Compositions and Methods for Stimulating Natural Killer Cells

Abstract

Compositions and methods for stimulating NK cell proliferation and cytotoxicity are described. Therapeutic compositions and methods using the expanded and stimulated NK cells are described.

Description

Technical Field

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 62 / 796,575, filed on Jan. 24, 2019, which is hereby incorporated by reference in its entirety.

[0002]

Background Art

[0003] ​Natural killer (NK) cell therapy is emerging as a treatment approach for cancer and, in some cases, for other diseases. Challenges to fully realizing the clinical potential of NK cell therapy include obtaining a large number of robust and healthy NK cells with high tumor cell cytotoxicity, the ability to target NK cells to disease targets, and, when introduced into patients, the ability to sustain NK cells sufficiently in vivo to achieve a therapeutic effect. This challenge is due in part to the fact that NK cell activity is tightly regulated by the balance of activating and inhibitory receptors, including immune checkpoints. For example, ligands for activating NK cell receptors are expressed only on stressed, transformed, or virus-infected cells, so the cytotoxic activity of NK cells targets such cells while sparing normal, healthy tissue. The cytotoxic activity of NK cells is further restricted by inhibitory ligands expressed on "self" cells. At the same time, inhibitory regulatory mechanisms that control the cytotoxicity of NK cells can be an avenue of attack by tumor cells that deploy various immunosuppressive interactions to prevent an immune attack. An example of how NK cells can resist tumor immunosuppression is the involvement of target cells marked with antibodies to induce antibody-dependent cell cytotoxicity (ADCC) in NK cells. As a result, the success of many new anti-tumor antibodies depends on the presence of more healthy NK cells within patients to support anti-tumor activity. Overall, in the field of NK cell therapy, there remains a need for approaches to obtain and stimulate a large number of healthy NK cells to achieve higher cytotoxicity and / or better ADCC functionality. SUMMARY OF THE INVENTION

[0004] Among the various aspects of the present disclosure, there is a feeder cell composition comprising at least one feeder cell, which comprises a fragment crystallizable (Fc) domain bound to the outer surface of the feeder cell. In some aspects, the at least one feeder cell further comprises one or more NK cell effector agents. In certain aspects, the at least one feeder cell comprises at least one NK cell effector agent, wherein the NK cell effector agent is IL-21. In another aspect, the at least one feeder cell further comprises at least two NK cell effector agents, wherein one of the at least two NK cell effector agents is IL-21.

[0005] Also disclosed herein is an NK cell growth composition that does not contain feeder cells and contains engineered particles, wherein the engineered particles comprise an Fc domain bound to the outer surface of the engineered particles of any of the preceding aspects. In some aspects, the engineered particles further comprise one or more NK cell effector agents. In some aspects, the engineered particles further comprise at least one NK cell effector agent, wherein the NK cell effector agent is IL-21. In another aspect, the engineered particles further comprise at least two NK cell effector agents, wherein one of the at least two NK cell effector agents is IL-21.

[0006] One aspect of the present disclosure is a therapeutic dose of NK cells grown in vitro and combined with an NK cell growth composition, the composition not containing feeder cells and containing at least one engineered particle, the engineered particle comprising an Fc domain bound to the outer surface of the engineered particle. In some aspects, the engineered particles further comprise one or more NK cell effector agents. In some aspects, the engineered particles further comprise at least one NK cell effector agent, wherein the NK cell effector agent is IL-21. In another aspect, the engineered particles further comprise at least two NK cell effector agents, wherein one of the at least two NK cell effector agents is IL-21.

[0007] One aspect of the present disclosure is a proliferated NK cell population exposed in vitro to an NK cell proliferation composition that does not contain feeder cells and contains at least one of the engineered plasma membrane (PM) particles disclosed herein. Another aspect of the present disclosure is a proliferated NK cell population exposed in vitro to an NK cell proliferation composition that contains at least one feeder cell, wherein the feeder cell contains an Fc domain bound to the outer surface of the feeder cells disclosed herein. Such methods optionally further include exposing the NK cells to one or more NK cell effector agents. The one or more cell effector agents may be dissolved in the cell culture medium and / or bound to the surface of the Fc-binding feeder cells or engineered PM particles disclosed herein.

[0008] Also disclosed herein is a method of treating, remitting, reducing, and / or inhibiting cancer or metastasis or an infectious disease, the method comprising administering to a subject in need thereof an effective amount of any NK cell proliferation composition of the present disclosure or an NK cell proliferation injection formulation of any preceding aspect. In one aspect, the NK cell proliferation composition or NK cell proliferation injection formulation can be administered in combination with or concomitantly with a therapeutic agent such as, for example, an anti-cancer therapeutic agent or an anti-viral agent or an antibiotic.

[0009] One aspect of the present disclosure is a method of preventing, reducing, alleviating, and / or inhibiting cancer recurrence or metastasis before or after stem cell transplantation, the method comprising administering to a subject in need thereof an effective amount of an NK cell proliferation composition, or an NK cell stimulation composition, or any proliferated NK cell population of the present disclosure exposed in vitro to any NK cell stimulation or proliferation injection formulation of the present disclosure. Any NK cell stimulation or proliferation composition or formulation of the present disclosure can be administered in combination with or separately from the stem cell transplantation.

[0010] One aspect of the present disclosure is a method of modulating a T cell repertoire, the method comprising administering to a subject in need thereof an effective amount of any of the disclosed expanded NK cell populations that have been exposed in vitro to an NK cell growth composition, or any of the disclosed NK cell growth infusion formulations.

[0011] One aspect of the present disclosure is a method of preventing, inhibiting, reducing, or alleviating acute or chronic graft-versus-host disease, the method comprising administering to a subject in need thereof an effective amount of any of the disclosed expanded NK cell populations that have been exposed in vitro to any of the disclosed NK cell growth compositions (one or more), or any of the disclosed NK cell growth infusion formulations.

[0012] Other aspects and features of the present disclosure are described in more detail below.

Brief Description of the Drawings

[0013]

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DETAILED DESCRIPTION OF THE INVENTION

[0014] The present disclosure provides compositions and formulations comprising NK cell stimulants, and their use for stimulating NK cells, and related methods of their use in various treatments described in more detail below.

[0015] Binding of the CD16 receptors (FcγRIIIa receptor (CD16a) and FcγRIIIb receptor (CD16b)) on NK cells can be a very powerful mechanism in NK cell stimulation. The Fc (fragment crystallizable region) domain of an antibody is recognized by CD16, and when the Fc domain binds to CD16, antibody-dependent cell cytotoxicity (ADCC) is induced. This disclosure describes an engineered stimulation of NK cells via CD16 binding to improve NK cell proliferation and enhance the cytotoxicity of NK cells. In other words, this disclosure contemplates stimulating NK cells using the Fc domain of an antibody, where the Fc domain has the ability to activate CD16 on NK cells and the Fc domain is presented to the NK cells while bound to feeder cells, plasma membrane (PM) particles, exosomes (EX), or a solid support. Fc-binding feeder cells, PM particles, exosomes, and solid supports can further include or be combined with other NK cell stimulating factors in various forms (e.g., membrane-bound or soluble IL-15, IL-21, 4-1BBL, other cytokines, or other stimulatory or inhibitory receptors, and other chemical moieties that are simultaneously involved in the corresponding signaling pathways). NK cells grown using the compositions according to the methods disclosed herein can exhibit higher cytotoxicity, higher CD16 expression, and / or improved ADCC functionality. Such NK cells are useful in therapeutic compositions and methods for treating human diseases and conditions including multiple types of cancer. Definitions

[0016] Unless otherwise defined, all technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this invention belongs. The following references provide one of ordinary skill in the art with many general definitions of terms used herein: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991). As used herein, the following terms have the following meanings ascribed to them unless otherwise specified.

[0017] When introducing elements of the present disclosure or preferred embodiments (s) thereof, the articles "a", "an", "the", and "said" are intended to mean that there are one or more elements. The terms "comprising", "including", "having" are intended to be inclusive and mean that additional elements may exist in addition to the recited elements.

[0018] Ranges can be expressed herein as from a particular value with "about" and / or to another particular value with "about". When such a range is expressed, another aspect includes one particular value and / or the other particular value. Similarly, when a value is expressed as an approximation by use of the preceding "about", it is to be understood that that particular value forms another embodiment. Further, it is to be further understood that each endpoint of each range is meaningful both in relation to the other endpoint and independent of the other endpoint. It is also to be understood that where a plurality of values are disclosed herein, each value is also disclosed herein as that particular value with "about" in addition to the value itself. For example, if the value "10" is disclosed, "about 10" is also disclosed. Also, as will be appropriately understood by those skilled in the art, it is to be understood that where a value is disclosed as "less than" that value, "greater than or equal to" that value and the possible ranges in between are also disclosed. For example, if the value "10" is disclosed, "10 or less" and "10 or greater" are also disclosed. Also, throughout this application, the data is presented in a plurality of different formats, and it is to be understood that this data represents ranges at endpoints and starting points, as well as in any combination of data points. For example, if a particular data point "10" and a particular data point 15 are disclosed, it is to be understood that being greater than, greater than or equal to, less than, less than or equal to, and equal to 10 and 15 is considered to be disclosed as well as between 10 and 15. It is also to be understood that each unit between two particular units is also disclosed. For example, if 10 and 15 are disclosed, 11, 12, 13, and 14 are also disclosed.

[0019] As used herein, the terms "optional" or "optionally" mean that the event or circumstance following may or may not occur, and that the description includes both the case where the event or circumstance occurs and the case where it does not occur.

[0020] As used herein, "N-terminal side" or "amino terminus" refers to the directionality of a peptide, polypeptide, or protein, and may not mean the N-terminus. In some aspects where chimeric or fusion peptides, polypeptides, or proteins are discussed, the N-terminal side may only refer to the components of the chimeric or fusion peptide, polypeptide, or protein and may not refer to the overall structure. For example, when discussing the Fc domain and it is described that the Fc domain is fused such that its amino terminus or N-terminal side faces intracellularly, what is contemplated herein is a chimeric or fusion peptide, polypeptide, or protein in which the signal anchor is at the N-terminus of the chimeric or fusion construct and actually spans the cell membrane. Thus, in such a chimera, the transmembrane anchor binds to the amino-terminal side of the Fc domain, and the directionality of the Fc domain is inverted relative to that of a typical Fc domain on a B cell (typically having a carboxy terminus that spans the cell membrane and an amino terminus that extends into the extracellular matrix).

[0021] The terms "peptide", "polypeptide", and "protein" are used interchangeably and refer to polymers of amino acid residues.

[0022] As used herein, the term "sequence identity" refers to a quantitative measure of the degree of identity between two sequences of substantially equal length. The percent identity of two sequences, whether nucleic acid or amino acid sequences, is calculated by dividing the number of exact matches between the two aligned sequences by the length of the shorter sequence and multiplying by 100. Approximate alignment of nucleic acid sequences is provided by the local homology algorithm of Smith and Waterman, Advances in Applied Mathematics 2:482-489 (1981). This algorithm can be applied to amino acid sequences by using a scoring matrix developed by Dayhoff, Atlas of Protein Sequences and Structure, M.O. Dayhoff ed., 5 suppl. 3:353-358, National Biomedical Research Foundation, Washington, D.C., USA and normalized by Gribskov, Nucl. Acids Res. 14(6):6745-6763 (1986). An exemplary implementation of this algorithm for determining percent sequence identity is provided by the Genetics Computer Group (Madison, Wis.) in the "BestFit" utility application. Other suitable programs for calculating percent identity or similarity between sequences are generally known in the art, for example, another alignment program is BLAST which is used with default parameters.For example, BLASTN and BLASTP can be used with the following default parameters: genetic code=standard; filter=none; strand=both; cutoff=60; expect=10; Matrix=BLOSUM62; Descriptions=50 sequences; sort by=HIGH SCORE; Databases=non-redundant,GenBank+EMBL+DDBJ+PDB+GenBank CDS translations+Swiss protein+Spupdate+PIR. Details of these programs can be found on the GenBank website. Generally, substitutions are conservative amino acid substitutions and are limited to exchanges within members of group 1: glycine, alanine, valine, leucine, and isoleucine; group 2: serine, cysteine, threonine, and methionine; group 3: proline; group 4: phenylalanine, tyrosine, and tryptophan; group 5: aspartic acid, glutamic acid, asparagine, and glutamine.

