WO2024036226A1

WO2024036226A1 - Method of treating autism with expanded natural killer cells

Info

Publication number: WO2024036226A1
Application number: PCT/US2023/071954
Authority: WO
Inventors: Paul Y. SONG; Sang Woo Park; Yong Man Kim
Original assignee: Nkmax Co., Ltd.; LOOTS, Eli
Priority date: 2022-08-10
Filing date: 2023-08-09
Publication date: 2024-02-15

Abstract

Provided herein is a method for treating autism or autism spectrum disorders. The method comprises identifying a subject and treating the subject with expanded natural killer cells (NKs). Also provided is a composition for treating autism or autism spectrum disorders. Natural killer (NK) cells have proven to be promising candidates for use in adoptive cell therapy (ACT) due to their high cytotoxicity and lower risk than T-cells. One general approach to NK ACT has been the administration of autologous NK cells expanded ex vivo.

Description

METHOD OF TREATING AUTISM WITH EXPANDED NATURAL KILLER

CELLS

INCORPORATION BY REFERENCE TO ANY PRIORITY APPLICATIONS

[0001] Any and all applications for which a foreign or domestic priority claim is identified in the Application Data Sheet as filed with the present application are hereby incorporated by reference under 37 CFR 1.57.

FIELD

[0002] The present disclosure relates to a method for treating autism and related disorders with natural killer cells.

BACKGROUND

[0003] Natural killer (NK) cells have proven to be promising candidates for use in adoptive cell therapy (ACT) due to their high cytotoxicity and lower risk than T-cells. One general approach to NK ACT has been the administration of autologous NK cells expanded ex vivo.

[0004] Autism is a neurodevelopmental disorder characterized by repetitive, stereotypical behaviors and impaired expressive communication.

SUMMARY OF THE INVENTION

[0005] This application is related to methods of producing high-purity natural killer cells, and a cell therapeutic composition for treating autism comprising high-purity natural killer cells and cytokines. Any features, structures, or steps disclosed herein can be replaced with or combined with any other features, structures, or steps disclosed herein, or omitted. Further, for purposes of summarizing the disclosure, certain aspects, advantages, and features of the inventions have been described herein. It is to be understood that not necessarily any or all such advantages are achieved in accordance with any particular embodiment of the inventions disclosed herein. No individual aspects of this disclosure are essential or indispensable.

[0006] In some embodiments, a method of treating autism in a subject is provided. In some embodiments, the method comprises identifying a subject, wherein the subject has autism: and administering to the subject a therapeutically effective amount of an autologous natural killer cell (NK) cell population.

[0007] In some embodiments, a method of treating autism spectrum disorder (ASD) in a subject is provided. In some embodiments, the method comprises identifying a subject, wherein the subject has ASD, and administering to the subject a therapeutically effective amount of an autologous natural killer cell (NK) cell population.

[0008] In some embodiments, a method of treating autism in a subject is provided. In some embodiments, the method comprises: identifying a subject, wherein the subject has autism; and administering to the subject an expanded NK cell population. In some embodiments, the NK cells are expanded by a method comprising: i) isolating at least one of CD56+ cells and/or CD3-/CD56+ cells from the PBMCs; ii) co-culturing the at least one of CD56+ cells and/or CD3-/CD56+ cells with a combination of feeder cells in the presence of at least two cytokines; iii) wherein the combination of feeder cells comprises irradiated Jurkat cells and irradiated Epstein-Barr virus transformed lymphocyte continuous line (EBV-LCL) cells; and iv) wherein the at least two cytokines comprise IL- 2 and IL-21.

[0009] In some embodiments, a method of treating autism spectrum disorder (ASD) in a subject is provided. In some embodiments, the method comprises: identifying a subject, wherein the subject has ASD; and administering to the subject an expanded NK cell population. In some embodiments, the NK cells are expanded by a method comprising: i) isolating at least one of CD56+ cells and/or CD3-/CD56+ cells from the PBMCs; ii) coculturing the at least one of CD56+ cells and/or CD3-/CD56+ cells with a combination of feeder cells in the presence of at least two cytokines; iii) wherein the combination of feeder cells comprises irradiated Jurkat cells and irradiated Epstein-Barr virus transformed lymphocyte continuous line (EBV-LCL) cells; and iv) wherein the at least two cytokines comprise IL-2 and IL-21.

[0010] In some embodiments, a method of cell therapy is provided, comprising: identifying a subject, wherein the subject has autism; and administering to the subject an expanded NK cell population. In some embodiments, the NK cells are expanded by a method comprising: i) isolating at least one of CD56+ cells and/or CD3-/CD56+ cells from the PBMCs; ii) co-culturing the at least one of CD56+ cells and/or CD3-/CD56+ cells with a combination of feeder cells in the presence of at least two cytokines; iii) wherein the combination of feeder cells comprises irradiated Jurkat cells and irradiated Epstein-Barr virus transformed lymphocyte continuous line (EBV-LCL) cells; and iv) wherein the at least two cytokines comprise IL-2 and IL-21.

[0011] In some embodiments, a method of cell therapy is provided, comprising: identifying a subject, wherein the subject has autism spectrum disorder (ASD); and administering to the subject an expanded NK cell population. In some embodiments, the NK cells are expanded by a method comprising: i) isolating at least one of CD56+ cells and/or CD3-/CD56+ cells from the PBMCs; ii) co-culturing the at least one of CD56+ cells and/or CD3-/CD56+ cells with a combination of feeder cells in the presence of at least two cytokines; iii) wherein the combination of feeder cells comprises irradiated Jurkat cells and irradiated Epstein-Barr virus transformed lymphocyte continuous line (EBV-LCL) cells; and iv) wherein the at least two cytokines comprise IL-2 and IL-21.

[0012] In some embodiments, a population of expanded NK cells is provided. In some embodiments, the NK cells were expanded by a method that comprises: i) isolating at least one of CD56+ cells and/or CD3-/CD56+ cells from the PBMCs; ii) co-culturing the at least one of CD56+ cells and/or CD3-/CD56+ cells with a combination of feeder cells in the presence of at least two cytokines; iii) wherein the combination of feeder cells comprises irradiated Jurkat cells and irradiated Epstein-Barr virus transformed lymphocyte continuous line (EBV-LCL) cells; and iv) wherein the at least two cytokines comprise IL- 2 and IL-21. In some embodiments, the population of expanded NK cells has been administered to a subject who has autism.

[0013] In some embodiments, a population of expanded NK cells is provided. In some embodiments, the NK cells were expanded by a method that comprises: i) isolating at least one of CD56+ cells and/or CD3-/CD56+ cells from the PBMCs; ii) co-culturing the at least one of CD56+ cells and/or CD3-/CD56+ cells with a combination of feeder cells in the presence of at least two cytokines; iii) wherein the combination of feeder cells comprises irradiated Jurkat cells and irradiated Epstein-Barr virus transformed lymphocyte continuous line (EBV-LCL) cells; and iv) wherein the at least two cytokines comprise IL- 2 and IL-21. In some embodiments, the population of expanded NK cells has been administered to a subject who has autism spectrum disorder (ASD).

[0014] In some embodiments, the amount of expanded NK cells administered to a subject is a therapeutically effective amount.

[0015] In some embodiments, the therapeutically effective amount of expanded NK cells comprises 2 x 10⁹ to 9 x 10⁹ cells. In some embodiments, the therapeutically effective amount of expanded NK cells comprises 1 x 10⁹ to 1 x IO¹⁰ cells. [0016] In some embodiments, IL-2 is added at a concentration of 50-1000 lU/mL during step ii).

[0017] In some embodiments, IL-21 is added at a concentration of 10-100 ng/mL during step ii).

[0018] In some embodiments, expansion of NK cells further comprises: coculturing the at least one of CD56+ cells and/or CD3-/CD56+ cells with the combination of feeder cells, in the presence of IL-2 for a first period; and co-cultunng the at least one of CD56+ cells and/or CD3-/CD56+ cells with the combination of feeder cells, in the presence of IL-21 for a second period.

[0019] In some embodiments, IL-21 is added more than once during Day 0-6 of the second period.

[0020] In some embodiments, IL-21 and the combination of feeder cells are added more than once during Day 0-6 of the second period.

[0021] In some embodiments, IL-21 is added more than once during the first six days of every fourteen-day cycle during the second period.

[0022] In some embodiments, the NK cells do not include a CAR.

[0023] In some embodiments, the NK cells do not include an engineered CAR.

[0024] In some embodiments, any of the above steps can have further steps added between them. In some embodiments, any one or more of the above steps can be performed concurrently or out of the order provided herein.

[0025] The method of any one of the preceding embodiments, wherein administration of the NK cells decreases neuroinflammation in the subject as compared to the level of neuroinflammation in the subject prior to administration of the NK cells. Also provided is a method of reducing neuroinflammation (e.g., inflammation in the brain related to autism) by administering a therapeutically effective amount of the expanded NK cells of the present disclosure, to a subject in need thereof. In some embodiments, the subject has autism. In some embodiments, decreased or reduced neuroinflammation is measured based on a decrease in one or more biomarkers of neuroinflammation, as described herein.

[0026] The method of any one of the preceding embodiments, wherein administration of the NK cells decreases neuroinflammation in the subject by up to about 100% as compared to the level of neuroinflammation in the subject prior to administration of the NK cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In addition to the features described above, additional features and variations will be readily apparent from the following descriptions of the drawings and exemplary embodiments. It is to be understood that these drawings depict typical embodiments and are not intended to be limiting in scope.

[0028] FIG. 1 is a flow chart depicting some non-limiting embodiments of a method of treating autism in a subject.

[0029] FIG. 2 is a flow chart depicting some non-limiting embodiments of a method of treating autism spectrum disorder (ASD) in a subject.

[0030] FIG. 3 is a flow chart depicting some non-limiting embodiments of a method of treating autism in a subject.

[0031] FIG. 4 is a flow chart depicting some non-limiting embodiments of a method of treating autism spectrum disorder (ASD) in a subject.

[0032] FIG. 5 is a flow chart depicting some non-limiting embodiments of a method of cell therapy.

[0033] FIG. 6 is a flow chart depicting some non-limiting embodiments of a population of expanded NK cells.

[0034] FIG. 7 is a flow chart depicting some non-limiting embodiments of a population of expanded NK cells.

[0035] FIG. 8A is a line graph depicting the average change in A|B-42 levels in the cerebrospinal fluid of subjects treated with different doses of NK cells.

[0036] FIG. 8B is a line graph depicting the change in A(3-42 levels in the cerebrospinal fluid of subjects treated with different doses of NK cells.

[0037] FIG. 9A is a line graph depicting the average change in A(3-42/40 ratio in the cerebrospinal fluid of subject’s treated with different doses of NK cells.

[0038] FIG. 9B is a line graph depicting the change in A(3-42/40 ratio in the cerebrospinal fluid of subject’s treated with different doses of NK cells.

[0039] FIG. 10 is a line graph depicting the average change in total Tau levels in the cerebrospinal fluid of subjects treated with different doses of NK cells.

[0040] FIG. 11A is a line graph depicting the average change in p-tau 181 levels in the cerebrospinal fluid of subjects treated with different doses of NK cells.

[0041] FIG. 1 IB is a line graph depicting the change in p-tau 181 levels in the cerebrospinal fluid of subjects treated with different doses of NK cells. [0042] FIG. 12A is a line graph depicting the average change in GFAP levels in the cerebrospinal fluid of subjects treated with different doses of NK cells.

[0043] FIG. 12B is a line graph depicting the change in GFAP levels in the cerebrospinal fluid of subjects treated with different doses of NK cells.

[0044] FIG. 13A is a line graph depicting the average change in NfL levels in the cerebrospinal fluid of subjects treated with different doses of NK cells.

[0045] FIG. 13B is a line graph depicting the change in NfL levels in the cerebrospinal fluid of subjects treated with different doses of NK cells.

[0046] FIG. 14A is a line graph depicting the average change in YKL-40 levels in the cerebrospinal fluid of subjects treated with different doses of NK cells.

[0047] FIG. 14B is a line graph depicting the change in YKL-40 levels in the cerebrospinal fluid of subjects treated with different doses NK cells.

[0048] FIG. 15A is a line graph depicting the average change in baseline CX3CL1 (Fractalkine) levels in the cerebrospinal fluid of subjects treated with different doses of NK cells.

[0049] FIG. 15B is a line graph depicting the change in baseline CX3CL1 (Fractalkine) levels in the cerebrospinal fluid of subjects treated with different doses NK cells.

[0050] FIG. 16A is a line graph depicting the average change in baseline IL-6 levels in the cerebrospinal fluid of subjects treated with different doses of NK cells.

[0051] FIG. 16B is a line graph depicting the change in baseline IL-6 levels in the cerebrospinal fluid of subjects treated with different doses of NK cells.

[0052] FIG. 17A is a line graph depicting the average change in baseline TNF- a levels in the cerebrospinal fluid of subjects treated with different doses of NK cells.

[0053] FIG. 17B is a line graph depicting the change in baseline TNF-a levels in the cerebrospinal fluid of subjects treated with different doses of NK cells.

[0054] FIG. 18A is a line graph depicting the average change in baseline IL-8 levels in the cerebrospinal fluid of subjects treated with different doses of NK cells.

[0055] FIG. 18B is a line graph depicting the change in baseline IL-8 levels in the cerebrospinal fluid of subjects treated with different doses of NK cells.

[0056] FIG. 19A is a line graph depicting the average change in baseline IL- 12/IL-23p40 levels in the cerebrospinal fluid of subjects treated with different doses of NK cells. [0057] FIG. 19B is a line graph depicting the change in baseline IL-12/IL- 23p40 levels in the cerebrospinal fluid of subjects treated with different doses of NK cells.

[0058] FIG. 20A is a line graph depicting the average change in baseline sTREM2 levels in the cerebrospinal fluid of subjects treated with different doses of NK cells.

[0059] FIG. 20B is a line graph depicting the change in baseline sTREM2 levels in the cerebrospinal fluid of subjects treated with different doses of NK cells.

[0060] FIG. 21A is a line graph showing the average expression level (percentage) of CX3CR1 in T cells in CSF of subjects treated with different doses of NK cells.

[0061] FIG. 21B is a line graph showing the expression level (percentage) of CX3CR1 in T cells in CSF of subjects treated with different doses of NK cells.

[0062] FIG. 22A is a line graph showing the average expression level (percentage) of CX3CR1 in NK cells in CSF of subjects treated with different doses of NK cells.

[0063] FIG. 22B is a line graph showing the expression level (percentage) of CX3CR1 in NK cells in CSF of subjects treated with different doses of NK cells.

[0064] FIG. 23A is a line graph showing the average expression level (percentage) of CX3CR1 in microglia in CSF of subjects treated with different doses of NK cells.

[0065] FIG. 23B is a line graph showing the expression level (percentage) of CX3CR1 in microglia in CSF of subjects treated with different doses of NK cells.

[0066] FIG. 24A is a bar graph depicting average NK cell activity in the plasma of subjects treated with different doses of NK cells.

[0067] FIG. 24B is a bar graph depicting NK cell activity in the plasma of subjects treated with different doses of NK cells.

[0068] FIG. 25 shows the Study Design for a dose escalation study of SNK01 administered to Alzheimer's Disease patients.

[0069] FIG. 26 shows a line graph depicting the change in A0-42 levels in the cerebrospinal fluid of subjects treated with different doses of NK cells.

[0070] FIG. 27 shows a line graph depicting the mean change from baseline for aggregate changes in Ap-42 levels in the cerebrospinal fluid of subjects treated with different doses of NK cells. [0071] FIG. 28 shows a line graph depicting the change in Ap-42/40 ratio in the cerebrospinal fluid of subjects treated with different doses of NK cells.

[0072] FIG. 29 shows a line graph depicting the mean change from baseline in Ap-42/40 ratio in the cerebrospinal fluid of subjects treated with different doses of NK cells.

[0073] FIG. 30 shows line graphs depicting the change in total tau levels in the cerebrospinal fluid of subjects treated with different doses of NK cells.

[0074] FIG. 31 shows a line graph depicting the change in p-tau 181 levels in the cerebrospinal fluid of subjects treated with different doses of NK cells.

[0075] FIG. 32 shows a line graph depicting the mean change from baseline in the aggregate change in p-tau 181 levels in the cerebrospinal fluid of subjects treated with different doses of NK cells.

[0076] FIG. 33 shows a line graph depicting the change in GFAP levels in the cerebrospinal fluid of subjects treated with different doses of NK cells.

[0077] FIG. 34 shows a line graph depicting the mean change from baseline in the aggregate change in GFAP levels in the cerebrospinal fluid of subjects treated with different doses of NK cells.

[0078] FIG. 35 shows a line graph depicting the change in NfL levels in the cerebrospinal fluid of subjects treated with different doses of NK cells.

[0079] FIG. 36 shows a line graph depicting the mean change from baseline in NfL levels in the cerebrospinal fluid of subjects treated with different doses of NK cells.

[0080] FIG. 37 shows a line graph depicting the change in YKL-40 levels in the cerebrospinal fluid of subjects treated with different doses of NK cells.

[0081] FIG. 38 shows a line graph depicting the mean change from baseline in YKL-40 levels in the cerebrospinal fluid of subjects treated with different doses of NK cells.

[0082] FIG. 39 shows a line graph depicting the change in baseline CX3CL1 (Fractalkine) levels in the cerebrospinal fluid of subjects treated with different doses of NK cells.

[0083] FIG. 40 shows line graphs depicting the change in baseline IL-6 levels in the cerebrospinal fluid of subjects treated with different doses of NK cells.

[0084] FIG. 41 shows line graphs depicting the change in baseline TNF-a levels in the cerebrospinal fluid of subjects treated with different doses of NK cells. [0085] FIG. 42 shows line graphs of Ap-42 changes in the plasma of subjects treated with NK cells.

[0086] FIG. 43 shows line graphs of A0-42/4O ratio changes in the plasma of subjects treated with NK cells.

[0087] FIG. 44 shows line graphs of changes in total Tau in the plasma of subjects treated with NK cells.

[0088] FIG. 45 shows line graphs of p-tau 181 changes in the plasma of subjects treated with NK cells.

[0089] FIG. 46 shows line graphs of GFAP changes in the plasma of subjects treated with NK cells.

[0090] FIG. 47shows line graphs of NfL changes in the plasma of subjects treated with NK cells.

[0091] FIG. 48 shows line graphs of YKL-40 changes in the plasma of subjects treated with NK cells.

[0092] FIG. 49 shows line graphs of TNF-a changes in the plasma of subjects treated with NK cells.

[0093] FIG. 50 shows line graphs of IL-8 changes in the plasma of subjects treated with NK cells.

[0094] FIG. 51 shows line graphs of IL-6 changes in the plasma of subjects treated with NK cells.

[0095] FIG. 52 shows line graphs of IL-1|3 changes in the plasma of subjects treated with NK cells.

[0096] FIG. 53 shows line graphs of IL-10 changes in the plasma of subjects treated with NK cells.

[0097] FIG. 54 shows line graphs of IFN-y changes in the plasma of subjects treated with NK cells.

[0098] FIG. 55 shows a line graph of the percentage of CD3+/CD56- T cells in the Leukocytes of subjects treated with NK cells.

[0099] FIG. 56 shows a line graph of the change from the baseline in frequency of CD3+/CD56- T cells in Leukocytes in subjects treated with NK cells.

[0100] FIG. 57 shows a line graph of the mean change from baseline in frequency of CD3+/CD56- T cells in Leukocytes in subjects treated with different doses of NK cells. [0101] FIG. 58 shows a line graph of the percentage of CD3+/CD56- T cells in Lymphocytes of subjects treated with NK cells.

[0102] FIG. 59 shows a line of the change from the baseline in frequency of CD3+/CD56- T cells in Lymphocytes in subjects treated with NK cells.

[0103] FIG. 60 shows a line graph of the mean change from baseline in frequency of CD3+/CD56- T cells in Lymphocytes in subjects treated with different doses of NK cells.

[0104] FIG. 61 shows a line graph of the percentage of CX3CR1+ cells in CD3-

CD56+ NK Cells from subjects treated with NK cells.

[0105] FIG. 62 shows a line graph of the change from the baseline in the percentage of CX3CR1+ cells in CD3-CD56+ NK Cells in subjects treated with NK cells.

[0106] FIG. 63 shows a line graph of the mean change from baseline in the percentage of CX3CR1+ cells in CD3-CD56+ NK Cells in subjects treated with different doses of NK cells.

[0107] FIG. 64 shows a line graph of the percentage of CX3CR1+ cells in CD3+CD56- T Cells from subjects treated with NK cells.

[0108] FIG. 65 shows a line graph of the change from the baseline in the percentage of CX3CR1+ cells in CD3+CD56- T Cells in subjects treated with NK cells.

[0109] FIG. 66 shows a line graph of the mean change from baseline in the percentage of CX3CR1+ cells in CD3+CD56- T Cells in subjects treated with different doses of NK cells.

[0110] FIG. 67 is a line graph depicting the change in total tau levels in the cerebrospinal fluid of subjects treated with different doses of NK cells.

DETAILED DESCRIPTION

[OHl] Provided herein are methods and compositions for treating autism and other ASDs comprising natural killer cells, which can be high purity and/or present in large amounts and/or especially active.

