RU2839664C2 - Compositions and methods for stimulating natural killer cells - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to biotechnology, specifically to agents stimulating natural (natural) killer (NK) cells, and can be used in medicine for treating cancer, as well as to inhibit acute or chronic graft-versus-host disease, virus reactivation and opportunistic infections. Disclosed is a composition containing a feeder cell designed for expression of a fused protein, containing membrane-bound inverted domain Fc, IL-21 and 4-1BBL, or a plasma membrane particle (PM) obtained from said feeder cell or an exosome. Fused protein contains a transmembrane domain bound to the amino-terminal of the membrane-bound inverted Fc domain and is configured to bind the amino-terminal of the inverted Fc domain to the outer surface of the engineered feeder cell, PM particle or exosome.

EFFECT: invention enables to increase the number and cytotoxicity of NK cells, which provides a therapeutic effect.

24 cl, 8 dwg, 5 ex

Description

This application claims priority to U.S. Provisional Application No. 62/796,575, filed January 24, 2019, which is incorporated herein by reference in its entirety.

AREA OF TECHNOLOGY

[1] This disclosure relates to compositions and methods for stimulating natural killer (NK) cells.

LEVEL OF TECHNOLOGY

[2] Natural killer (NK) cell therapy is emerging as an approach to treating cancer and possibly other diseases. Challenges associated with realizing the full clinical potential of NK cell therapy include obtaining large numbers of robust, healthy NK cells that are highly cytotoxic to tumor cells; the ability to target NK cells to the disease of choice; and maintaining NK cells in vivo after administration to the patient to achieve therapeutic effect. These challenges are explained in part by the fact that NK cell activity is tightly regulated by a balance of activating and inhibitory receptors, including immune checkpoints. For example, ligands for activating NK cell receptors are expressed only on stressed, transformed, or virally infected cells, so that the cytotoxic activity of NK cells is targeted to such cells to spare normal healthy tissue. The cytotoxic activity of NK cells is further limited by inhibitory ligands expressed on self-cells. At the same time, the inhibitory regulatory mechanisms that control NK cell cytotoxicity may be a route of attack for tumor cells, which employ various immunosuppressive interactions to prevent immune attack. An example of how NK cells counter tumor immunosuppression is the involvement of antibody-labeled target cells in the induction of antibody-dependent cellular cytotoxicity (ADCC) by NK cells. As a result, the success of many new antitumor antibodies depends on the presence of large numbers of healthy NK cells in the patient to maintain antitumor activity. Overall, there remains a need in the field of NK cell therapeutics for approaches to generate healthy NK cell numbers and to stimulate NK cells to achieve higher cytotoxicity and/or improve ADCC functionality.

BRIEF DESCRIPTION OF THE ESSENCE OF THE INVENTION

[3] Among the various aspects of the disclosure is a feeder cell composition comprising at least one feeder cell comprising a fragment of a crystallizable (Fc) domain that is associated with an 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 some aspects, the at least one feeder cell further comprises at least one NK cell effector agent, wherein the NK cell effector agent is IL-21. In some aspects, 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.

[4] Also disclosed are compositions for increasing the number of NK cells that do not contain feeder cells, comprising an engineered particle comprising an Fc domain linked to an outer surface of the engineered particle of any preceding aspect. In some aspects, the engineered particle further comprises one or more NK cell effector agents. In some aspects, the engineered particle further comprises at least one NK cell effector agent, wherein the NK cell effector agent is IL-21. In some aspects, the engineered particle further comprises at least two NK cell effector agents, wherein one of the at least two NK cell effector agents is IL-21.

[5] In one aspect of the disclosure, a therapeutic dose of NK cells is provided, comprising a plurality of NK cells expanded in vitro, in combination with a composition for increasing the number of NK cells, wherein the composition is free of feeder cells and comprises at least one engineered particle that comprises an Fc domain associated with an outer surface of the engineered particle. In some aspects, the engineered particle further comprises one or more NK cell effector agents. In some aspects, the engineered particle further comprises at least one NK cell effector agent, wherein the NK cell effector agent is IL-21. In some aspects, the engineered particle further comprises at least two NK cell effector agents, wherein one of the at least two NK cell effector agents is IL-21.

[6] In one aspect of the disclosure, an expanded population of NK cells is provided that has been exposed in vitro to a composition for expanding the number of NK cells, wherein the composition is free of feeder cells and comprises at least one engineered plasma membrane (PM) particle as disclosed herein. In another aspect of the disclosure, an expanded population of NK cells is provided that has been exposed in vitro to a composition for expanding the number of NK cells, wherein the composition comprises at least one feeder cell comprising an Fc domain associated with an outer surface of the feeder cell as disclosed herein. Such methods optionally further comprise exposing the NK cells to one or more NK cell effector agents. The one or more cellular effector agents may be in solution in the cellular medium and/or associated with the surface of an Fc-linked feeder cell or engineered PM particles as disclosed herein.

[7] Also disclosed herein are methods for "treating, alleviating, reducing, and/or inhibiting cancer, metastasis, or an infectious disease," comprising administering to a subject in need thereof an effective amount of any of the described NK cell increasing compositions or NK cell increasing infusion formulations of any of the preceding aspects. In one aspect, the NK cell increasing compositions or NK cell increasing infusion formulation may be combined with or co-administered with a therapeutic agent, such as, for example, an anti-cancer therapeutic agent or an antiviral or antibiotic agent.

[8] In one aspect of the disclosure, methods are disclosed for treating, ameliorating, reducing, and/or inhibiting cancer or metastasis before or after stem cell transplantation, 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 a composition for increasing the number of NK cells, or compositions that stimulate NK cells, or any disclosed infusion formulation for stimulating or expanding NK cells. Any of the described compositions or formulations for stimulating or expanding NK cells can be administered in combination with stem cell transplantation or alone.

[9] In one aspect of the disclosure, a method of modulating a T cell repertoire is provided 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 a composition for increasing the number of NK cells, or any disclosed infusion formulation for increasing the number of NK cells.

[10] In one aspect of the disclosure, there is provided a method for preventing, inhibiting, reducing, or ameliorating an acute or chronic graft-versus-host disease 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 a composition for increasing the number of NK cells, or any disclosed infusion formulation for increasing the number of NK cells.

[11] Other aspects and features of the disclosure are described in detail below.

BRIEF DESCRIPTION OF GRAPHIC MATERIALS

[12] Fig. 1A and 1B show a membrane-bound immune cell targeting ligand construct containing an uncleaved signaling anchor. Fig. 1A shows the structure of type I and type II integral membrane proteins that differ in orientation with respect to their N- and C-termini. Fig. 1B shows the structure of a chimeric NA-Fc protein used as a membrane-bound immune cell targeting ligand, consisting of a neuraminidase transmembrane domain that serves as a membrane anchor, a stalk region, and a human IgG' Fc region.

[13] Figure 2 shows alternative constructs of membrane-bound immune cell targeting ligands comprising an Fc domain containing a neuraminidase (NA) signaling anchor and an elongating NA stalk.

[14] Fig. 3 shows an example of the sequence of a membrane-associated immune cell targeting ligand (SEQID NO: 13), with the NA signal anchor fused to the Fc domain of IgG via an RS linker.

[15] Fig. 4 is a schematic diagram of Fc stimulation of NK cell.

[16] Figure 5 shows the involvement of CD16 via the Fc region in increasing the proliferation rate over 14 days. NK cells were expanded from PBMCs obtained from two donors [L54 (circles) or L44 (squares)] using either the CSTX002 (open symbols) or CSTX002-Fc (closed symbols) cell lines as feeder cells. The involvement of CD16 promotes the increase in NK cell proliferation. NK cells from both donors proliferated at similar rates when stimulated with IL21 alone (CSTX2) or IL21 and Fc (CSTX2-Fc) until day 14, at which point Fc-stimulated cultures divided at a higher rate than those stimulated with IL-21 alone.

[17] Figure 6 shows two graphs, each showing the cytotoxicity of NK cells expanded from a starting population of PBMCs obtained from a different donor. NK cells were expanded from PBMCs using either the CSTX002 (●) or CSTX002-Fc (■) cell lines as feeder cells. NK cells expanded from two different donors with CSTX002-Fc were found to have enhanced cytotoxicity toward SKOV3 cells.

[18] Figure 7 shows that NK cells from a weak responder donor expanded with Fc-linked feeder cells have enhanced cytotoxicity toward tumor targets that express membrane-linked Fc to mimic antibody-coated tumor cells that would trigger antibody-dependent cellular cytotoxicity (ADCC). NK cells were expanded from PBMCs using either the CSTX002 (●) or CSTX002-Fc (■) cell lines as feeder cells. NK cells expanded with CSTX002-Fc were found to have enhanced cytotoxicity toward SKOV3-Fc cells.

[19] Fig. 8 is a series of six (6) graphs, each showing comparative receptor expression by NK cells expanded with CSTX002 feeder cells with (CSTX002-Fc) and without (CSTX-002) membrane-bound Fc. NK cells were expanded from PBMCs derived from two donors L43 (●) or L44 (■) using either the CSTX002 or CSTX002-Fc cell lines as feeder cells. Expanded NK cells were analyzed for the expression of receptors considered important for cytotoxic function and homing. NK cells expanded with CSTX002-Fc have higher expression of CD16, NKp46, and CD62L.