[0023] Techniques for determining nucleic acid and amino acid sequence identity are known in the art. Typically, such techniques include determining the nucleotide sequence of mRNA in a gene and / or the amino acid sequence encoded thereby, and comparing these sequences to a second nucleotide or amino acid sequence. Genomic sequences can also be determined and compared in this way. Generally, identity refers to the exact nucleotide-to-nucleotide or exact amino acid-to-amino acid correspondence in two polynucleotide or polypeptide sequences. Two or more sequences (polynucleotide or amino acid) can be compared by determining their percent identity.

[0024] In the above-described cells and methods, various modifications can be made without departing from the scope of the present invention. Therefore, all matters included in the above description and the examples shown below are to be construed as illustrative and not in a limiting sense.

[0025] "Increase" can refer to any change that results in a greater amount of a symptom, disease, composition, condition, or activity. An increase can be any individual, median, or average increase in a state, symptom, activity, or composition that is statistically significant. Thus, an increase can be a 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, 95, or 100% increase, as long as the increase is statistically significant.

[0026] "Decrease" can refer to any change that results in a lesser amount of a symptom, disease, composition, condition, or activity. A substance is also understood to decrease the genetic output of a gene if the genetic output of the gene product containing that substance is less than the output of the gene product not containing that substance. Also, for example, a decrease can be a change such that the symptoms of a disorder become less severe than those observed in the past. A decrease can be any individual, median, or average decrease in a state, symptom, activity, or composition that is statistically significant. Thus, a decrease can be a 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, 95, or 100% decrease, as long as the decrease is statistically significant.

[0027] "Inhibit", "inhibiting", and "inhibition" mean reducing an activity, response, state, disease, or other biological parameter. This can include, but is not limited to, complete disappearance of an activity, response, state, or disease. It can also include, for example, a 10% reduction in an activity, response, state, or disease compared to a native or control level. Thus, the reduction can be 10, 20, 30, 40, 50, 60, 70, 80, 90, 100%, or any amount in between compared to a native or control level.

[0028] "Reduce", or other forms of the term (e.g., "reducing" or "reduction") means decreasing an event or characteristic (e.g., tumor growth). This is typically related to some reference or predicted value, i.e., it is relative, although it should be understood that reference to a reference value or relative value is not always required. For example, "reducing tumor growth" means reducing the rate of tumor growth compared to a reference or control.

[0029] "Prevent", or other forms of the term (e.g., "preventing" or "prevention") means stopping a particular event or characteristic, stabilizing or delaying the occurrence or progression of a particular event or characteristic, or minimizing the likelihood that a particular event or characteristic will occur. Prevention is typically more absolute than, for example, reduction, and thus does not require comparison to a control. As used herein, there are matters that can be reduced but not prevented, and there are matters that can be reduced and also prevented. Similarly, there are matters that can be prevented but not reduced, and there are matters that can be prevented and also reduced. When the terms reduction or prevention are used, it should be understood that the use of the other term is also explicitly disclosed unless otherwise specifically indicated.

[0030] The term "subject" refers to any individual that is the target of administration or treatment. The subject can be a vertebrate, such as a mammal. In one embodiment, the subject can be a human, non-human primate, cow, horse, pig, dog, or cat. Also, the subject can be a guinea pig, rat, hamster, rabbit, mouse, or mole. Thus, the subject can be a human or a veterinary patient. The term "patient" refers to a subject under the treatment of a clinician (e.g., a physician).

[0031] The term "therapeutically effective" refers to an amount of the composition used that is sufficient to relieve one or more causes or symptoms of a disease or disorder. Such relief only requires a decrease or change and does not necessarily require disappearance.

[0032] The term "treatment" refers to the medical management of a patient with the intent to cure, relieve, stabilize, or prevent a disease, pathologic condition, or disorder. This term includes active treatment, i.e., treatment specifically directed toward the improvement of a disease, pathologic condition, or disorder, and further includes causal treatment, i.e., treatment directed toward the removal of the cause of the related disease, pathologic condition, or disorder. In addition, this term includes palliative treatment, i.e., treatment designed for the relief of symptoms rather than the cure of the disease, pathologic condition, or disorder, and prophylactic treatment, i.e., treatment directed toward minimizing or partially or completely inhibiting the development of a related disease, pathologic condition, or disorder, and supportive therapy, i.e., treatment used to supplement another specific treatment directed toward the improvement of a related disease, pathologic condition, or disorder.

[0033] "Administration" to a subject includes any route by which an agent is introduced or delivered to the subject. Administration can be effected by any suitable route, including oral, topical, intravenous, subcutaneous, transcutaneous, transdermal, intramuscular, intra-articular, parenteral, arteriolar, intradermal, intraventricular, intracranial, intraperitoneal, intralesional, intranasal, rectal, vaginal, inhalation, implantable reservoir, parenteral (e.g., subcutaneous, intravenous, intramuscular, intra-articular, intra-synovial, intrasternal, intramedullary, intraperitoneal, intrahepatic, intralesional, and intracranial injection or infusion techniques), etc. As used herein, "concurrent administration", "combination administration", "simultaneous administration", or "administered simultaneously" means that the compounds are administered at the same time or are administered essentially immediately after one another. In the latter case, the two compounds are administered at times that are close enough that the observed results are indistinguishable from those achieved when the compounds are administered at the same time. "Systemic administration" refers to introducing or delivering an agent to a subject via a route that introduces or delivers the agent to a broad region (e.g., greater than 50% of the body) of the subject's body (e.g., via an entry into the circulatory or lymphatic system). In contrast, "local administration" refers to introducing or delivering an agent to a subject via a route that introduces or delivers the agent directly to the region immediately adjacent to the administration area or point of administration and does not introduce the agent systemically in a therapeutically significant amount. For example, a locally administered agent is readily detectable in the immediate vicinity of the point of administration but is undetectable or only detectably present in trace amounts at distal sites of the subject's body. Administration includes self-administration and administration by another person.

[0034] As used herein, "treating", "treatment", "treat", and grammatical variations thereof include administering a composition with the intent or purpose of partially or completely preventing, delaying, curing, recovering, alleviating, reducing, changing, correcting, remitting, improving, stabilizing, soothing, and / or reducing the intensity or frequency of one or more diseases or conditions, the symptoms of a disease or condition, or the underlying cause of a disease or condition. Treatment according to the present invention can be applied prophylactically, preventively, palliatively, or corrective. Preventive treatment is administered to a subject prior to onset (e.g., before overt signs of cancer), during initial onset (e.g., at the initial signs and symptoms of cancer), or after the onset of established cancer. Preventive administration can be performed from one or several days to several years before the symptoms of a disease or infection appear. (I) Fc fusion peptide

[0035] In one aspect, provided herein are solid supports comprising engineered feeder cells, engineered plasma membrane (PM) particles, engineered exosomes, engineered platelets (including, but not limited to, Fc-binding platelets), and engineered lymphocytes (e.g., lymphocytes engineered to express an Fc domain to stimulate NK cells (e.g., T cells)), as well as membrane-bound Fc fusion peptides (hereinafter referred to herein as Fc-binding feeder cells, Fc-binding PM particles, Fc-binding exosomes, Fc-binding platelets, and Fc-binding lymphocytes, respectively), wherein the Fc fusion peptide comprises a transmembrane peptide domain bound to the amino terminus of the Fc domain. In one aspect, the transmembrane domain of the Fc fusion peptide can comprise a cleaved or uncleaved signal-anchor sequence, e.g., the transmembrane domain of neuraminidase, a signal-anchor from parainfluenza virus hemagglutinin-neuraminidase, a signal-anchor from the transferrin receptor, a signal-anchor from the MHC class II invariant chain, a signal-anchor from P-glycoprotein, a signal-anchor from the asialoglycoprotein receptor, or a signal-anchor from neutral endopeptidase. In one example, the transmembrane domain comprises the parainfluenza virus hemagglutinin-neuraminidase (NA) peptide sequence. As schematically shown in FIG. 1, the transmembrane neuraminidase (NA) peptide domain is used to link or bind the Fc domain to the outer surface of the feeder cell. In other aspects, the transmembrane neuraminidase (NA) peptide domain is used to link or bind the Fc domain to the outer surface of PM nanoparticles, exosomes, or the solid support. The NA peptide domain consists of an N-terminal cytoplasmic tail, an uncleaved signal anchor that acts as a transmembrane domain, and a stalk region extending from the plasma membrane. It will be understood that the length of the stalk region can vary.

[0036] As used herein, the term "NA peptide domain" refers to a peptide sequence comprising at least a 50 - amino acid sequence of SEQ ID NO: 1 (MNPNQKITTIGSICLVVGLISLILQIGNIISIWISHSIQTGSQNHTGICN), or a sequence having at least about 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98, or 99% sequence identity to SEQ ID NO: 1.

[0037] The Fc domain is a ligand to which the NK - surface receptor CD16 (FcγRIII) binds. CD16 is one of the major receptors on NK cells. When CD16 binds to the Fc portion of an antibody (e.g., IgG1, IgG2, IgG3, and / or IgG4 Fc domain), this activates antibody - dependent cell - mediated cytotoxicity (ADCC) of NK cells. In another aspect, the Fc domain (IgG1, IgG2, IgG3, and / or IgG4) can also bind to the CD16 receptor on other immune cells (e.g., mast cells, macrophages, gamma - delta T cells), thereby similarly stimulating the proliferation of such cells and enhancing cytotoxicity. In another aspect, other types of cells can be engineered to be Fc - bound. Thus, the present disclosure also encompasses, for example, Fc - engineered platelets, Fc - bound primary tumor samples for use in tumor vaccines, and engineered iPSCs that express Fc.

[0038] In another aspect, other Fc immunoglobulin isotypes other than IgG (IgA, IgE, IgM) can be used to stimulate the corresponding different Fc receptors to stimulate other immune cell types. For example, the domain FcαRI (CD89) specifically binds to IgA on macrophages, neutrophils, and eosinophils, FcγRI (CD64) specifically binds to IgG on monocytes and macrophages, and FcεRII (CD23) specifically binds to IgE on B cells. Fc binds to and stimulates CD64 on monocytes or macrophages. Thus, using fusion peptides, Fc-binding feeder cells (FC), Fc-binding lymphocytes, Fc-engineered plasma membrane (PM) particles, Fc-engineered exosomes, and compositions containing them, it is also possible to proliferate mast cells and / or macrophages substantially according to the methods described herein for proliferating NK cells.

[0039] In one aspect, disclosed herein is a fusion peptide comprising an immunoglobulin Fc domain (e.g., IgG1, IgG2, IgG3, IgG4, IgA, and / or IgE Fc domain) fused to a transmembrane domain (e.g., an NA peptide domain as described above). The Fc domain(s) can be presented as a monomer, dimer, or multimer construct. In one aspect, the Fc domain(s) can be further modified to optimize or enhance antibody-mediated killing, NK cell recognition, and control the proliferation of activating Fc receptors. For example, the Fc domain(s) can be modified to increase its affinity for CD16. Thus, for example, the Fc domain(s) can contain one or more mutations such as T256A, K290A, S298A, E333A, K334A, L235V, F243L, R292P, Y300L, and / or P396L. Similarly, the Fc domain(s) can be further modified to increase the selectivity of binding to activating (IIIa) receptors and inhibitory Fc (IIb) receptors. Thus, for example, the Fc domain(s) can contain 1, 2, 3, 4, 5, 6, 7, 8, or more mutations, or alternative forms (e.g., S239D, I332E, A330L, F243L, R292P, V305I, and / or P396L). For example, in one aspect, the Fc domain can be modified to contain R292L, Y300L, V305I, and P396L. In another example, the Fc domain can be modified to contain S239D, I332E, and A330L. In another aspect, engineered variants of lower affinity Fc domains can be used to induce higher expression of CD16 on NK cells.

[0040] The transmembrane domain (e.g., the NA peptide domain) may be directly bound to the Fc domain via a chemical bond or indirectly bound via a linker. Direct chemical bonds include, for example, covalent bonds (e.g., peptide bonds, ester bonds, etc.) or alternatively non-covalent bonds (e.g., ionic, electrostatic, hydrogen, hydrophobic, van der Waals interactions, or π effects). Indirect binding can be achieved using a linker (i.e., a chemical group that connects one or more other chemical groups via at least one covalent bond). Suitable linkers include amino acids, peptides, nucleotides, nucleic acids, dimeric hinge-linked Fc, organic linker molecules (e.g., maleimide derivatives, N-ethoxybenzylimidazole, biphenyl-3,4’,5-tricarboxylic acid, p-aminobenzyloxycarbonyl, etc.), disulfide linkers, and polymeric linkers (e.g., PEG). The linker can contain one or more spacing groups, including but not limited to alkylene, alkenylene, alkynylene, alkyl, alkenyl, alkynyl, alkoxy, aryl, heteroaryl, aralkyl, aralkenyl, aralkynyl, etc. The linker may be neutral or have a positive or negative charge. Additionally, the linker may be cleavable such that the covalent bond of the linker connecting the linker to another chemical group can be decomposed or cleaved under certain conditions including pH, temperature, salt concentration, light, catalyst, or enzyme. In one embodiment, the NA peptide domain can be NA4-Fc Siadel (S239D / I332E / A330L).

