ cells with a percentage substantially similar to the percentage of CD3+ cells in a typical constant NKT cell colony. The CIK NKT cell colony 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 about 99% of CD3+ cells. In some embodiments, the CIK NKT cell colony comprises 50-100%, 70-100%, 85-100%, 90-100%, 95-100% or 98-100% of the CD3+ cells. In some embodiments, the CIK NKT cell colony comprises no less than 50%, no less than 70%, no less than 85%, no less than 90%, no less than 93% or no less than 95% of the CD3+ cells.

[0073] In some embodiments, a CIK NKT cell colony is provided herein, comprising CD56+CD3+ cells substantially similar to the percentage of CD56+CD3+ cells in a typical constant NKT cell colony. The CIK NKT cell colony 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 about 99% of CD56+CD3+ cells. In some embodiments, the CIK NKT cell colony comprises 50-100%, 70-100%, 85-100%, 90-100%, 95-100% or 98-100% of the CD56+CD3+ cells. In some embodiments, the CIK NKT cell population comprises no less than 50%, no less than 70%, no less than 85%, no less than 90%, no less than 93%, or no less than 95% of the CD56+CD3+ cells.

[0074] The cytotoxicity of CIK NKT cells

[0075] Optionally, the cytotoxicity of isolated or enriched natural killer cells can be assessed, for example, in a cytotoxicity assay using tumor cells (e.g., cultured K562, DAOY, THP-1, LN-19, U937, WERI-RB-1, U-118MG, HT-29, HCC2218, KG-1 or U266 tumor cells, etc.) 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.

[0076] Assays for assessing cytotoxicity, such as the MTT assay, are well known. This is a system based on the tetrazolium compound MTT. Briefly, after a treatment period in which target cells are exposed to CIK NKT cells, 10 μL of a freshly diluted MTT solution (2.5 mg mL -1 ) is added to each well, and the plate is incubated for 4 hours in a humidified 5% CO atmosphere at 37°C. At the end of the incubation period, the culture medium is removed, and the formazan product is dissolved in 100 μL of dimethyl sulfoxide. Using SUNRICE Tecan absorbance reader (Schoeller), cell viability is assessed by measuring the absorbance at 570nm. The compound concentration (IC50) that produces 50% cell growth inhibition is calculated by plotting the curve constructed by cell survival (%) relative to drug concentration (μM). The read value is converted into the percentage (cell survival percentage) of the control. Non-limiting methods of cell killing assays include sulforhodamine B (SRB) assays, neutral red (NR) assays (such as those described in www.rsc.org / suppdata / mt / c4 / c4mt00112e / c4mt00112e1.pdf, the entire contents of which are incorporated herein by reference).

[0077] The potency of CIK NKT cells in killing target cells can be evaluated with EC50. The EC50 used in the present disclosure refers to the ratio of the effector to the target used in the determination in which 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 cell is THP-1 and the EC50 is 4.64. In some embodiments, the target cell is DAOY and the IC50 is 3.69. In some embodiments, the target cell is K562 and the IC50 is 2.6.

[0078] Modified CIK NKT cells

[0079] Chimeric Antigen Receptor

[0080] CIK NKT cells produced as above can be further engineered to express chimeric antigen receptors (CAR) on the cell surface. Optionally, the CAR is specific to tumor-specific antigens. Tumor-specific antigens are described in US2013 / 0189268, WO 1999024566A1, US 7098008 and WO 2000020460 A1 by way of non-limiting examples, the entire contents of each of which are incorporated herein by reference. Tumor-specific antigens include, but are not limited to, NKG2D, CS1, CS2, 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 malignancies associated therewith can be found in Table 1.

[0081] Table 1: Tumor-specific antigens and associated malignancies

[0082]

[0083]

[0084]

[0085] In some embodiments, the CAR targets CD19, CD22 or CSPG-4.