DETAILED DESCRIPTION OF THE ESSENCE OF THE INVENTION

[20] This disclosure provides compositions and formulations comprising NK cell stimulating agents and related methods of using them to stimulate NK cells and in various treatment options, as described in more detail below.

[20] Engagement of the CD16 receptor (FcγRIIIa receptor (CD16a) and FcγRIIIb receptor (CD16b)) on NK cells is potentially a very potent mechanism for NK cell stimulation. The Fc domain (fragment of the crystallizable region) of an antibody is recognized by CD16, and binding of the Fc domain to CD16 induces antibody-dependent cellular cytotoxicity (ADCC). This disclosure describes engineered NK cell stimulation through CD16 engagement to improve NK cell proliferation and enhance NK cell cytotoxicity. In other words, this disclosure contemplates stimulating NK cells using an Fc domain of an antibody, wherein the Fc domain is a suitable agonist of CD16 on NK cells and is presented to the NK cells with the Fc domain associated with a feeder cell, a plasma membrane (PM) particle, an exosome (EX), or a solid carrier. Fc-associated feeder cells, PM particles, exosomes, and solid carriers may further comprise or be combined with other NK cell stimulating factors in various forms, such as membrane-bound or soluble IL-15, IL-21, 4-1BBL, other cytokines, or other chemical moieties that simultaneously interact with other stimulatory or inhibitory receptors and associated signaling pathways. NK cells expanded according to the methods and using the compositions described herein may exhibit increased cytotoxicity, increased CD16 expression, and/or improved ADCC activity. Such NK cells are useful in therapeutic compositions and methods for treating human diseases and conditions, including multiple types of cancer.

Definitions

[22] Unless otherwise defined, scientific and technical terms used in connection with this application shall have the meanings commonly understood by those skilled in the art to which this invention pertains. The following references provide one skilled in the art with a general definition of many of the terms used in this application: 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). In the context of this application, the following terms have the meanings ascribed to them unless otherwise defined.

[23] When introducing elements of this disclosure or preferred embodiments thereof, the articles "a," "an," "the," and "mentioned" are intended to indicate the existence of one or more elements. The terms "comprising," "including," and "having" are intended to be inclusive and mean that there may be additional elements other than those listed.

[24] As used herein, ranges may be expressed as from "about" one particular value and/or to "about" another particular value. When such a range is included, another embodiment includes from one particular value and/or to another particular value. Similarly, when values are expressed as approximations, using the preceding "about", it is to be understood that the particular value forms another embodiment. It is to be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint and independently of the other endpoint. It is also to be understood that there are a number of values disclosed herein, and that each value is also disclosed herein as "about" that particular value in addition to the value itself. For example, if the value "10" is disclosed, then "about 10" is also disclosed. It is also to be understood that when a value is disclosed, "less than or equal to" the value, "greater than or equal to" the value, and possible ranges between the values are also disclosed, as would be appropriately understood by one skilled in the art. For example, if the value "10" is disclosed, then "less than or equal to 10" is also disclosed, as is "greater than or equal to 10." It is also understood that the application provides data in several different formats, and that the data represents end points, start points, and ranges for any combination of data points. For example, if a specific data point of "10" and a specific data point of 15 are disclosed, it is understood that the values greater than, greater than or equal to, less than, less than or equal to, and equal to 10 and 15 are disclosed, as well as the range between 10 and 15. It should also be understood that each unit between the two specific units is also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

[25] As used herein, the terms "optional" or "optionally" mean that the event or circumstance described below may or may not occur and that the description includes instances in which the stated event or circumstance occurs and instances in which it does not occur.

[26] As used herein, the terms "N-terminal side" or "amino-terminal end" refer to the direction of a peptide, polypeptide, or protein and may not refer to the N-terminus. In some aspects, where a chimeric or hybrid peptide, polypeptide, or protein is described, the N-terminal side may refer only to a component of the chimeric or hybrid peptide, polypeptide, or protein, rather than the entire structure. For example, if an Fc domain is discussed and the Fc domain is described as fused to its amino-terminal end or N-terminal side facing the inside of the cell, as contemplated herein are chimeric or hybrid peptides, polypeptides, or proteins in which the signal anchor is at the N-terminus of the chimeric or hybrid construct, effectively spanning the cell membrane. Thus, in such a chimera, the transmembrane anchor is attached to the amino-terminal side of the Fc domain, with the Fc domain having the N-terminal side facing the cell, which is inverted relative to the Fc domain on a typical B cell, in which the carboxy-terminus typically spans the cell membrane and the amino-terminal end extends into the extracellular matrix.

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

[28] As used herein, the term "sequence identity" indicates a quantitative measure of the degree of identity between two sequences of substantially equal length. The percentage identity of two sequences, whether nucleic acid or amino acid sequences, is the number of exact matches between the two aligned sequences divided by the length of the shorter sequence and multiplied 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 using the 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 per Gribskov, Nucl. Acids Res. 14(6):6745–6763 (1986). A typical implementation of this algorithm for determining percent sequence identity is provided by the Genetics Computer Group (Madison, Wis.) in the program application “BestFit”. Other suitable programs for calculating percent identity or similarity between sequences are well known in the art, for example, another alignment program is BLAST, 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 web site. In general, the substitutions are conservative amino acid substitutions: restricted to substitutions 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: aspartate, glutamate, asparagine, and glutamine.

[29] Methods for determining nucleic acid and amino acid sequence identity are well known in the art. Typically, such methods involve determining the nucleotide sequence of the mRNA for a gene and/or determining the amino acid sequence it encodes and comparing these sequences to a second nucleotide or amino acid sequence. Genomic sequences can be determined and compared in a similar manner. In general, identity refers to an exact nucleotide-to-nucleotide or amino acid-to-amino acid correspondence between two polynucleotide or polypeptide sequences, respectively. Two or more sequences (polynucleotide or amino acid) can be compared by determining their percent identity.

[30] Since various changes can be made in the above described cells and methods without departing from the scope of the invention, it is intended that all matter contained in the above description and in the examples below be interpreted as illustrative and not limiting.

[31] "Increase" may refer to any change that results in an increase in a symptom, disease, composition, condition, or activity. An increase may be any individual, average, or mean increase in a condition, symptom, activity, composition by a statistically significant amount. Thus, an increase may 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.

[32] "Reduction" may refer to any change that results in a decrease in a symptom, disease, composition, condition, or activity. A substance is also said to decrease the genetic output of a gene when the genetic output of the gene product with the substance is less than the genetic output of the gene product without the substance. Also, for example, a reduction may be a change in the symptoms of a disorder such that the symptoms are less than previously observed. A reduction may be any individual, average, or median decrease in a condition, symptom, activity, composition by a statistically significant amount. Thus, a reduction may 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% reduction, as long as the reduction is statistically significant.

[33] “Inhibit,” “inhibits,” and “inhibition” mean a decrease in an activity, response, condition, disease, or other biological parameter. This may include, but is not limited to, a complete elimination of the activity, response, condition, or disease. It may also include, for example, a decrease in the activity, response, condition, or disease by 10% compared to a native or control level. Thus, the decrease may be 10, 20, 30, 40, 50, 60, 70, 80, 90, 100%, or any intermediate decrease compared to native or control levels.

[34] By "reduce" or other forms of the word such as "reduces" or "reduction" is meant a decrease in an event or characteristic (e.g. tumor growth). It is understood that this usually refers to some standard or expected value, in other words, it is a relative value, but it is not necessary to always refer to a standard or relative value. For example, "reduces tumor growth" means a decrease in the rate of tumor growth compared to a standard or control.

[35] By "prevent" or other forms of the word such as "prevents" or "prevention" is meant to stop a particular event or characteristic, to stabilize or retard the development or progression of a particular event or characteristic, or to minimize the likelihood that a particular event or characteristic will occur. Prevention does not require a comparison with control because it is generally more absolute than, for example, reduction. In this context, something may be reduced but not prevented, and also what is reduced may be prevented. Likewise, something may be prevented but not reduced, and also what is prevented may be reduced. It should be understood that if reduction or prevention is used, unless otherwise specifically stated, the use of another word is also a disclosure.

[36] The term "subject" refers to any individual that is the target of administration or treatment. The subject may be a vertebrate, such as a mammal. In one aspect, the subject may be a human, a non-human primate, a cow, a horse, a pig, a dog, or a cat. The subject may also be a guinea pig, a rat, a hamster, a rabbit, a mouse, or a mole. Thus, the subject may be a human or a veterinary patient. The term "patient" refers to a subject under treatment by a clinician, such as a physician.

[37] The term "therapeutically effective" refers to an amount of the composition used that is sufficient to alleviate one or more causes or symptoms of the disease or disorder. Such alleviation means only a reduction or modification, but not necessarily elimination.