[0041] In one embodiment, the linker can be a peptide linker. Examples of suitable peptide linkers are well known in the art and programs for designing linkers are readily available (see, for example, Crasto et al., Protein Eng., 2000, 13(5):309-312). The peptide linker can be a restriction site linker such as the short sequence RS, or a flexible amino acid linker (e.g., containing small non-polar or polar amino acids). Non-limiting examples of flexible linkers include LEGGGS (SEQ ID NO: 2), TGSG (SEQ ID NO: 3), GGSGGGSG (SEQ ID NO: 4), (GGGGS) 1-4 (SEQ ID NO: 5), GGGS (SEQ ID NO: 6) 1-4 , GSGGGG (SEQ ID NO: 7) 1-4 , and (Gly) 6-8 . Alternatively, the peptide linker may be a rigid amino acid linker. Examples of such linkers include (EAAAK) 1-4 (SEQ ID NO: 8), A(EAAAK) 2-5 A (SEQ ID NO: 9), PAPAP (SEQ ID NO: 10), and (AP) 6-8 . The Fc domain can bind to the N-terminus, C-terminus, and / or an internal position of the NA peptide.

[0042] In some embodiments, the Fc fusion peptide has an amino acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98, or 99% sequence identity to SEQ ID NO: 13. To target the placement of the Fc domain on the plasma membrane, a well-characterized membrane targeting domain from the influenza virus neuraminidase protein (NA) can be used. This domain consists of an N-terminal cytoplasmic tail, an uncleaved signal anchor that acts as a transmembrane domain, and a stalk region extending from the plasma membrane. FIGS. 1A and 1B are schematic diagrams showing the construction of a membrane-bound immune cell targeting ligand containing an uncleaved signal anchor sequence. FIG. 1A shows the structures of type I and type II complex membrane proteins and their respective signal anchors. FIG. 1B shows the structure of the uncleaved signal anchor from a type II complex membrane protein used in a membrane-bound immune cell targeting ligand. As shown in FIG. 1B, an exemplary but non-limiting construct according to the present disclosure is composed of an NA-Fc chimera in which the Fc domain (IgG1) is linked to the uncleaved NA stalk region via a short linker. Notably, the NA-Fc chimera can be inserted into a recombinant P / V / F virus to generate a novel oncolytic virus that is more specific for tumors than normal cells (due to the P / V mutation) and enhances ADCC by NK cells. FIG. 2 shows an alternative configuration of the NA-Fc chimera with increasing NA stalk length.

[0043] FIG. 3 shows one exemplary sequence of an NA-Fc chimera in which the Fc domain (IgG1) is linked to a 50-amino acid NA sequence by a short RS linker sequence to create a non-limiting example of an NA-Fc construct having the 279-amino acid sequence described below and shown in FIG. 3.

[0044]

[0045] As described above, the Fc region can contain, for example, one or more mutations selected from L234Y, L235V, L235Q, G236W, S239D, S239M, F243L, T256A, K290A, R292P, N297Q, S298A, Y300L, V305I, A330L, I332E, E333A, K334A, and / or P396L. Thus, specifically in this specification, leucine (L) or tyrosine (Y) at residue 234, leucine (L), glutamine, or valine (V) at residue 235, glutamine (G) or tryptophan (W) at residue 236, serine (S), methionine (M), or aspartic acid (D) at residue 239, and phenylalanine (F) or leucine (L) at residue 243, threonine (T) or alanine (A) at residue 256, histidine (H) or aspartic acid (D) at residue 268, aspartic acid (D) or glutamic acid (E) at residue 270, lysine (K) or alanine (A) at residue 290, arginine (R) or proline (P) at residue 292, serine (S) or alanine (A) at residue 298, asparagine or glutamine at residue 297, tyrosine (Y) or leucine (L) at residue 300, valine (V) or isoleucine (I) at residue 305, lysine (K) or aspartic acid (D) at residue 326, alanine (A), methionine (M), or leucine (L) at residue 330, and isoleucine (I) or glutamic acid (E) at residue 332, glutamic acid (E) or alanine (A) at residue 333, lysine (K), glutamic acid (E), or alanine (A) at residue 334, and / or proline (P) or leucine (L) at residue 396 are disclosed. It is specifically understood that there may be cases where none of the substitutions mentioned in this specification are present in the Fc region, or there may be cases where any one, or combinations of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 are present.Accordingly, in one aspect, the present specification discloses a fusion protein comprising substitutions in the Fc region at F243L, R292P, Y300L, V305I, and P396L, and the sequence of Na4-Fc comprises MNPNQKITTIGSICLVVGLISLILQIGNIISIWISHSIQTGSQNHTGICNQNIITYKNSTWVKDTTSVILTGNSSLCPIRGWAIYSKDNSIRIGSKGDVFVIREPFISCSHLECRTFFLTDKTHTCPPCPAPELLGGPSVFLLPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPPEEQYNSTLRVVSILTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPLVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 20), which comprises an Fc domain having the sequence GGPSVFLLPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPPEEQYNSTLRVVSILTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPLVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 25), and the Fc domain comprises a CH2 domain having the sequence GGPSVFLLPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPPEEQYNSTLRVVSILTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK (SEQ ID NO: 26) and a CH3 domain having the sequence GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPLVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 27).

[0046] In one embodiment, the Na4-Fc fusion comprises the S239D, I332E, and A330L substitutions and has the Fc domain of the sequence GGPDVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPLPEEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 21), which comprises a CH2 domain having the sequence GGPDVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPLPEEKTISKAK (SEQ ID NO: 22) and a CH2 domain having the sequence GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 24), and has the complete sequence of MNPNQKITTIGSICLVVGLISLILQIGNIISIWISHSIQTGSQNHTGICNQNIITYKNSTWVKDTTSVILTGNSSLCPIRGWAIYSKDNSIRIGSKGDVFVIREPFISCSHLECRTFFLTDKTHTCPPCPAPELLGGPDVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPLPEEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 23).

[0047] In one embodiment, the Na4-Fc fusion protein can comprise two Fc domains linked via a hinge region. For example, the Na-Fc fusion can comprise the sequence MNPNQKITTIGSICLVVGLISLILQIGNIISIWISHSIQTGSQNHTGICNQNIITYKNSTWVKDTTSVILTGNSSLCPIRGWAIYSKDNSIRIGSKGDVFVIREPFISCSHLECRTFFLTDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 28).

[0048] In another embodiment, the Fc domain can be an asymmetric variant. For example, one heavy chain Fc domain can comprise L234Y / L235Q / G236W / S239M / H268D / D270E / S298A, and the other Fc domain can comprise D270E / K326D / A330M / K334E.

[0049] Generally, any amino acid substitution is conservative, i.e., limited to exchanges within the members of Group 1: glycine, alanine, valine, leucine, and isoleucine; Group 2: serine, cysteine, threonine, and methionine; Group 3: proline; Group 4: phenylalanine, tyrosine, and tryptophan; and Group 5: aspartic acid, glutamic acid, asparagine, and glutamine.

[0050] The present disclosure also contemplates nucleic acids encoding any of the fusion proteins disclosed herein. For example, SEQ ID NO: 19 encodes the Na-Fc fusion shown in SEQ ID NO: 18, SEQ ID NO: 29 encodes the NA-Fc fusion shown in SEQ ID NO: 20, SEQ ID NO: 30 encodes the NA-Fc fusion shown in SEQ ID NO: 23, and SEQ ID NO: 31 encodes the NA-2xFc fusion shown in SEQ ID NO: 28. Additionally, vectors containing the claimed nucleic acids and cells containing such vectors are contemplated herein. Vectors and cells containing such vectors can be prepared using methods known in the art. (II) Engineered feeder cells, engineered plasma membrane particles, and engineered exosomes containing membrane-bound Fc

[0051] Compositions according to the present disclosure include compositions containing Fc-binding feeder cells (FC), compositions containing Fc-binding engineered plasma membrane (PM) particles, and compositions containing Fc-binding engineered exosomes. Fc-binding engineered PM particles include PM nanoparticles derived from Fc-binding feeder cells. Fc-binding engineered exosomes include exosomes or other extracellular vesicles derived from Fc-binding feeder cells, as described in more detail below. Alternatively, exosomes may be derived from other sources such as platelets and megakaryocytes.

[0052] As used herein, the term "Fc binding" is understood to refer to the attachment of the Fc domain in the reverse orientation (i.e., the amino terminus facing intracellularly) to the outer surface of a feeder cell or engineered particle via a transmembrane peptide. This can be achieved using the Fc fusion peptides disclosed herein. Thus, one aspect of the present disclosure provides a feeder cell composition comprising at least one feeder cell, i.e., a feeder cell comprising an Fc domain bound to the outer surface of the feeder cell, as further described in detail below. For example, the feeder cell can be genetically modified to express an Fc domain bound to the outer surface of the feeder cell, i.e., to express an Fc fusion peptide, as further described below. Another aspect of the present disclosure provides an NK cell expansion composition that does not contain feeder cells and comprises at least one Fc-binding engineered particle, i.e., an engineered particle comprising an Fc domain bound in the reverse orientation to the outer surface of a feeder cell. In some aspects, the feeder cell can be engineered to express a ligand (e.g., CD20) that can be tagged with a humanized antibody.

[0053] In the feeder cell composition, the at least one Fc-binding feeder cell optionally comprises at least one cellular NK cell effector agent. In one example, the Fc-binding feeder cell comprises one cellular NK cell effector that is IL-15 or IL-21. The Fc-binding feeder cell can comprise at least two or more different NK cell effector agents.

[0054] In the NK cell expansion composition that does not contain feeder cells, the Fc-binding engineered PM particles optionally comprise at least one cellular NK cell effector agent. In one example, the Fc-binding engineered particle comprises one cellular NK cell effector that is IL-15 or IL-21. The Fc-binding engineered PM particles can comprise at least two or more different NK cell effector agents.

[0055] In either the feeder cell composition or the feeder cell-free composition, where at least two NK cell effector agents are present, the second NK cell effector agent can be, for example, 41BBL. In either the feeder cell composition or the feeder cell-free NK cell expansion composition, where the feeder cells or engineered PM particles comprise one or more NK cell effector agents, the NK cell effector agents can be selected from 41BBL, IL-15, IL-2, IL-12, IL-18, IL-21, MICA, UBLP, 2sB4, LFA-1, a Notch ligand, a ligand for NKp46, or BCM1 / SLAMF2, a TLR ligand, and an NKG2D ligand, or a cytokine. In exemplary such compositions, the at least one additional NK cell effector agent is IL-15 or IL-21. (a) Fc-binding feeder cells

[0056] The present disclosure provides feeder cells comprising the Fc fusion peptides detailed above. NK cell feeder cells for use in the methods disclosed herein and for use in generating the PM particles and exosomes disclosed herein can be irradiated autologous or allogeneic peripheral blood mononuclear cells (PBMCs) or non-irradiated autologous or allogeneic PBMCs, RPMI8866, HFWT, 721.221, or K562 cells, as well as EBV-LCL, other non-HLA or low-HLA expressing cell lines, or patient-derived primary tumors that can be used as tumor vaccines. Fc-binding feeder cells can be prepared by transducing or transfecting feeder cells with any of the Fc fusion peptides described herein using standard transduction or transfection techniques well known in the art. For example, cDNA vectors for the Fc fusion peptides disclosed herein can be ligated into expression plasmids, thereby enabling expression in bacteria (E. coli), insects, or mammalian cells. The cDNA vectors can be tagged with FLAG or HIS. Suitable transfection methods include nucleofection (or electroporation), calcium phosphate-mediated transfection, cationic polymer transfection (e.g., DEAE-dextran or polyethyleneimine), viral transduction, virosome transfection, virion transfection, liposome transfection, cationic liposome transfection, immunoliposome transfection, non-liposomal lipid transfection, dendrimer transfection, heat shock transfection, magnetofection, lipofection, gene gun delivery, impalefection, sonoporation, optical transfection, and nucleic acid uptake enhanced with dedicated agents.Transformation methods are well known in the art (see, e.g., “Current Protocols in Molecular Biology” Ausubel et al., John Wiley & Sons, New York, 2003 or “Molecular Cloning: A Laboratory Manual” Sambrook & Russell, Cold Spring Harbor Press, Cold Spring Harbor, NY, 3rd edition, 2001). Alternatively, molecules can be introduced into cells by microinjection. For example, molecules can be injected into the cytoplasm or nucleus of the target cell. The amount of each molecule introduced into the cell can vary, but those skilled in the art are familiar with means for determining an appropriate amount.