[0086] In an embodiment, variant polypeptides are prepared using methods known in the art (e.g., oligonucleotide-mediated (site-directed) mutagenesis, alanine scanning, PCR mutagenesis). Site-directed mutagenesis (Carter, 1986; Zoller and Smith, 1987), cassette mutagenesis, restriction-selected mutagenesis (Wells et al., 1985) or other known techniques can be performed on cloned DNA to produce CD16 variants (Ausubel, 2002; Sambrook and Russell, 2001).

[0087] Optionally, the CAR targets an antigen associated with a particular cancer type. Optionally, the cancer is selected from leukemia (including acute leukemia (e.g., acute lymphocytic leukemia, acute myeloid leukemia (including granulocyte, promyelocyte, myelomonocytic, mononuclear 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 (Waldenstrom's macroglobulinemia), heavy chain disease, solid tumors including but not limited to sarcomas and carcinomas such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendothelioma sarcoma, synovioma, 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 carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatocarcinoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor, cervical cancer, testicular tumors, lung cancer, small cell lung cancer, bladder cancer, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, hemangioma, melanoma, neuroblastoma and retinoblastoma.

[0088] 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 replacements of amino acid replacements at amino acid residues leading to "non-essential" can be carried out in the CAR disclosed above. It can be such as the engineered CAR described in Patent Publication Nos. WO 2014039523, US20140242701, US20140274909, US20130280285 and WO 2014099671 (the entire contents of each of which are incorporated herein by reference). Optionally, the CAR is CD19 CAR, CD33 CAR or CSPG-4 CAR.

[0089] Additional Modifications - Cytokines

[0090] In some embodiments, the CIK NKT cells expressing CAR are further modified to express at least one cytokine. In a specific embodiment, the at least one cytokine is IL-2, IL-12, IL-15, IL-18, IL-21 or a variant thereof. In a preferred embodiment, the cytokine is IL-12. Representative polypeptides of IL-12 include or consist of the amino acid sequence shown in accession number IF45_A (https: / / www.ncbi.nlm.nih.gov / protein / 1F45_A) and the amino acid sequence shown in accession number IF45_B (https: / / www.ncbi.nlm.nih.gov / protein / 1F45_B).

[0091] Therapeutic applications

[0092] The present disclosure also provides a method for treating any type of cancer in a subject at any stage of the disease. Non-limiting examples of suitable cancers include carcinomas, melanomas, or sarcomas. In some embodiments, the present invention is used to treat cancers of hematopoietic origin, such as leukemias or lymphomas. In some embodiments, the cancer is a solid tumor.

[0093] In some embodiments, the method of treating any type of cancer in a subject comprises administering to the patient a therapeutically effective amount of CIK NKT cells, wherein the cancer is thereby treated. The CIK NKT cells are from a population of CIK NKT cells, wherein greater than 90% of the cells in the population express CD56 and CD3, and less than 10% of the cells in the population express Va24.

[0094] The present disclosure also provides a method of treating any type of viral infection, the method comprising administering a therapeutically effective amount of CIK NKT cells to a patient, wherein cancer is thereby treated. The CIK NKT cells are from a population of CIK NKT cells, wherein greater than 90% of the cells in the population express CD56 and CD3, and less than 10% of the cells in the population express Va24.

[0095] Also provided are methods of treating a subject in need thereof with the CIK NKT cells described herein. In some embodiments, the subject or patient suffers from cancer or an infectious disease (eg, a viral infection).

[0096] CIK NKT cells can be administered to an individual in an absolute number of cells, for example, about 1000 cells / injection up to about 10 billion cells / injection, for example about, at least about, or at most about 1×10 8 , 1×107 , 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 on) CIK NKT cells / injection or any range (including endpoints) between any two of the numbers. Therefore, the present disclosure also provides a composition comprising a plurality of CIK NKT cells, wherein the number of cells is 1×10 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 5×10 3 (and so on).

[0097] In other embodiments, about 1000 cells / injection / m can be administered to the individual. 2 Up to approximately 10 billion cells / injection / m 2 , such as about, at least about, or at most about 1×10 8 / m 2 , 1×10 7 / m 2 , 5×10 7 / m 2 , 1×10 6 / m 2 , 5×10 6 / m 2 , 1×10 5 / m 2 , 5×10 5 / m 2 , 1×10 4 / m 2 , 5×10 4 / m 2 , 1×10 3 / m 2 , 5×10 3 / m 2 (and so on) CIK NKT cells / injection or any range (including endpoints) between any two of the numbers.