[38] The term "treatment" refers to the medical management of a patient with the intent to cure, alleviate, stabilize, or prevent a disease, condition, or disorder. The term includes active treatment, that is, treatment specifically directed at ameliorating the disease, condition, or disorder, and also includes treatment of the cause, that is, treatment directed at eliminating the cause of the associated disease, condition, or disorder. Additionally, the term includes palliative treatment, that is, treatment intended to alleviate symptoms rather than cure the disease, condition, or disorder; prophylactic treatment, that is, treatment directed at minimizing or partially or completely suppressing the progression of the associated disease, condition, or disorder; and supportive treatment, that is, treatment used in addition to other specific therapy directed at ameliorating the associated disease, condition, or disorder.

[39] "Administration" to a subject includes any route of administration or delivery of the agent to the subject. Administration may be by any suitable route, including oral, topical, intravenous, subcutaneous, transdermal, intramuscular, intra-articular, parenteral, intra-arteriole, intradermal, intraventricular, intracranial, intraperitoneal, intralesional, intranasal, rectal, vaginal, by inhalation, via an implanted reservoir, parenterally (e.g., subcutaneous, intravenous, intramuscular, intra-articular, intra-synovial, intrasternal, intrathecal, intraperitoneal, intrahepatic, intralesional, and intracranial injection or infusion techniques), and the like. "Simultaneous administration," "combined administration," "simultaneous administration," or "administered simultaneously," as used herein, means that the compounds are administered at the same time or substantially immediately after each other. In the latter case, the two compounds are administered at a sufficiently close time that the observed results are indistinguishable from the results achieved by administering the compounds at the same time. "Systemic administration" refers to administration or delivery of an agent to a subject via a route that introduces or delivers the agent to widespread areas of the subject's body (e.g., more than 50% of the body), such as via entry into the circulatory or lymphatic systems. In contrast, "local administration" refers to administration or delivery of an agent to a subject via a route that introduces or delivers the agent to an area or area immediately adjacent to the site of administration and does not administer the agent systemically in a therapeutically effective amount. For example, agents administered locally are readily detectable in the immediate vicinity of the site of administration but are not detectable or are detectable in minute quantities in distal parts of the subject's body. Administration includes self-administration and administration by another person.

[40] The terms "treat," "treatment," and grammatical variations thereof as used herein include administering a composition with the intent or purpose of partially or completely preventing, delaying, treating, curing, alleviating, reducing, modifying, treating, ameliorating, alleviating, stabilizing, mitigating, and/or reducing the intensity or frequency of one or more diseases or conditions, a symptom of a disease or condition, or an underlying cause of a disease or condition. The therapeutic procedures of the invention may be administered preventively, prophylactically, palliatively, or curatively. Prophylactic treatment is administered to a subject prior to onset (e.g., prior to the onset of overt signs of cancer), during early onset (e.g., at the initial signs and symptoms of cancer), or after the established development of cancer. Prophylactic administration may occur day(s) or years before the onset of symptoms of a disease or infection.

(I) Fc-fusion peptides

[41] In one aspect of the present invention, engineered feeder cells, engineered plasma membrane particles (PM), engineered exosomes, engineered platelets (including, but not limited to, Fc-linked platelets), and engineered lymphocytes (such as, for example, lymphocytes (such as T cells) engineered to express Fc domains for stimulating NK cells), and solid supports comprising a membrane-linked Fc fusion peptide (referred to herein as Fc-linked feeder cells, Fc-linked PM particles, Fc-linked exosomes, Fc-linked platelets, and Fc-linked lymphocytes, respectively), are disclosed, wherein the Fc fusion peptide comprises a transmembrane peptide domain linked to the amino terminus of an Fc domain. In one aspect, the transmembrane domain of the Fc fusion peptide may comprise a cleaved or uncleaved signal anchor sequence, such as a neuraminidase transmembrane domain, a parainfluenza virus hemagglutinin-neuraminidase signal anchor, a transferrin receptor signal anchor, a signal anchor from an MHC class II invariant chain, a signal anchor from a P glycoprotein, a signal anchor from an asialoglycoprotein receptor, or a signal anchor from a neutral endopeptidase. In one example, the transmembrane domain comprises a parainfluenza virus hemagglutinin-neuraminidase (NA) peptide sequence. As schematically shown in Fig. 1, the neuraminidase (NA) transmembrane peptide domain is used to connect or link the Fc domain to the outer surface of a feeder cell. In other aspects, a neuraminidase (NA) transmembrane peptide domain is used to connect or link the Fc domain to the outer surface of a PM nanoparticle, exosome, or solid carrier. The NA peptide domain consists of an N-terminal cytoplasmic tail, an uncleaved signaling anchor that serves as a transmembrane domain, and a stalk region that extends from the plasma membrane. It should be understood that the length of the stalk region can vary.

[42] As used herein, the term "NA peptide domain" refers to a peptide sequence comprising at least fifty (50) amino acids of the sequence 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% identity to the sequence of SEQ ID NO: 1.

[43] The Fc domain is the ligand to which the NK cell surface receptor CD16 (FcγRIII) binds. CD16 is one of the primary receptors on NK cells, and when CD16 binds to the Fc portion of an antibody (e.g., the Fc domain of IgG1, IgG2, IgG3, and/or IgG4), it activates NK cell-mediated antibody-dependent cellular cytotoxicity (ADCC). In another aspect, the Fc domain (IgG1, IgG2, IgG3 and/or IgG4) can also bind the CD16 receptor on other immune cells such as mast cells, macrophages, gamma delta T cells; and thus will similarly stimulate the proliferation and enhance the cytotoxicity of such cells. In another aspect, other cell types can be engineered to be Fc-linked. Thus, the present disclosure also includes, for example, engineered platelets linked to Fc, primary tumor samples linked to Fc that can be used for tumor vaccines, and engineered iPSCs expressing Fc.

[44] In another aspect, other immunoglobulin Fc isotypes (IgA, IgE, IgM) other than IgG can be used to stimulate the corresponding different Fc receptors to stimulate other types of immune cells. For example, the FcαRI (CD89) domain specifically binds to IgA on macrophages, neutrophils, eosinophils; FcγRI (CD64) specifically binds to IgG on monocytes and macrophages; and FcεRII (CD23) specifically binds to IgE on B cells. Fc binds to CD64 on monocytes or macrophages and thereby stimulates them. Thus, fusion peptides, Fc-linked feeder cells (FCs), Fc-linked lymphocytes, Fc-linked engineered plasma membrane (PM) particles, Fc-linked engineered exosomes, and compositions containing them can also be used to increase the number of mast cells and/or macrophages, substantially in accordance with the methods described herein, to increase the number of NK cells.

[45] In one aspect, disclosed herein are fusion peptides comprising an immunoglobulin Fc domain (e.g., an IgG1, IgG2, IgG3, IgG4, IgA, and/or IgE Fc domain) fused to a transmembrane domain, such as an NA peptide domain, as described above. The Fc domain(s) can be provided as a monomeric, dimeric, or multimeric construct. In one aspect, the Fc domain(s) can be further modified to optimize or enhance antibody-mediated killing, NK cell recognition, and control of Fc activating receptor spreading. For example, the Fc domain(s) can be modified to increase affinity for CD16. Thus, for example, the Fc domain(s) may comprise one or more mutations, such as, for example, T256A, K290A, S298A, E333A, K334A, L235V, F243L, R292P, Y300L and/or P396L. Similarly, the Fc domain(s) may be further modified to increase the selectivity for binding to the activating Fc (IIIa) over the inhibitory (IIb) receptor. Thus, for example, the Fc domain(s) may comprise one, two, three, four, five, six, seven, eight or more mutations or alternative forms, such as, for example, S239D, I332E, A330L, F243L, R292P, V305I and/or P396L. For example, in one aspect, the Fc domain can be modified to include R292L, Y300L, V305I, and P396L. In another example, the Fc domain can be modified to include S239D, I332E, and A330L. In another aspect, engineered Fc domain mutants having lower affinity can be used to achieve higher CD16 expression on NK cells.

[46] A transmembrane domain, such as an NA peptide domain, may be linked directly to an Fc domain via a chemical bond or indirectly via a linker. A direct chemical bond is, for example, a covalent bond (e.g., a peptide bond, an ester bond, etc.) or, alternatively, a non-covalent bond (e.g., ionic, electrostatic, hydrogen, hydrophobic, van der Waal interactions, or π-effects). An indirect bond may be achieved using a linker, i.e., a chemical group that links one or more other chemical groups via at least one covalent bond. Suitable linkers include amino acids, peptides, nucleotides, nucleic acids, dimeric hinge Fc, organic linker molecules (e.g., maleimide derivatives, N-ethoxybenzylimidazole, biphenyl-3,4',5-tricarboxylic acid, para-aminobenzyloxycarbonyl, etc.), disulfide linkers, and polymeric linkers (e.g., PEG). The linker may include one or more intervening groups, including, but not limited to, alkylene, alkenylene, alkynylene, alkyl, alkenyl, alkynyl, alkoxy, aryl, heteroaryl, aralkyl, aralkenyl, aralkynyl, and the like. The linker may be neutral or carry a positive or negative charge. In addition, the linker may be cleavable, such that the covalent bond of the linker that connects the linker to another chemical group may be broken or cleaved under certain conditions, including pH, temperature, salt concentration, light, a catalyst, or an enzyme. In one aspect, the NA peptide domain may be NA4-Fc Siadel (S239D/I332E/A330L).