[0057] It will be appreciated that various molecules can be introduced into cells simultaneously or sequentially. For example, an Fc fusion peptide and one or more membrane-bound NK cell effector agents can be introduced into feeder cells simultaneously. Alternatively, one can be introduced first and the other molecule can be introduced intracellularly later. For example, feeder cells that have been transfected or transduced using an Fc fusion peptide can be further transfected using a membrane-bound NK cell effector agent such as IL-15 and / or IL-21 and / or 41BBL and / or infected as an EBV-LCL and / or other NK cell effector agent(s). Alternatively, feeder cells can be transfected or transduced simultaneously using an Fc fusion peptide and a membrane-bound NK cell effector agent (e.g., IL-15 and / or IL-21 and / or 41BBL and / or EBV-LCL and / or other NK cell effector agent). Alternatively, feeder cells that have been transfected or transduced in the past and express a membrane-bound NK cell effector agent such as IL-15 and / or IL-21 and / or 41BBL and / or infected as an EBV-LCL and / or other NK cell effector agent(s) can be transfected or transduced using an Fc fusion peptide. It will also be appreciated that other means such as chemical conjugation methods known in the art can be used to achieve membrane-bound Fc.

[0058] Generally, cells are maintained under conditions suitable for cell growth and / or maintenance. Suitable cell culture conditions are well known in the art and are described, for example, in Santiago et al., Proc. Natl. Acad. Sci. USA, 2008, 105:5809-5814; Moehle et al., Proc. Natl. Acad. Sci. USA, 2007, 104:3055-3060; Urnov et al., Nature, 2005, 435:646-651; and Lombardo et al., Nat. Biotechnol., 2007, 25:1298-1306. Those skilled in the art understand that methods for culturing cells are known in the art and can vary depending on the cell type and will in fact vary. Routine optimization can be used in all cases to determine the best technique for a particular cell type.

[0059] Fc-binding feeder cells can be used in cell culture to directly stimulate NK cells or to prepare PM particles or exosomes derived from the feeder cells. (b) Fc-binding PM particles

[0060] Fc-binding engineered PM particles include Fc-binding PM particles that can be prepared from Fc-binding NK cell feeder cells using well-known methods. PM particles are vesicles made from the plasma membrane of cells or artificially made vesicles (i.e., liposomes). PM particles can contain a lipid bilayer or simply a single lipid layer. PM particles can be prepared in monolayer, multilayer, or inverted forms. PM particles can be prepared from the Fc-binding feeder cells described herein using known plasma membrane preparation protocol(s) for preparing liposomes as described in U.S. Patent No. 9,623,082, the entire disclosure of which is incorporated herein by reference. In certain embodiments, the PM particles disclosed herein have an average diameter in the range of about 170 to about 300 nm. (c) Fc-binding exosomes

[0061] The Fc-binding exosomes disclosed herein can be prepared from exosome-secreting cells, which can be prepared from Fc-binding NK cell feeder cells using well-known methods. Exosomes are extracellular products of exosome-secreting cells, as described in U.S. Patent Application Publication No. 20170333479, the entire disclosure of which is incorporated herein by reference. Exosomes contain lipids and proteins, and the protein content found in a particular exosome depends on the cell(s) that produced it. The exosomes disclosed herein contain an Fc fusion peptide (i.e., one that binds to Fc) disclosed herein and, optionally, one or more stimulatory peptides (NK cell effector agents) present within the exosome membrane. Exosomes can be produced, for example, from cell lines that have been engineered to improve exosome formation or release. Such cell lines include, but are not limited to, the Fc-binding cell lines described in Section II(a) above. Non-limiting cell lines are Fc-binding K562-mb15-41BBL and Fc-binding K562. In certain embodiments, the exosomes disclosed herein have an average diameter in the range of about 30 to about 100 nm or up to about 160 nm. In one embodiment, the exosomes have an average diameter of about 60 to 80 nm. The ability of exosomes to achieve a smaller particle size than can be easily achieved with PM particles means that exosomes can be more readily adapted for use where a smaller size is preferred. For example, exosomes may be preferred for diffusion through physiological barriers, enhanced in vivo distribution through tissue compartments, or applications that require intravenous injection.

[0062] (III) Composition The present disclosure provides various NK cell proliferation compositions comprising the Fc-binding feeder cells disclosed above, and in other aspects, provides feeder cell-free NK cell proliferation compositions comprising one or more engineered Fc-binding particles such as the PM particles or exosomes disclosed above. Either the Fc-binding feeder cells or the engineered Fc-binding PM particles used in the composition may optionally further comprise at least one, two, or more different NK cell effector agents. In one aspect, one NK cell effector agent is IL-21, and in some aspects, one NK cell effector agent is IL-21 and a second NK cell effector agent is 41BBL. The Fc-binding feeder cells or the engineered Fc-binding PM particles may optionally comprise one or more of the additional NK cell effector agents disclosed above.

[0063] An NK cell proliferation composition comprising PM particles comprising a plasma membrane may further comprise a plurality of microparticles / nanoparticles, wherein the plasma membrane coats the plurality of microparticles and / or nanoparticles. The microparticles / nanoparticles can include magnetic microparticles, silica beads, polystyrene beads, latex beads, particle contrast agents, particle cancer therapeutics, or any combination thereof.

[0064] The present disclosure also contemplates an NK cell proliferation injection formulation comprising a combination of any of the NK cell proliferation compositions disclosed herein and a pharmaceutically acceptable carrier.

[0065] A pharmaceutical composition for treatment can be prepared, for example, as described in Remington: The Science and Practice of Pharmacy (19th ed.) ed. A. R. Gennaro, Mack Publishing Company, Easton, Pa. 1995, by combining Fc-binding feeder cells or engineered PM particles with a pharmaceutically acceptable carrier known in the art. Examples of pharmaceutically acceptable carriers include, but are not limited to, sterile water, saline, Ringer's solution, dextrose solution, and buffered solutions at physiological pH. For example, the pH of the solution is preferably about 5 to about 8, more preferably about 7 to about 7.5.

[0066] In addition to the selected molecule, the pharmaceutical composition can include a carrier, thickening agent, diluent, buffer, preservative, surfactant, etc. The pharmaceutical composition can also include one or more active ingredients such as antibacterial agents, anti-inflammatory agents, anesthetics, etc.

[0067] It will be apparent to those skilled in the art that a particular carrier may be more preferred, depending, for example, on the route of administration and the concentration of the composition being administered. The pharmaceutical composition can be appropriately prepared for administration via any of a plurality of known routes of administration to mammals, particularly humans, depending on whether local or systemic treatment is desired and on the area to be treated. Administration can be local (including eye, vagina, rectum, intranasal), oral, inhalation, or parenteral (e.g., by intravenous drip or injection), or subcutaneous, intraperitoneal, intramuscular, intracavitary, or transdermal injection.

[0068] Parenteral preparations include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcohol / aqueous solutions, emulsions, or suspensions (including physiological saline and buffered media). Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include liquids and nutrient replenishers, electrolyte replenishers (e.g., those based on Ringer's dextrose), etc. Preservatives and other additives, such as antimicrobial agents, antioxidants, chelating agents, and inert gases, may be present.

[0069] Preparations for topical administration may include ointments, lotions, creams, gels, drops, suppositories, sprays, liquids, and powders. Conventional pharmaceutical carriers, aqueous, powder, or oily bases, thickening agents, etc. may be required or desirable.

[0070] Compositions for oral administration include powders or granules, suspensions or solutions in water or non-aqueous media, capsules, sachets, or tablets. Thickening agents, flavoring agents, diluents, emulsifying agents, dispersion aids, or binders may be desirable.

[0071] Some compositions can be administered as pharmaceutically acceptable acid or base addition salts potentially formed by the reaction of inorganic acids (e.g., hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, and phosphoric acid) and organic acids (e.g., formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid, maleic acid, and fumaric acid), or by the reaction of inorganic bases (e.g., sodium hydroxide, ammonium hydroxide, potassium hydroxide) and organic bases (e.g., mono-, di-, trialkyl and aryl amines and substituted ethanolamines).

[0072] In this way, the NK cell growth injection preparation can be formulated for parenteral use, arterial injection, intravenous injection, artificial catheter-mediated injection, intravenous use, intraperitoneal use, subcutaneous injection, oral or local delivery.

[0073] In one aspect, the present disclosure contemplates any NK cell growth composition prepared in vivo or ex vivo as disclosed herein, which is administered or injected into a subject in need of NK cell growth. It is understood and contemplated herein that the injection can occur in vitro using a commercial source of NK cells or ex vivo from a donor source (e.g., an allogeneic or autologous donor source (i.e., the recipient subject receiving the expanded NK cells)).

[0074] In another aspect, the present disclosure contemplates an NK cell composition comprising an in vitro NK cell population contacted with an Fc-binding feeder cell composition as disclosed herein, or an Fc-binding NK cell growth composition without feeder cells as disclosed herein.

[0075] In another aspect, the present disclosure contemplates a source(s) of NK cells, including but not limited to peripheral blood, iPSC-derived NK cells, ESC-derived NK cells, NK cells having a polymorphism of the high-affinity Fc receptor Phe or Val at 158, and genetically modified NK cells.

[0076] In another aspect, the present disclosure contemplates a population of expanded NK cells that have been exposed in vitro to an NK cell expansion composition, the composition comprising at least one Fc-binding engineered particle disclosed herein, without feeder cells, and comprising at least two NK cell effector agents, wherein one of the at least two NK cell effector agents is IL-21 or IL-15. The expanded NK cell population may exhibit increased cytotoxicity as compared to unexpanded NK cells. In different aspects, the expanded NK cell population may exhibit cytotoxicity that is at least about 2-fold, 5-fold, or 10-fold that of unexpanded NK cells.

[0077] In another aspect, the present disclosure provides a composition comprising a therapeutic dose of NK cells comprising the expanded NK cell population disclosed herein, optionally in combination with a pharmaceutically acceptable carrier. The expanded NK cell population may exhibit higher CD16 as well as other advantageous properties (e.g., higher cytotoxicity and ADCC functionality). The amount of NK cells providing the therapeutic dose will vary depending on multiple factors as understood by those skilled in the art and is discussed, for example, in U.S. Patent No. 9,623,082. Factors include the age, sex, and diagnosis of the subject, as well as the route of administration, which may be, but is not limited to, oral, buccal, mucosal, and intravenous routes. For example, the therapeutic dose can be 1×10 4 / kg to 1×10 8 / kg per dose, which may be included in a single dose or divided into multiple doses. It will be understood that equivalents of the therapeutic doses as represented above may alternatively be expressed in terms of an amount per body surface area.

[0078] In another aspect, the present disclosure also provides an NK cell expansion medium formulation comprising any of the NK cell expansion compositions disclosed herein in combination with a cell culture medium solution comprising at least one soluble medium component (e.g., cytokine, IL-2, IL-12, IL-15, IL-18, IL-21, NAM, ascorbate, or any combination thereof).

[0079] (IV) Methods (a) Method for increasing the cytotoxicity of NK cells In one aspect, the present disclosure provides a method for increasing the cytotoxicity of NK cells by expanding an initial NK cell population using the NK cell expansion compositions or formulations disclosed herein. The methods of the present disclosure provide a simple expansion platform that avoids complex alternative processes for expansion, such as coating a solid support with monoclonal antibodies or using soluble cytokine(s) in solution. Instead, in the methods disclosed herein, the initial NK cell population is obtained from a donor and exposed to the NK cell expansion compositions disclosed herein. The exposure can be in vitro or in vivo. Figure 4 is a schematic diagram of Fc stimulation of NK cells according to the present disclosure. The NK cells are contacted with one or more Fc-binding feeder cells, Fc-binding PM particles, or Fc-binding exosomes, or any combination thereof. The exposed Fc domain binds to CD16 on the surface of the NK cells, resulting in the stimulation of the NK cells to proliferate faster and / or more efficiently, producing NK cells with higher anti-tumor cytotoxicity and NK cells with a more favorable overall phenotype.