[0112] The description herein is presented only for the purposes of illustration and description and is not intended to be exhaustive or to limit the invention to the precise forms disclosed. Many modifications and variations are possible in light of the teachings herein. It is contemplated that various combinations or sub combinations of the specific features and aspects of the embodiments disclosed herein may be made and still fall within one or more of the inventions. Further, the disclosure herein of any particular feature, aspect, method, property, characteristic, quality, attribute, element, or the like in connection with an embodiment can be used in all other embodiments set forth herein. Accordingly, it should be understood that various features and aspects of the disclosed embodiments can be combined with or substituted for one another in order to form varying modes of the disclosed inventions. Thus, it is intended that the scope of the present embodiments herein disclosed should not be limited by the particular disclosed embodiments described above. Moreover, while the invention is susceptible to various modifications, and alternative forms, specific examples thereof have been shown in the drawings and are herein described in detail. It should be understood, however, that the invention is not to be limited to the particular fonns or methods disclosed, but to the contrary, the invention is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the various embodiments described and the appended claims. Any methods disclosed herein need not be performed in the order recited. The methods disclosed herein include certain actions taken by a practitioner; however, they can also include any third-party instruction of those actions, either expressly or by implication. The ranges disclosed herein also encompass any and all overlap, sub-ranges, and combinations thereof.

[0113] The embodiments described herein were chosen and described in order to explain the principles of the invention and their practical application so as to activate others skilled in the art to utilize the invention and various embodiments and with various modifications as are suited to the particular use contemplated. Alternative embodiments will become apparent to those skilled in the art to which the invention is defined by the appended claims rather than the description and the exemplary embodiments described herein.

[0114] Conditional language, such as “can,” “could,” “might,” or “may,” unless specifically stated otherwise, or otherwise understood within the context as used, is generally intended to convey that certain embodiments include, while other embodiments do not include, certain features, elements, and/or steps. Thus, such conditional language is not generally intended to imply that features, elements, and/or steps are in any way required for one or more embodiments.

[0115] The terms “comprising,” “including,” “having,” and the like are synonymous and are used inclusively, in an open-ended fashion, and do not exclude additional elements, features, acts, operations, and so forth. Also, the term “or” is used in its inclusive sense (and not in its exclusive sense) so that when used, for example, to connect a list of elements, the term “or” means one, some, or all of the elements in the list. [0116] For purposes of this disclosure, certain aspects, advantages, and novel features are described herein. It is to be understood that not necessarily all such advantages may be achieved in accordance with any particular embodiment. Thus, for example, those skilled in the art will recognize that the disclosure may be embodied or carried out in a manner that achieves one advantage or a group of advantages as taught herein without necessarily achieving other advantages as may be taught or suggested herein.

[0117] Moreover, while illustrative embodiments are described herein, the scope of any and all embodiments having equivalent elements, modifications, omissions, combinations (e.g., of aspects across various embodiments), adaptations and/or alterations as would be appreciated by those in the art based on the present disclosure. The limitations in the claims are to be interpreted broadly based on the language employed in the claims and not limited to the examples described in the present specification or during the prosecution of the application, which examples are to be construed as non-exclusive. Further, the actions of the disclosed processes and methods may be modified in any manner, including by reordering actions and/or inserting additional actions and/or deleting actions. It is intended, therefore, that the specification and examples be considered as illustrative only, with a true scope and spirit being indicated by the claims and their full scope of equivalents.

[0118] The ranges disclosed herein also encompass any and all overlap, subranges, and combinations thereof. Language such as "‘up to,” “at least,” “greater than,” “less than,” “between” and the like includes the number recited. Numbers preceded by a term such as “about” or “approximately” include the recited numbers. For example, “about 5.0 cm” includes “5.0 cm.”

[0119] Autism is a neurodevel opmental disorder characterized by repetitive, stereoty pical behaviors and impaired expressive communication, which has since been folded into the broader classification of Autism Spectrum Disorders (ASDs). ASDs are complex neurodevelopmental disorders which are typically diagnosed within the first three years of life. ASD are characterized by significant impairments in social interaction and communicative skills, as well as restricted and stereoty ped behaviors and interests. ASD includes both Asperger’s syndrome and autism disorder, as well as pervasive developmental disorder not otherwise specified (PDD-NOS). As used herein, the terms autism, and ASD are interchangeable. Asperger’s Syndrome is a developmental disorder. Young people with Asperger’s Syndrome have a difficult time relating to others socially and their behavior and thinking patterns can be rigid and repetitive. Generally, children and teens with Asperger’s Syndrome can speak with others and can perform fairly well in their schoolwork. However, they have trouble understanding social situations and subtle forms of communication like body language, humor and sarcasm. They might also think and talk a lot about one topic or interest or only want to do a small range of activities. These interests can become obsessive and interfere with everyday life, rather than giving the child a healthy social or recreational outlet. Specific diagnosis is determined by the nature and severity of delays or deficits in communication and social interactions and the presence or absence of restricted and stereotyped behaviors/interests.

[0120] The broader background of immunogenetic factors related to ASD includes multiple networks of the immune system, such as pathways that regulate cytokines and NK cells, which together constitute a broad, endogenous environment of atypical immune regulation and response. Individuals with ASD may have endogenous anti-brain autoantibodies that correlate with aberrant development and impaired development. ASD- related immune dysregulation spans both innate and adaptive arms of the immune system. This includes an increased inflammatory cytokine milieu (e.g., IL-6, IL-8, and MCP-1), thus leading to an increased, pro-inflammatory Thl/Th2 ratio. Cytokines have been observed at atypical levels in the brain tissue, CSF, circulating blood, and GI tissues of subjects with ASD. These atypical levels can result in altered neuronal survival and proliferation. Similarly, cellular dysfunction observed in ASD may contribute to atypical CNS function in a number of ways including the production of cytokines, abnormal cell lysis and generation of brain-reactive antibodies. Abnormal levels of complement proteins and linkage to specific MHC molecules, have been repeatedly observed as in ASD, suggesting a role for immune function in synaptic pruning/plasticity in ASD. T-cell and natural killer cell populations may also be skewed, displaying a shift in cell subpopulations. Natural killer cells (NK cells) in particular show an increased baseline activity but a decreased response to activation, rendering the cells unable to properly respond to stimuli. Meltzer A, Van de Water J. The Role of the Immune System in Autism Spectrum Disorder. Neuropsychopharmacology. 2017 Jan;42(l):284-298. Doi: 10.1038/npp.2016. 158. Epub 2016 Aug 18. PMID: 27534269; PMCID: PMC5143489.

[0121] NK cells are one type of innate immune cells, which are known to non- specifically kill cancer, recognize and kill viruses, bacteria, and the like, and kill pathogens with enzymes such as perforin and granzyme or by Fas-FasL interaction. NK cells have also been reported to be able to kill activated T cells (Rabinovich B, et al., J Immunol, 2003: 170: 3572-3576). This discovery is of interest in autism where it has been found that nearly half of children with autism or an Autism Spectrum Disorder (ASD) suffer from low NK cell activity. (Vojdani, et al., Journal of Neuroimmunology., 2008: 205: 148-154). Additionally, it has been reported that low intracellular levels of glutathione, and interleukins, specifically interleukins may be responsible for this low NK cell activity.

[0122] Interleukins (IL) are a type of cytokine produced by leukocytes and other cells. IL production is a self-limited process. The messenger RNAs encoding most ILs are unstable and causes a transient synthesis. These molecules are rapidly secreted once synthesized. ILs play essential roles in the activation and differentiation of immune cells, as well as proliferation, maturation, migration, and adhesion. They also have pro- inflammatory and anti-inflammatory properties. ILs modulate growth, differentiation, and activation during inflammatory and immune responses. ILs consist of a large group of proteins that can elicit many reactions in cells and tissues by binding to high-affinity receptors in cell surfaces. They have both paracrine and autocrine function. ILs are also used in animal studies to investigate aspect related to clinical medicine. Cellular responses to interleukins include up- and down-regulatory mechanisms with the induction and participation of genes that encode inhibitors of the cytokine receptors. Interleukins have redundant functions. For instance, IL-4, IL-5, and IL-13 are B-cell growth factors and stimulate B-cell differentiation. Interleukins often influence other interleukin synthesis and actions. For instance, IL-1 promotes lymphocyte activation that leads to the release of IL- 2. Justiz Vaillant AA, Qurie A. Interleukin. 2021 Aug 30. In: StatPearls [Internet], Treasure Island (FL): StatPearls Publishing; 2022 Jan-. PMID: 29763015.

[0123] Interleukin-1 (IL-1) is secreted by macrophages, large granular lymphocytes, B cells, endothelium, fibroblasts, and astrocytes. T cells, B cells, macrophages, endothelium and tissue cells are the main IL-1 targets. IL-1 causes lymphocyte activation, macrophage stimulation, increased leukocyte/endothelial adhesion, fever due to hypothalamus stimulation, and release of acute phase proteins by the liver. It may also cause apoptosis in many cell types and cachexia.

[0124] Interleukin-2 (IL-2) is produced by T-cells. Its primary effects are T-cell proliferation and differentiation, increased cytokine synthesis, potentiating Fas-mediated apoptosis, and promoting regulatory T cell development. It causes proliferation and activation of NK cells and B-cell proliferation and antibody synthesis. Also, it stimulates the activation of cytotoxic lymphocytes and macrophages.

[0125] Interleukin-3 (IL-3) is produced by T cells and stem cells.. IL-3 functions as a multilineage colony -stimulating factor. [0126] Interleukin-4 (IL-4) is produced by CD4+T cells (Th2). IL-4 acts on both B and T cells. IL-4 is a B-cell growth factor and causes IgE and IgGl isotype selection. IL-4 causes Th2 differentiation and proliferation, and it inhibits IFN gamma-mediated activation on macrophages. IL-4 promotes mast cell proliferation in vivo.

[0127] Interleukin-5 (IL-5) is produced by CD4+T cells (Th2). IL-5 targets B cells. It causes B-cell growth factor and differentiation and IgA selection. IL-5 also causes eosinophil activation and increased production of these innate immune cells.

[0128] Interleukin-6 (IL-6) is produced by T and B lymphocytes, fibroblasts and macrophages. IL-6 targets B lymphocytes and hepatocytes. IL-6 results in B-cell differentiation and stimulation of acute phase proteins.

[0129] Interleukin-7 (IL-7) is produced by bone marrow stromal cells. IL-7 targets pre-B cells and T cells, causing B-cell and T-cell proliferation.

[0130] Interleukin-8 (IL-8) is produced by monocytes and fibroblasts. IL-8 targets neutrophils, basophils, mast cells, macrophages, and keratinocytes. IL-8 results in neutrophil chemotaxis, angiogenesis, superoxide release, and granule release.

[0131] Interleukin-9 (IL-9) is produced by Th9, Th2, Thl7, mast cells, NKT cells, and regulatory T cells. IL-9 enhances T-cell survival, mast cell activation and synergy with erythropoietin.

[0132] Interleukin- 10 (IL-10) is produced by Th2 cells. IL-10 targets Thl cells. IL- 10 inhibits IL-2 and interferon gamma, decreases antigen presentation, and MHC class II expression of dendritic cells, co-stimulatory molecules on macrophages. IL-10 also downregulates pathogenic Thl 7 cell responses and inhibits IL-12 production by macrophages.

[0133] Interleukin- 11 (IL-11) is produced by bone marrow stromal cells and fibroblasts. IL- 11 targets hemopoietic progenitors and osteoclasts. IL- 11 promotes osteoclast formation, colony stimulating factor, raised platelet count in vivo, and inhibition of pro-inflammatory cytokine production.

[0134] Interleukin- 12 (IL-12) is produced by monocytes. IL-12 targets T cells. IL-12 causes induction of Thl cells and is a potent inducer of interferon gamma production by T lymphocytes and NK cells.

[0135] Interleukin- 13 (IL-13) is produced by CD4+T cells (Th2), NKT cells and mast cells. IL-13 acts on monocytes, fibroblasts, epithelial cells and B cells. IL-13 promotes B-cell growth and differentiation, stimulates isotype switching to IgE, increased mucus production by epithelial cells, and increased collagen synthesis by fibroblasts. IL- 13inhibits pro-inflammatory cytokine production. IL- 13 works together with IL-4 in producing biologic effects associated with allergic inflammation and in defense against parasites.

[0136] Interleukin- 14 (IL-14) is produced by T cells. IL-14 stimulates activated B cell proliferation and inhibits immunoglobulin secretion.

[0137] Interleukin-15 (IL-15) is produced by monocytes, epithelium, and muscles. IL-15 targets T cells and activated B cells. IL-15 causes the proliferation B cells, T cells, NK cell memory, and CD8+ T cell proliferation.

[0138] Interleukin-16 (IL- 16) is produced by eosinophils and CD8+T cells. IL-

16 targets CD4+ T cells. IL-16 causes CD4+ T cell chemoattraction.

[0139] Interleukin- 17 (IL-17) is produced by Th-17. IL-17 acts on epithelial and endothelial cells. IL-17 promotes the release of IL-6 and other pro-inflammatory cytokines. IL-17 also enhances the activities of antigen-presenting cells and stimulates chemokine synthesis by endothelial cells. There are six members in the IL- 17 family: IL- 17A, IL-17B, IL-17C, IL-17D, IL-17E, and IL-17F.

[0140] IL-17A is heightened in mothers following infections during pregnancy. IL-17A alters brain development in the fetus. IL-17A also changes the mother’s gut microbiome. The microbiome rearrangement affects the fetus’ immune system by altering the chromatin landscape in CD4 positive T cells. This change in immune development primes the offspring for inflammatory attacks of the gut after birth. IL-17A promotes tumorigenesis, metastasis, and viral infection by constraining NK cell antitumor and antiviral activity via inhibition of NK cell maturation. The ablation of IL- 17 A signaling increases terminally mature CD27-CDl lb+ NK cells, whereas constitutive IL-17A signaling reduces terminally mature NK cells. IL-17A suppresses IL-15-induced phosphorylation of STAT5 via up-regulation of S0CS3 in NK cells, leading to inhibition of NK cell terminal maturation. Therefore, IL- 17 A acts as the checkpoint during NK cell terminal maturation, which suggests potential interventions to defend against tumors and infections. Wang et. al, PNAS 2019: 116 (35) 17409-17418.

[0141] Interleukin- 18 (IL-18) is produced by macrophages, hepatocytes, and keratinocytes. IL- 18 targets a co-factor in Thl cell induction. IL- 18 causes interferon gamma production and enhances NK cell activity.

[0142] Interleukin- 19 (IL-19) is produced by Th2 lymphocytes. IL-19 targets resident vascular cells in addition to immune cells. IL-19 is an anti-inflammatory molecule. IL- 19 promotes immune responses mediated by regulatory lymphocytes and has substantial activity on microvascular.

[0143] Interleukin-20 (IL-20) is produced by immune cells and activated epithelial cells. IL-20 targets epithelial cells. IL-20 plays a vital role in the cellular communication between epithelial cells and the immune system under inflammatory conditions.

[0144] Interleukin-21 (IL-21) is produced by NK cells and CD4+ T lymphocytes. IL-21 targets various immune cells of innate and the adaptive immune systems. IL-21 promotes B and T lymphocyte proliferation and differentiation. It enhances NK cell activity.

[0145] Interleukin-22 (IL-22) is primarily produced by T cells but is also produced by different cells in both innate and acquired immunities. IL-22 inhibits IL-4 production. IL-22 also has essential functions in mucosal surface protection and tissue repair.

[0146] Interleukin-23 (IL-23) is produced by macrophages and dendritic cells. IL-23 targets T cells causing maintenance of IL- 17 producing T cells.

[0147] Interleukin-24 (IL-24) is produced by monocytes, T and B cells. IL-24 causes cancer-specific cell death, wound healing, and protects against bacterial infections and cardiovascular diseases.

[0148] Interleukin-25 (IL-25) is produced by dendritic cells. IL-25 targets various types of cells, including Th2 cells IL-25 stimulates the synthesis of Th2 cytokine profile including IL-4 and IL-13.

[0149] Interleukin-26 (IL-26) is strongly associated inflammatory activity. IL-

26 is produced by Thl7 cells. IL-26 targets epithelial cells and intestinal epithelial cells. IL-26 induces IL-10 expression, stimulates the production of IL-l-beta, IL-6, and IL-8 and causes Thl7 cell generation.

[0150] Interleukin-27 (IL-27) is produced by T cells. IL-27 activates STAT-1 and STAT-3, which regulates immune responses. IL-27 stimulates IL-10 production. IL-

27 is a pro-inflammatory molecule and upregulates t pe-2 interferon synthesis by natural killer cells.

[0151] Interleukin-28 (IL-28) is produced by regulatory T-cells. IL-28 targets keratinocytes and melanocytes. IL-28 stimulates cell presentation of viral antigens to CD8+T lymphocytes. IL-28 also upregulates TLR-2 and TLR-3 expression. IL-28 enhances the keratinocyte capacity to recognize pathogens in the healthy skin. [0152] Interleukin-29 (IL-29) is a type-3 interferon that is produced by virus- infected cells, dendritic cells, and regulatory T-cells. IL-29 upregulates viral protective responses. Virus-infected cells may regulate IL-29 genome.

[0153] Interleukin-30 (IL-30) is produced by monocytes in response to TLR agonists including bacterial LPS. IL-30 acts on monocytes, macrophages, dendritic cells, T and B lymphocytes, natural killer cells, mast cells, and endothelial cells.

[0154] Interleukin-31 (IL-31) is produced by Th2 cells and dendritic cells. IL- 31 is a proinflammatory cytokine and a chemotactic factor that direct polymorphonuclear cells, monocytes, and T cells to inflammatory lesions. IL-31 induces chemokines production and synthesis of IL-6, IL-16, and IL-32.

[0155] Interleukin-32 (IL-32) is a pro-inflammatory molecule produced by NK cells and monocytes. IL-32 induces the synthesis of various cytokines including IL-6, and IL- 1 beta. It inhibits IL- 15 production.

[0156] Interleukin-33 (IL-33) is produced by mast cells and Th2 lymphocytes. IL-33 targets various innate and immune cells including dendritic cells and T and B lymphocytes. IL-32 mediates Th2 responses and therefore participates in the protection against parasites and type-I hypersensitivity reaction.

[0157] Interleukin-34 (IL-34) is produced by various phagocytes and epithelial cells synthesize Interleukin-34 (IL-34). IL-34 enhances IL-6 production and participates in the differentiation and development of antigen-presenting cells including microglia.

[0158] Interleukin-35 (IL-35) is produced by regulatory B cells. IL-35 is involved in lymphocyte differentiation. IL-35 exhibits an immune-suppressive effect.

[0159] Interleukin-36 (IL-36) is produced by phagocytes. IL-36 targets T lymphocytes and NK cells regulating the IFN-y synthesis. IL-36 stimulates the hematopoiesis and expression of both MHC class I and II molecules as well as intracellular adhesion molecules (ICAM)-l.

[0160] Interleukin-37 (IL-37) plays an essential role in the regulation of the innate immunity causing immunosuppression. IL-37 is produced by phagocytes and organs including the uterus, testis, and thymus. IL-37 upregulates immune responses and inflammation in autoimmune disorders.

[0161] Interleukin-38 (IL-38) is produced by the placenta, tonsil's B lymphocytes, spleen, skin, and thymus. IL-38targets T cells and inhibits the synthesis of IL- 17 and IL-22. [0162] Interleukin (IL-39) B lymphocytes mainly produce IL-39. It acts on neutrophils inducing their differentiation or expansion.

[0163] Interleukin-40 (IL-40) is produced in the bone marrow, fetal liver, and by activated B cells. IL-40 plays a vital role in the development of humoral immune responses.

[0164] Cytokines are proteins made in response to pathogens and other antigens that regulate and mediate inflammatory and immune responses. Cytokines stimulate switching of antibody isotypes in B cells, differentiation of helper T cells into Th-1 and Th- 2 subsets, and activation of microbicidal mechanisms in phagocytes. Cellular responses to cytokines are stimulated and regulated by external signals or high-affinity receptors. For example, stimulation of B-cells by pathogens leads to increased expression of cytokine receptors. Most cytokines act either on the same cell that secretes the cytokine, for instance, IL-2 produced by T cells operates on the same T cells that made it or on a nearby cell. Besides, cytokines may enter the circulation and act far from the site of production, for example, IL-1 is an endogenous pyrogen that works on the central nervous system (CNS) and causes fever. Small quantities of a cytokine are needed to occupy receptors and elicit biologic effects.

[0165] Enhanced IL-17A is correlated with dysregulated immune response in kids with ASD.

[0166] In order to obtain the therapeutic effect of NK-mediated cells in autism, aNK cells having high purity can be useful. However, it is not easy to obtain alarge amount of blood from the autistic patient, and of the proportion of NK cells in the blood is small, only about 5 to 20%. This is one reason it has been difficult for using the NK cells as an immunotherapeutic agent.

[0167] In some embodiments, it is desirable to effectively expand and proliferate only the NK cells, but in a conventional method of proliferating NK cells, various expensive cytokines need to be used at a high concentration, thus the corresponding therapy is only available to some financially stable patients. Further, according to conventional methods of proliferating NK cells, other types (e.g., T cells, B cells, etc.) of immune cells may be present together with the NK cells, and allogeneic administration of the NK cells containing T cells may cause a graft versus host disease (GVHD) and allogeneic administration of the NK cells containing B cells to blood-type incompatible subjects may cause a passenger B-lymphocyte syndrome, and thus, the therapeutic effect in autism is not maximized. [0168] Further, in addition to expanding and proliferating NK cells, it is desirable to highly maintain the functions of NK cells until the expanded and proliferated NK cells are actually used. As a result, the development of a composition capable of promoting the proliferation of the NK cells, increasing production of cytokines such as TNFa, INFy and GM-CSF derived from the NK cells, and increasing activity of the NK cells is sought.