[47] In one aspect, the linker may be a peptide linker. Examples of suitable peptide linkers are well known in the art, and programs for creating linkers are readily available (see, e.g., Crasto et al., Protein Eng., 2000, 13(5):309-312). The peptide linker may be a restriction site linker, such as a short RS sequence, 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 can be a rigid amino acid linker. Such linkers include (EAAAK)1-4 (SEQ ID NO: 8), A(EAAAK)2-5A (SEQ ID NO: 9), PAPAP (SEQ ID NO: 10), and (AP)6-8. The Fc domain can be linked to the N-terminus, the C-terminus, and/or the interior of the NA peptide.

[48] In some aspects, the Fc fusion peptide has an amino acid sequence that has 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 NOS:13. To target the Fc domain to the plasma membrane, the membrane targeting domain from the well-characterized influenza virus neuraminidase (NA) protein can be used, which consists of an N-terminal cytoplasmic tail, an uncleaved signal anchor that serves as a transmembrane domain, and a stalk region that extends from the plasma membrane. Fig. 1A and 1B schematically show a membrane-bound immune cell targeting ligand construct comprising an uncleaved signaling anchor. Fig. 1A shows the structure of type I and type II integral membrane proteins and the signaling anchors for each. Fig. 1B shows the structure of an uncleaved signaling anchor of a type II integral 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 consists of an NA-Fc chimera in which the Fc (IgG1) domain is linked to the uncleaved portion of the NA stalk by 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 specific for tumor cells compared to normal cells (due to P/V mutations) and can enhance NK cell-mediated ADCC. Figure 2 shows alternative NA-Fc chimera constructs with increasing NA stalk length.

[49] Fig. 3 shows one example of an NA-Fc chimera sequence with an Fc domain (IgG1) linked by a short RS binding sequence to a 50 amino acid NA sequence to produce a non-limiting example of an NA-Fc construct having a 279 amino acid sequence, which is provided below and shown in Fig. 3:

[50] MNPNQKITTIGSICLVVGLISLILQIGNIISIWISHSIQTGSQNHTGICNRSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLT VLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 13). As noted in FIG. 2, the NA-Fc construct may comprise an NA targeting domain (SEQ ID NO: 1), a linker (e.g., an RS linker), a DKTHTCPPCPAPELL (SEQ ID NO: 11) or TCPPCPAPELL (SEQ ID NO: 12) hinge region, and an Fc region GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 14) including a CH2 domain GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK (SEQ ID NO: 15) and the CH3 domain GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 16) 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: 14, 15, or 16. It is evident and contemplated herein that the NA-Fc chimera can include a membrane targeting domain of any length from a well-characterized influenza virus neuraminidase (NA) protein, including MNPNQKITTIGSICLVVGLISLILQIGNIISIWISHSIQTGSQNHTGICNQNIITYKNSTWVKDTTSVILTGNSSLCPIRGWAIYSKDNSIRIGSKGDVFVIREPFISCSHLECRTFFLT (SEQ ID NO: 17), 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% identity to the sequence of SEQ ID NO: 17. In one aspect, the NA-Fc fusion may comprise MNPNQKITTIGSICLVVGLISLILQIGNIISIWISHSIQTGSQNHTGICNQNIITYKNSTWVKDTTSVILTGNSSLCPIRGWAIYSKDNSIRIGSKGDVFVIREPFISCSHLECRTFFLTDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEV KFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTK NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 18), which is encoded by a nucleic acid sequence ATGAATCCAAATCAGAAAATAACAACCATTGGATCAATCTGTCTGGTAGTCGGACTAATTAGCCTAATATTGCAAATAGGGAATATAATCTCAATATGGATTAGCCATTCAATTCAAACTGGAAGTCAAA ACCATACTGGAATATGCAACCAAAACATCATTACCTATAAAAATAGCACCTGGGTAAAGGACACAACTTCAGTGATATTAACCGGCAATTCATCTCTTTGTCCCATCCGTGGGTGGGCTATATACAGCAAA GACAATAGCATAAGAATTGGTTCCAAAGGAGACGTTTTTGTCATAAGAGAGCCCTTTATTTCATGTTCTCACTTGGAATGCAGGACCTTTTTTCTGACCGACAAAACTCACACATGCCCACCGTGCCCAG CACCTGAACTCCTGGGGGGACCGTCAGTCTTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAG TTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGG AGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGGAGGAGATGACCAAGAAC CAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCT TCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCACGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATAA (SEQ ID NO: 19).

[51] As noted above, the Fc region may contain one or more mutations, such as, for example, L234Y, L235V, L235Q, G236W, S239D, S239M, F243L, T256A, K290A, R292P, N297Q, S298A, Y300L, V305I, A330L, I332E, E333A, K334A, and/or P396L. Thus, this document specifically describes Fc regions comprising 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 aspartate (D) at residue 239, and phenylalanine (F) or leucine (L) at residue 243, threonine (T) or alanine (A) at residue 256, histidine (H) or aspartate (D) at residue 268, aspartate (D) or glutamate (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 aspartate (D) at residue 326, alanine (A), methionine (M) or leucine (L) at residue 330 and isoleucine (I) or glutamate (E) at residue 332, glutamate (E) or alanine (A) at residue 333, lysine (K), glutamate (E) or alanine (A) at residue 334 and/or proline (P) or leucine (L) at residue 396. In particular, it is intended that none, any substitution, or combination of two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, or seventeen of the substitutions mentioned herein may be present in the Fc region. Accordingly, in one aspect described herein are fusion proteins comprising an Fc region substitution of F243L, R292P, Y300L, V305I, and P396L, wherein the Na4-Fc sequence comprises MNPNQKITTIGSICLVVGLISLILQIGNIISIWISHSIQTGSQNHTGICNQNIITYKNSTWVKDTTSVILTGNSSLCPIRGWAIYSKDNSIRIGSKGDVFVIREPFISCSHLECRTFFLTDKTHTCPPCPAPELLGGPSVFLLPPKPKDTLMISRTPEVTCVVVDVSHEDPEV KFNWYVDGVEVHNAKTKPPEEQYNSTLRVVSILTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTK NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPLVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 20); which contains an Fc domain with the sequence GGPSVFLLPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPPEEQYNSTLRVVSILTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPLVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 25) which includes a CH2 domain with the sequence GGPSVFLLPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPPEEQYNSTLRVVSILTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK (SEQ ID NO: 26) and a CH3 domain with the sequence GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPLVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 27).

[52] In one aspect, the Na4-Fc fusion comprises the substitutions S239D, I332E, and A330L having an Fc domain with the sequence GGPDVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPLPEEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 21) that comprises a CH2 domain with the sequence GGPDVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPLPEEKTISKAK (SEQ ID NO: 22) and the CH2 domain with the sequence GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 24) and the full sequence MNPNQKITTIGSICLVVGLISLILQIGNIISIWISHSIQTGSQNHTGICNQNIITYKNSTWVKDTTSVILTGNSSLCPIRGWAIYSKDNSIRIGSKGDVFVIREPFISCSHLECRTFFLTDKTHTCPPCPAPELLGGPDVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEV KFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPLPEEKTISKAKGQPREPQVYTLPPSREEMTK NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 23).

[53] In one aspect, the Na4-Fc fusion protein may comprise 2 Fc domains linked via a hinge region. For example, the Na-Fc fusion may comprise the sequence MNPNQKITTIGSICLVVGLISLILQIGNIISIWISHSIQTGSQNHTGICNQNIITYKNSTWVKDTTSVILTGNSSLCPIRGWAIYSKDNSIRIGSKGDVFVIREPFISCSHLECRTFFLTDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAV EWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLT VLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 28).

[54] In another aspect, the Fc domains may be asymmetric variants, such that one Fc domain of the heavy chain may comprise L234Y/L235Q/G236W/S239M/H268D/D270E/S298A, while another Fc domain comprises D270E/K326D/A330M/K334E.

[55] In general, any amino acid substitutions are conservative: for example, limited to substitutions 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; and group 5: aspartate, glutamate, asparagine, and glutamine.

[56] This disclosure also contemplates a nucleic acid encoding any fusion protein described herein, such as, for example, SEQ ID NO: 19 encoding the Na-Fc fusion as set forth in SEQ ID NO: 18; SEQ ID NO: 29 encoding the Na-Fc fusion as set forth in SEQ ID NO: 20; SEQ ID NO: 30 encoding the Na-Fc fusion as set forth in SEQ ID NO: 23; and SEQ ID NO: 31 encoding the Na-2xFc fusion as set forth in SEQ ID NO: 28. Additionally contemplated herein are vectors comprising such a nucleic acid according to a claim and a cell comprising such a vector. Vectors and cells comprising 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.

[57] Compositions according to this description include compositions comprising Fc-linked feeder cells (FC), compositions comprising Fc-linked engineered plasma membrane (PM) particles, and compositions comprising Fc-linked engineered exosomes. Fc-linked engineered plasma membrane particles comprise PM nanoparticles derived from Fc-linked feeder cells. Fc-linked engineered exosomes comprised exosomes or other extracellular vesicles derived from Fc-linked feeder cells, as also described in more detail below. Alternatively, exosomes can be derived from other sources, such as platelets and megakaryocytes.