[0080] As shown in FIG. 4, the composition to be contacted with NK cells can include any Fc-binding feeder cell or Fc-engineered PM particle or Fc-engineered exosome disclosed herein. The engineered PM particles can be Fc-binding PM particles. In one embodiment, the optionally present NK cell effector agent is IL-21 or IL-15. The optionally present second NK cell effector agent can be selected from 41BBL, IL-2, IL-12, IL-15, IL-18, IL-21, MICA, UBLP, 2B4, LFA-1, Notch ligand, NKp46, or a ligand for BCM1 / SLAMF2, TLR ligand, and NKG2D ligand. In one embodiment, the second NK cell effector agent is 41BBL. The composition can further include at least one additional (i.e., third, fourth, fifth, etc.) NK cell effector agent selected from IL-2, IL-12, IL-15, IL-18, IL-21, MICA, UBLP, 2sB4, LFA-1, Notch ligand, NKp46, or a ligand for BCM1 / SLAMF2, TLR ligand, and NKG2D ligand. NK cell expansion carried out in this way can achieve expansion far exceeding several-fold (about 3-4 fold) in 10 days. Rather, NK cell expansion according to the methods of the present invention is in 16 days, at least about 100-fold, about 200-fold, about 300-fold, about 400-fold, about 500-fold, about 600-fold, about 700-fold, about 800-fold, about 900-fold, about 1100-fold, about 1200-fold, about 1300-fold, about 1400-fold, about 1500-fold, about 1600-fold, about 1700-fold, about 1800-fold, about 1900-fold, about 2000-fold increase in the number of NK cells, and can achieve more than that over longer times. Thus, the methods of the present disclosure are useful for the scale-up production of NK cells.Sources of NK cells can be peripheral blood, splenic NK cells, lymphocyte preparations (e.g., buffy coats), iPSC-derived NK cells, ESC-derived NK cells, and genetically modified / engineered NK cells, or any genetically modified NK cells (including, but not limited to, NK cells derived from polymorphisms of Fc receptors (e.g., Phe or Val at position 158) (e.g., as known in the art, e.g., as described in Blood (1997) 90:1109-14, and J Clin Invest. (1997) 100:1059-70)). Such sources of genetically modified NK cells can be engineered using methods known in the art. Alternatively, NK cells may be derived from a cell donor having the desired polymorphism, and the provided cells are used as an initial NK cell population and expanded by using the methods and compositions described herein. Thus, "genetically modified" in this context encompasses natural NK cells having polymorphisms. This method can be applied to NK cells of human origin or from other animals.

[0081] Furthermore, the methods of the present disclosure have the additional advantage of providing cells with higher cytotoxicity and ADCC functionality. An initial NK cell population grown according to the methods of the present disclosure exhibits a proliferated NK cell population having at least about 2 times the cytotoxicity of the initial NK cell population, at least about 4 times the cytotoxicity of the initial NK cell population, at least about 5 times the cytotoxicity of the initial NK cell population, at least about 8 times the cytotoxicity of the initial NK cell population, or at least about 10 times the cytotoxicity of the initial NK cell population. Additionally, NK cells grown according to the methods of the present disclosure exhibit higher cytotoxicity against ADCC-capable targets. Higher expression of ADCC-related proteins (e.g., in non-limiting examples, CD16, or other NK cell ligands (e.g., in non-limiting examples, NKG2D, NKp46, CD62L)) can be used to evaluate the relative cytotoxicity of the proliferated NK cells compared to non-proliferated NK cells or NK cells grown under other conditions. Markers such as NKG2D and NKp46 are indicators of NK cells in an activated state. Together, these markers can provide a signal of increased cytotoxicity even when cytotoxicity cannot be directly evaluated. For example, the proliferated NK cell populations disclosed herein can exhibit increased killing of tumor targets or secrete higher amounts of anti-tumor cytokines (IFN, TNF) compared to non-proliferated NK cells. In another aspect, the proliferated NK cell populations disclosed herein can exhibit increased expression of NKG2D, NKp46, and CD16 compared to non-proliferated NK cells. Various means for detecting the amount of specific proteins to evaluate the activation state of NK cells are known in the art and can be used, including spectroscopic methods such as flow cytometry, or immunodetection methods such as Western blot, enzyme-linked immunosorbent assay (ELISA), protein immunoprecipitation, immunoelectrophoresis, or immunostaining.

[0082] In addition, the proliferated NK cell populations disclosed herein have improved ability to withstand cryopreservation and can retain viability and cytotoxicity after freezing and thawing.

[0083] As shown in FIGS. 2 and 3, the composition of NK cells grown in Fc-expressing feeder cells exhibits higher cytotoxicity against SKOV3 ovarian cancer target cells. The composition of NK cells grown in Fc domain-expressing feeder cells has an enhanced phenotype with increased CD16, NKp46, and CD62L. These NK cells with an enhanced phenotype may have an enhanced therapeutic effect. The increase in CD16 may increase the ability to bind to antibody-coated target cells. The increase in NKp46 may increase the ability to bind to activating ligands. The increase in CD62L as an L-selectin ligand may enhance the transport of NK cells to the lymph or bone marrow compartment.

[0084] (b) Treatment method The compositions and methods disclosed herein can be used in various therapeutic, diagnostic, industrial, and research applications. In some embodiments, the present disclosure can be used for the treatment of cancer. Thus, in one embodiment, described herein is a method of treating, inhibiting, reducing, and / or preventing cancer, cancer recurrence, or metastasis, or an infectious disease such as a viral or bacterial infection in a subject, the method comprising administering to a subject in need thereof an effective amount of the composition or the expanded NK cell population described herein.

[0085] Cancer can be selected from, but is not limited to, blood cancer, lymphoma, colorectal cancer, colon cancer, lung cancer, head and neck cancer, ovarian cancer, prostate cancer, testicular cancer, kidney cancer, skin cancer, cervical cancer, pancreatic cancer, and breast cancer. In one embodiment, cancer includes solid tumors. In another embodiment, cancer is selected from acute myeloid leukemia, myelodysplastic syndrome, chronic myeloid leukemia, acute lymphoblastic leukemia, myelofibrosis, multiple myeloma. In another embodiment, cancer is selected from leukemia, lymphoma, sarcoma, carcinoma, and can be derived from bone marrow, brain, lung, breast, pancreas, liver, head and neck, skin, reproductive organs, prostate, colon, liver, kidney, intra-abdominal, bone, joint, eye.

[0086] In another aspect, the treatment methods include methods for preventing, inhibiting, reducing or alleviating cancer recurrence or metastasis after stem cell transplantation; methods for regulating the T cell repertoire after stem cell transplantation; general methods for regulating the immune repertoire; methods for preventing, inhibiting, reducing or alleviating acute or chronic graft-versus-host disease; and methods for preventing, inhibiting, reducing or alleviating viral reactivation (e.g., reactivation of herpes simplex virus type 1 (HSV-1), herpes simplex virus type 2 (HSV-2), cytomegalovirus (CMV), varicella-zoster virus (VZV), Epstein-Barr virus (EBV), adenovirus, adeno-associated virus, parvovirus, JC virus, and / or BK virus), each method comprising administering to a subject in need thereof an effective amount of the composition described herein or the expanded NK cell population.