[0169] The term “treat” and “treatment” includes therapeutic treatments, prophylactic treatments, and applications in which one reduces the risk that a subject will develop a disorder or other risk factor. Treatment does not require the complete curing of a disorder and encompasses embodiments in which one reduces symptoms or underlying risk factors. For example, in some embodiments a treatment may reduce one or more symptoms of autism or ASD. In some embodiments, the symptom or disorder is avoiding eye contact, a subject failing to respond to their own name by nine months, a lack of facial expressions by nine months, failure to play in interactive games by 12 months, use of few or no gestures (e.g., waiving good-bye) by 12 months, failure to share interests with others their same age by 15 months, failure to point out interesting objects or circumstances by 18 months, failure to notice when others are hurt or upset by 24 months, lack of empathy, failure to notice other children playing by 36 months, failure to join other children playing by 36 months, failure to play make believe by 48 months, failure to sing, dance, or act by 60 months. In some embodiments, the symptom includes lining up toys or other objects, getting upset when order is changed, repeating words and phrases over and over, echolalia, playing with toys the same way every time, intensely focusing on parts of objects, getting upset by minor changes, having obsessive interests, having to follow certain routines, obsessive compulsive, flaps hands, rocks body, or spins self in circles, or has unusual reactions to the way things sound, smell taste, look, or feel. In some embodiments, the autistic subject has delayed language skills, delayed movement skills, delayed cognitive skills, delayed learning skills, hyperactivity, impulsivity, inattentive behavior, epilepsy, seizure disorder, unusual eating or sleeping habits, gastrointestinal issues, constipation, unusual mood or emotional reactions, anxiety, stress, excessive worry, lack of fear, or greater fear than expected. In some embodiments, one or more of the autism or ASD symptoms disclosed herein are treated by administration of NK cells. In some embodiments, treatment of an autism or ASD symptom does not require 100% reversal of the disease state. As used herein, slowing, stopping, and/or reversing one or more symptoms will also qualify as a treatment. [0170] The term “prevent” does not require the 100% elimination of the possibility of an event. Rather, it denotes that the likelihood of the occurrence of the event has been reduced in the presence of the compound or method.

[0171] Numbers preceded by a term such as “approximately”, “about”, and “substantially” as used herein include the recited numbers (e.g., about 10% = 10%), and also represent an amount close to the stated amount that still performs a desired function or achieves a desired result. For example, the terms “approximately”, “about”, and “substantially” may refer to an amount that is within less than 10% of, within less than 5% of, within less than 1% of, within less than 0.1% of, and within less than 0.01% of the stated amount.

[0172] The term “generally” as used herein represents a value, amount, or characteristic that predominantly includes or tends toward a particular value, amount, or characteristic. As an example, in certain embodiments, the term “generally uniform” refers to a value, amount, or characteristic that departs from exactly uniform by less than 20%, less than 15%, less than 10%, less than 5%, less than 1%, less than 0.1%, and less than 0.01%.

[0173] As used herein, the term “YKL-40” (also known as Chitinase 3-like 1) refers to a glycoprotein produced by inflammatory, cancer and stem cells. YKL-40 is elevated in the brain and cerebrospinal fluid (CSF) in several neurological and neurodegenerative diseases associated with inflammatory processes.

[0174] As used herein, the term “CX3CL1” or “Fractalkine” refers to a chemokine expressed mainly in neurons in the CNS. Soluble CX3CL1 has a chemoattractive effect for monocytes, natural killer cells, and lymphocyte cells. Receptor CX3CR1 is expressed in microglia, astrocytes, T cells and NK cells. The interaction between CX3CL1 and CX3CR1 has both beneficial and detrimental consequences throughout the activation of various pathways within microglia. Therefore, correct functionality of the CX3CL1/CX3CR1 axis is crucial for the maintenance of brain homeostasis, and especially for dealing with microglia-mediated inflammation in the CNS. CX3CL1 acts as a regulator of microglia activation in response to brain injury or inflammation.

[0175] In some embodiments, the cell therapeutic composition may include a therapeutically effective amount of cell therapeutic agent for treatment of diseases. The term “therapeutically effective amount” means an amount of an active ingredient or a cell therapeutic composition which induces biological or medical responses in tissue systems, animals, or humans which are considered by researchers, veterinarians, physicians, or other clinicians, and includes an amount of inducing alleviation of symptoms of diseases or disorders to be treated. It will be apparent to those skilled in the art that the cell therapeutic agent included in the cell therapeutic composition may be changed according to a desired effect. Therefore, the optimal content of the cell therapeutic agent may be easily determined by those skilled in the art, and may be adjusted according to various factors including a ty pe of disease, seventy of the disease, contents of other ingredients contained in the composition, a type of formulation, and an age, a weight, a general health condition, a gender, and a diet of a patient, an administration time, an administration route, a secretion ratio of the composition, a treatment period, and simultaneously used drugs. It is important to include an amount capable of obtaining a maximum effect by a minimum amount without side effects by considering all of the factors. For example, in some embodiments, the cell therapeutic composition may include a cell therapeutic agent of 1 x 10⁶ to 5 x 10⁸ cells per kg of body weight. In some embodiments, the cell therapeutic composition may include a cell therapeutic agent of 1 x 10⁶ to 1 x 10¹² cells.

[0176] Autism spectrum disorder (ASD) is a neurological and developmental disorder that affects how people interact with others, communicate, learn, and behave. Although autism can be diagnosed at any age, it is described as a “developmental disorder” because symptoms generally appear in the first two years of life.

[0177] In some embodiments, a method of treating autism in a subject is provided. In some embodiments, the method comprises: identifying a subject, wherein the subject has autism; and administering to the subject an expanded natural killer (NK) cell population. In some embodiments, the NK cells are expanded by a method comprising: i) isolating at least one of CD56+ cells and/or CD3-/CD56+ cells from the PBMCs; ii) coculturing the at least one of CD56+ cells and/or CD3-/CD56+ cells with a combination of feeder cells in the presence of at least two cytokines; iii) wherein the combination of feeder cells comprises irradiated Jurkat cells and irradiated Epstein-Barr virus transformed lymphocyte continuous line (EBV-LCL) cells; and iv) wherein the at least two cytokines comprise IL-2 and IL-21.

[0178] In some embodiments, a method of cell therapy is provided, comprising: identifying a subject, wherein the subject has autism; and administering to the subject an expanded NK cell population. In some embodiments, the NK cells are expanded by a method comprising: i) isolating at least one of CD56+ cells and/or CD3-/CD56+ cells from the PBMCs; ii) co-culturing the at least one of CD56+ cells and/or CD3-/CD56+ cells with a combination of feeder cells in the presence of at least two cytokines; iii) wherein the combination of feeder cells comprises irradiated Jurkat cells and irradiated Epstein-Barr virus transformed lymphocyte continuous line (EBV-LCL) cells; and iv) wherein the at least two cytokines comprise IL-2 and IL-21.

[0179] FIG. 1 is a flow chart depicting some non-limiting embodiments of a method of treating autism in a subject. In some embodiments, a method of treating autism in a subject 100 is disclosed. In some embodiments, the method comprises identifying a subject, wherein the subject has autism, at block 101 (with reference to FIG. 1); and administering to the subject an expanded natural killer (NK) cell population, at block 102. In some embodiments, the NK cells were expanded by isolating at least one of CD56+ cells and/or CD3-/CD56+ cells from the PBMCs (in block 103) and co-culturing the at least one of CD56+ cells and/or CD3-/CD56+ cells with a combination of feeder cells in the presence of at least two cytokines (in block 104). In some embodiments, the combination of feeder cells comprises irradiated Jurkat cells and irradiated Epstein-Barr virus transformed lymphocyte continuous line (EBV-LCL) cells (in block 105). In some embodiments, the at least two cytokines comprise IL-2 and IL-21 (in block 106). In some embodiments, the at least two cytokines comprise any two or more of IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL- 21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, IL- 34, IL-35, IL-36, IL-37, IL-38, IL-39, or IL-40.

[0180] In some embodiments, a method of treating autism spectrum disorder (ASD) in a subject is provided. In some embodiments, the method comprises: identifying a subject, wherein the subject has ASD; and administering to the subject an expanded natural killer (NK) cell population. In some embodiments, the NK cells are expanded by a method comprising: i) isolating at least one of CD56+ cells and/or CD3-/CD56+ cells from the PBMCs; ii) co-culturing the at least one of CD56+ cells and/or CD3-/CD56+ cells with a combination of feeder cells in the presence of at least two cytokines; iii) wherein the combination of feeder cells comprises irradiated Jurkat cells and irradiated Epstein-Barr virus transformed lymphocyte continuous line (EBV-LCL) cells; and iv) wherein the at least two cytokines comprise IL-2 and IL-21.

[0181] FIG. 2 is a flow chart depicting some non-limiting embodiments of a method of treating autism spectrum disorder (ASD) in a subject.

[0182] In some embodiments, a method of treating autism spectrum disorder (ASD) in a subject 200 is disclosed. In some embodiments, the ASD is autism, high- functioning autism, Asperger’s syndrome, Pervasive developmental disorder — not otherwise specified (PDD-NOS), and/or another ASD. In some embodiments, the method comprises identifying a subject, wherein the subject has ASD, at block 201; and administering to the subject an expanded natural killer (NK) cell population, at block 202. In some embodiments, the NK cells were expanded by isolating at least one of CD56+ cells and/or CD3-/CD56+ cells from the PBMCs (in block 203) and co-culturing the at least one of CD56+ cells and/or CD3-/CD56+ cells with a combination of feeder cells in the presence of at least two cytokines (in block 204). In some embodiments, the combination of feeder cells comprises irradiated Jurkat cells and irradiated Epstein-Barr virus transformed lymphocyte continuous line (EBV-LCL) cells (in block 205). In some embodiments, the at least two cytokines comprise IL-2 and IL-21 (in block 206). In some embodiments, the at least two cytokines comprise any two or more of IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL- 21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, IL- 34, IL-35, IL-36, IL-37, IL-38, IL-39, or IL-40.

[0183] In some embodiments, a method of treating autism in a subject is provided. In some embodiments, the method comprises identifying a subject, wherein the subject has autism: and administering to the subject a therapeutically effective amount of an autologous natural killer cell (NK) population.

[0184] FIG. 3 is a flow chart depicting some non-limiting embodiments of a method of treating autism in a subject.

[0185] In some embodiments, a method of treating autism in a subject 300 is provided. In some embodiments, the method comprises identifying a subject, wherein the subject has autism, at block 301; and administering to the subject a therapeutically effective amount of an autologous natural killer cell (NK) population, at block 302.

[0186] In some embodiments, a method of treating autism spectrum disorder (ASD) in a subject is provided. In some embodiments, the method comprises identifying a subject, wherein the subject has ASD; and administering to the subject a therapeutically effective amount of an autologous natural killer cell (NK) population.

[0187] FIG. 4 is a flow chart depicting some non-limiting embodiments of a method of treating autism spectrum disorder (ASD) in a subject.

[0188] In some embodiments, a method of treating autism spectrum disorder (ASD) in a subject 400 is provided. In some embodiments, the ASD is autism, high- functioning autism, Asperger’s syndrome, Pervasive developmental disorder — not otherwise specified (PDD-NOS), and/or another ASD. In some embodiments, the method comprises identifying a subject, wherein the subject has ASD (at block 401); and administering to the subject a therapeutically effective amount of an autologous natural killer cell (NK) population (at block 402).

[0189] In some embodiments, a method of cell therapy is provided, comprising: identifying a subject, wherein the subject has autism; and administering to the subject an expanded NK cell population. In some embodiments, the NK cells are expanded by a method comprising: i) isolating at least one of CD56+ cells and/or CD3-/CD56+ cells from the PBMCs; ii) co-culturing the at least one of CD56+ cells and/or CD3-/CD56+ cells with a combination of feeder cells in the presence of at least two cytokines; iii) wherein the combination of feeder cells comprises irradiated Jurkat cells and irradiated Epstein-Barr virus transformed lymphocyte continuous line (EBV-LCL) cells; and iv) wherein the at least two cytokines comprise IL-2 and IL-21. In some embodiments, the two cytokines comprise IL-2 and IL-21. In some embodiments the two cytokines comprise any two of IL-

I, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, IL-34, IL-35, IL-36, IL-37, IL-38, IL-39, or IL-40.

[0190] FIG. 5 is a flow chart depicting some non-limiting embodiments of a method of cell therapy.

[0191] In some embodiments, a method of cell therapy 500 is provided, comprising: identifying a subject at block 501, wherein the subject has autism; and administering to the subject an expanded NK cell population, at block 502. In some embodiments, the NK cells are expanded by a method comprising: i) isolating at least one of CD56+ cells and/or CD3-/CD56+ cells from the PBMCs, at block 503; ii) co-culturing the at least one of CD56+ cells and/or CD3-/CD56+ cells with a combination of feeder cells in the presence of at least two cytokines, at block 504; iii) wherein the combination of feeder cells comprises irradiated Jurkat cells and irradiated Epstein-Barr vims transformed ly mphocyte continuous line (EBV-LCL) cells, at block 505; and iv) wherein the at least two cytokines comprise IL-2 and IL-21 (at block 506). In some embodiments, the at least two cytokines comprise IL-2 and IL-21. In some embodiments the at least two cytokines comprise any two or more of IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-

I I, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL- 24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, IL-34, IL-35, IL-36, IL- 37, IL-38, IL-39, or IL-40. [0192] In some embodiments, a population of expanded NK cells is provided. In some embodiments, the NK cells were expanded by a method that comprises: i) isolating at least one of CD56+ cells and/or CD3-/CD56+ cells from the PBMCs; ii) co-culturing the at least one of CD56+ cells and/or CD3-/CD56+ cells with a combination of feeder cells in the presence of at least two cytokines; iii) wherein the combination of feeder cells comprises irradiated Jurkat cells and irradiated Epstein-Barr virus transformed lymphocyte continuous line (EBV-LCL) cells; and iv) wherein the at least two cytokines comprise IL- 2 and IL-21. In some embodiments, the population of expanded NK cells has been administered to a subject who has autism.

[0193] FIG. 6 is a flow chart depicting some non-limiting embodiments of a population of expanded NK cells.

[0194] In some embodiments, a population of expanded NK cells is provided 600. In some embodiments, the NK cells were expanded by a method that comprises: i) isolating at least one of CD56+ cells and/or CD3-/CD56+ cells from the PBMCs, at block 601; ii) co-culturing the at least one of CD56+ cells and/or CD3-/CD56+ cells with a combination of feeder cells in the presence of at least two cytokines, at block 602; iii) wherein the combination of feeder cells comprises irradiated Jurkat cells and irradiated Epstein-Barr virus transformed lymphocyte continuous line (EBV-LCL) cells (in block 603); and iv) wherein the at least two cytokines comprise IL-2 and IL-21 (in block 604). In some embodiments, the at least two cytokines comprise any two or more of IL-1, IL-2, IL- 3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-1 1 , IL-12, IL-13, IL-14, IL-15, IL-16, IL- 17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL- 30, IL-31, IL-32, IL-33, IL-34, IL-35, IL-36, IL-37, IL-38, IL-39, or IL-40. In some embodiments, the population of expanded NK cells has been administered to a subject who has autism (in block 604). In some embodiments, the ASD is autism, high-functioning autism, Asperger’s syndrome, Pervasive developmental disorder — not otherwise specified (PDD-NOS), and/or another ASD.

[0195] In some embodiments, a population of expanded NK cells is provided. In some embodiments, the NK cells were expanded by a method that comprises: i) isolating at least one of CD56+ cells and/or CD3-/CD56+ cells from the PBMCs; ii) co-culturing the at least one of CD56+ cells and/or CD3-/CD56+ cells with a combination of feeder cells in the presence of at least two cytokines; iii) wherein the combination of feeder cells comprises irradiated Jurkat cells and irradiated Epstein-Barr virus transformed lymphocyte continuous line (EBV-LCL) cells; and iv) wherein the at least two cytokines comprise IL- 2 and IL-21. In some embodiments, the population of expanded NK cells has been administered to a subject who has autism spectrum disorder (ASD).

[0196] FIG. 7 is a flow chart depicting some non-limiting embodiments of a population of expanded NK cells.

[0197] In some embodiments, a population of expanded NK cells is provided 700. In some embodiments, the NK cells were expanded by a method that comprises: i) isolating at least one of CD56+ cells and/or CD3-/CD56+ cells from the PBMCs, at block 701; ii) co-culturing the at least one of CD56+ cells and/or CD3-/CD56+ cells with a combination of feeder cells in the presence of at least two cytokines, at block 702; iii) wherein the combination of feeder cells comprises irradiated Jurkat cells and irradiated Epstein-Barr virus transformed lymphocyte continuous line (EBV-LCL) cells (in block 703); and iv) wherein the at least two cytokines comprise IL-2 and IL-21 (in block 704). In some embodiments, the at least two cytokines comprise any two or more of IL-1, IL-2, IL- 3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL- 17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL- 30, IL-31, IL-32, IL-33, IL-34, IL-35, IL-36, IL-37, IL-38, IL-39, or IL-40. In some embodiments, the population of expanded NK cells has been administered to a subject who has ASD (in block 704).

[0198] In some embodiments, the therapeutically effective amount of autologous NK cells is administered about everv 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 1 , 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 days, or for a range that is defined by any two of the preceding values. For example, in some embodiments, the therapeutically effective amount of autologous NK cells is administered between about every 1 and 31, 1 and 30, 1 and 29, 1 and 28, 1 and 21, 1 and 14, 1 and 7, 3 and 31, 3 and 30, 3 and 29, 3 and 28, 3 and 21, 3 and 14, 3 and 7, 5 and 31, 5 and 30, 5 and 29, 5 and 28, 5 and 21, 5 and 14, 5 and 7, 7 and 31, 7 and 30, 7 and 29, 7 and 28, 7 and 21, 7 and 14, 14 and 31, 14 and 30, 14 and 29, 14 and 28, and 14 and 21 days. In some embodiments, it is less than once a month.

[0199] In some embodiments, the therapeutically effective amount of autologous NK cells is administered about every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months, or for a range that is defined by any two of the preceding values. For example in some embodiments, In some embodiments, the therapeutically effective amount of autologous NK cells is administered between about every 1 and 12, 1 and 10, 1 and 8, 1 and 6, 1 and 4, 1 and 3, 3 and 12, 3 and 10, 3 and 8, 3 and 6, 4 and 12, 4 and 10, 4 and 8, 4 and 6, 6 and 12, 6 and 10, 6 and 8, 8 and 12, or 8 and 10 months.

[0200] In some embodiments, the therapeutically effective amount of autologous NK cells is administered about every' 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 years, or in a range that is defined by any two of the preceding values. For example, in some embodiments, the therapeutically effective amount of autologous NK cells is administered between about every 1 and 10, 1 and 7, 1 and 5, 1 and 3, 3 and 10, 3 and 7, 3 and 5, 5 and 10, or 5 and 7 years.

[0201] In some embodiments, the therapeutically effective amount of autologous NK cells that is administered differs with the subject’s age. For example, in some embodiments, the administration of therapeutically effective amount of autologous NK cells occurs with a decreased frequency as the subject ages. In some embodiments, the frequency of autologous NK cell administration decreases by about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% as the autism subject ages, or is decreased by a range that is defined by any two of the preceding claims. For example, in some embodiments, the frequency of autologous NK cell administration decreases by between about 1% and 100%, 1% and 95%, 1% and 90%, 1% and 75%, 1% and 50%, 1% and 25%, 1% and 10%, 1% and 5%, 5% and 100%, 5% and 95%, 5% and 90%, 5% and 75%, 5% and 50%, 5% and 25%, 5% and 10%, 10% and 100%, 10% and 95%, 10% and 90%, 10% and 75%, 10% and 50%, 10% and 25%, 25% and 100%, 25% and 95%, 25% and 90%, 25% and 75%, 25% and 50%, 50% and 100%, 50% and 95%, 50% and 90%, 50% and 75%, 75% and 100%, 75% and 95%, 75% and 90%, 90% and 100%, or 90% and 95%, as the autism subject ages. In some embodiments, the frequency of autologous NK cell administration decreases by about 1-fold, 2-fold, 3-fold, 4-fold, 5- fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold, or by a range that is defined by any two of the preceding values. For example, in some embodiments, the frequency of autologous NK cell administration decreases by between about 1-fold an 10-fold, 1-fold and 7-fold, 1-fold and 5-fold, 1-fold and 3-fold, 3-fold and 10-fold, 3-fold and 7-fold, 3-fold and 5-fold, 5- fold and 10-fold, 5-fold and 7-fold, and 7-fold and 10-fold.

[0202] In some embodiments, the administration of therapeutically effective amount of autologous NK cells occurs with an increased frequency as the subject ages. For example, in some embodiments, the administration of therapeutically effective amount of autologous NK cells occurs with an increased frequency as the subject ages. In some embodiments, the frequency of autologous NK cell administration increases by about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% as the autism subject ages, or is increased by a range that is defined by any two of the preceding claims. For example, in some embodiments, the frequency of autologous NK cell administration increases by between about 1 % and 100%, 1 % and 95 %, 1% and 90%, 1% and 75%, 1% and 50%, 1% and 25%, 1% and 10%, 1% and 5%, 5% and 100%, 5% and 95%, 5% and 90%, 5% and 75%, 5% and 50%, 5% and 25%, 5% and 10%, 10% and 100%, 10% and 95%, 10% and 90%, 10% and 75%, 10% and 50%, 10% and 25%, 25% and 100%, 25% and 95%, 25% and 90%, 25% and 75%, 25% and 50%, 50% and 100%, 50% and 95%, 50% and 90%, 50% and 75%, 75% and 100%, 75% and 95%, 75% and 90%, 90% and 100%, or 90% and 95%, as the autism subject ages. In some embodiments, the frequency of autologous NK cell administration increases by about 1- fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold, or by a range that is defined by any two of the preceding values. For example, in some embodiments, the frequency of autologous NK cell administration increases by between about 1-fold an 10- fold, 1-fold and 7-fold, 1-fold and 5-fold, 1-fold and 3-fold, 3-fold and 10-fold, 3-fold and 7-fold, 3-fold and 5-fold, 5-fold and 10-fold, 5-fold and 7-fold, and 7-fold and 10-fold.