[58] As used herein, the term "Fc-linked" should be understood to refer to the linkage of the Fc domain in an inverted orientation (i.e., the amino-terminal end facing the interior of the cell) to the outer surface of a feeder cell or engineered particle via a transmembrane peptide. This can be achieved using the Fc fusion peptides described herein. Accordingly, one aspect of the disclosure provides a feeder cell composition comprising at least one Fc-linked feeder cell, i.e., a feeder cell comprising an Fc domain linked to the outer surface of the feeder cell, as described in more detail below. For example, a feeder cell can be genetically modified to express an Fc domain linked to the outer surface of the feeder cell, i.e., to express an Fc fusion peptide, as further described below. Another aspect of the disclosure provides a composition for increasing the number of NK cells that does not contain feeder cells, which includes at least one Fc-linked engineered particle, i.e., an engineered particle comprising an Fc domain linked in an inverted orientation to the outer surface of a feeder cell. In some aspects, the feeder cells can be engineered to express a ligand that can be labeled with a humanized antibody (such as, for example, CD20).

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

[60] In a composition for increasing the number of NK cells that does not contain feeder cells, the Fc-linked engineered PM particles optionally contain at least one NK cell effector agent. In one example, the Fc-linked engineered particle contains one NK cell effector agent, which is IL-15 or IL-21. The Fc-linked engineered PM particles can contain at least two or more different NK cell effector agents.

[61] In either a feeder cell composition or a feeder cell-free composition in which at least two NK cell effector agents are present, the second NK cell effector agent can be, for example, 41BBL. In either a feeder cell composition or a feeder cell-free composition for expanding NK cells in which 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, Notch ligand, ligands for NKp46 or BCM1/SLAMF2, TLR ligands and NKG2D ligands, or a cytokine. In such a typical composition, at least one additional NK cell effector agent is IL-15 or IL-21.

(a) Fc-linked feeder cells

[62] Provided herein are feeder cells comprising an Fc fusion peptide as described in detail 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 either irradiated autologous or allogeneic peripheral blood mononuclear cells (PBMC) or non-irradiated autologous or allogeneic PBMC, RPMI8866, HFWT, 721.221 or K562, as well as EBV-LCL, other non-HLA or low HLA cell lines, or primary tumors derived from patients that can be used as a tumor vaccine. Fc-linked feeder cells can be produced by transfecting or transducing feeder cells with any Fc fusion peptide as described herein using standard transduction or transfection techniques well known in the art. For example, the cDNA vectors for the Fc fusion peptides described herein can be ligated into an expression plasmid that provides for expression in bacterial (E. coli), insect, or mammalian cells. The cDNA vector can be tagged with a FLAG or HIS tag. Suitable transfection methods include nucleofection (or electroporation), calcium phosphate-mediated transfection, cationic polymer (e.g., DEAE-dextran or polyethyleneimine) transfection, viral transduction, viral transfection, virion transfection, liposome transfection, cationic liposome transfection, immunoliposome transfection, non-liposomal transfection, dendrimer transfection, heat shock transfection, magnetofection, lipofection, gene gun delivery, impalefection, sonoporation, optical transfection, and proprietary agent-enhanced nucleic acid uptake. Transfection techniques are well known in the art (see, for example, “Current Protocols in Molecular Biology” by Ausubel et al., John Wiley & Sons, New York, 2003 or “Molecular Cloning: A Laboratory Manual” by Sambrook & Russell, Cold Spring Harbor Press, Cold Spring Harbor, NY, 3rd edition, 2001). Alternatively, molecules can be introduced into a cell by microinjection. For example, molecules can be introduced into the cytoplasm or nucleus of the cells of interest. The amount of each molecule introduced into a cell can vary, but those skilled in the art are familiar with methods for determining an appropriate amount.

[63] It is understood that different molecules can be introduced into the cell simultaneously or sequentially. For example, an Fc fusion peptide and one or more “membrane-bound NK cell effector agents” can be introduced simultaneously into a feeder cell. Alternatively, one molecule can be introduced first and then the other molecule(s) can be introduced into the cell. For example, feeder cells, immediately after transfection or transduction with the Fc fusion peptide, can be further transfected with membrane-bound NK cell effector agents such as IL-15 and/or IL-21 and/or 41BBL and/or infected with EBV-LCL and/or other NK cell effector agent(s). Alternatively, feeder cells can be simultaneously transfected or transduced with an Fc fusion peptide and membrane-bound NK cell effector agents such as IL-15 and/or IL-21 and/or 41BBL and/or infected with EBV-LCL and/or other NK cell effector agent(s). Alternatively, feeder cells previously transfected or transduced and expressing membrane-bound NK cell effector agents such as IL-15 and/or IL-21 and/or 41BBL and/or infected with EBV-LCL and/or other NK cell effector agent(s) can be transfected or transduced with 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 obtain membrane-bound Fc.

[64] Typically, the cell is cultured under conditions suitable for the growth and/or maintenance of the cells. 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 will appreciate that cell culture techniques are known in the art and can and will vary depending on the cell type. In all cases, routine optimization can be used to determine the best methods for a particular cell type.

[65] Fc-linked feeder cells can be used in cell culture to directly stimulate NK cells or can be used to produce PM particles or feeder cell-derived exosomes.

(b) Fc-linked PM particles

[66] Fc-linked engineered PM particles include Fc-linked PM particles that can be prepared from Fc-linked feeder cells for NK cells using well-known methods. The PM particles are vesicles made from the plasma membrane of a cell or artificially created (e.g., liposomes). The PM particle can comprise a lipid bilayer or simply a single lipid layer. The PM particles can be prepared in a monolayer, multilayer, or inverted form. The PM particles can be prepared from Fc-linked feeder cells as described herein using known plasma membrane preparation protocols or liposome preparation protocols, such as those described in U.S. Patent No. 9,623,082, the entire disclosure of which is incorporated herein by reference. In some aspects, the PM particles disclosed herein have an average diameter of from about 170 nm to about 300 nm.

(b) Fc-linked exosomes

[67] Fc-linked exosomes as described herein can be obtained from exosome-secreting cells, which can be obtained from Fc-linked NK cell feeder cells using well-known methods, wherein the exosome is an extracellular product of the exosome-secreting cells, as described in U.S. Patent App. Pub. No. 20170333479, the entire disclosure of which is incorporated herein by reference. Exosomes contain lipids and proteins, and the identity of the proteins found in a particular exosome depends on the cells that produce them. The exosomes disclosed herein comprise an Fc fusion peptide as described herein (i.e., are Fc-linked), and optionally one or more stimulatory peptides (NK cell effector agents) present in the membrane of the exosome. Exosomes can be obtained, for example, from cell lines engineered for enhanced exosome formation or release. Such cell lines include, but are not limited to, Fc-linked cell lines as described above in Section II(a). Non-limiting cell lines include Fc-linked K562-mb15-41BBL and Fc-linked K562. In some aspects, the exosomes disclosed herein have an average diameter of from about 30 nm to about 100 nm, or up to about 160 nm. In one aspect, the average diameter of the exosomes is about 60-80 nm. The ability of exosomes to achieve particle sizes smaller than that readily achieved by PM particles means that exosomes can be more readily adapted for applications where a smaller size is advantageous. For example, exosomes may be advantageous in applications requiring diffusion across physiological barriers, enhanced biodistribution across tissue compartments, or intravenous injection.

(III) Compositions

[68] The disclosure provides various compositions for increasing the number of NK cells comprising Fc-linked feeder cells as described above, and in other aspects, compositions for increasing the number of NK cells that do not comprise feeder cells, comprising one or more Fc-linked engineered particles, such as PM particles or exosomes, as described above. Any of the Fc-linked feeder cells or Fc-linked engineered PM particles used in the compositions 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 the second is 41BBL. The Fc-linked feeder cells or Fc-linked engineered PM particles optionally comprise one or more additional NK cell effector agents as described above.

[69] A composition for increasing the number of NK cells that comprises a PM particle 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 may include magnetic microparticles, silica beads, polystyrene beads, latex beads, a particulate contrast agent, a particulate therapeutic agent, or any combination thereof.

[70] This application also provides an infusion formulation for increasing the number of NK cells comprising any of the compositions for increasing the number of NK cells disclosed herein in combination with a pharmaceutically acceptable carrier.

[71] Therapeutic pharmaceutical compositions can be prepared by combining Fc-linked feeder cells or engineered PM particles with a pharmaceutically acceptable carrier known in the art, as described, for example, in Remington: The Science and Practice of Pharmacy (19th ed.) ed. A. R. Gennaro, Mack Publishing Company, Easton, Pa. 1995. 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 from about 5 to about 8, and more preferably from about 7 to about 7.5.

[72] The pharmaceutical compositions may include carriers, thickeners, diluents, buffers, preservatives, surface-active agents, and the like in addition to the selected molecule. The pharmaceutical compositions may also include one or more active ingredients such as antimicrobials, anti-inflammatory agents, anesthetics, and the like.