[0098] In other embodiments, CIK NKT cells can be administered to such an individual in an absolute number of cells, for example, about 1000 cells up to about 10 billion cells per kilogram of the individual can be administered to the individual, for example, about, at least about, or at most about 1×10 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 5×10 3 (and so on) CIK NKT cells per kilogram of said individual or any range (including endpoints) between any two of said numbers.

[0099] In other embodiments, the total dose can be calculated by measuring the body surface area m 2 to calculate, which includes about 1×10 11 , 1×10 10 , 1×10 9 , 1×10 8 , 1×10 7 Each m 2 Or any range between any two of the above numbers (including the endpoints). About 1.6 to about 1.8 m 2 In a preferred embodiment, about 1 billion to about 3 billion CIK NKT cells are administered to a patient. In other embodiments, the amount of CIK NKT cells injected per dose can be determined by m of body surface area. 2 To calculate, it includes 1×10 11 , 1×10 10 , 1×10 9 , 1×10 8 , 1×10 7 Each m 2 The average human body surface area is 1.6-1.8m 2 .

[0100] In other embodiments, CIK NKT cells can be administered to such an individual in an absolute number of cells, for example, about 1000 cells up to about 10 billion cells per kilogram of the individual can be administered to the individual, for example, about, at least about, or at most about 1×10 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 5×10 3 (and so on) CIK NKT cells per kilogram of said individual or any range (including endpoints) between any two of said numbers.

[0101] CIK NKT cells can be administered once to a cancer patient, or they can be administered 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 during treatment, 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 between any two of the recited numbers (including the endpoints).

[0102] In some embodiments, CIK NKT cells are administered in the form of a composition comprising CIK NKT cells and a culture medium (e.g., human serum or its equivalent). In some embodiments, the culture medium comprises human serum albumin. In some embodiments, the culture medium comprises human plasma. In some embodiments, the culture medium comprises about 1% to about 15% human serum or a human serum equivalent. In some embodiments, the culture medium comprises about 1% to about 10% human serum or a human serum equivalent. In some embodiments, the culture medium comprises about 1% to about 5% human serum or a human serum equivalent. In some embodiments, the culture medium comprises about 2.5% human serum or a human serum equivalent. In some embodiments, the serum is human AB serum. In some embodiments, serum substitutes acceptable for human treatment are used instead of human serum. Such serum substitutes may be known in the art or developed in the future. Although concentrations of human serum exceeding 15% may be used, concentrations greater than 5% are expected to be costly. In some embodiments, CIK NKT cells are administered in the form of a composition comprising CIK NKT cells and an isotonic liquid solution that supports cell viability. In some embodiments, CIK NKT cells are administered in a composition that has been reconstituted from a cryopreserved sample.

[0103] Pharmaceutically acceptable compositions comprising CIK NKT cells may include a variety of carriers and excipients. A variety of aqueous carriers may be used, such as buffered saline, etc. These solutions are sterile and generally free of undesirable substances. Suitable carriers and excipients and their preparations 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 biologically or otherwise non-adverse material, that is, the material is applied to a subject without causing adverse biological effects or interacting with other components of the pharmaceutical composition contained therein in a harmful manner. If applied to a subject, the carrier is optionally selected to minimize the 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 pharmaceutically acceptable. Pharmaceutical compositions will generally include reagents for buffering and preservation in storage and may include buffers and carriers for appropriate transportation, depending on the route of administration.

[0104] These compositions for use in vivo or in vitro can be sterilized by sterilization techniques used for cells. The compositions may contain acceptable auxiliary substances required to approximate physiological conditions, such as pH adjusters and buffers, toxicity adjusters, etc., such as sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, etc. The cell concentration in these formulations and / or other reagents can vary and will be selected based primarily on body fluid volume, viscosity, body weight, etc., depending on the specific mode of administration selected and the needs of the subject.