[0087] Any treatment method of the present disclosure may further comprise administering to the subject an additional therapeutic agent or regimen (concurrently, simultaneously, or as a single formulation) in combination with an effective amount of the composition or expanded NK cell population described herein. The additional therapeutic agent may be a drug-based conditioning regimen such as Cy-Flu, Bu-Flu, Flu-Mel, or the like, or similar with dosage or administration adjustments. Alternatively, the additional therapeutic agent may be, but is not limited to, a graft-versus-host (GvHD) prophylaxis agent such as cyclophosphamide. Alternatively, the additional therapeutic agent or regimen may be selected from chemotherapy agents and regimens such as, by way of non-limiting example, those known by the acronyms CHOP, FLAG (including FLAG-Ida or FLAG-IDA or IDA-FLAG or Ida-FLAG, and FLAG-Mito or FLAG-MITO or Mito-FLAG or MITO-FLAG or FLANG), IA or IAC, or 7+3. For example, herein, the methods of the present disclosure for inhibiting, reducing, and / or preventing cancer metastasis and / or recurrence are intended to include the administration of any anti-cancer agent known in the art including, but not limited to: abemaciclib, abiraterone acetate, Abitrexate (methotrexate), Abraxane (paclitaxel albumin-stabilized nanoparticle formulation), ABVD, ABVE, ABVE-PC, AC, AC-T, Adcetris (brentuximab vedotin), ADE, Ado-trastuzumab emtansine, Adriamycin (doxorubicin hydrochloride), afatinib malate, Afinitor (everolimus), Akynzeo (netupitant·palonosetron hydrochloride), Aldara (imiquimod), aldesleukin, Alecensa (alectinib), alectinib, alemtuzumab, Alimta (pemetrexed disodium), Aliqopa (copanlisib hydrochloride), Alkeran injection (melphalan hydrochloride), Alkeran tablets (melphalan), Aloxi (palonosetron hydrochloride), Alunbrig (brigatinib), Ambochlorin (chlorambucil), Amboclorin (chlorambucil), amifostine, aminolevulinic acid, anastrozole, aprepitant,Aredia (pamidronate disodium), Arimidex (anastrozole), Aromasin (exemestane), arsenic trioxide, Arzerra (ofatumumab), asparaginase Erwinia chrysanthemi, atezolizumab, Avastin (bevacizumab), avelumab, axitinib, azacitidine, Bavencio (avelumab), BEACOPP, Becenum (carmustine), Beleodaq (belinostat), belinostat, bendamustine hydrochloride, BEP, Besponsa (inotuzumab ozogamicin), bevacizumab, bexarotene, Bexxar (tositumomab and iodine I131 tositumomab), bicalutamide, BiCNU (carmustine), bleomycin, blinatumomab, Blincyto (blinatumomab), bortezomib, Bosulif (bosutinib), bosutinib, brentuximab vedotin, brigatinib, BuMel, busulfan, Busulfex (busulfan), cabazitaxel, Cabometyx (cabozantinib-S-malate), cabozantinib-S-malate, CAF, Campath (alemtuzumab), Camptosar (irinotecan hydrochloride), capecitabine, CAPOX, Carac (fluorouracil topical), carboplatin, carboplatin-paclitaxel, carfilzomib, Carmubris (carmustine), carmustine, carmustine implant, Casodex (bicalutamide), CEM, ceritinib, Cerubidine (daunorubicin hydrochloride), Cervarix (recombinant HPV bivalent vaccine), cetuximab, CEV, chlorambucil, chlorambucil-prednisone, CHOP, cisplatin, cladribine, Clafen (cyclophosphamide), clofarabine, Clofarex (clofarabine), Chloral (clofarabine), CMF, cobimetinib, Cometriq (cabozantinib-S-malate), copanlisib hydrochloride, COPDAC, COPP, COPP-ABV, Cosmegen (dactinomycin), Cotellic (cobimetinib), crizotinib, CVP, cyclophosphamide, Cyfos (ifosfamide), Cyramza (ramucirumab), cytarabine, cytarabine liposome, Cytosar-U (cytarabine), Cytoxan (cyclophosphamide),Dabrafenib, Dacarbazine, Dacogen (Decitabine), Dactinomycin, Daratumumab, Darzalex (Daratumumab), Dasatinib, Daunorubicin Hydrochloride, Daunorubicin Hydrochloride and Cytarabine Liposome, Decitabine, Defibrotide Sodium, Defitelio (Defibrotide Sodium), Degarelix, Denileukin Diftitox, Denosumab, DepoCyt (Cytarabine Liposome), Dexamethasone, Dexrazoxane Hydrochloride, Dinutuximab, Docetaxel, Doxil (Doxorubicin Hydrochloride Liposome), Doxorubicin Hydrochloride, Doxorubicin Hydrochloride Liposome, Dox-SL (Doxorubicin Hydrochloride Liposome), DTIC-Dome (Dacarbazine), Durvalumab, Efudex (Fluorouracil Topical), Elitek (Rasburicase), Ellence (Epirubicin Hydrochloride), Elotuzumab, Eloxatin (Oxaliplatin), Eltrombopag Olamine, Emend (Aprepitant), Empliciti (Elotuzumab), Enasidenib Mesylate, Enzalutamide, Epirubicin, EPOCH, Erbitux (Cetuximab), Eribulin Mesylate, Eribedge (Bismuth Subsalicylate), Erlotinib Hydrochloride, Arwinase (Asparaginase Erwinia chrysanthemi), Ethyol (Amifostine), Etopophos (Etoposide Phosphate), Etoposide, Etoposide Phosphate, Evacet (Doxorubicin Hydrochloride Liposome), Everolimus, Evista (Raloxifene Hydrochloride), Evomela (Melphalan Hydrochloride), Exemestane, 5-FU (Fluorouracil Injection), 5-FU (Fluorouracil Topical), Fareston (Toremifene), Faridulak (Panobinostat), Femara (Letrozole), Filgrastim, Fludara (Fludarabine Phosphate), Fludarabine Phosphate, Fluoroplex (Fluorouracil Topical), Fluorouracil Injection, Fluorouracil Topical, Flutamide, Folex (Methotrexate), Folex PFS (Methotrexate), FOLFIRI, FOLFIRI - Bevacizumab, FOLFIRI - Cetuximab, FOLFIRINOX, FOLFOX,Folotyn (Pralatrexate), FU-LV, Fluvestrant, Gardasil (Recombinant Quadrivalent HPV Vaccine), Gardasil 9 (Recombinant Nonavalent HPV Vaccine), Gazyna (Obinutuzumab), Gefitinib, Gemcitabine Hydrochloride, Gemcitabine-Cisplatin, Gemcitabine-Oxaliplatin, Gemtuzumab Ozogamicin, Gemzar (Gemcitabine Hydrochloride), Gilotrif (Afatinib Malate), Glivec (Imatinib Mesylate), Gliadel (Carmustine Implant), Gliadel Wafer (Carmustine Implant), Glucarpidase, Goserelin Acetate, Halaven (Eribulin Mesylate), Hemangeol (Propranolol Hydrochloride), Herceptin (Trastuzumab), Recombinant Bivalent HPV Vaccine, Recombinant Nonavalent HPV Vaccine, Recombinant Quadrivalent HPV Vaccine, Hycamtin (Topotecan Hydrochloride), Hydrea (Hydroxyurea), Hydroxyurea, Hyper-CVAD, Ibrance (Palbociclib), Ibritumomab Tiuxetan, Ibrutinib, ICE, Iclusig (Ponatinib Hydrochloride), Idamycin (Idarubicin Hydrochloride), Idarubicin Hydrochloride, Idelalisib, Idhifa (Enasidenib Mesylate), Ifex (Ifosfamide), Ifosfamide, Ifosfamidum (Ifosfamide), IL-2 (Aldesleukin), Imatinib Mesylate, Imbruvica (Ibrutinib), Imfinzi (Durvalumab), Imiquimod, Imlygic (Talimogene Laherparepvec), Inlyta (Axitinib), Inotuzumab Ozogamicin, Recombinant Interferon Alfa-2b, Interleukin-2 (Aldesleukin), Intron A (Recombinant Interferon Alfa-2b), Iodine I131 Tositumomab and Tositumomab, Ipilimumab, Iressa (Gefitinib), Irinotecan Hydrochloride, Irinotecan Hydrochloride Liposome, Istodax (Romidepsin), Ixabepilone, Ixazomib Citrate, Ixempra (Ixabepilone), Jakafi (Ruxolitinib Phosphate), JEB, Jevtana (Cabazitaxel), Kadcyla (Ado-Trastuzumab Emtansine), Keoxifene (Raloxifene Hydrochloride), Kepivance (Palfermin),Keytruda (pembrolizumab), Kisqali (ribociclib), Kimria (tisagenlecleucel), Kyprolis (carfilzomib), lanreotide acetate, lapatinib tosylate, Lartruvo (olaratumab), lenalidomide, lenvatinib mesylate, Lenvima (lenvatinib mesylate), letrozole, leucovorin calcium, Leukeran (chlorambucil), leuprolide acetate, Leustatin (cladribine), Levulan (aminolevulinic acid), Linfolizin (chlorambucil), LipoDox (doxorubicin hydrochloride liposome), Lomustine, Lonsurf (trifluridine tipiracil hydrochloride), Lupron (leuprolide acetate), Lupron Depot (leuprolide acetate), Lupron Depot-Ped (leuprolide acetate), Lynparza (olaparib), Marqibo (vincristine sulfate liposome), Matulane (procarbazine hydrochloride), mechlorethamine hydrochloride, megestrol acetate, Mekinist (trametinib), melphalan, melphalan hydrochloride, mercaptopurine, mesna, Mesnex (mesna), Methazolastone (temozolomide), methotrexate, methotrexate LPF (methotrexate), methylnaltrexone bromide, Mexate (methotrexate), Mexate-AQ (methotrexate), midostaurin, mitomycin C, mitoxantrone hydrochloride, Mitozytrex (mitomycin C), MOPP, Mozobil (plerixafor), Mustargen (mechlorethamine hydrochloride), Mutamycin (mitomycin C), Myleran (busulfan), Mylosar (azacitidine), Mylotarg (gemtuzumab ozogamicin), nanoparticle paclitaxel (paclitaxel albumin-stabilized nanoparticle formulation), Navelbine (vinorelbine tartrate), necitumumab, nelarabine, Neosar (cyclophosphamide), neratinib maleate, Nerlynx (neratinib maleate), netupitant and palonosetron hydrochloride, Neulasta (pegfilgrastim), Neupogen (filgrastim), Nexavar (sorafenib tosylate), Nilandron (nilutamide), nilotinib,Nilutamide, Ninlaro (Ixazomib citrate), Niraparib tosylate hydrate, Nivolumab, Norvadex (Tamoxifen citrate), Nplate (Romiplostim), Obinutuzumab, Odomzo (Sonidegib), OEPA, Ofatumumab, OFF, Olaparib, Olalatumab, Omacetaxine, Sin-Mepezasinate, Oncaspar (Pegaspargase), Ondansetron Hydrochloride, Onivyde (Irinotecan Hydrochloride Liposome), Ontak (Denileukin Diftitox), Opdivo (Nivolumab), OPPA, Osimertinib, Oxaliplatin, Paclitaxel, Paclitaxel Albumin-Stabilized Nanoparticle Formulation, PAD, Palbociclib, Parifermin, Paroxetine Hydrochloride, Paroxetine Hydrochloride and Netupitant, Pamidronate Disodium, Panitumumab, Panobinostat, Paraplat (Carboplatin), Paraplatin (Carboplatin), Pazopanib Hydrochloride, PCV, PEB, Pegaspargase, Pegfilgrastim, Peginterferon Alfa-2b, PEG-Intron (Peginterferon Alfa-2b), Pembrolizumab, Pemetrexed Disodium, Perjeta (Pertuzumab), Pertuzumab, Platinol (Cisplatin), Platinol-AQ (Cisplatin), Preloxafol, Pomalidomide, Pomalist (Pomalidomide), Ponatinib Hydrochloride, Portrazza (Necitumumab), Pralatrexate, Prednisone, Procarbazine Hydrochloride, Proleukin (Aldesleukin), Pralia (Denosumab), Promacta (Eltrombopag Olamine), Propranolol Hydrochloride, Provenge (Sipuleucel-T), Purinethol (Mercaptopurine), Purixan (Mercaptopurine), Radium Chloride 223, Raloxifene Hydrochloride, Ramucirumab, Rasburicase, R-CHOP, R-CVP, Recombinant Human Papillomavirus (HPV) Bivalent Vaccine, Recombinant Human Papillomavirus (HPV) Nonavalent Vaccine, Recombinant Human Papillomavirus (HPV) Quadrivalent Vaccine, Recombinant Interferon Alfa-2b, Regorafenib, Relistor (Methylnaltrexone Bromide), R-EPOCH, Revlimid (Lenalidomide), Rheumatrex (Methotrexate), Ribociclib, R-ICE, Rituxan (Rituximab), RituxanHycela (rituximab and human hyaluronidase), rituximab, rituximab and human hyaluronidase, romidepsin, romiplostim, Rubidomycin (daunorubicin hydrochloride), Rubraca (rucaparib camsylate), rucaparib camsylate, luxolitinib phosphate, Rydapt (midostaurin), Sclerosol intrapleural aerosol (talc), siltuximab, sipuleucel-T, Somatuline depot (lanreotide acetate), sonidegib, sorafenib tosylate, Sprycel (dasatinib), STANFORD V, sterile talc powder (talc), Steritalc (talc), stibarga (regorafenib), sunitinib malate, Sutent (sunitinib malate), Sylatron (peginterferon alfa-2b), Sylvant (siltuximab), Synribo (omacetaxine mepesuccinate), Tabloid (thioguanine), TAC, tafinlar (dabrafenib), tagrisso (osimertinib), talc, talimogene laherparepvec, tamoxifen citrate, TarabinePFS (Cytarabine), Tarceva (Erlotinib Hydrochloride), Targretin (Bexarotene), Tasigna (Nilotinib), Taxol (Paclitaxel), Taxotere (Docetaxel), Tecentriq (Atezolizumab), Temodal (Temozolomide), Temozolomide, Temsirolimus, Thalidomide, Thalomid (Thalidomide), Thioguanine, Thiotepa, Chisagenleclucel, Tolak (Topical Fluorouracil), Topotecan Hydrochloride, Toremifene, Torisel (Temsirolimus), Tositumomab and Iodine I131 Tositumomab, Totect (Dexrazoxane Hydrochloride), TPF, Trabectedin, Trametinib, Trastuzumab, Treanda (Bendamustine Hydrochloride), Trifluridine / Tipiracil Hydrochloride, Trisenox (Arsenic Trioxide), Tykerb (Lapatinib Tosylate), Unituxin (Dinutuximab), Uridine Triacetate, VAC, Vandetanib, VAMP, Varubi (Rolapitant Hydrochloride), Vectibix (Panitumumab), VeIP, Velban (Vinblastine Sulfate), Velcade (Bortezomib), Velsar (Vinblastine Sulfate), Bemlafenib, Venetoclax, Venetoclax, Verzenio (Abemaciclib), Viadur (Leuprolide Acetate), Vidaza (Azacitidine), Vinblastine Sulfate, VincasarPFS (vincristine sulfate), vincristine sulfate, vincristine sulfate liposome, vinorelbine tartrate, VIP, besimodegib, Vistogard (uridine triacetate), Voraxaze (glucarpidase), vorinostat, Votrient (pazopanib hydrochloride), Vyxeos (daunorubicin hydrochloride and cytarabine liposome), Wellcovorin (leucovorin calcium), Zelboraf (vemurafenib), Zevalin (ibritumomab tiuxetan), Zinecard (dexrazoxane hydrochloride), Ziv-aflibercept, Zofran (ondansetron hydrochloride), Zoladex (goserelin acetate), zoledronic acid, Zolinza (vorinostat), Zometa (zoledronic acid), Zydelig (idelalisib), Kadcyla (ado-trastuzumab emtansine), and / or Zytiga (abiraterone acetate). Also contemplated herein are chemotherapeutic agents that are PD1 / PDL1 blocking inhibitors (e.g., ramucirumab, nivolumab, pembrolizumab, pidilizumab, BMS-936559, atezolizumab, durvalumab, or avelumab).

[0088] Alternatively, the additional therapeutic agent can be an antiviral agent selected from, but not limited to, 5-substituted 2-deoxyuridine analogs, nucleoside analogs, (non-nucleoside) pyrophosphate analogs, nucleoside reverse transcriptase (RT) inhibitors (NRTIs), non-nucleoside reverse transcriptase inhibitors (NNRTIs), protease inhibitors (PIs), and integrase inhibitors, entry inhibitors, and acyclic guanosine analogs, acyclic nucleoside phosphonate (ANP) analogs, hepatitis C virus (HCV) NS5A and NS5B inhibitors, as well as influenza virus inhibitors, immunostimulants, interferons, oligonucleotides, and antimitotic inhibitors. Non-limiting examples of antiviral agents are acyclovir, famciclovir, valacyclovir, penciclovir, ganciclovir, ritonavir, lopinavir, saquinavir, etc.; cimetidine; ranitidine; captopril; metformin; bupropion; fexofenadine; oxcarbazepine; levetiracetam; tramadol; or any isomers, tautomers, analogs, polymorphs, solvates, derivatives, or pharmaceutically acceptable salts thereof.

[0089] Alternatively, the additional therapeutic agent can be an antibiotic selected from, but not limited to, penicillin, tetracycline, cephalosporin, lincomycin, macrolide, sulfonamide, glycopeptide, aminoglycoside, and carbapenem. Non-limiting examples of antiviral agents are amoxicillin, doxycycline, cephalexin, ciprofloxacin, clindamycin, metronidazole, azithromycin, sulfamethoxazole and trimethoprim, clavulanic acid, and levofloxacin.