[73] It will be apparent to those skilled in the art that certain carriers may be preferred depending, for example, on the route of administration and the concentration of the composition being administered. The pharmaceutical composition may be suitably formulated for administration by any of a number of known routes of administration to mammals, especially humans, depending on whether local or systemic treatment is desired and the area to be treated. Administration may be local (including ophthalmic, vaginal, rectal, intranasal), oral, by inhalation, or parenteral, such as by intravenous drip or injection, or subcutaneous, intraperitoneal, intramuscular, intracavitary or transdermal administration.

[74] Parenteral preparations include sterile aqueous or non-aqueous solutions, suspensions and emulsions. Examples of non-aqueous vehicles are propylene glycol, polyethylene glycol, vegetable oils such as olive oil and organic esters for injection such as ethyl oleate. Aqueous vehicles include water, alcoholic/aqueous solutions, emulsions or suspensions including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, Ringer's lactate or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (e.g., Ringer's dextrose) and the like. Preservatives and other additives such as, for example, antimicrobials, antioxidants, chelating agents and inert gases and the like may also be present.

[75] Formulations for topical application may include ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickening agents and the like may be necessary or desirable.

[76] 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, emulsifiers, dispersing agents or binders may be desirable.

[77] Some of the compositions can potentially be administered in the form of pharmaceutically acceptable acid addition salts or base addition salts formed by reaction with inorganic acids such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid and phosphoric acid, and organic acids such as 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 reaction with an inorganic base such as sodium hydroxide, ammonium hydroxide, potassium hydroxide, and organic bases such as mono-, di-, trialkylamines and arylamines, and substituted ethanolamines.

[78] Thus, the infusion formulation for increasing the number of NK cells can be formulated for parenteral infusion, arterial infusion, venous infusion, catheter-mediated infusion, intravenous, intraperitoneal, subcutaneous injection, oral or topical delivery.

[79] In one aspect, this disclosure contemplates any composition for expanding NK cells, produced in vitro or ex vivo, as described herein, administered or infused into a subject in need of expanding NK cells. Obviously, and as contemplated herein, infusion may occur in vitro with a commercial source of NK cells or ex vivo from a donor source (such as, for example, an allogeneic donor or an autologous donor source (i.e., the recipient subject receiving the expanded NK cells)).

[80] In another aspect, this disclosure contemplates an NK cell composition comprising an in vitro population of NK cells contacted with an Fc-linked feeder cell composition as disclosed herein, or an Fc-linked NK cell enhancing composition that does not comprise feeder cells as disclosed herein.

[81] In another aspect, this disclosure contemplates sources of NK cells that include, but are not limited to, peripheral blood, iPSC-derived NK cells, ESC-derived NK cells, NK cells having the Phe or Val high affinity Fc receptor polymorphism at 158, and genetically modified NK cells.

[82] In another aspect, this disclosure provides an expanded population of NK cells exposed in vitro to a composition for expanding the number of NK cells, wherein the composition is free of feeder cells and comprises at least one Fc-linked engineered particle as disclosed herein 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 population of NK cells can exhibit increased cytotoxicity compared to non-expanded NK cells. In various aspects, the expanded population of NK cells can exhibit cytotoxicity that is at least about 2, 5, or 10 times greater than non-expanded NK cells.

[83] In another aspect, the disclosure provides a composition comprising a therapeutic dose of NK cells, comprising an expanded population of NK cells as disclosed herein, optionally in combination with a pharmaceutically acceptable carrier. The expanded population of NK cells may exhibit higher levels of CD16 and other advantageous properties such as higher cytotoxicity and ADCC functionality. The amount of NK cells that provides a therapeutic dose will vary depending on a number of factors that are appreciated by those skilled in the art and are discussed, for example, in U.S. Patent No. 9,623,082. Factors include the age, gender, 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, a therapeutic dose may be from 1×10 ⁴ /kg to 1×10 ⁸ /kg per dose, which may be included in a single dose or divided into several doses. It should be understood that the therapeutic dose equivalent, as specified above, may alternatively be expressed in an amount per total body surface area.

[84] In another aspect, this disclosure also provides NK cell enhancing media compositions comprising any NK cell enhancing composition as disclosed herein in combination with a cell medium solution comprising at least one soluble medium component such as a cytokine, IL-2, IL-12, IL-15, IL-18, IL-21, NAM, ascorbate, or any combination thereof.

(IV) Methods

(a) Methods for enhancing NK cell cytotoxicity

[85] In one aspect, the disclosure provides a method for increasing NK cell cytotoxicity by expanding a starting population of NK cells using an NK cell expansion composition or formulation as disclosed herein. The disclosed methods provide a simple expansion platform that avoids the complex alternative expansion process of, for example, coating a solid support with a monoclonal antibody and using soluble cytokine(s) in solution. Instead, in the methods disclosed herein, a starting population of NK cells is obtained from a donor and exposed to an NK cell expansion composition as disclosed herein. The exposure may occur in vitro or in vivo. Fig. 4 is a schematic diagram of Fc stimulation of an NK cell in accordance with the disclosure. NK cells are contacted by one or more Fc-linked feeder cells, Fc-linked PM particles, or Fc-linked exosomes, or any combination thereof. The exposed Fc domain binds to CD16 on the NK cell surface, resulting in stimulation of NK cells to proliferate more rapidly and/or efficiently, producing NK cells with higher antitumor toxicity and NK cells with a more desirable overall phenotype.

[86] As shown in Fig. 4, the NK cell contacting composition may comprise any of the Fc-linked feeder cells or Fc-linked engineered PM particles or Fc-linked engineered exosomes disclosed herein. The engineered PM particles may be Fc-linked PM particles. In one aspect, the optional NK cell effector agent present is IL-21 or IL-15. The optional second NK cell effector agent present may be selected from 41BBL, IL-2, IL-12, IL-15, IL-18, IL-21, MICA, UBLP, 2B4, LFA-1, Notch ligand, ligands for NKp46 or BCM1/SLAMF2, TLR ligands, and NKG2D ligands. In one aspect, the optional NK cell effector agent present is 41BBL. The composition may further comprise 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, 2B4, LFA-1, Notch ligand, NKp46 or BCM1/SLAMF2 ligands, TLR ligands, and NKG2D ligands. The expansion of NK cells accomplished in this manner may be much greater than several fold (about 3-4 fold) in 10 days. In contrast, the expansion of NK cells according to the present methods can reach 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 up to 2000-fold increase in the number of NK cells in 16 days or more in a longer period of time. Thus, the disclosed methods are useful for large-scale production of NK cells. Sources of NK cells may be peripheral blood, spleen NK cells, lymphocyte preparations such as buffy coat, 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 Fc receptor polymorphisms such as Phe or Val at position 158, such as those known in the art and described, for example, in Blood (1997) 90:1109-14, and J Clin Invest. (1997) 100:1059-70. Such genetically modified sources of NK cells may be constructed using methods known in the art. Alternatively, NK cells can be obtained from a cell donor that carries the desired polymorphism, and the donor cells can be used as a starting population of NK cells that are targeted by the methods and composition described herein. Thus, in this context, "genetically modified" includes naturally occurring NK cells that carry the polymorphism. The method can be applied to NK cells of humans or other animals.

[87] Furthermore, the disclosed methods have the additional advantage of providing cells with increased cytotoxicity and ADCC functionality. A parent population of NK cells expanded according to the disclosed methods yields an expanded population of NK cells that exhibits at least about 2-fold the cytotoxicity of the parent population of NK cells, at least about 4-fold the cytotoxicity of the parent population of NK cells, at least about 5-fold the cytotoxicity of the parent population of NK cells, at least about 8-fold the cytotoxicity of the parent population of NK cells, or at least about 10-fold the cytotoxicity of the parent population of NK cells. Moreover, NK cells expanded according to the disclosed methods exhibit increased cytotoxicity for a target that may be ADCC competent. Higher expression of ADCC associated proteins such as, in a non-limiting example, CD16; or other NK cell ligands such as, in a non-limiting example, NKG2D, NKp46, CD62L, can be used to assess the relative cytotoxicity of expanded NK cells compared to non-expanded NK cells or NK cells expanded under other conditions. Markers such as NKG2D, NKp46, etc. are indicators of NK cells in an activated state. In combination, the markers can serve as a signal of increased cytotoxicity even if the cytotoxicity cannot be directly assessed. For example, an expanded NK cell population as disclosed herein can exhibit increased killing of tumor targets or secrete higher amounts of anti-tumor cytokines (IFN, TNF) compared to non-expanded NK cells. In another aspect, an expanded population of NK cells as disclosed herein may exhibit increased expression of NKG2D, NKp46, and CD16 compared to non-expanded NK cells. Various means for determining the amount of a particular protein to assess the activation state of NK cells are known in the art, including spectrometric methods such as flow cytometry or immunodetection methods such as Western blotting, enzyme-linked immunosorbent assay (ELISA), protein immunoprecipitation; immunoelectrophoresis, or immunostaining.

[88] Furthermore, the expanded NK cell population as disclosed herein may exhibit improved ability to tolerate cryopreservation, maintaining viability and cytotoxicity, as well as subsequent freezing and thawing.