[0203] In some embodiments, the amount of expanded NK cells administered to a subject is a therapeutically effective amount. In some embodiments, the therapeutically effective amount of expanded NK cells comprises about Ixl O⁶, 1x10⁷, Ixl O⁸, 2 l 0⁸, 3x10⁸, 4xl0⁸, 5xl0⁸, 6xl0⁸, 7xl0⁸, 8xl0⁸, 9xl0⁸, IxlO⁹, 2xl0⁹, 3xl0⁹, 4xl0⁹, 5xl0⁹, 6xl0⁹, 7xl0⁹, 8xl0⁹, 9xl0⁹, IxlO¹⁰, 2xlO¹⁰, 3xl0¹⁰, 4xlO¹⁰, 5xl0¹⁰, 6xlO¹⁰, 7xlO¹⁰, 8xl0¹⁰, 9xlO¹⁰, IxlO¹¹, 2xlOⁿ, 3x10“, 4xlOⁿ, 5xl0ⁿ, 6xlOⁿ, 7x10“ 8xl0ⁿ, 9xlOⁿ, or IxlO¹² NK cells, or is a range that is defined by any two of the preceding values. For example, in some embodiments, the therapeutically effective amount of expanded NK cells comprises between about IxlO⁶ and IxlO¹², IxlO⁶ and IxlO¹¹, IxlO⁶ and IxlO¹⁰, IxlO⁶ and IxlO⁹,

IxlO⁶ and IxlO⁸, IxlO⁶ and IxlO⁷, IxlO⁷ and IxlO¹², IxlO⁷ and IxlO¹¹, IxlO⁷ and IxlO¹⁰,

IxlO⁷ and IxlO⁹, IxlO⁷ and IxlO⁸, IxlO⁸ and IxlO¹², IxlO⁸ and IxlO¹¹, IxlO⁸ and IxlO¹⁰,

IxlO⁸ and IxlO⁹, IxlO⁹ and IxlO¹², IxlO⁹ and IxlO¹¹, IxlO⁹ and IxlO¹⁰, IxlO¹⁰ and IxlO¹², and IxlO¹⁰ and lxl0ⁿNK cells.

[0204] In some embodiments, IL-2 is added at a concentration of 50-1000 lU/mL during step ii). [0205] In some embodiments IL-21 is added at a concentration of 10-100 ng/mL during step ii).

[0206] In some embodiments, expansion of NK cells further comprises: coculturing the at least one of CD56+ cells and/or CD3-/CD56+ cells with the combination of feeder cells, in the presence of IL-2 for a first period; and co-culturing the at least one of CD56+ cells and/or CD3-/CD56+ cells with the combination of feeder cells, in the presence of IL-21 for a second period.

[0207] In some embodiments, IL-21 is added more than once during Day 0-6 of the second period.

[0208] In some embodiments, IL-21 and the combination of feeder cells are added more than once during Day 0-6 of the second period.

[0209] In some embodiments, IL-21 is added more than once during the first six days of every fourteen-day cycle during the second period.

[0210] In some embodiments, the NK cells do not include a chimeric antigen receptor (CAR).

[0211] In some embodiments, the NK cells do not include an engineered CAR.

[0212] In any method of the present disclosure, in some embodiments, the NK cells to be administered can be NK cells that have been expanded with any suitable option for expanding NK cells. In some embodiments, the NK cells are autologous (e.g., autologous to the subject to which the NK cells are administered). In some embodiments, the NK cells are or comprise SNK01. (See www(dot)sec(dot)gov/ix?doc=/Archives/edgar/data/l 845459/000110465923074785/gfor- 2023033 lxs4a (dot) htm, which is incorporated by reference herein as to SNK01). As used herein, “SNK01” denotes SNK01 autologous NK cells produced by NKGen Biotech, Inc. (Irvine, CA). In some embodiments, the NK cells are or comprise SNK01 autologous cells, produced by NKGen Biotech, Inc. (Irvine, CA). Suitable options for expanding NK cells are provided in, e.g., PCT publication No. WO 2019/152663, which is incorporated by reference in its entirety herein. In some embodiments, the NK cells are allogeneic (e.g., allogeneic to the subject to which the NK cells are administered).

[0213] In some embodiments, any of the above steps can have further steps added between them. In some embodiments, any one or more of the above steps can be performed concurrently or out of the order provided herein.

[0214] A method for producing high-purity NK cells without using expensive cytokines has been developed. After CD56+ cells are isolated from peripheral blood mononuclear cells, when the CD56+ cells isolated from peripheral blood mononuclear cells are co-cultured with feeder cells in the presence of cytokines, high-purity CD56+ NK cells could be produced. Also, a cell therapeutic composition for treating autism comprising NK cells which are effectively usable for autologous and allogeneic therapy is provided herein. As a result, when a specific cytokine was added to CD56+ NK cells isolated from peripheral blood mononuclear cells, high survival rate and high activity were exhibited. Therefore, in some embodiments, the treatment of autism involves or includes a method for expanding NK cells and to provide a cell therapeutic composition for the treatment of autism comprising expanded peripheral blood-derived CD56+ NK cells.

[0215] According to some embodiments, a method for producing high-purity NK cells may include: isolating peripheral blood mononuclear cells (PBMCs) from a blood sample (“First Isolation Step”); isolating cells selected from a group consisting of CD56+ cells and CD3-/CD56+ cells from the peripheral blood mononuclear cells (“Second Isolation Step”); and co-culturing the cells selected from a group consisting of CD56+ cells and CD3-/CD56+ cells together with feeder cells in the presence of cytokine (“Culturing Step”). Each step is described in greater detail herein. The CD3-/CD56+ cells produced according to the disclosed method may exhibit not only higher purity and higher activity, but also other distinguished characteristics, such as having different surface markers or activated receptors, for example, one or more from CD16, CD25, CD27, CD28, CD69, CD94/NKG2C, CD94/NKG2E, CD266, CD244, NKG2D, KIR2S, KIR3S, Ly94D, NCRs, IFN-a, IFN-b,CXCR3, CXCR4, CX3CR1, CD62L and CD57, as compared with NK cells produced from peripheral blood mononuclear cells without isolating CD56+ cells.

First Isolation Step

[0216] In the present specification, the “blood sample” may be, but not limited to, whole blood of the peripheral blood or leukocytes isolated from the peripheral blood using leukapheresis. Further, the peripheral blood may be obtained from a normal person, a patient having a risk of autism, or a autism patient, but the source of the peripheral blood is not limited thereto.

[0217] In the present specification, the term “leukapheresis” may refer to a method of selectively removing (isolating) leukocytes from the collected blood and then giving the blood to a patient again, and in some embodiments, the leukocytes isolated by the method may be used without additional methods such as a Ficoll-Hypaque density gradient method. [0218] In the present specification, the term “peripheral blood mononuclear cell” may be used interchangeably with “PBMC”, “mononuclear cell” or “monocyte”, and may refer to a mononuclear cell isolated from the peripheral blood which is generally used for anti-autism immunotherapy. The peripheral blood mononuclear cells may be obtained from the collected human blood using known methods such as a Ficoll-Hypaque density gradient method.

[0219] In some embodiments, the peripheral blood mononuclear cells may be autologous, but allogeneic peripheral blood mononuclear cells may also be used for producing high-purity NK cells for immunotherapy according to methods described herein. Further, in some embodiments, the peripheral blood mononuclear cells may be obtained from a normal person, but the peripheral blood mononuclear cells may be also obtained from a patient having a risk of autism and/ or a autism patient.

[0220] In the present specification, the term “CD56+ cells” may be used interchangeably with “CD56+ NK cells”, or “CD56+ natural killer cells”, and the term “CD3-/CD56+ cells” may be used interchangeably with “CD3-/CD56+ NK cells.” The CD56+ cells or CD3-/CD56+ cells may include cells in which CD56 glycoprotein on the cell surface is expressed, or further, cells in which CD3 glycoprotein is not expressed while the CD56 glycoprotein is expressed. Even the same type of immune cells may have differences in CD type attached to the cell surface and expression rate and thus, the functions thereof may be different.

Second Isolation Step

[0221] In some embodiments, the isolating of the CD56+ natural killer cells from the blood sample may be performed by an isolating method using at least one selected from the group consisting of CD56 microbeads and CD3 microbeads, or an isolating method using equipment such as CliniMACSs, a flow cytometry cell sorter, etc.

[0222] For example, the isolating method using the CD56 microbeads and/or the CD3 microbeads may be performed by adding the CD56 microbeads to PBMCs and then removing non-specific binding, or performed by adding the CD3 microbeads to the PBMCs to remove specific binding and then adding the CD56 microbeads again to remove non-specific binding. In some instances, through isolating CD56+ cells and/or CD3- /CD56+ cells from PBMCs, T cells or other non-natural killer cells may be removed. Culturing Step

[0223] In the present specification, the term “cytokine” may refer to an immunoactive compound that is usable to induce the peripheral blood mononuclear cells to differentiate into NK cells.

[0224] In some embodiments, the cytokine may be interleukin-2 (IL-2), IL-15, IL-21, FMS-like tyrosine kinase 3 ligand (F1I3-L), a stem cell factor (SCF), IL-7, IL-18, IL-4, ty pe I interferons, a granulocyte-macrophage colony-stimulating factor (GM-CSF), and an insulin-like growth factor 1 (IGF 1), but not limited thereto.

[0225] In some embodiments, the cytokine may be used at a concentration of 50-1,000, 50-900, 50-800, 50-700, 50-600, 50-550, 100-550, 150-550, 200-550, 250-550, 300-550, 350-550, 400-550, 450-550 lU/mL. Conventional methods of proliferating NK cells utilize high concentrations of various cytokines. Conversely, in some embodiments of the method of proliferating NK cells described herein, since two types of feeder cells may be used with the high-purity CD56+ cells, NK cells with high yield and high purity may be proliferated using only low concentrations of one cytokine.

[0226] In the present specification, the term “feeder cell” may refer to a cell that does not divide and proliferate, but has metabolic activity to produce various metabolites and thus, helps the proliferation of target cells.

[0227] In some embodiments, the feeder cells may be at least one selected from the group consisting of irradiated Jurkat cells, irradiated Epstein-Ban virus transformed lymphocyte continuous line (EBV-LCL) cells, and PBMC, HFWT, RPMI 1866, Daudi, MM-170, K562 or cells genetically modified by targeting K562 (for example, K562-mbIL- 15-41BB ligand). For example, in one embodiment, the feeder cells may be the irradiated Jurkat cells and the EBV-LCL cells.

[0228] In the present specification, the term “Jurkat cell” or “Jurkat cell line” may refer to a blood cancer (immortalized acute T cell leukemia) cell line, which has been developed by Dr. Arthur Weiss of the University of California at San Francisco. Jurkat cells, in which various chemokine receptors are expressed and capable of producing IL-2, have not generally been considered as a possible candidate of the feeder cells for immunotherapy because MFIC class I, which is a natural killer cell activation inhibitor, is highly expressed on the cell surface thereof. The Jurkat cells may be obtained from the ATCC (ATCC TIB-152).

[0229] In the present specification, the term “EBV-LCL cell” or “EBV-LCL cell line” refers to an Epstein-Barr virus transformed lymphocyte continuous line (EBV- LCL) (D.M.Koelle et al., J Clin Invest, 1993: 91: 961-968), which is a B cell line that is made by infecting human B cells with Epstein-Barr virus in a test tube. The EBV-LCL cells may be directly prepared and used in a general laboratory by a method of adding cyclosporine A in a process of infecting EBV in the PBMC. In some embodiments, the EBV-LCL cell may be prepared by following steps. 30 x 10⁶ PBMCs are added in 9 mL of a culture medium, the mixture is added in a T 25 culture flask, and then 9 mL of an EBV supernatant is added. 80 pL of cyclosporine A (50 pg/mL) is added and then cultured at 37°C. After 7 days of culture, a half of supernatant is removed, a fresh culture medium is added, and then 40 pL of cyclosporine A is added. The same process may be repeated once every 7 days until 28 days of culture. The cell line may be usable after 28 days of culture, and from this time, the cell line may be cultured in the culture medium without adding cyclosporine A.

[0230] The Jurkat cells and the EBV-LCL cells may be used as the feeder cells after irradiation. In some embodiments, the irradiated Jurkat cells and the irradiated EBV- LCL cells may be included at a content ratio of 1:0. 1-5, 1:0. 1-4, 1:0.1-3, 1:0.1-2, 1:0. 1-1.5, L0.5-1.5, 1:0.75-1.25, 0.1-5:l, 0. 1-4:1, 0. 1-3: 1, 0.1-2: 1, 0.1-1.5: 1, 0.5-1.5: 1 or 0.75-1.25: 1. For example, the irradiated Jurkat cells and the irradiated EBV-LCL cells may be included at a content ratio of 1 : 1.

[0231] In some embodiments, the irradiated Jurkat cells and the irradiated EBV-LCL cells may be obtained by treating with irradiation of 50-500, 50-400, 50-300, 50-200, 50-150, 70-130, 80-120 or 90-1 10 Gy. For example, the irradiated Jurkat cells and/or the irradiated EBV-LCL cells may be obtained by treating Jurkat cells and/or EBV- LCL cells with irradiation of 100 Gy.

[0232] In some embodiments, the culturing may be performed for 1-50, 1-42, 1-40, 1-35, 1-20, 1-19, 1-18, 1-17, 1-16, 1-15 or 1-14 days.

[0233] In some embodiments, the culturing step may further include following steps: co-culturing with the feeder cells and a first cytokine (“first culturing step”); and further co-culturing after addition of a second cytokine (“second culturing step”)

[0234] The second culturing step may include adding the second cytokine once or more between day 0-6 of culturing. For example, the second culturing step may include adding the second cytokine once on each of day 0 and day 3 of culturing.

[0235] The second culturing step may include adding the second cytokine and the feeder cells during the first 6 days of the cycle of 14 days of culturing. For example, the second culturing step may include adding the feeder cells during a 14 days cycle, and adding the second cytokine on day 3 and 6 of each cycle once each.

[0236] In some embodiments, the first cytokine may be IL-2. In some embodiments, the second cytokine may be IL-21. In some embodiments, the second cytokine may be used at the concentration of 10-1000, 10-500, 10-100, 20-100, 30-100, 40- 100, 50-100 or 10-50 ng/mL. In some embodiments, culturing with the addition of the second cytokine once or more during day 0-6 may exhibit superior proliferation and/or activity. In some embodiments, culturing with the addition of the feeder cells and the second cytokine for six days in the cycle of 14 days may exhibit superior proliferation and/or activity.

[0237] In some embodiments, the co-culturing may be performed by including the peripheral blood mononuclear cells and the feeder cells (for example, the Jurkat cells and the EBV-LCL cells) at a mixing ratio of 1:1-100, 1: 1-90, 1: 1-80, 1: 1-70, 1: 10-65, 1:20- 65, 1:30-65, 1:40-65, 1:50-65 or 1 :55-65.

[0238] The co-culturing may be performed in a medium and any suitable media generally used for induction and proliferation of the peripheral blood mononuclear cells to the NK cells in the art may be used without a limitation as such a medium. For example, an RPML1640, DMEM, x-vivolO, x-vivo20, or cellgro SCGM medium may be used as such a medium. In addition, the culture conditions such as a temperature may follow any suitable culture conditions of the peripheral blood mononuclear cells known in the art.

[0239] In some embodiments, within the produced NK cells, a ratio or purity of the CD56+ NK cells may be 85% or more, 90% or more, or 95% or more, or 98% or more with respect to the whole cells. In some embodiments, within the produced NK cells, a ratio of T cells to whole cells may be 15% or less, 10% or less, 5% or less, 2% or less, 1% or less.

[0240] In some embodiments, the cytokines IL-2 and IL-21 are capable of supporting expansion of a CD3-/CD56+, or CD56+ population in vitro. In some embodiments, the population of CD3-/CD56+ or CD56+ cells expanded with IL-2 and IL- 21 possesses an NK cell phenotype.

[0241] In some embodiments, the method of treatment of autism involves culturing and/or expanding cells in tine with one or more of the approaches outlined in U.S. Pat. No. 10,590,385.

[0242] In the present specification, the term “peripheral blood-derived” may mean that the cells are derived from “whole blood of the peripheral blood” or “leukocytes isolated from the peripheral blood using leukapheresis.” The peripheral blood derived CD56+ NK cells may be used interchangeably with peripheral blood mononuclear cell (PBMC) derived CD56+ NK cells.

[0243] In some embodiments, the term “subject” refers to a mammal which is a subject for treatment, observation, or testing, and preferably, a human. The subject may be a patient of AUTISM, but not limited thereto.

[0244] In some embodiments, in the case of an adult, the cell therapeutic composition may be administered once to several times a day The cell therapeutic composition may be administered every day or in a 2-180 day interval, the cell therapeutic agent included in the composition may include 1 x 10^fi to 1 x 10¹¹ peripheral blood-derived CD56+ natural killer cells, for example, about 1 x 10⁶ to 1 x 10⁸ NK cells per kg of body weight. In some preferred embodiments, the cell therapeutic agent included in the composition may include 2 x 10⁹ to 9 x 10⁹ peripheral blood-derived CD56+ natural killer cells. In some embodiments, the peripheral blood-derived CD56+ natural killer cells in the cell therapeutic composition are at least about 90% pure. In some embodiments, the cytokine is IL-2 at a concentration ranging from about 50 - 50,000 lU/ml.

[0245] In some embodiments, the cell therapeutic composition of the present invention may be administered by any suitable method, such as administration through a rectal, intravenous, intraarterial, intraperitoneal, intramuscular, intrastemal, percutaneous, topical, intraocular, or intradermal route. In some embodiments, the NK cells included in the composition may be allogenic, i.e. obtained from a person other than the subject being treated. In some embodiments, the person may be a normal person or a patient with autism. In some embodiments, the NK cells included in the composition may be autologous, i.e. obtained from the subject being treated.

[0246] In some embodiments the subject has autism or an Autism Spectrum Disorder (ASD).

[0247] In some embodiments, identifying a subject with autism comprises a medical diagnosis of autism and/or an ASD. In some embodiments, diagnosis of autism comprises assessment of a child’s developmental history and behavior. In some embodiments, the diagnosis is made in subjects between 6 and 36 months old. In some embodiments, the diagnosis is made in subjects about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 months old, or in a range that is defined by any two of the preceding values. For example, in some embodiments, the diagnosis is made in subjects between about 6 and 36, 6, and 30, 6, and 24, 6, and 18, 6 and 12, 9 and 36, 9, and 30, 9, and 24, 9 and 18, 9 and 12, 12 and 36, 12 and 30, 12 and 24, or 12 and 18 months old.

[0248] Natural killer cells (NK cells) are one type of innate immune cells, which are known to non-specifically kill cancer, recognize and kill viruses, bacteria, and the like, and kill pathogens with enzymes such as perforin and granzyme or by Fas-FasL interaction.

[0249] In some embodiments the NK cells administered to the patient are autologous to the subject. In some embodiments, the NK cells administered to the patient are allogenic with respect to the subject. In some embodiments, the NK cells administered are derived from a healthy subject. In some embodiments, the NK cells administered are derived from a subject, with disease such as a subject with autism.

[0250] In some embodiments, the NK cell population has undergone expansion prior to administration. In some embodiments, an autologous NK cell population was expanded in vitro prior to administration. In some embodiments, an allogenic NK cell population was expanded in vitro prior to administration. In some embodiments NK cell expansion is accomplished by feeder cells. In some embodiments, NK cell expansion is accomplished by cytokine stimulation. In some embodiments, NK cell expansion is accomplished by both cytokines and feeder cells. In some embodiments, expansion of NK cells results in a population with a high purity of NK cells.

[0251] In some embodiments, the ratio of CD56+ NK cells to whole cells (purity) may be 85% or more, 90% or more, 95% or more, or 98% or more.

[0252] In some embodiments, the composition may not include T cells, or may include only trace amount of T cells. For example, the ratio of T cells to whole cells in the composition may be less than 15%, less than 10%, less than 5%, less than 2%, less than 1% or less.

[0253] In some embodiments, the NK cells are co-administered with a cytokine. In some embodiments the cytokine is IL-2, IL-21, IL-15, Flt3-L, IL-7, SCF, IL-18, IL-4, type I IFN, GM-CSF, IGF I, or any combinations thereof. In some embodiments, the cytokine may be used at a concentration of 18-180,000, 20-100,000, 50-50,000, 50-1,000, 50-900, 50-800, 50-700, 50-600, 50-550, 100-550, 150-550, 200-550, 250-550, 300-550, 350-550, 400-550, 450-550 lU/rnL. When the cytokine is used in these ranges, it may suppress apoptosis of the NK cells included in the treatment composition and increase activity of the NK cells.

[0254] In the present specification, the term “cell therapeutic agent” refers to a medicine which is used for treatment, diagnosis, and prevention through a series of actions, such as proliferating and screening autologous, allogeneic, and xenogeneic living cells in vitro for restoring functions of cells and tissues or changing biological characteristics of the cells by other methods. The cell therapeutic agents have been regulated as medical products from 1993 in USA and 2002 in Korea. These cell therapeutic agents may be largely classified into two fields, that are, first, stem cell therapeutic agents for tissue regeneration or recovery of organ functions, and second, immune cell therapeutic agents for regulation of immune responses, such as inhibition of the immune response or enhancement of the immune response in vivo.