[0105] In one embodiment, CIK NKT cells are administered to a patient along with one or more other therapies or agents for the cancer being treated. In some embodiments, the one or more other therapies for the cancer being treated include, for example, antibodies, radiation, chemotherapy, stem cell transplantation, or hormone therapy.

[0106] In some embodiments, CIK NKT cells and other cancer agents / therapies are administered simultaneously or approximately simultaneously (e.g., about 1, 5, 10, 15, 20, or 30 minutes apart from each other). In some embodiments, the CIK NKT cells and the other cancer agents / therapies are administered sequentially. In some embodiments, the other cancer therapy / agent is administered one, two, or three days after the CIK NKT cells are administered.

[0107] In one embodiment, the other cancer agent is an antibody. In one embodiment, CIK NKT cells are administered in combination with antibodies targeting diseased cells. In one embodiment, CIK NKT cells and antibodies are administered to the patient together (e.g., in the same preparation); administered separately to the patient (e.g., administered simultaneously with different preparations) or may be administered separately, for example, at different dosing schedules or at different times of the day. When administered separately, the antibody may be administered by any suitable route (e.g., intravenous or intratumoral injection).

[0108] In some embodiments, CIK NKT cells of the present disclosure are used in combination with therapeutic antibodies and / or other anti-cancer agents. Therapeutic antibodies can be used to target cells expressing markers associated with cancer or associated with tumors. Examples of cancer therapeutic monoclonal antibodies are shown in Table 2.

[0109] Table 2. Illustrative Therapeutic Monoclonal Antibodies Examples of FDA-approved Therapeutic Monoclonal Antibodies

[0110]

[0111]

[0112] Examples of FDA-approved therapeutic monoclonal antibodies

[0113]

[0114]

[0115] Such CIK NKT cell administration can be carried out simultaneously with the administration of the monoclonal antibody, or in a sequential manner. In some embodiments, the CIK NKT cell is administered to the subject after the subject is treated with the monoclonal antibody. Alternatively, the CIK NKT cell can be administered at the same time as the monoclonal antibody (e.g., within 24 hours).

[0116] In some embodiments, CIK NKT cells are administered intravenously. In some embodiments, the CIK NKT cells are infused directly into the bone marrow.

[0117] Thus, the present disclosure provides a method of treating cancer or viral infection in a patient in need thereof, the method comprising administering to the patient a therapeutically effective amount of CIK NKT cells from a population of CIK NKT cells using the methods disclosed herein, thereby treating cancer.

[0118] Reagent test kit

[0119] Also disclosed is a kit for treating cancer or infectious diseases using a composition comprising the amount of CIK NKT cells described herein. In some embodiments, the kit described in the present disclosure may further comprise at least one monoclonal antibody.

[0120] In certain embodiments, the kit may include additional compounds, such as therapeutically active compounds or drugs, which are administered before, simultaneously with, or after the administration of CIK NKT cells. Examples of such compounds include antibodies, vitamins, minerals, fludrocortisone, ibuprofen, lidocaine, quinidine, chemotherapeutic agents, etc.

[0121] In various embodiments, the instructions for using the kit will include instructions for using the kit components in the treatment of cancer or infectious diseases. The instructions may further include information on how to CIK NKT cells (e.g., thaw and / or culture). The instructions may further include guidance on dosage and frequency of administration.

[0122] In certain embodiments, the kit further comprises one or more containers containing one or more compositions described herein, such as compositions comprising CIK NKT cells as described herein. Optionally, associated with such containers may be labels indicating that the kit is for use in treating cancer, such as those described herein. Optionally, the label also includes a notice in a form prescribed by a government agency regulating the production, use or sale of drugs or biological products, which reflects the approval of the government agency for production, use or sale for human administration.

[0123] While the present disclosure and working examples illustrate the production and use of CIK NKT cells derived from umbilical cord blood samples, one of ordinary skill in the art will appreciate that CIK NKT cells may also be generated from other hematopoietic progenitor cell samples using methods similar to those described herein.