[89] The NK cell composition expanded with Fc domain expressing feeder cells exhibits enhanced cytotoxicity against SKOV3 ovarian cancer target cells as shown in Fig. 2 and Fig. 3. The NK cell compositions expanded with Fc domain expressing feeder cells exhibit an enhanced phenotype with increased levels of CD16, NKp46, and CD62L. Such NK cells with an enhanced phenotype may have enhanced therapeutic efficacy. Increased levels of CD16 may allow for increased ability to interact with antibody-coated target cells. Increased NKp46 may have greater ability to bind activating ligands. Increased levels of CD62L as an L-selectin ligand may enhance delivery of NK cells to lymphatic or bone compartments.

(b) Therapeutic methods

[90] The compositions and methods disclosed herein can be used for a variety of therapeutic, diagnostic, industrial, and research purposes. In some aspects, this disclosure can be used to treat cancer. Accordingly, in one aspect, disclosed herein are methods for "treating, inhibiting, reducing, and/or preventing cancer, cancer recurrence, metastasis, or an infectious disease such as a viral infection or bacterial infection in a subject, comprising administering to a subject in need thereof an effective amount of a composition or expanded population of NK cells as described herein.

[91] The cancer may be selected from, but is not limited to, hematological 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 aspect, the cancer comprises a solid tumor. In another aspect, the cancer is selected from acute myeloid leukemia, myelodysplastic syndrome, chronic myeloid leukemia, acute lymphoblastic leukemia, myelofibrosis, multiple myeloma. In another aspect, the cancer is selected from leukemia, lymphoma, sarcoma, carcinoma and can occur in the bone marrow, brain, lung, breast, pancreas, liver, head and neck, skin, reproductive tract, prostate, colon, liver, kidney, intraperitoneal space, bone, joint, eye.

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

[93] Any of the disclosed methods of treatment may further comprise administering to the subject (sequentially, simultaneously, or as a single formulation) an additional therapeutic agent or regimen in combination with an effective amount of the composition or expanded population of NK cells as described herein. The additional therapeutic agent may be a drug-based formulation regimen such as Cy-Flu, Bu-Flu, Flu-Mel, or the like, with an adjustment in dosage or dosing. Alternatively, the additional therapeutic agent may be a prophylactic agent against graft-versus-host disease (GvHD), such as, but not limited to, cyclophosphamide. Alternatively, the additional therapeutic agents or regimens may be selected from chemotherapeutic agents and regimens such as, in a non-limiting example, those known by the abbreviations 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 +3. For example, it is contemplated herein that the disclosed methods for inhibiting, reducing, and/or preventing metastasis and/or recurrence of cancer may include administering any anticancer agent known in the art, including, but not limited to, Abemaciclib, Abiraterone Acetate, Abitrexate (Methotrexate), Abraxane (a composition of nanoparticles stabilized by an albumin and paclitaxel conjugate), ABVD, ABVE, ABVE-PC, AC, AC-T, Adcetris (Brentuximab Vedotin), ADE, Ado-Trastuzumab Emtansine, Adriamycin (Doxorubicin Hydrochloride), Afatinib Dimaleate, Afinitor (Everolimus), Akynzeo (Netupitant and 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), Arranon (Nelarabine), Arsenic Trioxide, Arzerra (Ofatumumab), Erwinia chrysanthemi Asparaginase, 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 I 131 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--For topical use, Carboplatin, CARBOPLATIN-TAXOL, Carfilzomib, Carmubris (Carmustine), Carmustine, Carmustine implant, Casodex (Bicalutamide), CEM, Ceritinib, Cerubidine (Daunorubicin Hydrochloride), Cervarix (Recombinant bivalent HPV vaccine), Cetuximab, CEV, Chlorambucil, CHLORAMBUCIL-PREDNISONE, CHOP, Cisplatin, Cladribine, Clafen (Cyclophosphamide), Clofarabine, Clofarex (Clofarabine), Clolar (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 Liposomes, Cytosar-U (Cytarabine), Cytoxan (Cyclophosphamide), Dabrafenib, Dacarbazine, Dacogen (Decitabine), Dactinomycin, Daratumumab, Darzalex (Daratumumab), Dasatinib, Daunorubicin Hydrochloride, Daunorubicin Hydrochloride and CytarabineDecitabine Liposomes, Defibrotide Sodium, Defitelio (Defibrotide Sodium), Degarelix, Denileukin Diftitox, Denosumab, DepoCyt (Cytarabine Liposomes), Dexamethasone, Dexrazoxane Hydrochloride, Dinutuximab, Docetaxel, Doxil (Doxorubicin Hydrochloride Liposomes), Doxorubicin Hydrochloride, Doxorubicin Hydrochloride Liposomes, Dox-SL (Doxorubicin Hydrochloride Liposomes), 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 Hydrochloride , EPOCH, Erbitux (Cetuximab), Eribulin Mesylate, Erivedge (Vismodegib), Erlotinib Hydrochloride, Erwinaze (Asparaginase) Erwinia chrysanthemi), Ethyol (Amifostine), Etopophos (Etoposide Phosphate), Etoposide, Etoposide Phosphate, Evacet (Doxorubicin Hydrochloride Liposomes), Everolimus, Evista , (Raloxifene Hydrochloride), Evomela (Melphalan Hydrochloride), Exemestane, 5-FU (Fluorouracil Injection), 5-FU (Fluorouracil--Topical, Fareston (Toremifene), Farydak (Panobinostat), Faslodex (Fulvestrant), FEC, 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, Fulvestrant, Gardasil (Recombinant quadrivalent HPV vaccine), Gardasil 9 (Recombinant non-valent HPV vaccine), Gazyva (Obinutuzumab), Gefitinib, Gemcitabine Hydrochloride, GEMCITABINE-CISPLATIN, GEMCITABINE-OXALIPLATIN, Gemtuzumab Ozogamicin, Gemzar (Gemcitabine Hydrochloride), Gilotrif (Afatinib Dimaleate), Gleevec (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 Non-Avalent 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 Alpha-2b, Interleukin-2 (Aldesleukin), Intron A (Recombinant Interferon Alpha-2b), Iodine I 131 Tositumomab and Tositumomab, Ipilimumab, Iressa (Gefitinib), Irinotecan Hydrochloride, Irinotecan Hydrochloride Liposomes, Istodax (Romidepsin), Ixabepilone, Ixazomib Citrate, Ixempra (Ixabepilone), Jakafi (Ruxolitinib Phosphate), JEB, Jevtana (Cabazitaxel), Kadcyla (Ado-Trastuzumab Emtansine), Keoxifene (Raloxifene Hydrochloride), Kepivance (Palifermin), Keytruda (Pembrolizumab), Kisqali (Ribociclib), Kymriah (Tisagenlecleucel), Kyprolis (Carfilzomib), Lanreotide Acetate, Lapatinib Ditosylate, 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 Liposomes), Lomustine, Lonsurf (Trifluridine and Tipiracil Hydrochloride), Lupron (Leuprolide Acetate), Lupron Depot (Leuprolide Acetate), Lupron Depot-Ped (Leuprolide Acetate), Lynparza (Olaparib), Marqibo (Vincristine Sulfate Liposomes), 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 (Albumin Paclitaxel Conjugate Nanoparticle Composition), 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 Monohydrate, Nivolumab, Nolvadex (Tamoxifen Citrate), Nplate (Romiplostim), Obinutuzumab, Odomzo (Sonidegib), OEPA, Ofatumumab, OFF, Olaparib, Olaratumab, Omacetaxine Mepesuccinate, Oncaspar (Pegaspargase), Ondansetron Hydrochloride, Onivyde (Irinotecan Hydrochloride Liposomes), Ontak (Denileukin Diftitox), Opdivo (Nivolumab), OPPA, Osimertinib, Oxaliplatin, Paclitaxel, Albumin Paclitaxel Conjugate Nanoparticle Formulation, PAD, Palbociclib, Palifermin, Palonosetron Hydrochloride, Palonosetron Hydrochloride and Netupitant, Pamidronate Disodium, Panitumumab, Panobinostat, Paraplat (Carboplatin), Paraplatin (Carboplatin), Pazopanib Hydrochloride, PCV, PEB, Pegaspargase, Pegfilgrastim, Peg-Interferon Alfa-2b, PEG-Intron (Peg-Interferon Alfa-2b), Pembrolizumab, Pemetrexed Disodium, Perjeta (Pertuzumab), Pertuzumab, Platinol (Cisplatin), Platinol-AQ (Cisplatin), Plerixafor, Pomalidomide, Pomalyst (Pomalidomide), Ponatinib Hydrochloride, Portrazza (Necitumumab), Pralatrexate, Prednisone, Procarbazine Hydrochloride , Proleukin (Aldesleukin), Prolia (Denosumab), Promacta (Eltrombopag Olamine), Propranolol Hydrochloride, Provenge (Sipuleucel-T), Purinethol (Mercaptopurine), Purixan (Mercaptopurine), Radium 223 Dichloride, Raloxifene Hydrochloride, Ramucirumab, Rasburicase, R-CHOP, R-CVP, Recombinant Bivalent Human Papillomavirus (HPV) Vaccine, Recombinant Non-Novalent Human Papillomavirus (HPV) Vaccine, Recombinant Quadrivalent Human Papillomavirus (HPV) Vaccine, Recombinant Interferon Alpha-2b, Regorafenib, Relistor (Methylnaltrexone Bromide), R-EPOCH, Revlimid (Lenalidomide), Rheumatrex (Methotrexate), Ribociclib, R-ICE, Rituxan (Rituximab), Rituxan Hycela (Rituximab and Human Hyaluronidase), Rituximab, Rituximab and , Human Hyaluronidase, Rolapitant Hydrochloride, Romidepsin, Romiplostim, Rubidomycin (Daunorubicin Hydrochloride), Rubraca (Rucaparib Camsylate), Rucaparib Camsylate, Ruxolitinib 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), Stivarga (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, Tarabine PFS (Cytarabine), Tarceva (Erlotinib Hydrochloride), Targretin (Bexarotene), Tasigna (Nilotinib), Taxol (Paclitaxel), Taxotere (Docetaxel), Tecentriq, (Atezolizumab), Temodar (Temozolomide), Temozolomide, Temsirolimus, Thalidomide, Thalomid (Thalidomide), Thioguanine, Thiotepa, Tisagenlecleucel, Tolak (Fluorouracil--Topical, Topotecan Hydrochloride, Toremifene, Torisel (Temsirolimus), Tositumomab and Iodine I 131 Tositumomab, Totect (Dexrazoxane Hydrochloride), TPF, Trabectedin, Trametinib, Trastuzumab, Treanda (Bendamustine Hydrochloride), Trifluridine and Tipiracil Hydrochloride, Trisenox (Arsenic Trioxide), Tykerb (Lapatinib Ditosylate), Unituxin (Dinutuximab), Uridine Triacetate, VAC, Vandetanib, VAMP, Varubi (Rolapitant Hydrochloride), Vectibix (Panitumumab), VeIP, Velban (Vinblastine Sulfate), Velcade (Bortezomib), Velsar (Vinblastine Sulfate), Vemurafenib, Venclexta (Venetoclax), Venetoclax, Verzenio (Abemaciclib), Viadur (Leuprolide Acetate), Vidaza (Azacitidine), Vinblastine Sulfate, Vincasar PFS (Vincristine Sulfate), Vincristine Sulfate, Vincristine Sulfate Liposomes, Vinorelbine Tartrate, VIP, Vismodegib, Vistogard (Uridine Triacetate), Voraxaze (Glucarpidase), Vorinostat, Votrient (Pazopanib Hydrochloride), Vyxeos (Daunorubicin Hydrochloride and Cytarabine Liposomes), Wellcovorin (Leucovorin Calcium), Xalkori (Crizotinib), Xeloda (Capecitabine), XELIRI, XELOX, Xgeva (Denosumab), Xofigo (Radium 223 Dichloride), Xtandi (Enzalutamide), Yervoy (Ipilimumab), Yondelis (Trabectedin), Zaltrap (Ziv-Aflibercept), Zarxio (Filgrastim), Zejula (Niraparib Tosylate Monohydrate), 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), Zykadia (Ceritinib), and/or Zytiga (Abiraterone Acetate). This document also discusses chemotherapeutic agents that are PD1/PDL1 blockade inhibitors (such as, for example, lambrolizumab, nivolumab, pembrolizumab, pidilizumab, BMS-936559, Atezolizumab, Durvalumab, or Avelumab).