[0090] (V) Kit A further aspect of the disclosure is a kit comprising at least one of the fusion peptides detailed above, and and / or at least one of the Fc-binding feeder cells, and / or at least one of the Fc-binding engineered particles (PM particles and / exosomes) detailed above Provided. The fusion peptide can be provided in a suitable container together with other kit components (e.g., cell reagents, cell growth media, selection media, protein purification reagents, buffers, etc.). The kits provided in this specification generally include instructions for performing the methods detailed below. The instructions contained in the kit may be affixed to the packaging material or included as an accompanying document. The instructions are typically written materials, i.e., printed materials, but are not limited to this. In the present disclosure, any medium capable of storing such instructions and transmitting them to the end user is contemplated. Such media include, but are not limited to, electronic storage media (e.g., magnetic disks, tapes, cartridges, chips), optical media (e.g., CD ROM), etc. As used herein, the term "instructions" can include the address of an Internet site that provides the instructions. This application also provides, for example, the following inventions. [1] A fusion protein comprising a transmembrane domain bound to the amino terminus of an Fc domain. [2] The fusion protein according to [1] above, wherein the transmembrane domain comprises a signal anchor sequence selected from the transmembrane domain of neuraminidase, the signal anchor from parainfluenza virus hemagglutinin-neuraminidase, the signal anchor from the transferrin receptor, the signal anchor from the MHC class II invariant chain, the signal anchor from P-glycoprotein, the signal anchor from the asialoglycoprotein receptor, and the signal anchor from neutral endopeptidase. [3] The fusion protein according to [1] above, wherein the transmembrane domain comprises a parainfluenza virus hemagglutinin-neuraminidase (NA) peptide sequence. [4] The fusion protein according to [3] above, wherein the parainfluenza virus hemagglutinin-neuraminidase (NA) peptide sequence comprises a sequence having at least about 81% sequence identity to SEQ ID NO: 1 or SEQ ID NO: 17. [5] The fusion protein according to [4] above, wherein the parainfluenza virus hemagglutinin-neuraminidase (NA) peptide sequence comprises a sequence having at least about 95% sequence identity to SEQ ID NO: 1 or SEQ ID NO: 17. [6] The fusion protein according to [1] above, wherein the Fc domain comprises an immunoglobulin Fc domain selected from IgG1, IgG2, IgG3, IgG4, IgA, and IgE. [7] The fusion protein according to [1] above, further comprising a peptide linker between the transmembrane domain and the Fc domain. [8] A nucleic acid encoding the fusion protein according to any one of [1] to [7] above. [9] A vector comprising the nucleic acid according to [8] above.

[10] A cell comprising the vector according to [9] above.

[11] An engineered plasma membrane (PM) particle or exosome comprising the fusion protein according to any one of [1] to [7] above.

[12] An NK cell proliferation composition comprising a feeder cell, an engineered PM particle, or a membrane-bound inverted Fc domain bound to the outer surface of an exosome.

[13] The NK cell proliferation composition according to

[12] above, comprising engineered PM particles or engineered exosomes that substantially do not contain feeder cells.

[14] The NK cell proliferation composition according to

[13] above, wherein the engineered particles further comprise at least one NK cell effector agent.

[15] The NK cell proliferation composition according to

[12] above, further comprising at least one NK cell effector agent.

[16] The NK cell proliferation composition according to

[14] or

[15] above, wherein the at least one NK cell effector agent is IL-21 or IL-15.

[17] The NK cell proliferation composition according to

[16] above, further comprising a second NK cell effector agent, wherein the second NK cell effector agent is 41BBL.

[18] The NK cell proliferation composition according to

[12] above, wherein the engineered PM particles comprise plasma membrane vesicles purified from NK cell feeder cells transfected or transduced with a fusion protein containing a transmembrane domain bound to an Fc domain, and the at least one NK cell effector agent.

[19] The NK cell proliferation composition according to

[12] above, wherein the engineered PM particles comprise exosomes derived from NK cell feeder cells transfected with a fusion protein containing a transmembrane domain bound to an Fc domain, and the at least one NK cell effector agent.

[20] The NK cell proliferation composition according to

[17] above, further comprising at least one additional NK cell effector agent, wherein the at least one additional NK cell effector agent is a cytokine, an adhesion molecule, or an NK cell activator, and the at least one additional NK cell effector agent is selected from IL-15, IL-2, IL-12, IL-18, IL-21, MICA, UBLP, 2sB4, LFA-1, Notch ligand, NKp46, or a ligand for BCM1 / SLAMF2, a TLR ligand, and an NKG2D ligand.

[21] The NK cell proliferation composition according to

[12] above, wherein the engineered particles are plasma membrane particles comprising a plasma membrane, the composition further comprises a solid surface, and the plasma membrane coats at least a portion of the solid surface.

[22] The NK cell proliferation composition according to

[21] above, wherein the solid surface contains at least one of magnetic microparticles, silica beads, polystyrene beads, latex beads, microstructures, contrast agents, and / or cancer therapeutic agents.

[23] An NK cell proliferation injection preparation comprising the NK cell proliferation composition according to any one of

[12] to

[22] above and a pharmaceutically acceptable carrier.

[24] The NK cell proliferation injection preparation according to

[23] above, wherein the preparation is selected from parenteral, arterial injection, intravenous injection, artificial catheter-mediated injection, intravenous, intraperitoneal, subcutaneous injection, oral, and topical preparations.

[25] The NK cell proliferation injection preparation according to

[24] above, which is injected into a subject in need of in vivo NK cell proliferation.

[26] An NK cell composition comprising an in vitro NK cell population in contact with the NK cell proliferation composition according to

[12] above.

[27] The NK cell composition according to

[26] above, further comprising at least one NK cell effector agent.

[28] The NK cell composition according to

[27] above, wherein the at least one NK cell effector agent is IL-21 or IL-15.

[29] The NK cell composition according to

[27] above, wherein the at least one NK cell effector agent is soluble.

[30] A proliferated NK cell population exposed in vitro to the NK cell proliferation composition according to

[26] above, wherein the composition contains feeder cells or contains at least one engineered particle and does not contain feeder cells, and the feeder cells or engineered particles contain an Fc domain bound to their outer surface.

[31] The proliferated NK cell population according to

[32] above, wherein the NK cells have increased cytotoxicity compared to non-proliferated NK cells.

[32] The proliferated NK cell population according to

[30] above, wherein the cytotoxicity of the proliferated NK cells is at least about 2 times the cytotoxicity of non-proliferated NK cells.

[33] The proliferated NK cell population according to

[30] above, wherein the cytotoxicity of the proliferated NK cells is at least about 5 times the cytotoxicity of non-proliferated NK cells.

[34] The proliferated NK cell population according to

[30] above, wherein the cytotoxicity of the proliferated NK cells is at least about 10 times the cytotoxicity of non-proliferated NK cells.

[35] A composition comprising a therapeutic dose of NK cells comprising the expanded NK cell population according to any one of

[30] to

[34] above and a pharmaceutically acceptable carrier.

[36] The expanded NK cell population according to

[30] above or the composition according to

[35] above, further comprising at least one NK cell effector agent.

[37] The composition according to

[36] above, wherein the at least one NK cell effector agent is selected from IL-15, IL-2, IL-12, IL-18, IL-21, MICA, UBLP, 2sB4, LFA-1, Notch ligand, ligand for NKp46, or BCM1 / SLAMF2, TLR ligand, and NKG2D ligand.

[38] The composition according to

[37] above, wherein the at least one NK cell effector agent is soluble.

[39] The composition according to

[37] above, further comprising a second NK cell effector agent, wherein the second NK cell effector agent is 41BBL.

[40] A method for treating, remitting, reducing, and / or inhibiting cancer or metastasis or an infectious disease in a subject, comprising administering to the subject in need thereof an effective amount of the composition or expanded NK cell population according to any one of

[12] to

[39] above, optionally in contact with an NK cell population.

[41] The method according to

[40] above, wherein the cancer is selected from the group consisting of tumor, blood cancer, lymphoma, leukemia, acute myeloid leukemia, myelodysplastic syndrome, chronic myeloid leukemia, acute lymphoblastic leukemia, myelofibrosis, multiple myeloma, colorectal cancer, colon cancer, lung cancer, head and neck cancer, ovarian cancer, pancreatic cancer, liver cancer, skin cancer, prostate cancer, kidney cancer, intraperitoneal cancer, and breast cancer.

[42] A method for inhibiting, reducing, and / or preventing recurrence of cancer or metastasis before or after stem cell transplantation, comprising administering to the subject in need thereof an effective amount of the composition or expanded NK cell population according to any one of

[12] to

[39] above.

[43] The method according to any one of

[40] to

[42] above, further comprising administering to the subject at least one cancer therapeutic agent in combination with an effective amount of the composition or expanded NK cell population.

[44] The method according to

[43] above, wherein the at least one cancer therapeutic agent is selected from chemotherapeutic agents, drug-based regimens, or combinations thereof.

[45] The method according to

[44] above, wherein the chemotherapeutic agent is selected from CHOP, FLAG, 7+3), and the drug-based preparation regimen is selected from Cy-Flu, Bu-Flu, and Flu-Mel.

[46] A method for modulating the T cell repertoire during or after stem cell transplantation, the method comprising administering to a subject in need thereof an effective amount of the composition according to any one of

[12] to

[39] above or a proliferated NK cell population.

[47] A method for preventing, inhibiting, reducing, or alleviating acute or chronic graft-versus-host disease, the method comprising administering to a subject in need thereof an effective amount of the composition according to any one of

[12] to

[39] above or a proliferated NK cell population.

[48] The method according to

[47] above for preventing, inhibiting, reducing, or alleviating acute or chronic graft-versus-host disease, further comprising administering to the subject a GvHD prophylactic agent in combination with an effective amount of the composition or the proliferated NK cell population.

[49] A method for preventing, inhibiting, reducing, or alleviating viral reactivation, the method comprising administering to a subject in need thereof an effective amount of the composition according to any one of

[12] to

[39] above or a proliferated NK cell population.

[50] The method according to

[49] above for preventing, inhibiting, reducing, or alleviating viral reactivation, wherein the viral infection includes herpes simplex virus type 1 (HSV-1), herpes simplex virus type 2 (HSV-2), cytomegalovirus (CMV), varicella-zoster virus (VZV), Epstein-Barr virus (EBV), adenovirus, adeno-associated virus, parvovirus, JC virus, and BK virus.

[51] A method for preventing, inhibiting, reducing, or alleviating opportunistic infection, the method comprising administering to a subject in need thereof an effective amount of the composition according to any one of

[12] to

[39] above or a proliferated NK cell population.

[52] An NK cell growth medium preparation comprising the NK cell growth composition according to

[12] above and at least one medium component.

[53] The NK cell growth medium preparation according to

[52] above, further comprising at least one additional component selected from cytokines, IL-2, IL-12, IL-18, NAM, reducing agents, human platelets, human platelet lysate, insulin, and ascorbate.

[54] The expanded NK cell population according to any one of

[30] to

[34] above, wherein the expanded NK cells show an increased secretion of anti-tumor cytokines as compared to non-expanded NK cells.

[55] The expanded NK cell population according to any one of

[30] to

[34] above, wherein the expanded NK cells show an increased expression of NKG2D, NKp46, and CD16 as compared to non-expanded NK cells.

[56] A cryopreserved therapeutic dose of the expanded NK cell population according to any one of

[30] to

[34] above, wherein the expanded NK cells continue to survive after thawing.

[57] A method for increasing the cytotoxicity of NK cells, comprising exposing an initial NK cell population to an NK cell proliferation composition in vitro, the composition comprising at least one feeder cell or engineered particle, the at least one feeder cell or engineered particle comprising at least two NK cell effector agents, one of the at least two NK cell effector agents being IL-21, and an Fc domain bound to the outer surface of the feeder cell or engineered particle.

[58] The method according to

[57] above, wherein the NK cell proliferation composition comprises at least one feeder cell having a membrane-bound Fc domain.

[59] The method according to

[57] above, wherein the NK cell proliferation composition comprises an engineered particle selected from PM particles having a membrane-bound Fc domain and exosomes having a membrane-bound Fc domain.

[60] The method according to

[57] above, further comprising obtaining an expanded NK cell population having increased cytotoxicity as compared to the initial NK cell population.

[61] The method according to

[57] above, wherein the cytotoxicity of the expanded NK cells is at least about 2-fold that of the initial NK cell population.

[62] The method according to

[57] above, wherein the cytotoxicity of the expanded NK cells is at least about 5-fold that of the initial NK cell population.

[63] The method according to

[57] above, wherein the cytotoxicity of the expanded NK cells is at least about 10-fold that of the initial NK cell population.

[64] The source of NK cells to be proliferated or stimulated may include peripheral blood (PBMC, apheresis, leukopak, buffy coat), iPSC-derived NK cells, ESC-derived NK cells, NK cells having a polymorphism of a high-affinity Fc receptor having Phe or Val at position 158, and genetically modified NK cells, according to any one of the above items

[40] to

[51] or

[57] to

[63] .