[0255] The cell therapeutic composition described herein may be formulated in a suitable fomr together with a pharmaceutically acceptable carrier suitable or generally used for cell therapy. The “pharmaceutically acceptable” refers to a composition which is physiologically acceptable and does not generally cause an allergic reaction such as gastrointestinal disorders, dizziness, or the like, or similar reactions thereto, when being administered to the human body. The pharmaceutically acceptable carrier may include, for example, parenteral administration carries such as water, suitable oils, saline, aqueous glucose and glycol, and the like, and further include stabilizers and preservatives. The suitable stabilizer includes an antioxidant such as sodium hydrogen sulfite, sodium sulfite, or ascorbic acid, sucrose, albumin, or the like. The suitable preservative includes DMSO, glycerol, ethylene glycol, sucrose, trehalose, dextrose, polyvinylpyrrolidone, or the like.

[0256] The cell therapeutic composition may also be administered by any device in which the cell therapeutic agent may move to the target cell.

[0257] In some embodiments, the NK cells are not engineered to express a T cell receptor (TCR) or CAR. In some embodiments, the NK cells are not engineered to express additional stimulatory or co-stimulatory domains. In some embodiments, the NK cells are not engineered to express an antigen binding domain. In some embodiments, the NK cells are not engineered to express additional members of the NKG2 family. In some embodiments, the NK cells are expanded without additional engineering steps.

[0258] Some embodiments have been described wherein treatment of ASDs with expanded NK cells leads to a slowing of the progression of the disease, improvement of symptoms or a reversal in the progression of ASD as measured by any suitable tests and criteria, and/or by measuring the change in one or more CSF biomarkers from the beginning to the end of a study or treatment method. For example, in certain embodiments, the treatment with expanded NK cells leads to an improvement in the symptoms or reversal in the progression of disease of at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100%, including ranges between any of the listed values. In some embodiments, the CSF biomarker is a core CSF biomarker, a CSF inflammatory biomarker, a CSF immune cell chemokine ligand, a CSF innate immune receptor, and/or any combination thereof. In some embodiments, the CSF inflammatory marker comprises glial fibrillary acidic protein (GFAP), YKL-40, IL-12/IL-23p40, IL-6, IL-8, TNF-a, IL-10, GM-CSF, IL-10, INF-y, and/or any combination thereof. In some embodiments, the CSF immune cell chemokine ligand comprises CX3CL1 (Fractalkine). In some embodiments, the CSF innate immune receptor biomarker comprises soluble TREM2. In some embodiments, the CSF biomarker comprises, without limitation, one or more of CD3+/CD56- T cells (e.g., % CD3+/CD56- T cells in leukocytes and/or lymphocytes), % CX3CR1+ cells in CD3-CD56+NK Cells, and % CX3CR1+ cells in CD3+/CD56- T cells.

[0259] In some embodiments, a method of treating ASD in a subj ect is provided 200 (with reference to Fig. 2). In some embodiments, the method comprises: identifying a subject, wherein the subject has ASD, at block 201; and administering to the subject an expanded natural killer (NK) cell population, at block 202. In some embodiments, the NK cells are expanded by a method comprising: i) isolating at least one of CD56+ cells and/or CD3-/CD56+ cells from the PBMCs, at block 203; ii) co-culturing the at least one of CD56+ cells and/or CD3-/CD56+ cells with a combination of feeder cells in the presence of at least two cytokines, at block 204; iii) wherein the combination of feeder cells comprises irradiated Jurkat cells and irradiated Epstein-Barr virus transformed lymphocyte continuous line (EBV-LCL) cells, at block 205; and iv) wherein the at least two cytokines comprise IL-2 and IL-21, at block 206. In some embodiments, the method further comprises administering one or more behavioral assessments to the subject. In some embodiments, the behavioral assessment is administered before and/or after administration of the NK cells. In some embodiments, the behavioral assessment comprises assessment of social interaction and communicative skills as described herein. In some embodiments, the method further comprises detecting and/or quantifying one or more biomarkers of neuroinflammation. In some embodiments, the biomarker is a CSF or plasma biomarker. In some embodiments, the CSF biomarker is a core CSF biomarker, a CSF inflammatory biomarker, a CSF immune cell chemokine ligand, a CSF innate immune receptor, and/or any combination thereof. In some embodiments, the CSF inflammatory marker comprises glial fibrillary acidic protein (GFAP), YKL-40, IL-12/IL-23p40, IL-6, IL-8, TNF-a, IL-10, GM-CSF, IL-10, INF-y, and/or any combination thereof. In some embodiments, the CSF immune cell chemokine ligand comprises CX3CL1 (Fractalkine). In some embodiments, the CSF biomarker comprises, without limitation, one or more of CD3+/CD56- T cells (e.g., % CD3+/CD56- T cells in leukocytes and/or lymphocytes), % CX3CR1+ cells in CD3-CD56+ NK Cells, and % CX3CR1+ cells in CD3+/CD56- T cells. In some embodiments, the CSF innate immune receptor biomarker comprises soluble TREM2. Plasma inflammatory markers included YKL-40, IL-ip, IL-6, IL-8, IL-10, TNF-a, and/or INF-y, or any combination thereof. In some embodiments, the one or more biomarkers is detected and/or quantified before and/or after administration of the NK cells. In some embodiments, administration of the expanded NK cell population results in an improvement in the subject’s performance on one or more assessments described herein. In some embodiments, administration of the expanded NK cell population results in an increase or decrease in one or more CSF and/or plasma biomarkers of neuroinflammation. In some embodiments, the one or more biomarkers comprises Glial Fibrillary Acidic Protein (GFAP). In some embodiments, the CSF biomarker comprises, without limitation, one or more of CD3+/CD56- T cells (e.g., % CD3+/CD56- T cells in leukocytes and/or lymphocytes), % CX3CR1+ cells in CD3-CD56+ NK Cells, and % CX3CR1+ cells in CD3+/CD56- T cells. Plasma inflammatory markers include, without limitation, YKL-40, IL-ip, IL-6, IL-8, IL-10, TNF-a, and/or INF-y, or any combination thereof. In some embodiments, administration of the expanded NK cell population results in an increase in IL-8. In some embodiments, administration of the expanded NK cell population results in a decrease in GFAP, YKL-40, CX3CL1 (Fractalkine), IL-6, TNF-a, IL-12/IL-23p40, and/or sTREM2. In some embodiments, administration of the expanded NK cell population results in decreased neuroinflammation.

[0260] In some embodiments, administration of the expanded NK cell population results in an increase or decrease in one or more CSF and/or plasma biomarkers of neuroinflammation. Plasma inflammatory biomarkers include, without limitation, YKL- 40, IL-ip, IL-6, IL-8, IL-10, TNF-a, and/or INF-y, or any combination thereof. In some embodiments, the CSF biomarker comprises, without limitation, one or more of CD3+CD56- T cells (e.g., % CD3+CD56- T cells in leukocytes and/or lymphocytes), % CX3CR1+ cells in CD3-CD56+ NK Cells, and % CX3CR1+ cells in CD3+/CD56- T cells. In some embodiments, administration of the expanded NK cell population results in an increase in or a stable level of (e.g., lack of a decrease in) IL-8 (e.g., as measured in CSF or plasma). In some embodiments, administration of the expanded NK cell population results in an increase or a stable level of CSF IL-8 in, in about, or in at least 20%, 30%, 40%, 50%, 60%, 70%, or 80%, or a percentage in a range defined by any two of the preceding values (e.g., 20-80%, 30-80%, 40-70%, 50-80%, etc.) of the treated subjects, up to about 12 weeks, or more, after the last dose. In some embodiments, administration of the expanded NK cell population results in an increase in or a stable level of CSF or plasma IL-8 in, in about, or in at least 20%, 30%, 40%, 50%, 60%, 70%, or 80%, or a percentage in a range defined by any two of the preceding values (e.g., 20-80%, 30-80%, 40-70%, 50- 80%, etc.) of the treated subjects, from 1-12 weeks, or more, after the last dose.

[0261] In some embodiments, administration of the expanded NK cell population results in a decrease in or a stable level of (e g., lack of an increase in) GFAP, YKL-40, CX3CL1 (Fractalkine), IL-6, TNF-a, IL-12/IL-23p40, and/or sTREM2 (e.g., as measured in CSF or plasma). In some embodiments, administration of the expanded NK cell population results in a decrease or a stable level of CSF or plasma GFAP, and/or YKL- 40 in, in about, or in at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or about 100%, or a percentage in a range defined by any two of the preceding values (e.g., 20-100%, 30- 90%, 40-80%, 60-100%, 30-100%, etc.) of the subjects, up to about 12 weeks, or more, after the last dose. In some embodiments, administration of the expanded NK cell population results in a decrease or a stable level of CSF or plasma GFAP, and/or YKL-40 in, in about, or in at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or about 100%, or a percentage in a range defined by any two of the preceding values (e.g., 20-100%, 30- 90%, 40-80%, 60-100%, 30-100%, etc.) of the subjects, from 1-12 weeks, or more, after the last dose. In some embodiments, administration of the expanded NK cell population results in a decrease or a stable level of CSF GFAP, and/or YKL-40 in at least about 30% and up to about 100% of the treated subjects, from 1-12 weeks or more after the last dose. In some embodiments, administration of the expanded NK cell population results in a decrease or a stable level of plasma GFAP, and/or YKL-40 in at least about 50% and up to about 100% of the treated subjects, at least up to about 12 weeks, after the last dose. In some embodiments, administration of the expanded NK cell population results in decreased neuroinflammation. In some embodiments, this decrease in neuroinflammation treats ASD including, for example, autism.

[0262] In methods of treating ASD of the present disclosure, in some embodiments, administration of the expanded NK cell population results in an improvement in, or stable CSF and/or plasma levels of protein biomarkers and/or neuroinflammation markers over the course of treatment. As used herein, an improvement or stable level of a biomarker or neuroinflammation marker denotes the level of the biomarker or neuroinflammation marker not showing a change that is associated with or with worsening of the disease (e.g., ASDs), as described herein, over the relevant time period. In some embodiments, administration of the expanded NK cell population results in an improvement in, or stable CSF and/or plasma levels of one or more protein biomarkers for neuroinflammation (e.g., GFAP and/or YKL-40). In some embodiments, administration of the expanded NK cell population results in an improvement, or stable CSF and/or plasma levels of one or more protein biomarkers for neuroinflammation (e.g., GFAP and/or YKL-40) in, in about, or in at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or about 100%, or a percentage in a range defined by any two of the preceding values (e.g., 20-100%, 30-90%, 40-80%, 60-100%, 30-100%, etc.) of the treated subjects, up to about 12 weeks, or more, after the last dose. In some embodiments, administration of the expanded NK cell population results in an improvement, or stable CSF and/or plasma levels of one or more protein biomarkers for neuroinflammation (e.g., GFAP and/or YKL-40) in, in about, or in at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or about 100%, or a percentage in a range defined by any two of the preceding values (e.g., 20-100%, 30-90%, 40-80%, 60-100%, 30-100%, etc.) of the treated subjects, from 1-12 weeks, or more, after the last dose. In some embodiments, administration of the expanded NK cell population results in an improvement, or stable CSF levels of one or more protein biomarkers for neuroinflammation (e.g., GFAP and/or YKL-40) in at least about 30% and up to about 100% of the treated subjects, from 1-12 weeks or more after the last dose. In some embodiments, administration of the expanded NK cell population results in an improvement, or stable plasma levels of one or more protein biomarkers for neuroinflammation (e.g., GFAP and/or YKL-40) in at least about 50% and up to about 100% of the treated subjects, at least up to about 12 weeks after the last dose.

[0263] In any method of treating ASD of the present disclosure, in some embodiments, administration of the expanded NK cell population results in an improvement in, or stable CSF and/or plasma levels of one or more neuroinflammation markers (e.g., GFAP and/or YKL-40). In some embodiments, administration of the expanded NK cell population results in an improvement, or stable CSF and/or plasma levels of one or more neuroinflammation markers (e.g., GFAP and/or YKL-40) in, in about, or in at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or about 100%, or a percentage in a range defined by any two of the preceding values (e.g., 20-100%, 30-90%, 40-80%, 60- 100%, 30-100%, etc.) of the treated subjects, up to about 12 weeks, or more, after the last dose. In some embodiments, administration of the expanded NK cell population results in an improvement, or stable CSF and/or plasma levels of one or more neuroinflammation markers (e.g., GFAP and/or YKL-40) in, in about, or in at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or about 100%, or a percentage in a range defined by any two of the preceding values (e.g., 20-100%, 30-90%, 40-80%, 60-100%, 30-100%, etc.) of the treated subjects, from 1-12 weeks, or more, after the last dose. In some embodiments, administration of the expanded NK cell population results in an improvement, or stable CSF levels of one or more neuroinflammation markers (e.g., GFAP and/or YKL-40) in at least about 30% and up to about 90% of the treated subjects, from 1-12 weeks or more after the last dose. In some embodiments, administration of the expanded NK cell population results in an improvement, or stable plasma levels of one or more neuroinflammation markers (e.g., GFAP and/or YKL-40) in at least about 50% and up to about 75% of the treated subjects, at least up to about 12 weeks, after the last dose.

[0264] In any method of treating ASD of the present disclosure, in some embodiments, a subject shows rebound from an improvement (e.g., reversing or halting an improvement) in, or from stable CSF and/or plasma levels of one or more neuroinflammation markers (e.g., GFAPand/or YKL-40) after administration of the expanded NK cell population is terminated. In some embodiments, rebound from an improvement or stable CSF levels of one or more neuroinflammation markers (e.g., GFAP and/or YKL-40) after administration of the expanded NK cell population is terminated is observed in, in about, or in at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or about 100%, or a percentage in a range defined by any two of the preceding values (e.g., 20- 100%, 30-90%, 40-80%, 60-100%, 30-100%, etc.) of the treated subjects. In some embodiments, rebound from an improvement or stable CSF levels of one or more neuroinflammation markers (e.g., GFAP and/or YKL-40) after administration of the expanded NK cell population is terminated is observed in at least about 20% and up to about 100% of the treated subjects.

[0265] In some embodiments, the subject's level of neuroinflammation decreases following one or more administrations of the NK cells (e.g., the expanded NK cells). In some embodiments, the subject’s level of neuroinflammation decreases by about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 25, 30, 40, 50, 60, 70, 75, 80, 90, or 100%, or by an amount in a range that is defined by any two of the preceding values, following one or more administrations of the NK cells. For example, in some embodiments, the subject’s level of neuroinflammation decreases by between about 1-100, 1-75, 1-50, 1-25, 1-10, 10-100, 10- 75, 10-50, 10-25, 25-100, 25-75, 25-50, 50-100, 50-75, or 75-100%, following one or more administrations of the NK cells. The level of neuroinflammation can be measured using any suitable option. In some embodiments, the level of neuroinflammation is measured by assaying the level or change in level of one or more markers of inflammation in plasma or CSF, e.g., as described herein.

Numbered Arrangements

[0266] Some embodiments provided herein are described by way of the following provided numbered arrangements and also provided as possible combinations or overlapping embodiments:

1. A method of treating autism in a subject, the method comprising: a. identifying a subject, wherein the subject has autism ; and b. administering to the subject an expanded natural killer (NK) cell population, wherein the NK cells are expanded by a method comprising: i) isolating at least one of CD56+ cells and/or CD3-/CD56+ cells from the PBMCs; ii) co-culturing the at least one of CD56+ cells and/or CD3-/CD56+ cells with a combination of feeder cells in the presence of at least two cytokines; iii) wherein the combination of feeder cells comprises irradiated Jurkat cells and irradiated Epstein-Barr virus transformed lymphocyte continuous line (EBV-LCL) cells; and iv) wherein the at least two cytokines comprise TL-2 and TL-21 .

2. A method of treating Autism Spectrum Disorder (ASD) in a subject, the method comprising: a. identifying a subject, wherein the subject has ASD ; and b. administering to the subject an expanded natural killer (NK) cell population, wherein the NK cells are expanded by a method comprising: i) isolating at least one of CD56+ cells and/or CD3-/CD56+ cells from the PBMCs; ii) co-culturing the at least one of CD56+ cells and/or CD3-/CD56+ cells with a combination of feeder cells in the presence of at least two cytokines; iii) wherein the combination of feeder cells comprises irradiated Jurkat cells and irradiated Epstein-Barr virus transformed lymphocyte continuous line (EBV-LCL) cells; and iv) wherein the at least two cytokines comprise IL-2 and IL-21.

3. A method of treating autism in a subject, the method comprising: a. identifying a subject, wherein the subject has autism; and b. administering to the subject a therapeutically effective amount of an autologous NK cell population.

4. A method of treating ASD in a subject, the method comprising: a. identifying a subject, wherein the subject has ASD; and b. administering to the subject a therapeutically effective amount of an autologous NK cell population.

5. A method of cell therapy comprising: a. identifying a subject, wherein the subject has autism; and b. administering to the subject an expanded NK cell population, wherein the NK cells are expanded by a method comprising: i) isolating at least one of CD56+ cells and/or CD3-/CD56+ cells from the PBMCs; n) co-culturmg the at least one of CD56+ cells and/or CD3-/CD56+ cells with a combination of feeder cells in the presence of at least two cytokines; iii) wherein the combination of feeder cells comprises irradiated Jurkat cells and irradiated Epstein-Barr virus transformed lymphocyte continuous line (EBV-LCL) cells; and iv) wherein the at least two cytokines comprise IL-2 and IL-21.

6. The method of any one of the preceding arrangements, wherein the ASD is autism, high-functioning autism, Asperger’s syndrome, and/or Pervasive developmental disorder — not otherwise specified (PDD-NOS).

7. The method of any one of the preceding arrangements, wherein the amount of expanded NK cells administered to a subj ect is a therapeutically effective amount.

8. The method of any one of the preceding arrangements, wherein the therapeutically effective amount of expanded NK cells comprises 2 x 10⁹ to 9 x 10⁹ cells.

9. The method of any one of the preceding arrangements, wherein the therapeutically effective amount of expanded NK cells comprises 1 x 10⁹ to 1 x IO¹⁰ cells.

10. The method of any one of the preceding arrangements, wherein IL-2 is added at a concentration of 50-1000 lU/rnL during step ii). 11. The method of any one of the preceding arrangements, wherein IL-21 is added at a concentration of 10-100 ng/mL during step ii).

12. The method of any one of the preceding arrangements, further comprising: co-culturing the at least one of CD56+ cells and/or CD3-/CD56+ cells with the combination of feeder cells, in the presence of IL-2 for a first period; and co-culturing the at least one of CD56+ cells and/or CD3-/CD56+ cells with the combination of feeder cells, in the presence of IL-21 for a second period.

13. The method of any one of the preceding arrangements, wherein IL-21 is added more than once during Day 0-6 of the second period.

14. The method of any one of the preceding arrangements, wherein IL-21 and the combination of feeder cells are added more than once during Day 0-6 of the second period.

15. The method of any one of the preceding arrangements, wherein IL-21 is added more than once during the first six days of every fourteen-day cycle during the second period.

16. The method of any one of the preceding arrangements, wherein IL-21 and the combination of feeder cells are added more than once during Day 0-6 of the second period.

17. The method of any one of the preceding arrangements, wherein IL-21 is added more than once during the first six days of every fourteen-day cycle during the second period.

18. The method of any one of the preceding arrangements, wherein the amount of expanded NK cells administered to a subject is a therapeutically effective amount.

19. The method of any one of the preceding arrangements, wherein the therapeutically effective amount of expanded NK cells comprises 2 x 10⁹ to 9 x 10⁹ cells.

20. The method of any one of the preceding arrangements, wherein IL-2 is added at a concentration of 50-1000 lU/rnL during step ii).

21. The method of any one of the preceding arrangements, wherein IL-21 is added at a concentration of 10-100 ng/mL during step ii).

22. The method of any one of the preceding arrangements, further comprising: co-culturing the at least one of CD56+ cells and/or CD3-/CD56+ cells with the combination of feeder cells, in the presence of IL-2 for a first period; and co-culturing the at least one of CD56+ cells and/or CD3-/CD56+ cells with the combination of feeder cells, in the presence of IL-21 for a second period.

23. The method of any one of the preceding arrangements, wherein the NK cells do not include a CAR. 24. The method of any one of the preceding arrangements, wherein the NK cells do not include an engineered CAR.

25. A population of expanded NK cells, wherein the NK cells were expanded by a method that comprises: i) isolating at least one of CD56+ cells and/or CD3-/CD56+ cells from the PBMCs; li) co-culturing the at least one of CD56+ cells and/or CD3-/CD56+ cells with a combination of feeder cells in the presence of at least two cytokines; iii) wherein the combination of feeder cells comprises irradiated Jurkat cells and irradiated Epstein-Barr virus transformed lymphocyte continuous line (EBV- LCL) cells; and iv) wherein the at least two cytokines comprise IL-2 and IL-21; and wherein the population of expanded NK cells has been administered to a subject who has autism.

26. A population of expanded NK cells, wherein the NK cells were expanded by a method that compnses: i) isolating at least one of CD56+ cells and/or CD3-/CD56+ cells from the PBMCs; ii) co-culturing the at least one of CD56+ cells and/or CD3-/CD56+ cells with a combination of feeder cells in the presence of at least two cytokines; iii) wherein the combination of feeder cells comprises irradiated Jurkat cells and irradiated Epstein-Barr vims transformed lymphocyte continuous line (EBV- LCL) cells; and iv) wherein the at least two cytokines comprise IL-2 and IL-21; and wherein the population of expanded NK cells has been administered to a subject who has ASD.

27. The population of expanded NK cells of any one of the preceding arrangements, wherein the amount of expanded NK cells administered to a subject is a therapeutically effective amount.

28. The population of expanded NK cells of any one of the preceding arrangements, wherein the therapeutically effective amount of expanded NK cells comprises 2 x 10⁹ to 9 x 10⁹ cells.

29. The population of expanded NK cells of any one of the preceding arrangements, wherein IL-2 is added at a concentration of 50-1000 lU/mL during step ii). 30. The population of expanded NK cells of any one of the preceding arrangements, wherein IL-21 is added at a concentration of 10-100 ng/mL during step ii).