[0124] Materials, compositions and components are disclosed, which can be used in the disclosed methods and compositions, can be used in combination with them, can be used in their preparation or can be the products thereof. These and other materials are disclosed herein, and it should be understood that when combinations, subsets, interactions, groups, etc. of these materials are disclosed, although specific references to each of the various individual and collective combinations and arrangements of these compounds may not be explicitly disclosed, each is specifically considered and described herein. For example, if a method is disclosed and discussed and a variety of modifications (including the method) that can be made to a variety of molecules are discussed, unless there is an explicit statement to the contrary, each combination and arrangement of the method and possible modifications will be specifically considered. Similarly, any subset or combination of these is also specifically considered and disclosed. This concept applies to all aspects of the present disclosure, including but not limited to steps in the method using the disclosed composition. Therefore, if there are multiple additional steps that can be performed, it should be understood that each of these additional steps can be performed with any specific method step or combination of method steps disclosed, and each such combination or subset of combinations is specifically considered and should be considered disclosed.

[0125] Example

[0126] The present disclosure includes the following non-limiting embodiments.

[0127] Embodiment 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.

[0128] Embodiment 2. The population of CIK NKT cells according to embodiment 1, wherein the CIK NKT cells can kill target cells in the absence of α-galactosylceramide (Gal-Cer).

[0129] Embodiment 3. The population of CIK NKT cells according to embodiment 1, wherein the target cell is a cancer cell.

[0130] Embodiment 4. The population of CIK NKT cells according to embodiment 1, wherein the cancer cell line is selected from the group consisting of myeloid leukemia cells, medulloblastoma cells and monocytes.

[0131] Embodiment 5. The population of CIK NKT cells according to embodiment 3, wherein the cancer cells are selected from K562 cells, Daudi cells, DAOY cells and THP-1 cells.

[0132] Embodiment 6. The population of CIK NKT cells according to embodiments 2-5, wherein the CIK NKT cells kill a plurality of target cells with an EC50 between 1.0 and 10.0.

[0133] Embodiment 7. The population of CIK NKT cells according to embodiment 6, wherein the CIK NKT cells can kill the target cells with an EC50 that is not less than 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.

[0134] Embodiment 8. A composition comprising a plurality of CIK NKT cells from the population of CIK NKT cells according to any one of embodiments 1-7 and a physiologically acceptable excipient.

[0135] Embodiment 9. A kit for treating cancer, comprising a plurality of CIK NKT cells from the population of CIK NKT cells according to any one of embodiments 1-7, wherein the kit further comprises a container and / or a label indicating that the kit is for treating cancer.

[0136] Embodiment 10. A method for enriching CIK NKT cells from an umbilical cord blood sample, the method comprising: isolating monocytes from the umbilical cord blood sample; and contacting the isolated monocytes with one or more reagents, thereby enriching CIK NKT cells, the reagents being selected from IL-7, ALT-803 or IL-15, FLT3 ligand and Gal-Cer.

[0137] Embodiment 11. The method of embodiment 10, wherein the IL-7, if present, is at a concentration ranging from 5 to 20 ng / mL.

[0138] Embodiment 12. The method of embodiment 10 or 11, wherein the ALT-803, if present, is at a concentration ranging from 100 to 300 ng / mL.

[0139] Embodiment 13. The method of any one of embodiments 10-12, wherein the FLT3 ligand, if present, is at a concentration ranging from 5 to 20 ng / mL.

[0140] Embodiment 14. The method of any one of embodiments 10-13, wherein the Gal-Cer is present in a concentration range of 2 to 10 μg / mL.

[0141] Embodiment 15. The method of embodiment 10, wherein the method further comprises isolating the enriched CIK NKT cells from the remainder of the cord blood sample.

[0142] Embodiment 16. The method according to embodiment 15, wherein the method further comprises contacting the isolated CIK NKT cells with anti-CD3, anti-CD28 and IL2 to expand the CIK NKT cells.

[0143] Embodiment 17. The method according to embodiment 16, wherein the method further comprises contacting the isolated CIK NKT cells with Gal-Cer.