Example

[0091] Example 1: Binding of CD16 via the Fc region enhances the growth rate after day 14 The K562 cell line, i.e., a cell line expressing 41BBL and membrane-bound IL-21 (“CSTX-002”), was obtained.

[0092] Separate samples of K562 cells were transfected with NA-Fc to produce Fc-binding K562 cells (“CSTX002-Fc”).

[0093] Peripheral blood mononuclear cells (PBMCs) were obtained from leukocyte sources from two different donors (L43 and L44) and divided into multiple aliquots. For one sample of PBMCs from each donor, the proliferation of NK cells was tested in the presence of CSTX002, and for another sample, the proliferation of NK cells was tested in the presence of CSTX002-Fc.

[0094] PBMCs isolated from buffy coats by Ficoll-Paque density gradient were co-cultured and grown in SCGM CellGro medium supplemented with 10% FBS and 100 U / mL IL-2 and mitomycin C-treated or irradiated feeder cells (either CSTX002 cells or CSTX002-Fc cells) at a ratio of 1 feeder cell per NK cell. The cells were incubated in 5% CO 2It was maintained at 37°C in a humidified atmosphere. From the 5th day of culture, the medium was changed every other day, and half of the medium was replaced with fresh medium supplemented with 100 U of IL2. Cells were counted every other day, and the culture content was checked regularly from the 7th day.

[0095] Figure 5 is a graph showing the proliferation of NK cells against the number of culture days, indicating that the binding of CD16 via the Fc region enhances the proliferation rate after the 14th day. NK cells were proliferated from PBMC obtained from 2 donors (L54 (circle) or L44 (square)). Either CSTX002 (open symbols) or CSTX002-Fc cell line (filled symbols) was used as feeder cells. The proliferation of NK cells is increased by the binding of CD16. NK cells from both donors proliferated at the same rate until the 14th day in response to stimulation with IL21 alone or IL21 and Fc, and on the 14th day, the Fc-stimulated cultures separated at an increased rate compared to IL21 alone. Example 2: Cytotoxicity of natural killer cells grown in the presence of Fc-binding feeder cells

[0096] CSTX002 cells and CSTX002-Fc cells were prepared as described in Example 1. The cytotoxicity assay was performed as follows. The ovarian cancer-derived target cell line SKOV3 transfected with green fluorescent protein (GFP) was used as a target for measuring the anti-tumor cytotoxicity of effector NK cells. Target cells were cultured alone (control wells) or co-cultured with NK cells at the indicated effector-to-target (E:T) ratio of 0.5×10 6 cells / mL and incubated at 37°C for 45 minutes in a 5% CO 2 atmosphere. The cells were then centrifuged and resuspended in Annexin V labeling buffer containing Annexin V-PacBlue antibody, incubated at 4°C for 15 minutes, and then analyzed by flow cytometry. Cytotoxicity was determined based on the absolute amount (VTC E:T ) of viable target cells (GFP+ / Annexin V-) remaining in each well with the effector, and referenced against the average VTC (VTC T対照 ) in the "target only" control wells. Cytotoxicity E:T (%) = (VTC E:T / average VTC T対照 ) * 100

[0097] Figure 6 presents two graphs, each showing the cytotoxicity of NK cells expanded from PBMC obtained from two different donors (L43 and L44). For each donor, NK cells were expanded from PBMC and either the CSTX002 (●) or CSTX002-Fc (■) cell line was used as feeder cells. For each of the two different donors, it was found that NK cells expanded using CSTX002-Fc had increased cytotoxicity against SKOV3. Example 3: Increased cytotoxicity of natural killer cells derived from PBMC of donors with poor response

[0098] PBMCs were obtained from donors who, when stimulated with CSTX002 cells (without Fc addition) in the past, were observed to have no cytotoxicity against SKOV3 cells. NK cells were expanded from PBMCs that were previously observed to have a poor response, and either the CSTX002 (●) or CSTX002-Fc (■) cell line was used as feeder cells as described in Example 1. Next, for the two different NK cell populations obtained, cytotoxicity was tested as described in Example 1, except that SKOV3 transformed to express Fc was used. Figure 7 is a graph of the cytotoxicity of NK cells expanded from PBMCs using either CSTX002 (●) or CSTX002-Fc (■). The results shown in Figure 7 indicate that PBMC-derived NK cells from donors with a poor cytotoxic response against SKOV3 cells respond well when expanded using Fc-binding feeder cells (CSTX002-Fc), which shows cytotoxicity against tumor targets with a bound Fc domain in comparison to PBMC-derived NK cells from the same poorly responsive donors expanded using feeder cells not bound to Fc (CSTX002). NK cells expanded using CSTX002-Fc are thought to be better involved in antibody-dependent cell cytotoxicity and exhibit higher killing activity against tumor targets bound to the antibody.

[0099] Example 4: Preferred Receptor Expression NK cells derived from PBMC obtained from 2 donors [L43 (●) or L44 (■)] were grown as described in Example 1 using either CSTX002 (open symbols) or CSTX002-Fc cell line (filled symbols) as feeder cells. Subsequently, the obtained NK cells were analyzed. Figure 8 is a series of six graphs, each showing a comparison of receptor expression by NK cells grown using CSTX002 feeder cells with or without membrane-bound Fc. NK cells derived from PBMC obtained from 2 donors [L43 (●) or L44 (■)] were grown using either CSTX002 (open symbols) or CSTX002-Fc cell line (filled symbols) as feeder cells. For the grown NK cells, the expression of receptors considered important for cytotoxic function or homing was analyzed. NK cells grown using CSTX002-Fc had higher expression of CD16, NKp46, and CD62L than NK cells grown using CSTX002.

[0100] Example 5: Fc-Binding Plasma Membrane Particles Procedure K562 cells, i.e., a cell line expressing 41BBL and membrane-bound IL-21, are processed as described in U.S. Patent No. 9,623,082 to obtain PM-mb21-41BBL plasma membrane vesicles, i.e., "CSTX002" particles or PM21 particles. Briefly, K562 is cultured in RPMI medium supplemented with 10% FBS, and the culture is scaled up to 1 L. The cells are harvested by centrifugation at 1000 × g, washed with cold PBS containing 10 mM EDTA, and resuspended in lysis buffer (50 mM HEPES, pH 7.4, protease inhibitor cocktail). The cells are disrupted, and the lysate solution is centrifuged at 300 × g for 15 minutes to remove remaining whole cells. The crude plasma membrane is separated from cytosolic components by centrifugation at 4°C for 30 minutes. The crude membrane is resuspended and further purified using a sucrose density gradient to obtain pure plasma membrane vesicles designated PM-mb21-41BBL.

[0101] Transfect separate samples of K562 cells to express Fc and produce Fc-binding K562, and then process this as described above to obtain Fc-binding PM-mb21-41BBL plasma membrane vesicles, or "CSTX002-Fc" particles.

[0102] Peripheral blood mononuclear cells (PBMCs) are obtained from a single donor and divided into multiple samples. NK cell proliferation from PBMCs is tested in the presence of CSTX002 membrane particles or CSTX002-Fc membrane particles. The amount of each membrane particle used is 200 μg of membrane protein per mL of culture. PBMCs isolated from blood by Ficoll-Paque density gradient are grown in SCGM CellGro medium supplemented with 10% FBS and 100 U / mL of IL-2. The cells are maintained at 37 °C in a humidified atmosphere containing 5% CO2. From day 5 of the culture, the medium is changed every other day, replacing half of the medium with fresh medium and replacing the amount of membrane removed by the medium change. The cells are counted every other day and the culture content is checked on days 7, 10, and 14.

[0103] The cytotoxicity assay is performed as described in Example 1. NK cells grown with CSTX002-Fc will show an increase in cytotoxicity against SKOV3 cells. sequence SEQ ID NO: 29 Sequence number 30 Sequence number 31

Claims

**Claim 1** a) feeder cells, said feeder cells comprising a membrane-bound inverted Fc domain bound to the outer surface of said feeder cells; b) engineered plasma membrane (PM) particles, said engineered PM particles comprising a membrane-bound inverted Fc domain bound to the outer surface of said engineered PM particles, or c) engineered exosomes, said engineered exosomes comprising a membrane-bound inverted Fc domain bound to the outer surface of said engineered exosomes A natural killer (NK) cell proliferation composition comprising: further comprising at least one NK cell effector agent; wherein said membrane-bound inverted Fc domain is part of a fusion protein comprising a transmembrane domain bound to the amino terminus of the Fc domain, and the amino terminus of said Fc domain faces said outer surface of said feeder cells, said engineered PM particles or said engineered exosomes; wherein said NK cell proliferation composition results in proliferated NK cells that exhibit enhanced cytotoxicity as measured by (1) increased secretion of anti-tumor cytokines and / or (2) increased expression of CD62L, CD16, NKp46 and / or NKG2D, and the cytotoxicity of said proliferated NK cells is at least 2-fold, at least 5-fold or at least 10-fold that of non-proliferated NK cells. An NK cell proliferation composition. **Claim 2** The NK cell proliferation composition according to claim 1, comprising engineered PM particles or engineered exosomes that do not contain feeder cells. **Claim 3** The NK cell proliferation composition according to claim 2, comprising said engineered PM particles, wherein said at least one NK cell effector agent is at least one membrane-bound NK cell effector agent comprising IL-21 or IL-15. **Claim 4** The NK cell proliferation composition according to claim 3, wherein said at least one membrane-bound NK cell effector agent comprises IL-21. **Claim 5** The NK cell proliferation composition according to claim 4, further comprising a second membrane-bound NK cell effector agent, wherein said second membrane-bound NK cell effector agent is 4-1BBL. **Claim 6** The NK cell proliferation composition according to claim 1, wherein said at least one NK cell effector agent comprises IL-21 or IL-15, and said fusion protein comprises the amino acid sequence of SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 23 or SEQ ID NO:

28. **Claim 7**: The NK cell proliferation composition according to claim 1, comprising a membrane-bound inverted Fc domain to which the engineered PM particles are bound on the outer surface of the engineered PM particles, wherein the at least one NK cell effector agent comprises IL-21. **Claim 8** The NK cell proliferation composition according to claim 7, wherein the engineered PM particles further comprise membrane-bound 4-1BBL. **Claim 9** The NK cell proliferation composition according to claim 5, further comprising at least one additional NK cell effector agent, wherein the at least one additional NK cell effector agent is a cytokine, an adhesion molecule, or an NK cell activator, and the at least one additional NK cell effector agent is selected from IL-15, IL-2, IL-12, IL-18, IL-21, MICA, UBLP, 2sB4, LFA-1, a ligand for Notch ligand, NKp46 or BCM1 / SLAMF2, a TLR ligand, and an NKG2D ligand. **Claim 10** The composition according to any one of claims 1 to 9, for use in treating, remitting, reducing, and / or inhibiting cancer or metastasis or an infectious disease in a subject. **Claim 11** The composition according to claim 10, wherein the cancer is selected from the group consisting of tumor, blood cancer, lymphoma, leukemia, acute myeloid leukemia, myelodysplastic syndrome, chronic myeloid leukemia, acute lymphoblastic leukemia, myelofibrosis, multiple myeloma, colorectal cancer, colon cancer, lung cancer, head and neck cancer, ovarian cancer, pancreatic cancer, liver cancer, skin cancer, prostate cancer, kidney cancer, intraperitoneal cancer, and breast cancer. **Claim 12** The composition according to any one of claims 1 to 9, for use in inhibiting, reducing, and / or preventing recurrence of cancer or metastasis before or after stem cell transplantation. **Claim 13** The composition according to claim 10, used in combination with at least one cancer therapeutic agent. **Claim 14** The composition according to claim 12, used in combination with at least one cancer therapeutic agent. **Claim 15** The composition according to claim 13 or 14, wherein the at least one cancer therapeutic agent is selected from a chemotherapeutic agent, a drug-based preparation regimen, or a combination thereof. **Claim 16** The composition according to claim 15, wherein the chemotherapeutic agent is selected from CHOP, FLAG, and 7+3, and the drug-based preparation regimen is selected from Cy-Flu, Bu-Flu, and Flu-Mel. **Claim 17** The composition according to any one of claims 1 to 9 for use in preventing, inhibiting, reducing or alleviating acute or chronic graft-versus-host disease.

18. The composition according to claim 17 for use in combination with a prophylactic agent for graft-versus-host disease.

19. The composition according to any one of claims 1 to 9 for use in preventing, inhibiting, reducing or alleviating viral reactivation.

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