31. The population of expanded NK cells of any one of the preceding arrangements, further comprising: co-culturing the at least one of CD56+ cells and/or CD3-/CD56+ cells with the combination of feeder cells, in the presence of IL-2 for a first period; and co-culturing the at least one of CD56+ cells and/or CD3-/CD56+ cells with the combination of feeder cells, in the presence of IL-21 for a second period.

32. The population of expanded NK cells of any one of the preceding arrangements, wherein IL-21 is added more than once during Day 0-6 of the second period.

33. The population of expanded NK cells of any one of the preceding arrangements, wherein IL-21 and the combination of feeder cells are added more than once during Day 0- 6 of the second period.

34. The population of expanded NK cells of any one of the preceding arrangements, wherein IL-21 is added more than once during the first six days of every fourteen-day cycle during the second penod.

35. The method of any one of the preceding arrangements, wherein the expanded NK cell population or the NK cell population is or comprises SNK01.

36. The population of any one of the preceding arrangements, wherein the expanded NK cells are or comprise SNK01.

EXAMPLES

[0267] The following examples are provided to illustrate certain particular features and/or embodiments. These examples should not be construed to limit the disclosure to the particular features or embodiments described.

Example 1. Production of CD56+ natural killer (NK) cells

[0268] CD56+ cells and CD3-/CD56+ cells are isolated from PBMCs by the following method. First, the PBMCs are isolated from the blood using a Ficoll-Hypaque density gradient method and then the cells are counted.

Example 1-1. Preparation for producing CD56+ cells

[0269] The counted PBMCs are added with a MACS buffer (lx PBS+0.5% HSA) and suspended, and added with CD 6 microbeads (Miltenyi Biotec) to be 1 to 20 pL per 1.0 x 10⁷ PBMCs, and then incubated at 2 to 8°C for 5 to 30 minutes. After incubation, the MACS buffer is added and mixed, and then the mixture is centrifuged (600 x g) to precipitate the cells. After centrifugation, a supernatant is removed, and the cells are suspended by adding the MACS buffer and added in a column connected to a MACS separator. The MACS buffer is passed through the column to remove non-specific binding. The column is separated from the MACS separator and transferred to a 15 mL conical tube, and then added with the MACS buffer to isolate CD56+ cells attached to the column.

Example 1-2, Preparation for producing CD3-/CD56+ cells

[0270] The counted PBMCs are added with a MACS buffer (lx PBS±0.5% HSA) and suspended, and added with CD3 microbeads (Miltenyi Biotec) to be 1 to 20 pL per 1.0 x 10⁷ PBMCs, and then incubated at 2 to 8°C for 5 to 30 minutes. After incubation, the MACS buffer is added and mixed, and then the mixture is centrifuged (600 x g) to precipitate the cells. After centrifugation, a supernatant is removed, and the cells are suspended by adding the MACS buffer and added in a column connected to a MACS separator The MACS buffer passed through the column to collect CD3- cells. The collected CD3- cells are added with a MACS buffer (lx PBS+0.5% HSA) and suspended, and added with CD56 microbeads (Miltenyi Biotec) to be 1 to 20 pL per 1.0 x 10' CD3- cells, and then incubated at 2 to 8°C for 5 to 30 minutes. After incubation, the MACS buffer is added and mixed, and then the mixture is centrifuged (600 x g) to precipitate the cells. After centrifugation, a supernatant is removed, and the cells are suspended by adding the MACS buffer and added in a column connected to a MACS separator. The MACS buffer is passed through the column to remove non-specific binding. The column is separated from the MACS separator and transferred to a 15 mL conical tube, and then added with the MACS buffer to isolate CD3-/CD56+ cells attached to the column.

Example 1-3, Production of NK cells using the CD56+ cells and CD3-/CD56+ cells

[0271] The CD56+ cells or the CD3-/CD56+ cells isolated from the PBMCs as in Examples 1-1 and 1-2 are added in a RPML1640 medium containing FBS 10% added with IL-2 at a concentration of 500 lU/mL together with prepared combination of feeder cells (Jurkat cells and EBV-LCL cells) irradiated with 100 Gy radiation and then cocultured in an incubator at 37°C and 5% CO2. The ratio of (CD56+ cells and/or CD3- /CD56+ cells): (Jurkat cells): (EBV-LCL cells) is about 1 :30:30. [0272] Meanwhile, the Jurkat cells are obtained from ATCC (ATCC TIB- 152), and the EBV-LCL cells are prepared by the following method: 30 x 10⁶ PBMCs are added in 9 mL of a culture medium, the mixture is added in a T 25 culture flask, and then 9 m of an EBV supernatant is added. 80 pL of cyclosporine A is added and then cultured at 37°C. After 7 days of culture, a half of supernatant is removed, a fresh culture medium is added, and then 40 L of cyclosporine A is added. The same process as the 7th day is repeated once every 7 days until 28 days of culture. The cell line is usable after 28 days of culture, and from this time, the cell line is cultured in the culture medium without adding cyclosporine A.

Example 2. Production of CD56+ natural killer (NK) cells (TL-2/IL-21 treated)

[0273] NK cells are produced using same method of Example 1 (1-1 to 1-3), except for adding IL-2 (500 lU/mL) and IL-21 (50ng/mL) instead of IL-2 (500 lU/mL).

Comparative Example 1. Production of natural killer (NK) cells without the CD56+ cells isolation step (IL-2 treated)

[0274] PBMCs are isolated from the blood using a Ficoll-Hypaque density gradient method. The PBMCs are added in a RPMI-1640 medium containing FBS 10% added with IL-2 at a concentration of 500 lU/mL together with prepared feeder cells (Jurkat cells and EBV-LCL cells) irradiated with 100 Gy radiation and then co-cultured in an incubator at 37°C and 5% CO2.

Comparative Example 2, Production of natural killer (NK) cells without the CD56+ cells isolation step (IL-2/IL-21 treated)

[0275] NK cells are produced using same method of Comparative Example 1, except for adding IL-2 (500 lU/mL) and IL-21 (50ng/mL) instead of IL-2 (500 lU/mL).

Comparative Examples 3&4, Production of natural killer (NK) cells without the CD56+ cells isolation step

[0276] NK cells are produced using similar methods of Comparative Examples 1&2, respectively, except for that a ratio of PBMC: (Jurkat cells): (EBV-LCL cells) is l :0.5:0.5. Experimental Example 5, Treatment of autism patients with NK cells

[0277] CD56+ NK cells are produced according to the method of Examples 1,

2 and Comparative Examples 1, 2 for 18 days, except that PBMCs of autism patients are used. With respect to each of the NK cells cultured in a CO2 incubator according to Examples 1, 2 and Comparative Examples 1, 2, on Day 6 of culture in a T 25 culture flask, cells are inoculated into a 350 mL bag on the basis of the cell number of 1.0 x 10⁵ to 2.0 x IO⁶ /mL and further cultured for 4 days. On Day 10 of culture, the cells are inoculated into a 1 L bag on the basis of the cell number of 1.0 x 105 to 2.0 x 10⁶ /mL and then further cultured for 4 days. Finally, on Day 14 of culture, the cells are inoculated into a 1 L bag on the basis of the cell number of 1.0 x 10⁵ to 2.0 x 10⁶ /mL and then further cultured for 3 to 6 days.

[0278] Autism patients are grouped randomly and marked. The control group will not be injected with NK cells. The NK cell-treated group is injected six times with between about 1x10⁹ and IxlO¹⁰ NK cells and 500 lU/mL of IL-2 at weekly intervals intravenously. NK cells are added repeatedly until improvement in autism symptoms is achieved.

[0279] Cognitive and motor functions of the patient are monitored at 1, 3, 6, 12 months. After 12 months, the NK cell-treated group exhibit improved cognitive and motor functions. Social interaction and/or communicative skills, and/or stereotyped behavior and/or interests of the patient are assessed after treatment (e.g., 1, 3, 6, 12 months after treatment). After 12 months, the NK cell-treated group exhibit improved social interaction and/or communicative skill, and/or behavior and/or interests.

[0280]

Example 6, Single center, open-label, phase 1 study to evaluate the safety, tolerability, and exploratory efficacy of SNK01 in subjects with mild cognitive impairment (MCI) and Alzheimer’s Disease (AD) (Study SNK01-MX04)

[0281] It is predicted that neuroinflammation plays a role in autism. The data show how an autologous natural killer cell therapy could be used to treat disorders involving neuroinflammation, and as autism involves neuroinflammation, it follows that the examples are predictive that the therapy will be effective for treating autism.

[0282] CSF specimens collected from subjects participating in a single center, open-label, phase 1 study to evaluate the safety, tolerability, and preliminary efficacy of SNK01 (autologous natural killer cell), as a single agent, in subjects with Alzheimer’s disease, were used to Examine the level and change of AD biomarkers and cytokine/chemokine proteins by treatment of 3 different doses of SNK01 (www(dot)sec(dot)gov/ix?doc=/Archives/edgar/data/l 845459/000110465923074785/gfor -2023033 lxs4a (dot) htm). Alzheimer’s disease is a dual proteinopathy characterized by extracellular deposits of fibrillar amyloid-beta peptides and aggregates of the phosphorylated microtubule-associated protein tau in neurofibrillary tangles. Amyloid Beta 42 (A042), A042/A04O ratio, tau proteins which includes total tau (t-tau) and phosphorylated tau (p-tau 181), Neurofilament light (NIL), cytokine, chemokine for Fractalkine (CX3CL1), Glial fibrillary acidic protein (GFAP) and Chitinase-3 -like protein 1 (YKL-40) were evaluated.

[0283] The quantification of the markers was conducted using Meso Scale Discovery (MSD) multiplexed sandwich immunoassays. MSD assays are designed to measure levels of peptide and protein in biological samples. The multiplexed assays use electrochemiluminescent labels that are conjugated to detection antibodies. The labels allow for ultra-sensitive detection. Analytes in the sample bind to capture antibodies immobilized on the working electrode surface and recruitment of the detection antibodies conjugated with electrochemiluminescent labels. Electricity is applied to the electrodes by an MSD instrument leading to light emission by the conjugated labels. Light intensity is then measured to quantify analytes in the sample.

[0284] To identify the Maximum Tolerated Dose (MTD) of SNK01, the subj ects were given SNK01 in an open-label setting per the following treatment plan, using a 3 + 3 design (Table 1):

[0285] Cohort 1 - 1.0 x 10⁹ cells; infusions Q3W, total of four doses.

[0286] Cohort 2 - 2.0 x 10⁹ cells; infusions Q3W, total of four doses.

[0287] Cohort 3 - 4.0 x 10⁹ cells; infusions Q3W, total of four doses.

Table 1

[0288] Once the MTD was identified, a final cohort, Cohort 4, including 12 subjects, received the MTD to study safety, tolerability, and preliminary efficacy

[0289] Subjects were assessed at visit 1 to establish a baseline prior to the first dose of SNK01. Table 2 shows the mean and median baseline profile of subjects prior to the first dose of SNK01.

Table 2

[0290] Table 3 shows a comparison of the baseline profile of study subjects as compared to the profile of Alzheimer’s disease subjects as reported in the art.

Table 3

[0291] Subjects were then assessed again at visit 5 (week 11), 1 week after the last dose of SNK01. Subjects were assessed once again at visit 6 (week 22), 12 weeks after the last dose of SNK01.

[0292] Assessments included detecting and or quantifying one or more biomarkers, immunophenotyping, genotyping, evaluating NK cell activity, performing one or more cognitive assessments. Alzheimer’s Disease biomarkers, pro-inflammatory biomarkers, and anti-inflammatory biomarkers were evaluated in the subjects’ CSF and plasma. CSF core biomarkers included amyloid beta 42, amyloid beta 40, amyloid beta 42/40 ratio, total Tau (Tt-tau), phosphorylated Tau (p-Tau), p-Tau 181, and neurofilament light (NfL). CSF inflammatory marker included glial fibrillary acidic protein (GFAP), YKL-40, IL-12/IL-23p40, IL-6, IL-8, TNF-a, IL-10, GM-CSF, IL-10, and INF-y. CSF immune cell chemokine ligand included CX3CL1 (Fractalkme). CSF innate immune receptor biomarker included soluble TREM2 Plasma biomarkers included amyloid beta 42, amyloid beta 40, amyloid beta 42/40 ratio, total tau (t-tau), phosphorylated tau (p-tau), Glial Fibrillary Acidic Protein (GFAP), and neurofilament light (NfL). Plasma inflammatory markers included YKL-40, IL-10, IL-6, IL-8, IL-10, TNF-a, and INF-y.

[0293] Immunophenotyping was performed on the subjects’ CSF and whole blood. SNP genotyping of APOE and TREM2. NK cell activity was analyzed using NK Vue.

[0294] FIG. 8A is a line graph depicting the average change in A0-42 levels in the cerebrospinal fluid of subjects treated with different doses of NK cells.

[0295] FIG. SB is a line graph depicting the change in A0-42 levels in the cerebrospinal fluid of subjects treated with different doses of NK cells.

[0296] As can be seen in FIG. 8A and FIG. 8B, A0-42 levels increase in the cerebrospinal fluid of subjects treated with NK cells.

[0297] FIG. 9A is a line graph depicting the aggregate change in A0-42/4O ratio in the cerebrospinal fluid of subjects treated with different doses of NK cells.

[0298] FIG. 9B is a line graph depicting the change in A0-42/4O ratio in the cerebrospinal fluid of subjects treated with different doses of NK cells.

[0299] As can be seen in FIG. 9A and FIG. 9B, A0-42/4O ratio increase in the cerebrospinal fluid of subjects treated with NK cells.

[0300] FIG. 10 is a line graph depicting the average change in total Tau levels in the cerebrospinal fluid of subjects treated with different doses of NK cells.

[0301] FIG. 67 is a line graph depicting the change in total tau levels in the cerebrospinal fluid of subjects treated with different doses of NK cells.

[0302] As can be seen in FIGs. 10 and 67, total tau levels may decrease in the cerebrospinal fluid of subjects treated with NK cells.

[0303] FIG. 11A is a line graph depicting the average change in p-tau 181 levels in the cerebrospinal fluid of subjects treated with different doses of NK cells. [0304] FIG. 11B is a line graph depicting the change in p-tau levels in the cerebrospinal fluid of subjects treated with different doses of NK cells.

[0305] As can be seen in FIG. 11A and FIG. 11B, p-tau levels may decrease in the cerebrospinal fluid of subjects treated with NK cells.

[0306] FIG. 12A is a line graph depicting the average change in GFAP levels in the cerebrospinal fluid of subjects treated with different doses of NK cells.

[0307] FIG. 12B is a line graph depicting the change in GFAP levels in the cerebrospinal fluid of subjects treated with different doses of NK cells.

[0308] As can be seen in FIG. 12A and FIG. 12B, total neuroinflammation, as indicated by GFAP levels in the cerebrospinal fluid of subjects treated with NK cells, may decrease.

[0309] FIG. 13A is a line graph depicting the average change in NfL levels in the cerebrospinal fluid of subjects treated with different doses of NK cells.

[0310] FIG. 13B is a line graph depicting the change in NfL levels in the cerebrospinal fluid of subjects treated with different doses of NK cells.

[0311] As can be seen in FIG. 13A and FIG. 13B, NfL levels may decrease in the cerebrospinal fluid of subjects treated with NK cells.

[0312] Table 4 shows the mean change in Ap-42/40, Ap-42. total tau, p-tau, GFAP, and NfL, from baseline at week 11 and week 22 of the study.

Table 4

[0313] FIG. 14A is a line graph depicting the aggregate change in YKL-40 levels in the cerebrospinal fluid of subjects treated with different doses of NK cells.

[0314] FIG. 14B is a line graph depicting the change in YKL-40 levels in the cerebrospinal fluid of subjects treated with different doses NK cells.

[0315] As can be seen in FIG. 14A and FIG. 14B, neuroinflammation, as indicated by YKL-40 levels in the cerebrospinal fluid of subjects treated with NK cells, may change from baseline following NK cell administration.

[0316] FIG. 15A is a line graph depicting the aggregate change in baseline CX3CL1 (Fractalkine) levels in the cerebrospinal fluid of subjects treated with different doses of NK cells.

[0317] FIG. 15B is a line graph depicting the change in baseline CX3CL1 (Fractalkine) levels in the cerebrospinal fluid of subjects treated with different doses NK cells.

[0318] As can be seen in FIG. 15A and FIG. 15B, CX3CL1 (Fractalkine) levels in the cerebrospinal fluid of subjects treated with NK cells may decrease from baseline following NK cell administration.

[0319] FIG. 16A is a line graph depicting the average change in baseline IL-6 levels in the cerebrospinal fluid of subjects treated with different doses of NK cells.

[0320] FIG. 16B is a line graph depicting the change in baseline IL-6 levels in the cerebrospinal fluid of subjects treated with different doses of NK cells.

[0321] As can be seen in FIG. 16A and FIG. 16B, IL-6 levels in the cerebrospinal fluid of subjects treated with NK cells may decrease from baseline Follow ing NK cell administration.

[0322] FIG. 17A is a line graph depicting the average change in baseline TNF- a levels in the cerebrospinal fluid of subjects treated with different doses of NK cells. [0323] FIG. 17B is a line graph depicting the change in baseline TNF-a levels in the cerebrospinal fluid of subjects treated with different doses of NK cells.

[0324] As can be seen in FIG. 17B, TNF-a levels in the cerebrospinal fluid of subjects treated with NK cells may decrease from baseline following NK cell administration.

[0325] FIG.18A is a line graph depicting the average change in baseline IL-8 levels in the cerebrospinal fluid of subjects treated with different doses of NK cells.

[0326] FIG. 18B is a line graph depicting the change in baseline IL-8 levels in the cerebrospinal fluid of subjects treated with different doses of NK cells.

[0327] As can be seen in FIG. 18A and FIG. 18B, IL-8 levels in the cerebrospinal fluid of subjects treated with NK cells may change from baseline following NK cell administration.

[0328] FIG. 19A is a line graph depicting the average change in baseline IL- 12/IL-23p40 levels in the cerebrospinal fluid of subjects treated with different doses of NK cells.

[0329] FIG. 19B is a line graph depicting the change in baseline IL-12/IL- 23p40 levels in the cerebrospinal fluid of subjects treated with different doses of NK cells.

[0330] As can be seen in FIG. 19A and FIG. 19B, IL-12/IL-23p40 ratio in the cerebrospinal fluid of subjects treated with NK cells may decrease from baseline following NK cell administration.

[0331] FIG. 20A is a line graph depicting the average change in baseline sTREM2 levels in the cerebrospinal fluid of subjects treated with different doses of NK cells.

[0332] FIG. 20B is a line graph depicting the change in baseline sTREM2 levels in the cerebrospinal fluid of subjects treated with different doses of NK cells.

[0333] As can be seen in FIG. 20A and FIG. 20B, sTREM2 levels in the cerebrospinal fluid of subjects treated with NK cells may decrease from baseline following NK cell administration.

[0334] T able 5 shows the mean change in YKL-40, CX3 CL 1 , TNF -a, IL-6, IL-

8, IL-12/IL-23p40, and sTREM2, from baseline at week 11 and week 22 of the study.

[0335] FIG. 21A is a line graph showing the aggregate expression level (percentage) of CX3CR1 in T cells in CSF of subjects treated with different doses of NK cells.

[0336] FIG. 21B is a line graph showing the expression level (percentage) of CX3CR1 in T cells in CSF of subjects treated with different doses of NK cells.

[0337] As can be seen in FIG. 21A and FIG. 21B, the percentage of CX3CR1+ cells in CD3-CD56+ T-cells may increase following NK cell administration.

[0338] FIG. 22A is a line graph showing the aggregate expression level (percentage) of CX3CR1 in NK cells in CSF of subjects treated with different doses of NK cells.

[0339] FIG. 22B is a line graph showing the expression level (percentage) of CX3CR1 in NK cells in CSF of subjects treated with different doses of NK cells. [0340] As can be seen in FIG. 22A and FIG. 22B, the percentage of CX3CR1+ cells in CD3-CD56+ NK cells may increase following NK cell administration.

[0341] FIG. 23A is a line graph showing the expression level (percentage) of CX3CR1 in microglia in CSF of subjects treated with different doses of NK cells.

[0342] FIG. 23B is a line graph showing the expression level (percentage) of CX3CR1 in microglia in CSF of subjects treated with different doses of NK cells.

[0343] As can be seen in FIG. 23A and FIG. 23B, the percentage of CX3CR1+ cells in microglia may change following NK cell administration.

[0344] FIG. 24A is a bar graph depicting NK cell activity the plasma of subj ects treated with different doses of NK cells.

[0345] FIG. 24B is a bar graph depicting NK cell activity in the plasma of subjects treated with different doses of NK cells.

[0346] As can be seen in FIG. 24A and FIG. 24B, the NK cell activity may increase following NK cell administration.

Example 7 Use of Expanded Non-Genetically Modified Natural Killer Cells (SNK01) with Enhanced Cytotoxicity in Patients with Alzheimer's Disease — Interim Report of a Phase I Trial

[0347] It is predicted that neuroinflammation plays a role in autism. The data show how an autologous natural killer cell therapy could be used to treat disorders involving neuroinflammation, and as autism involves neuroinflammation it follows that the examples are predictive that the therapy will be effective for treating autism.

[0348] Purpose/Objectives The accumulation of misfolded proteins is known to elicit a cascade of neuroinflammation by CNS-resident or infiltrating immune cells, resulting in neuronal cell death in Alzheimer’s disease (AD). It is now recognized that only clearing these proteins may not be the best treatment strategy for AD.