[0144] Embodiment 18. The method according to embodiment 17, wherein the Gal-Cer is present in the form of a CD1d tetramer loaded with Gal-Cer.

[0145] Embodiment 19. The method of embodiment 16, wherein the anti-CD3 antibody is present in an amount of 5 ng / mL to 60 ng / mL.

[0146] Embodiment 20. The method of any one of embodiments 16-19, wherein the anti-CD28 antibody is present in an amount of 0.1 μg / mL to 2 μg / mL.

[0147] Embodiment 21. The method of any one of embodiments 16-20, wherein the IL-2 is present at a concentration of 50 ng / mL to 500 ng / mL.

[0148] Embodiment 22. The method of any one of embodiments 16-21, wherein the production of CIK NKT cells does not include interferon-γ.

[0149] Embodiment 23. A method of treating cancer or viral infection in a patient in need thereof, the method comprising administering to the patient a therapeutically effective amount of CIK NKT cells from the population of CIK NKT cells of any one of embodiments 1-7, thereby treating cancer.

[0150] Embodiment 24. The method of embodiment 23, wherein about 1 x 10 8 About 1x10 11 cells are administered to the patient.

[0151] Embodiment 25. The method of embodiment 23, wherein the cancer is selected from leukemia, lymphoma, polycythemia vera, multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, sarcoma and carcinoma.

[0152] Embodiment 26. The method of embodiment 25, wherein the cells are administered to the patient by a route selected from intravenous, intraperitoneal, and subcutaneous.

[0153] Embodiment 27. A method according to any one of embodiments 23-26, wherein the method further comprises administering an antibody.

[0154] Embodiment 28. A population of CIK NKT cells produced by the method of any one of embodiments 10-27.

[0155] Embodiment 29. A population of CIK NKT cells according to embodiment 1, wherein the CIK NKT cells express CAR and / or cytokines.

[0156] Example

[0157] 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 will similarly allow one to successfully perform the following examples.

[0158] Example 1: Phenotype of CIK NKT cells

[0159] Ficoll-Paque and Sepmate from Stem Cell Technology were used TM , cord blood mononuclear cells (Comics) were isolated from cord blood samples by density gradient method. Cord blood mononuclear cells were incubated with Gal-Cer (5 μg / mL), FLT3-L (10 ng / mL), IL-7 (10 ng / mL) and ALT-803 (175 ng / mL) in RPMI medium with 10% human AB serum for two weeks to enrich NKT. NKT cells were isolated by affinity chromatography using reagents from Miltenyi Biotec, Germany. Subsequently, the isolated cells were amplified in NK Macs medium with 10% human AB serum in the presence of anti-CD3 antibody (20 ng / mL), anti-CD28 antibody (0.5 μg / mL) and IL2 (200 ng / mL) for overnight activation of them by CD1d tetramers loaded with Gar-Cer. CD1d tetramer loaded with Gar-Cer was obtained from Prolmmune, Oxford, UK, and the amount used was such that Gar-Cer was present at 100 ng / mL. After one week of expansion, the cells were stained with antibodies recognizing CD3, CD56, or Va24. The 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 have low Va24 expression, but the expression of CD3 and CD56 remains intact.

[0160] Example 2: CIK NKT cell cytotoxicity is independent of Gal-Cer

[0161] The cytotoxicity of NKT-CIK cells prepared as described in Example 2 was evaluated against the cancer cell line DAOY. DAOY cells were incubated with Gal-Cer at 1 μg / mL overnight. 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 cells at various effector to target ratios as shown (the highest ratio was 32:1) for 4 hours. The killing of DAOY cells was measured by cell lysis, which is represented by the amount of CalcienAM dye released from the cells. Figure 3A The amount of CalcienAM dye released from DAOY cells by CB-NKT CIK is shown. The data are expressed as % 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 target cells were not loaded with Gal-Cer, indicating that Gal-Cer treatment did not confer specificity in killing.