[0349] Natural Killer (NK) cells are an essential part of the innate immune system that have been shown pre-clinically to slow progression of amyloid deposition as well as to decrease neuroinflammation by recognizing and eliminating autoreactive immune cells and damaged neurons. SNK01 is a first-in-kind, autologous non-genetically modified NK cell product wdth high cytotoxicity and over 90% activating receptor expression. It can be consistently produced from any patients for clinical use. A clinical trial was carried out to try to demonstrate that SNK01 can be safely infused to reduce neuroinflammation by crossing the blood brain barrier (BBB) in AD patients. [0350] Materials & Methods In this Phase 1 dose escalation study (Study SNK01-MX04, NCT04678453), SNK01 was administered intravenously (IV) every three weeks for a total of 4 treatments using a 3+3 dose escalation design [low dose (1 x 10⁹ cells), medium dose (2 x 10⁹ cells), and high dose (4 x 10⁹ cells)] in subjects with either mild, moderate or severe AD confirmed by MRI and PET scans. Assessment of baseline severity was based on the CDR-SB score.

[0351] Cognitive assessments (CDR-SB (Clinical Dementia Rating-Sum of Box), ADAS-Cog (Alzheimer's disease assessment scale-cognitive subscale), and MMSE (Mini-Mental State Examination)) and CSF analyses (by electrochemiluminescent multiplexed immunoassays) were performed at baseline and at one week and 12 weeks after the final dose (Weeks 11 and 22, respectively) (See Study Design, FIG. 25).

[0352] FIG. 25 shows the study design for the SNK01 infusion assessment including screening, timing, and dosing of infusions, cognitive assessment and CSF biomarkers.

[0353] Ten subjects with mild (n=5) and moderate to severe AD (n=5) were enrolled in the three dose-escalation cohorts. Median age was 79 (56-85). Baseline median scores for CDR-SB, ADAS-Cog, and MMSE were 9 (4-18), 27.5 (18-65), and 14 (2-23), respectively. SNK01 was successfully activated/expanded from all enrolled subjects’ peripheral blood and then administered. No treatment related adverse events have been observed to date.

[0354] Treatment with SNK01 showed changes in some CSF biomarker levels when tested 1 week after the last dose (FIGs. 26-38). Some subjects maintained this treatment effect and biomarker levels when tested at 12 weeks after the last dose. Especially, subject 014 treated with high dose showed a marked improvement of cognition by cognitive assessments as well as favorable changes in GFAP and p-taul81 levels (FIGs. 31, 33).

[0355] FIG. 26 shows a line graph depicting the change in A0-42 levels in the cerebrospinal fluid of subjects treated with different doses of SNK01.

[0356] FIG. 27 summarizes FIG. 26 and shows a line graph depicting the mean change from baseline in A [>-42 levels in the cerebrospinal fluid of subjects treated with different doses of SNK01 grouped according to dosage. The underlying data includes the data plotted in FIG. 8B.

[0357] FIG. 28 shows a line graph depicting the change in Afl-42/40 ratio in the cerebrospinal fluid of subjects treated with different doses of SNK01. [0358] FIG. 29 summarizes FIG. 28 and shows a line graph depicting the mean change from baseline in A -42/40 ratio in the cerebrospinal fluid of subjects treated with different doses of SNK01 grouped according to dosage. The underlying data includes the data plotted in FIG. 9B. Decreased ratio of A 42/40 is a strong marker of Alzheimer's disease and can be detected early in the disease progression, even before clinical dementia occurs.

[0359] FIG. 30 shows line graphs depicting the change in total Tau levels in the cerebrospinal fluid of subjects treated with different doses of SNK01. Left panel shows changes in the subjects over time. Right panel shows the mean change over time, grouped according to dosage. CSF t-tau increase in AD patients may be caused by damaged neurons and the formation of tau tangles in the CNS in relation to neurodegeneration. Increases in total tau protein, as well as phosphorylated tau (p-tau), are also seen in CSF of AD patients. The underlying data includes the data plotted in FIG. 67.

[0360] FIG. 31 shows a line graph depicting the change in p-tau 181 levels in the cerebrospinal fluid of subjects treated with different doses of SNK01.

[0361] FIG. 32 summarizes FIG. 31.

[0362] FIG. 33 shows a line graph depicting the change in GFAP levels in the cerebrospinal fluid of subjects treated with different doses of SNK01.

[0363] FIG. 34 summarizes FIG. 33 and shows a line graph depicting the mean change from baseline in GFAP levels in the cerebrospinal fluid of subjects treated with different doses of SNK01 grouped according to dosage. The underlying data includes the data plotted in FIG. 12B. Glial fibrillary acidic protein (GFAP) is a marker of reactive astrogliosis that increases in the cerebrospinal fluid (CSF) and blood of individuals with Alzheimer disease (AD). GFAP correlates with astroglia activation. GFAP has been proposed as a biomarker of Alzheimer's disease (AD). GFAP expression correlates with A(3 plaque density. CSF concentration is elevated in AD.

[0364] FIG. 35 shows a line graph depicting the change in NfL levels in the cerebrospinal fluid of subjects treated with different doses of SNK01.

[0365] FIG. 36 summarizes FIG. 35 and shows a line graph depicting the mean change from baseline in NfL levels in the cerebrospinal fluid of subjects treated with different doses of SNK01 grouped according to dosage. The underlying data includes the data plotted in FIG. 13B. Cerebrospinal fluid (CSF) neurofilament light (NfL) is a biomarker of neurodegeneration in Alzheimer's disease (AD), the levels of which are significantly elevated in AD. [0366] FIG. 37 shows a line graph depicting the change in YKL-40 levels in the cerebrospinal fluid of subjects treated with different doses of SNK01.

[0367] FIG. 38 summarizes FIG. 37 and shows a line graph depicting the mean change from baseline in YKL-40 levels in the cerebrospinal fluid of subjects treated with different doses of NK cells grouped according to dosage. The underlying data includes the data plotted in FIG. 14B. YKL-40 (Chitinase 3-like I) is increased in CSF of Alzheimer’s disease (AD) and frontotemporal lobar degeneration (FTLD) patients and is considered a potential neuroinflammatory biomarker.

[0368] Table 6 summarizes the results of this study.

Table 6

[0369] Conclusions: SNK01 appeared to be safe and well tolerated. SNK01 showed clinical activity in AD. In addition, based on the CSF biomarker data, SNK01 given via peripheral IV seems to reduce p-tau!81 and neuroinflammation in a dose dependent manner by crossing the blood brain barrier. Finally, there appears to be a rebound effect in these biomarkers when SNK01 treatment is discontinued.

Example 8 Neuroinflammatory markers in plasma

[0370] It is predicted that neuroinflammation plays a role in autism. An autologous natural killer cell therapy could be used to treat disorders involving neuroinflammation, and as autism involves neuroinflammation, it follows that the effects on neuroinflammation are predictive that the therapy will be effective for treating autism.

[0371] Objective and Method: Alzheimer’s disease (AD) is a dual proteinopathy characterized by extracellular deposits of fibrillar amyloid-beta peptides and aggregates of the phosphorylated microtubule-associated protein tau in neurofibrillary tangles. [0372] In the literature, sensitivity and specificity may be observed for AD neuropathological change in plasma biomarkers related to amyloid, tau, and neurodegeneration. Blood biomarkers indicative of AD pathology are altered in both preclinical and symptomatic stages of the disease. Distinctive biomarkers may be suitable for the identification of AD pathology or monitoring of disease progression. Blood biomarkers that correlate with changes in cognition and atrophy during the course of the disease are used in clinical trials to identify successful interventions and thereby accelerate the development of efficient therapies. Lower plasma Ap42/A|340 ratio and higher phosphorylated tau (p-taul81), Glial fibrillary acidic protein (GFAP), and Neurofilament light (NfL) are associated with cognitive decline and increased Ap-PET load. (Smirnov et al. 2022, Ashton et al.2022, Chatterjee et al. 2023)

[0373] Plasma from subjects with AD who participated in the autologous NK cell therapy (SNK01) study were collected and used to access the cell therapy treatment responses. Blood was drawn from a forearm vein into EDTA citrate vacutainer tubes and centrifuged at 1000 xg for 10 min at 4 °C in a tabletop centrifuge within 1 h or less of blood draw. Plasma was separated and aliquoted into polypropylene cryotubes, snap frozen and stored at - 80° until biomarkers analyses were conducted.

[0374] Quantification of the presence of the AD biomarkers and proteins in plasma were conducted to evaluate the effect of three levels of dosage of SNK01 treatments for mild, moderate and severe AD subjects. The biomarkers panel included Amyloid Beta 42 (Ap42), AP42/AP40 ratio, p-tau 181 , NfL, GFAP, Chitinase-3-like protein l(YKL-40), Interleukin 6 (IL-6) and Tumor necrosis factor a (TNF-a).

[0375] The quantification of the markers was conducted using Meso Scale Discovery (MSD) multiplexed sandwich immunoassays. MSD assays are designed to measure levels of peptide and protein in biological samples. The multiplexed assays use electrochemiluminescent labels that are conjugated to detect antibodies. The labels allow for ultra-sensitive detection. Analytes in the sample bind to capture antibodies immobilized on the working electrode surface and recruitment of the detection antibodies conjugated with electrochemiluminescent labels. Electricity is applied to the electrodes by an MSD instrument leading to light emission by the conjugated labels. Light intensity is then measured to quantify analytes in the sample.

[0376] Background of the markers: Plasma A|342/Ap40 ratio is a diagnostic biomarker of AD during both predementia and dementia stages with comparable correlation to level of CSF A042/AP4O ratio. The ratios reflect AD-type pathology better, whereas decline in A042 is also associated with non- AD subcortical pathologies. Studies suggested that the ratios rather than A(342 can be used in the clinical work-up of AD. (Janelidze et al. 2016, Wilczynska el at. 2021)

[0377] The tangles characteristic of AD are made up of filaments formed from an abnormally phosphorylated form of tau called phospho-tau (p-tau). P-tau is believed to reflect neurofibrillary pathology. Level of plasma p-tau 181 correlates to CSF p-tau 181, tau PET and cognitive impairment (Janelidze et al. 2016, Tatebe et al. 2017, Mielke et al. 2018, Yang et al. 2018)

[0378] NfL, an intermediate filament protein expressed exclusively in neurons, has emerged as a promising blood-based biomarker of neurodegeneration in several neurological disorders, including AD. In sporadic AD, the concentrations of NfL in CSF and blood are significantly increased in both the prodromal and dementia stages of the disease, in which they associate with cognitive decline and disease-related structural brain changes. Consistent observations found that the concentration of NfL in plasma correlates positively with those in CSF, suggesting that NfL in blood is likely to originate from the central nervous system (CNS). In addition, NfL in both CSF and plasma were higher in patients with mild cognitive impairment (MCI) and AD dementia compared to cognitive unimpaired subjects suggesting that NfL tracks neurodegeneration. (Khalil et al., 2018, Mattsson et al., 2017, 2019; Olsson et al., 2019; Zetterberg et al., 2016)

[0379] GFAP is an intermediate filament structural protein involved in cytoskeleton assembly and integrity, expressed in high abundance in activated glial cells. Neuronal stress, caused by either disease or injury, evokes astrocyte activation as a response, including hypertrophy, proliferation, and increased GFAP expression. GFAP is a marker of reactive astrogliosis that increases in CSF and blood of individuals with Alzheimer disease (AD) (Ganne, Akshatha et al. 2022)

[0380] YKL-40 is an inflammatory marker considered as a potential biomarker of dementia, neoplastic diseases, and chronic inflammation. It is elevated in the brain, CSF and in serum in several neurological and neurodegenerative diseases associated with inflammatory processes. YKL-40 is a highly sensitive and specific marker that differentiates healthy individuals from patients with Alzheimer’s, vascular or mixed dementia. Studies shown that the increase in peripheral blood YKL-40 concentration in AD results from the activation of proinflammatory cells due to cell death caused by the accumulation of beta amyloid. YKL-40 correlated with the concentrations of other markers (t-tau and A(342/A|340), the severity of dementia as reflected by negative correlation with the MMSE score, and the parameters of inflammation (Llorens et al. Molecular Neurodegeneration 2017, Wilczynska et al. 2021)

[0381] Interleukin 6 (IL-6) is upregulated in AD brain and plasma, correlates positively with brain inflammation and inversely with MMSE scores. IL-6 is a component of early-stage amyloid plaque formation in AD brains and has been implicated in tau phosphorylation, synapse loss, and learning deficits in mice. IL-6 is increased in both CSF and plasma of mild cognitive impairment (MCI) and AD patients compared to healthy individuals (Silva et al. 2021)

[0382] Tumor necrosis factor a (TNF-a) plays an essential role in the cytokine cascade during neuroinflammation response. The levels of TNF-a are significantly elevated in blood and CNS of patients with AD. The role of TNF-a in AD pathology was further suggested by studies in which significant elevation of TNF-a levels in the CSF and serum of patients with AD correlated with disease progression. (Chang et al. 2017)

[0383] References:

Ganne, Akshatha et al. “Glial Fibrillary Acidic Protein: A Biomarker and Drug Target for Alzheimer's Disease.” Pharmaceutics vol. 14,7 1354. 26 Jun. 2022 doi: 10.3390/pharmaceuticsl4071354

Wilczynska K, Maciejczyk M, Zalewska A, Waszkiewicz N. Serum Amyloid Biomarkers, Tau Protein and YKL-40 Utility in Detection, Differential Diagnosing, and Monitoring of Dementia. Front Psychiatry. 2021 Sep 13;12:725511. doi: 10.3389/fpsyt.202L 725511. PMID: 34589009; PMCID: PMC8473887.

Ashton, N.J., Janelidze, S., Mattsson-Carlgren, N. et al. Differential roles of A [342/40. p- tau231 and p-tau217 for Alzheimer’s trial selection and disease monitoring. Nat Med 28, 2555-2562 (2022).

Smirnov DS, Ashton NJ, Blennow K, Zetterberg H, Simren J, Lantero-Rodriguez J, Karikari TK, Hiniker A, Rissman RA, Salmon DP, Galasko D. Plasma biomarkers for Alzheimer's Disease in relation to neuropathology and cognitive change. ActaNeuropathol. 2022 Apr;143(4):487-503. doi: 10.1007/s00401-022-02408-5. Epub 2022 Feb 23. PMID: 35195758; PMCID: PMC8960664.

Janelidze S, Mattsson N, Palmqvist S, et al. Plasma P-taul81 in Alzheimer's disease: relationship to other biomarkers, differential diagnosis, neuropathology and longitudinal progression to Alzheimer's dementia. Nature Medicine. 2020 Mar;26(3):379-386. DOI: 10.1038/s41591-020-0755-l. PMID: 32123385.

Llorens, F., Thiine, K., Tahir, W. et al. YKL-40 in the brain and cerebrospinal fluid of neurodegenerative dementias. Mol Neurodegeneration 12, 83 (2017).

Xinyu Li, Weiren Wang; Levels of IL-6 in peripheral blood and cerebrospinal fluid of Alzheimer's disease: a meta-analysis. International Journal of Frontiers in Medicine. ISSN 2706-6819 Vol.5, Issue 2: 53-60, DOI: 10.25236/IJFM.2023.050210 Chatterjee P, Pedrini S, Doecke JD, Thota R, Villemagne VL, Dore V, Singh AK, Wang P, Rainey-Smith S, Fowler C, Taddei K, Sohrabi HR, Molloy MP, Ames D, Maruff P, Rowe CC, Masters CL, Martins RN; AIBL Research Group. Plasma A[}42/40 ratio, p-taul81, GFAP, and NfL across the Alzheimer's disease continuum: A cross-sectional and longitudinal study in the AIBL cohort. Alzheimers Dement. 2023 Apr;19(4): 1117-1134. doi: 10.1002/alz.12724. Epub 2022 Jul 21. PMTD: 36574591.

Tatebe, H., T. Kasai, T. Ohmichi, Y. Kishi, T. Kakeya, M. Waragai, M. Kondo, D. Allsop and T. Tokuda (2017). "Quantification of plasma phosphorylated tau to use as a biomarker for brain Alzheimer pathology: pilot case-control studies including patients with Alzheimer's disease and down syndrome." Mol Neurodegener 12(1): 63.

Mielke, M. M., C. E. Hagen, J. Xu, X. Chai, P. Vemuri, V. J. Lowe, D. C. Airey, D. S. Knopman, R. O. Roberts, M. M. Machulda, C. R. Jack, Jr., R. C. Petersen and J. L. Dage (2018). "Plasma phospho-taul81 increases with Alzheimer's disease clinical severity and is associated with tau- and amyloid-positron emission tomography." Alzheimers Dement 14(8): 989-997.

Yang, C. C., M. J. Chiu, T. F. Chen, H. L. Chang, B. H. Liu and S. Y. Yang (2018). "Assay of Plasma Phosphorylated Tau Protein (Threonine 181) and Total Tau Protein in Early - Stage Alzheimer's Disease." J Alzheimers Dis 61(4): 1323-1332.

Stocker H, Beyer L, Perna L, Rujescu D, Holleczek B, Beyreuther K, Stockmann J, Schottker B, Gerwert K, Brenner H. Association of plasma biomarkers, p-taul81, glial fibrillary acidic protein, and neurofilament light, with intermediate and long-term clinical Alzheimer's disease risk: Results from a prospective cohort followed over 17 years. Alzheimers Dement. 2023 Jan;19(l):25-35. doi: 10.1002/alz. 12614. Epub 2022 Mar 2. PMID: 35234335.

Andersson E, Janelidze S, Lampinen B, Nilsson M, Leuzy A, Stomrud E, Blennow K, Zetterberg H, Hansson O. Blood and cerebrospinal fluid neurofilament light differentially detect neurodegeneration in early Alzheimer's disease. Neurobiol Aging. 2020 Nov;95: 143-153. doi: 10.1016/j.neurobiolaging.2020.07.018. Epub 2020 Jul 25. PMID: 32810755; PMCID: PMC7649343.

Khalil M., Teunissen C.E., Otto M., Piehl F., Sormani M.P., Gattringer T., Barro C., Kappos L., Comabella M., Fazekas F., Petzold A., Blennow K., Zetterberg H., Kuhle J. Neurofilaments as biomarkers in neurological disorders. Nat. Rev. Neurol. 2018;14:577- 589. [PubMed: 30171200]

Zetterberg H., Skillback T., Mattsson N., Trojanowski J.Q., Portelius E., Shaw L.M. Association of cerebrospinal fluid neurofilament light concentration with Alzheimer disease progression. JAMA Neurol. 2016;73:60-67. [PMCID: PMC5624219] [PubMed: 26524180]

Lyra e Silva, N.M., Goncalves, R.A., Pascoal, T.A. et al. Pro-inflammatory interleukin-6 signaling links cognitive impairments and peripheral metabolic alterations in Alzheimer’s disease. Transl Psychiatry 11, 251 (2021).

Chang, R., Yee, K. L., & Sumbria, R. K. (2017). Tumor necrosis factor a Inhibition for Alzheimer's Disease. Journal of central nervous system disease, 9, 1179573517709278.

Example 9. Single center, open-label, phase 1 study to evaluate the safety, tolerability', and exploratory efficacy of SNK01 in subjects with mild cognitive impairment (MCI) and Alzheimer’s Disease (AD) (Study SNK01-MX04)

[0384] It is predicted that neuroinflammation plays a role in autism. The data show how an autologous natural killer cell therapy could be used to treat disorders involving neuroinflammation, and as autism involves neuroinflammation, it follows that the examples are predictive that the therapy will be effective for treating autism.

[0385] This non-limiting example shows the results of a phase 1 study to evaluate the safety, tolerability, and exploratory efficacy of SNK01 in subjects with mild cognitive impairment (MCI) and Alzheimer’s Disease (AD), as described in Examples 6 and 7. This example includes the subjects and data described in Example 6, and further adds additional subjects and corresponding data, as described in Example 7. Measurement of marker levels were carried out as described in Examples 6-8.

[0386] Table 7 summarizes some of the results of this example and shows the percentage of patients with positive outcomes in their plasma samples. It shows the number of patients with a “stable or improved” outcome over the total number of patients.

Table 7

[0387] FIG. 39 shows line graph depicting the change in baseline CX3CL1 (Fractalkine) levels in the cerebrospinal fluid of subjects treated with different doses of SNK01. Left panel shows changes in the subjects over time. Right panel shows the mean change over time, grouped according to dosage. The underlying data includes the data plotted in FIG. 15B.

[0388] Decreased CX3CL1 concentrations are found in the CSF of AD patients compared to non-AD patients. CX3C chemokine ligand 1 (CX3CL1, also named fractalkine) plays an important role in reducing neuromflammation and is highly expressed in the main area of pathological changes in AD, such as the hippocampus and cerebral cortex, and the expression level of CX3CL1 reflects the progression of the disease. The activation of microglial CX3CR1, the sole receptor for CX3CL1, reduces the activation of microglia, which contribute to the neuronal damage characteristic of AD. Therefore, alterations of CX3CR1 production in microglia can translate into the enhancement or inhibition of CX3CL1 anti-inflammatory effect.

[0389] FIG. 40 shows line graphs depicting the change in baseline IL-6 levels in the cerebrospinal fluid of subjects treated with different doses of SNK01. Left panel shows changes in the subjects over time. Right panel shows the mean change over time, grouped according to dosage. The underlying data includes the data plotted in FIG. 16B.

[0390] FIG. 41 shows line graphs depicting the change in baseline TNF-a levels in the cerebrospinal fluid of subjects treated with different doses of SNK01. Left panel shows changes in the subjects over time. Right panel shows the mean change over time, grouped according to dosage. The underlying data includes the data plotted in FIG. 17B

[0391] FIG. 42 shows line graphs of A|3-42 changes in the plasma of subjects treated with SNK01. Right panel shows the mean change over time, grouped according to dosage.