[0162] Figure 3B The cytotoxicity of NKT-CIK cells derived from cord blood as described above (CB-CIK NKT cells) was compared to PBiNKT cells (iNKT cells isolated from peripheral blood) on THP1 cells expressing luciferase. PBiNKT was isolated in the same manner as CD-CIK NKT cells (see Example 1), except that the source was peripheral blood instead of cord blood. THP1 cells were co-cultured with CB-CIK NKT cells or PBiNKT cells at various effector to target ratios as shown, 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 effective than PBiNKT cells in killing THP-1.

[0163] Example 3: Cytotoxicity of CIK NKT cells on K562 and Daudi cells

[0164] The ability of the CB-NKT CIK cells obtained above to kill the luciferase-expressing cancer cell lines DAOY, Daudi and K562 was determined (respectively at Figure 4 iNKT cells isolated from peripheral blood were used as controls ( Figure 4). Cell killing assays were performed after co-culturing cancer cells and effector cells (CB CIK NKT or PBiNKT) for 4 hours. Cancer cell killing was measured by the percentage of cell lysis, which is represented by the loss of luciferase in these cancer cell lines. Various effector to target ratios were used as indicated, up to 32:1. The results indicate that CB-CIKNKT cells kill target cells in a non-CD1d / MHC restricted manner.

[0165] Preferred embodiment:

[0166] 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.

[0167] 2. The population of CIK NKT cells according to item 1, wherein the CIK NKT cells can kill target cells in the absence of α-galactosylceramide (Gal-Cer).

[0168] 3. The population of CIK NKT cells according to item 1, wherein the target cell is a cancer cell.

[0169] 4. The population of CIK NKT cells according to item 1, wherein the cancer cell line is selected from myeloid leukemia cells, medulloblastoma cells and monocytes.

[0170] 5. The population of CIK NKT cells according to item 3, wherein the cancer cells are selected from K562 cells, Daudi cells, DAOY cells and THP-1 cells.

[0171] 6. The population of CIK NKT cells according to item 2, wherein the CIK NKT cells kill a plurality of the target cells with an EC50 between 1.0 and 10.0.

[0172] 7. The population of CIK NKT cells according to item 6, wherein the CIK NKT cells can kill the target cells with an EC50 that is not less than 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.

[0173] 8. A composition comprising a plurality of CIKNKT cells from the population of CIK NKT cells according to any one of item 1 and a physiologically acceptable excipient.

[0174] 9. A kit for treating cancer, comprising a plurality of CIK NKT cells from the population of CIK NKT cells according to any one of item 1, wherein the kit further comprises a container and / or a label indicating that the kit is for treating cancer.

[0175] 10. A method for enriching CIK NKT cells from an umbilical cord blood sample, the method comprising:

[0176] isolating mononuclear cells from the cord blood sample; and

[0177] The isolated monocytes are contacted with one or more agents selected from IL-7, ALT-803 or IL-15, FLT3 ligand, and Gal-Cer to enrich for CIK NKT cells.

[0178] 11. The method according to item 10, wherein the IL-7, if present, has a concentration ranging from 5 to 20 ng / mL.

[0179] 12. The method according to item 10, wherein the ALT-803, if present, is in a concentration range of 100 to 300 ng / mL.

[0180] 13. The method according to item 10, wherein the FLT3 ligand, if present, is at a concentration ranging from 5 to 20 ng / mL.

[0181] 14. The method according to item 10, wherein the Gal-Cer is present in a concentration range of 2 to 10 μg / mL.

[0182] 15. The method according to item 10, wherein the method further comprises isolating the enriched CIK NKT cells from the remainder of the umbilical cord blood sample.

[0183] 16. The method according to item 15, wherein the method further comprises contacting the isolated CIK NKT cells with anti-CD3, anti-CD28 and IL2 to expand the CIK NKT cells.

[0184] 17. The method according to item 16, wherein the method further comprises contacting the isolated CIK NKT cells with Gal-Cer.

[0185] 18. The method according to item 17, wherein the Gal-Cer is present in the form of a CD1d tetramer loaded with Gal-Cer.

[0186] 19. The method according to item 16, wherein the anti-CD3 antibody is present in an amount of 5 ng / mL to 60 ng / mL.

[0187] 20. The method according to item 16, wherein the anti-CD28 antibody is present in an amount of 0.1 μg / mL to 2 μg / mL.

[0188] 21. The method according to item 16, wherein the IL-2 is present at a concentration of 50 ng / mL to 500 ng / mL.

[0189] 22. The method according to item 16, wherein the production of CIK NKT cells does not include interferon-γ.

[0190] 23. A method for treating cancer or viral infection in a patient in need thereof, the method comprising administering to the patient a therapeutically effective amount of CIK NKT cells from the population of CIK NKT cells of any one of items 1-7, thereby treating cancer.

[0191] 24. The method according to item 23, wherein about 1 x 10 8 About 1x10 11 cells are administered to the patient.

[0192] 25. The method according to item 23, wherein the cancer is selected from leukemia, lymphoma, polycythemia vera, multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, sarcoma and carcinoma.

[0193] 26. The method according to item 25, wherein the cells are administered to the patient by a route selected from intravenous, intraperitoneal and subcutaneous.

[0194] 27. The method according to item 23, wherein the method further comprises administering an antibody.

[0195] 28. A population of CIK NKT cells produced by the method according to item 10.

[0196] 29. A population of CIK NKT cells according to item 1, wherein the CIK NKT cells express CAR and / or cytokines.

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 according to claim 1 , wherein the CIK NKT cells can kill target cells in the absence of α-galactosylceramide (Gal-Cer). The population of CIK NKT cells according to claim 1 , wherein the target cell is a cancer cell. 4 . The population of CIK NKT cells according to claim 2 , wherein the CIK NKT cells kill a plurality of the target cells with an EC50 between 1.0 and 10.

0. 5 . The population of CIK NKT cells according to claim 4 , wherein the CIK NKT cells can kill the target cell with an EC50 that is not less than 90% and not more than 110% of the EC50 at which the CIK NKT cells kill the target cell in the presence of Gal-Cer. 6 . A composition comprising a plurality of CIK NKT cells derived from the population of CIK NKT cells according to claim 1 and a physiologically acceptable excipient.

7. A kit for culturing CIK NKT cells from a blood sample, the kit comprising i) IL-7, ii) ALT-803 or IL-15, iii) FLT3 ligand, and iv) Gal-Cer.

8. A cell culture mixture comprising monocytes and i) IL-7, ii) ALT-803 or IL-15, iii) FLT3 ligand, and iv) Gal-Cer.

9. The cell culture mixture of claim 8, wherein the monocytes comprise 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.

10. The cell culture mixture according to claim 8, wherein the mononuclear cells are derived from an umbilical cord blood sample. The cell culture mixture according to claim 8 , wherein the mononuclear cells are derived from a peripheral blood sample.

12. The cell culture mixture according to claim 8, wherein the concentration of IL-7 ranges from 5 to 20 ng / mL.

13. The cell culture mixture according to claim 8, wherein the concentration of ALT-803 ranges from 100 to 300 ng / mL.

14. The cell culture mixture of claim 8, wherein the FLT3 ligand is present in a concentration range of 5 to 20 ng / mL. The cell culture mixture according to claim 8 , wherein the Gal-Cer is present in a concentration range of 2 to 10 μg / mL. The cell culture mixture according to claim 8 , wherein the Gal-Cer is present in the form of a CD1d tetramer loaded with Gal-Cer.

17. The cell culture mixture according to claim 8, wherein the cell culture mixture further comprises anti-CD3 antibody, anti-CD28 antibody and IL2.

18. The cell culture mixture of claim 17, wherein the anti-CD3 antibody is present in an amount of 5 ng / mL to 60 ng / mL.

19. The cell culture mixture of claim 17, wherein the anti-CD28 antibody is present in an amount of 0.1 μg / mL to 2 μg / mL.

20. The cell culture mixture of claim 17, wherein the IL-2 is present at a concentration of 50 ng / mL to 500 ng / mL.

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