[0392] FIG. 43 shows line graphs of AP-42/40 ratio changes in the plasma of subjects treated with SNK01. Right panel shows the mean change over time, grouped according to dosage. [0393] FIG. 44 shows line graphs of changes in total tau in the plasma of subjects treated with SNK01. Right panel shows the mean change over time, grouped according to dosage.

[0394] FIG. 45 shows line graphs of p-tau 181 changes in the plasma of subjects treated with SNK01. Right panel shows the mean change over time, grouped according to dosage.

[0395] FIG. 46 shows line graphs of GFAP changes in the plasma of subjects treated with SNK01. Right panel shows the mean change over time, grouped according to dosage.

[0396] FIG. 47 shows line graphs of NfL changes in the plasma of subjects treated with SNK01. Right panel shows the mean change over time, grouped according to dosage.

[0397] FIG. 48 shows line graphs of YKL-40 changes in the plasma of subjects treated with SNK01. Right panel shows the mean change over time, grouped according to dosage.

[0398] FIG. 49 shows line graphs of TNF-a changes in the plasma of subjects treated with SNK01. Right panel shows the mean change over time, grouped according to dosage.

[0399] FIG. 50 shows line graphs of IL-8 changes in the plasma of subjects treated with SNK01. Right panel shows the mean change over time, grouped according to dosage.

[0400] FIG. 51 shows line graphs of IL-6 changes in the plasma of subjects treated with SNK01.

[0401] FIG. 2 shows line graphs of IL-1J3 changes in the plasma of subjects treated with SNK01. Right panel shows the mean change over time, grouped according to dosage.

[0402] FIG. 53 shows line graphs of IL-10 changes in the plasma of subjects treated with SNK01. Right panel shows the mean change over time, grouped according to dosage.

[0403] FIG. 54 shows line graphs of IFN-y changes in the plasma of subjects treated with SNK01. Right panel shows the mean change over time, grouped according to dosage. Example 10 CSF Immunophenotype Markers

[0404] It is predicted that neuroinflammation plays a role in autism. The data show how an autologous natural killer cell therapy could be used to treat disorders involving neuroinflammation, and as autism involves neuroinflammation, it follows that the examples are predictive that the therapy will be effective for treating autism.

[0405] OBJECTIVE AND METHOD: Alzheimer’s disease (AD) is characterized by extracellular deposits of fibrillar amyloid-beta peptides and aggregates of the phosphorylated microtubule-associated protein tau in neurofibrillary' tangles. These proteins accumulate in the brain causes chronic deposition and lead to an inflammatory cascade involving alterations in the cross talks between glial cells and neurons (Yan 2021). Studies have shown T cells contribute indirectly to neuroinflammation by secreting proinflammatory mediators via direct crosstalk with glial cells and infiltrating the brain. (Dai 2020, Chen 2023). It is widely accepted that neuroinflammation in AD is driven by microglia and astrocytes while T cells are believed to be key mediators of the inflammatory response. While neuroinflammation can be a potentially beneficial defense mechanism that initially protects the brain by inhibiting diverse pathogens and clearing cellular debris, persistent inflammation can adversely affect neuronal plasticity, impair memory, and is generally considered as a main driver of tissue damage in neurodegenerative disorders (Kwon 2020). NK cells have been shown to have a protective role in other diseases caused by autoreactive T cells through cytokine production and direct killing of T cells.

[0406] Cerebrospinal fluid (CSF) from subjects with AD who participated in the autologous NK cell therapy (SNK01) were collected for the assessment of cell therapy treatment responses. Immunophenotyping of the immune cell subset frequencies and receptor expressions was done flow cytometrically. Immunophenotyping by flow cytometry was performed to analyze the expression of cell markers in a single-cell suspension from a sample of biofluid. The process identifies cells based on the types of antigens present on the cell surface or expressed intracellularly.

[0407] CSF samples were incubated with specific fluorophore-conjugated antibodies directed against the antigens of the receptor’s molecules and protein molecules. The conjugated antibodies bind to the corresponding specific antigens that are presented on each single cell. After washing away the unbound antibodies, cells were then analyzed using a flow cytometer. The flow cytometer combines fluidics, optics, and electronics to convert target expressions to a measurable signal output. Briefly, the fluidics system is responsible for the acquisition and direction of cells into a stream, which enables the analysis of single cells. The optics system consists of lasers, filters, and detectors; lasers excite the fluorophores, filters direct the path of light, and detectors convert the light into an electronic signal. Lastly, the electronic component processes the output from the detector and digitizes the information for subsequent analysis using flow cytometry data analysis software to determine the quasi-quantitation of the targeted immune cell subset frequencies and receptor expressions.

[0408] Results of the immunophenotyping are shown in FIGs. 55-66.

[0409] FIG. 55 shows a line graph of the percentage of CD3+CD56- T cells in the Leukocytes of subjects treated with NK cells.

[0410] FIG. 56 shows a line graph of the change from the baseline in the frequency of CD3+CD56- T cells in Leukocytes in subjects treated with NK cells.

[0411] FIG. 57 shows a line graph of the mean change from baseline in the frequency of CD3+CD56- T cells in Leukocytes in subjects treated with different doses of NK cells.

[0412] FIG. 58 shows a line graph of the percentage of CD3+CD56- T cells in Lymphocytes of subjects treated with NK cells.

[0413] FIG. 9 shows a line of the change from the baseline in the frequency of CD3+CD56- T cells in Lymphocytes in subjects treated with NK cells.

[0414] FIG. 60 shows a line graph of the mean change from baseline in the frequency of CD3+CD56- T cells in Lymphocytes in subjects treated with different doses of NK cells.

[0415] FIG. 61 shows a line graph of the percentage of CX3CR1+ cells in CD3-

CD56+ NK Cells from subjects treated with NK cells.

[0416] FIG. 62 shows a line graph of the change from the baseline in CX3CR1+ cells in CD3-CD56+ NK Cells in subjects treated with NK cells.

[0417] FIG. 63 shows a line graph of the mean change from baseline in the percentage of CX3CR1+ cells in CD3-CD56+ NK Cells in subjects treated with different doses of NK cells.

[0418] FIG. 4 shows a line graph of the percentage of CX3CR1+ cells in CD3+CD56- T Cells from subjects treated with NK cells.

[0419] FIG. 65 shows a line graph of the change from the baseline in the percentage of CX3CR1+ cells in CD3+CD56- T Cells in subjects treated with NK cells. [0420] FIG. 66 shows a line graph of the mean change from baseline in the percentage of CX3CR1+ cells in CD3+CD56- T Cells in subjects treated with different doses of NK cells.

BACKGROUD OF THE MARKERS

[0421] The chronic deposit and buildup of amyloid proteins has been found to illicit a pro-inflammatory dysregulation of the adaptive immune system (Yan 2021) with increased T cell autoreactivity to amyloid proteins (Monsonego 2003, Mate 2015). T cells have very high CXCR3 expression and migrate to brain via CXCR3 to CXCL10 positive astrocytes that are associated with protein deposits. (Liu 2019, Xia 2000). CD4+ and CD8+ T cells cause autoimmune inflammation and damage neurons in the brain (Monsonego 2003, Mate 2015, Lindestram Arelehamm 2022). NK cells can secrete interferon gamma to activate macrophages and microglia to phagocytose misfolded proteins amyloid-beta and tau tangles. (Earls 2020, Marsh 2019). SNK01 cells also traffic into the brain due to their high expression of CXCR3 and are chemoattracted by CXCL10 positive astrocytes. SNK01 can identify and eliminate autoreactive T cells to reduce neuroinflammation. (Rabinovich 2003, Lu 2007, Nielsen 2014, Gross 2016, Schuster 2016).

[0422] In the brain, the CX3CR1 receptor is predominantly expressed in microglia. Its ligand is the secreted soluble form of fractalkine (CX3CL1) and is constitutively expressed by neurons. CX3CL1 exerts an inhibitory signal, maintaining microglia in a resting state. (Hemonnot 2019). CX3CL1 is an essential chemokine, for regulating adhesion and chemotaxis through binding to CX3CR1, which plays a critical role in the crosstalk between glial cells and neurons by direct or indirect ways in the central nervous system (CNS). CX3CL1/CX3CR1 axis regulates microglial activation and function, neuronal survival and synaptic function by controlling the release of inflammatory cytokines and synaptic plasticity in the course of neurological disease. CX3CL1/CX3CR1 is necessary for the brain to maintain the homeostasis and effectively ameliorate inflammatory response in damaged brain via regulating the balance of pro- and anti-inflammatory cytokines such as tumor necrosis factor-alpha (TNF-a), interleukin-6 (IL-6). CX3CL1/CX3CR1 binding promotes microglial activation and phagocytosis, thereby promoting the clearance of extracellular amyloid beta (A(3) plaque, and attenuating p-tau. (Luo 2019, Subbarayan 2022)

[0423] References: Yan et. al, Dysregulation of the Adaptive Immune System in Patients with Early-Stage Parkinson’s Disease - Neurol Neuroimmunol Neuroinflamm (2021) 8:31036

Dai L, Shen Y. Insights into T-cell dysfunction in Alzheimer's disease. Aging Cell. 2021 Dec;20(12):el3511. doi: 10.1111/acel.13511. Epub 2021 Nov 1. PMID: 34725916; PMCID: PMC8672785.

Chen, X., Firulyova, M., Manis, M. et al. Microglia-mediated T cell infiltration drives neurodegeneration in tauopathy. Nature 615, 668-677 (2023).

Kwon et. al, - Neuroinflammation in neurodegenerative disorders: the roles of microglia and astrocytes Translational Neurodegeneration (2020) 9:42

Monsonego et. al, Increased T cell reactivity to amyloid B proteins in older humans and patients with Alzheimers’ disease (2003) - J. Clin. Invest 112:415

Mate Function and Redox State of Peritoneal Leukocytes as Preclinical and Prodromic Markers in a Longitudinal Study of Triple-Transgenic Mice for Alzheimer’s Disease- Journal of Alzheimer’s Disease 43 (2015) 213-226

Lindestram Arlehamm et. al., a-Synucl ein-specific T cell reactivity is associated with preclinical and early Parkinson’s disease - Nature Communication (2020) 11: 1875

Earls et al., NK cells clear a-synuclein and the depletion of NK cells exacerbates synuclein pathology in a mouse model of a-synucleinopathy. PNAS (2020) vol. 117:3 1762

Marsh et al., The adaptive immune system restrains Alzheimer’s disease pathogenesis by modulating microglial function PNAS | Published online February 16, 2016 | E1316

Rabinovich et. al., Activated, But Not Resting, T Cells Can Be Recognized and Killed by Syngeneic NK Cells J Immunol 2003; 170:3572-3576

Lu, et. al. Regulation of Activated CD4+ T Cells by NK Cells via the Qa-1-NKG2A Inhibitory Pathway Immunity (2007) 26, 593-604

Nielsen N, et. al.. Cytotoxicity of CD56bright NK Cells towards Autologous Activated CD4+ T Cells Is Mediated through NKG2D, LFA-1 and TRAIL and Dampened via CD94/NKG2A. (2012) PLoS ONE 7(2): e31959.

Gross et., al., Impaired NK-mediated regulation of T-cell activity in multiple sclerosis is reconstituted by IL-2 receptor modulation PNAS (2016) e2973

Schuster IS, Coudert JD, Andoniou CE, Degli-Esposti MA. "Natural Regulators" : NK Cells as Modulators of T Cell Immunity. Front Immunol. 2016 Jun 14;7:235. doi: 10.3389/fimmu.2016.00235. PMID: 27379097; PMCID: PMC4905977.

Hemonnot AL, Hua J, Ulmann L, Hirbec H. Microglia in Alzheimer Disease: Well-Known Targets and New Opportunities. Front Aging Neurosci. 2019 Aug 30;l 1:233. doi: 10.3389/fnagi.2019.00233. PMID: 31543810; PMCID: PMC6730262. Piao Luo, Shi-feng Chu, Zhao Zhang, Cong-yuan Xia, Nai-hong Chen, Fractalkine/CX3CR1 is involved in the cross-talk between neuron and glia in neurological diseases, Brain Research Bulletin, Volume 146, 2019, Pages 12-21, ISSN 0361-9230,

Meena S. Subbarayan, Aurelie Joly-Amado, Paula C. Bickford, Kevin R. Nash, CX3CL1/CX3CR1 signaling targets for the treatment of neurodegenerative diseases, Pharmacology & Therapeutics, Volume 231, 2022, 107989, ISSN 0163-7258,

[0424] The foregoing description of the exemplary embodiments has been presented only for the purposes of illustration and description and is not intended to be exhaustive or to limit the invention to the precise forms disclosed. Many modifications and variations are possible in light of the above teaching. It is contemplated that various combinations or sub combinations of the specific features and aspects of the embodiments disclosed above may be made and still fall within one or more of the inventions. Further, the disclosure herein of any particular feature, aspect, method, property, characteristic, quality, attribute, element, or the like in connection with an embodiment can be used in all other embodiments set forth herein. Accordingly, it should be understood that various features and aspects of the disclosed embodiments can be combined with or substituted for one another in order to form varying modes of the disclosed inventions. Thus, it is intended that the scope of the present inventions herein disclosed should not be limited by the particular disclosed embodiments described above. Moreover, while the invention is susceptible to various modifications, and alternative forms, specific examples thereof have been shown in the drawings and are herein descnbed in detail. It should be understood, however, that the invention is not to be limited to the particular forms or methods disclosed, but to the contrary, the invention is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the various embodiments described and the appended claims. Any methods disclosed herein need not be performed in the order recited. The methods disclosed herein include certain actions taken by a practitioner; however, they can also include any third-party instruction of those actions, either expressly or by implication. The ranges disclosed herein also encompass any and all overlap, sub-ranges, and combinations thereof.

[0425] The embodiments were chosen and descnbed in order to explain the principles of the invention and their practical application so as to activate others skilled in the art to utilize the invention and various embodiments and with various modifications as are suited to the particular use contemplated. Alternative embodiments will become apparent to those skilled in the art to which the invention is defined by the appended claims rather than the foregoing description and the exemplary embodiments described therein.

Claims

WHAT IS CLAIMED IS:

1. A method of treating autism in a subject, the method comprising: a. identifying a subject, wherein the subject has autism ; and b. administering to the subject an expanded natural killer (NK) cell population, wherein the NK cells are expanded by a method comprising: i) isolating at least one of CD56+ cells and/or CD3-/CD56+ cells from the PBMCs; ii) co-culturing the at least one of CD56+ cells and/or CD3-/CD56+ cells with a combination of feeder cells in the presence of at least two cytokines; iii) wherein the combination of feeder cells comprises irradiated Jurkat cells and irradiated Epstein-Barr virus transformed lymphocyte continuous line (EBV-LCL) cells; and iv) wherein the at least two cytokines comprise IL-2 and IL-21.

2. A method of treating Autism Spectrum Disorder (ASD) in a subject, the method comprising: a. identifying a subject, wherein the subject has autism ; and b. administering to the subject an expanded natural killer (NK) cell population, wherein the NK cells are expanded by a method comprising: i) isolating at least one of CD56+ cells and/or CD3-/CD56+ cells from the PBMCs; ii) co-culturing the at least one of CD56+ cells and/or CD3 /CD56+ cells with a combination of feeder cells in the presence of at least two cytokines; iii) wherein the combination of feeder cells comprises irradiated Jurkat cells and irradiated Epstein-Barr virus transformed lymphocyte continuous line (EBV-LCL) cells; and iv) wherein the at least two cytokines comprise IL-2 and IL-21.

-n-

4. A method of treating ASD in a subject, the method comprising: a. identifying a subject, wherein the subject has autism; and b. administering to the subject a therapeutically effective amount of an autologous NK cell population.

5. A method of cell therapy comprising: a. identifying a subject, wherein the subject has autism; and b. administering to the subject an expanded NK cell population, wherein the NK cells are expanded by a method comprising: i) isolating at least one of CD56+ cells and/or CD3-/CD56+ cells from the PBMCs; ii) co-culturing the at least one of CD56+ cells and/or CD3-/CD56+ cells with a combination of feeder cells in the presence of at least two cytokines; in) wherein the combination of feeder cells comprises irradiated Jurkat cells and irradiated Epstein-Barr virus transformed lymphocyte continuous line (EBV-LCL) cells; and iv) wherein the at least two cytokines comprise IL-2 and IL-21.

6. The method of any one of the preceding claims, wherein the ASD is autism, high-functioning autism, Asperger’s syndrome, and/or Pervasive developmental disorder — not otherwise specified (PDD-NOS).

7. The method of any one of the preceding claims, wherein the amount of expanded NK cells administered to a subject is a therapeutically effective amount.

8. The method of any one of the preceding claims, wherein the therapeutically effective amount of expanded NK cells comprises 2 x 10⁹ to 9 x 10⁹ cells.

9. The method of any one of the preceding claims, wherein the therapeutically effective amount of expanded NK cells comprises 1 x 10⁹ to 1 x IO¹⁰ cells.

10. The method of any one of the preceding claims, wherein IL-2 is added at a concentration of 50-1000 lU/mL during step ii).

11. The method of any of the preceding claims, wherein IL-21 is added at a concentration of 10-100 ng/mL during step ii).

12. The method of any one of the preceding claims, further comprising: co-culturing the at least one of CD56+ cells and/or CD3-/CD56+ cells with the combination of feeder cells, in the presence of IL-2 for a first period; and co-culturing the at least one of CD56+ cells and/or CD3-/CD56+ cells with the combination of feeder cells, in the presence of IL-21 for a second period.

13. The method of any one of the preceding claims, wherein IL-21 is added more than once during Day 0-6 of the second period.

14. The method of any one of the preceding claims, wherein IL-21 and the combination of feeder cells are added more than once during Day 0-6 of the second period.

15. The method of any one of the preceding claims, wherein IL-21 is added more than once during the first six days of every' fourteen-day cycle during the second period.

16. The method of any one of the preceding claims, wherein IL-21 and the combination of feeder cells are added more than once during Day 0-6 of the second period.

17. The method of any one of the preceding claims, wherein IL-21 is added more than once during the first six days of every' fourteen-day cycle during the second period.

18. The method of any one of the preceding claims, wherein the amount of expanded NK cells administered to a subject is a therapeutically effective amount.

19. The method of any one of the preceding claims, wherein the therapeutically effective amount of expanded NK cells comprises 2 x 10⁹ to 9 x 10⁹ cells.

20. The method of any one of the preceding claims, wherein IL-2 is added at a concentration of 50-1000 lU/mL during step ii).

21. The method of any one of the preceding claims, wherein IL-21 is added at a concentration of 10-100 ng/mL during step ii).

22. The method of any one of the preceding claims, further comprising: co-culturing the at least one of CD56+ cells and/or CD3-/CD56+ cells with the combination of feeder cells, in the presence of IL-2 for a first period; and co-culturing the at least one of CD56+ cells and/or CD3-/CD56+ cells with the combination of feeder cells, in the presence of IL-21 for a second period.

23. The method of any one of the preceding claims, wherein the NK cells do not include a CAR.

24. The method of any one of the preceding claims, wherein the NK cells do not include an engineered CAR.

25. A population of expanded NK cells, wherein the NK cells were expanded by a method that comprises: i) isolating at least one of CD56+ cells and/or CD3-/CD56+ cells from the PBMCs; ii) co-culturing the at least one of CD56+ cells and/or CD3-/CD56+ cells with a combination of feeder cells in the presence of at least two cytokines; iii) wherein the combination of feeder cells comprises irradiated Jurkat cells and irradiated Epstein-Barr virus transformed lymphocyte continuous line (EBV- LCL) cells; and iv) wherein the at least two cytokines comprise IL-2 and IL-21; and wherein the population of expanded NK cells has been administered to a subject who has autism.

26. A population of expanded NK cells, wherein the NK cells were expanded by a method that comprises: i) isolating at least one of CD56+ cells and/or CD3-/CD56+ cells from the PBMCs; ii) co-culturing the at least one of CD56+ cells and/or CD3-/CD56+ cells with a combination of feeder cells in the presence of at least two cytokines;

-SO- iii) wherein the combination of feeder cells comprises irradiated Jurkat cells and irradiated Epstein-Barr vims transformed lymphocyte continuous line (EBV- LCL) cells; and iv) wherein the at least two cytokines comprise IL-2 and IL-21; and wherein the population of expanded NK cells has been administered to a subject who has ASD.

27. The population of expanded NK cells of any one of the preceding claims, wherein the amount of expanded NK cells administered to a subject is a therapeutically effective amount.

28. The population of expanded NK cells of any one of the preceding claims, wherein the therapeutically effective amount of expanded NK cells comprises 2 x 10⁹ to 9 x 10⁹ cells.

29. The population of expanded NK cells of any one of the preceding claims, wherein IL-2 is added at a concentration of 50-1000 lU/mL during step ii).

30. The population of expanded NK cells of any one of the preceding claims, wherein IL-21 is added at a concentration of 10-100 ng/mL during step ii).

31. The population of expanded NK cells of any one of the preceding claims, further comprising: co-culturing the at least one of CD56+ cells and/or CD3-/CD56+ cells with the combination of feeder cells, in the presence of IL-2 for a first period; and co-culturing the at least one of CD56+ cells and/or CD3-/CD56+ cells with the combination of feeder cells, in the presence of IL-21 for a second period.

32. The population of expanded NK cells of any one of the preceding claims, wherein IL-21 is added more than once during Day 0-6 of the second period.

33. The population of expanded NK cells of any one of the preceding claims, wherein IL-21 and the combination of feeder cells are added more than once during Day 0- 6 of the second period.

34. The population of expanded NK cells of any one of the preceding claims, wherein IL-21 is added more than once during the first six days of every fourteen-day cycle during the second period.

35. The method of any one of the preceding claims, wherein the expanded NK cell population or the NK cell population is or comprises SNK01.

36. The population of any one of the preceding claims, wherein the expanded NK cells are or comprise SNK01.