[94] Alternatively, the additional therapeutic agent may be an antiviral agent selected from, but not limited to, a 5-substituted 2-deoxyuridine analog, a nucleoside analog, a (non-nucleoside) pyrophosphate analog, a nucleoside reverse transcriptase inhibitor (NRTI) and a non-nucleoside reverse transcriptase inhibitor (NNRTI), a protease inhibitor (PI) and integrase inhibitor, an integrase inhibitor and an acyclic guanosine analog, an acyclic nucleoside phosphonate (ANP) analog, an inhibitor of NS5A and NS5B of hepatitis C virus (HCV) and an inhibitor of influenza virus, an immunostimulant, an interferon, an oligonucleotide, and an antimitotic inhibitor. Non-limiting examples of antiviral agents include acyclovir, famciclovir, valacyclovir, penciclovir, ganciclovir, ritonavir, lopinavir, saquinavir, etc.; cimetidine; ranitidine; captopril; metformin; bupropion; fexofenadine; oxcarbazepine; leveteracetam; tramadol; or any of their isomers, tautomers, analogs, polymorphs, solvates, derivatives, or pharmaceutically acceptable salts.

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

[96]

(V) Sets

[97] A further aspect of the disclosure provides kits comprising at least one of the fusion peptides as detailed above and/or at least one of the Fc-linked feeder cells and/or at least one Fc-linked engineered particle (PM particle and/or exosome) as described above. The fusion peptides can be provided in suitable containers along with other kit components such as cellular reagents, cell growth media, selection media, protein purification reagents, buffers, and the like. The kits provided herein typically include instructions for performing the methods detailed below. The instructions included in the kits can be affixed to the packaging material or can be included as a package insert. Although the instructions are typically written or printed materials, they are not limited to such. The disclosure contemplates any medium capable of storing such instructions and communicating them to an end user. 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. The term "instructions" as used herein may include the address of an Internet site that provides the instructions.

EXAMPLES

Example 1: Engagement of CD16 via the Fc region in increased proliferation rate over 14 days

[98] The K562 cell line, a cell line expressing 41BBL and membrane-associated IL-21 (“CSTX-002”), was generated.

[99] A separate sample of K562 cells was transfected with NA-Fc to generate Fc-coupled K562 cells (“CSTX002-Fc”).

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

[101] PBMCs isolated from leukocytes using a Ficoll-Paque density gradient were grown in CellGro SCGM 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, were co-cultured at a ratio of 1 feeder cell per NK cell. Cells were cultured at 37°C in a humidified incubator with 5% CO2. Starting on day 5, culture media were changed every other day, replacing half of the medium with fresh medium supplemented with 100 U IL2. Cells were counted every other day, and culture status was checked regularly starting on day 7.

[102] Figure 5 shows a graph of NK cell growth as a function of days of culture, showing that engagement of CD16 via the Fc site increases the proliferation rate over 14 days. NK cells were expanded from PBMCs obtained from two donors, L54 (circles) or L44 (squares), using the CSTX002 (open symbols) or CSTX002-Fc (closed symbols) cell lines as feeder cells. Engagement of CD16 enhances NK cell proliferation. NK cells from both donors proliferated at similar rates when stimulated with IL21 alone or IL21 and Fc until day 14, at which point Fc-stimulated cultures divided at a higher rate than those stimulated with IL21 alone.

Example 2: Natural killer cell cytotoxicity is enhanced in the presence of Fc-linked feeder cells

[103] CSTX002 cells and CSTX002-Fc cells were prepared as described in Example 1. Cytotoxicity assays were performed as follows. The target ovarian cancer-derived cell line SKOV3 transfected with green fluorescent protein (GFP) was used as targets to measure the antitumor cytotoxicity of effector NK cells. Target cells were cultured alone (control wells) or co-cultured at a concentration of 0.5× ¹⁰⁶ cells/mL with NK cells at the indicated effector-to-target (E:T) ratios for 45 min at 37°C, under 5% CO2. Cells were then centrifuged and resuspended in Annexin V labeling buffer containing the Annexin V-PacBlue antibody and incubated for 15 min at 4°C before flow cytometric analysis. Cytotoxicity was determined based on the absolute number of viable target cells (GFP+/Annexin V-) remaining in each effector well (VTCE:T) and was normalized to the mean VTC in target-only control wells (VTCT ctrl).

Cytotoxicity ^E:T (%) = (VTC ^E:T / Average VTC ^{T ctrl} .)*100

[104] Figure 6 shows two graphs, each showing the cytotoxicity of NK cells expanded from PBMCs obtained from two different donors (L43 and L44). For each donor, NK cells were expanded from PBMCs using either the CSTX002 (●) or CSTX002-Fc (■) cell lines as feeder cells. NK cells expanded from each of the two different donors with CSTX002-Fc were found to have enhanced cytotoxicity to SKOV3 cells.

Example 3: Cytotoxicity of natural killer cells from PBMCs from a poorly responsive donor

[105] PBMCs obtained from a donor previously shown to lack cytotoxicity towards SKOV3 cells when expanded by stimulation with CSTX002 cells (no added Fc). NK cells were expanded from PBMCs previously shown to be weakly responsive using either the CSTX002 (●) or CSTX002-Fc (■) cell lines as feeder cells as described in Example 1. The two different resulting NK cell populations were then tested for cytotoxicity as also described in Example 1, but using SKOV3 transformed to express Fc. Figure 7 is a graph of the cytotoxicity of NK cells expanded from PBMCs using either CSTX002 (●) or CSTX002-Fc (■). The results shown in Fig. 7, demonstrate that PBMC-derived NK cells from a donor with weak cytotoxic reactivity against SKOV3 cells respond well when expanded with Fc-coupled feeder cells (CSTX002-Fc), exhibiting cytotoxicity towards tumor targets that have an Fc domain coupled compared to those PBMC-derived NK cells from the same weak donor expanded with non-Fc-coupled feeder cells (CSTX002). NK cells expanded with CSTX002-Fc will be better engaged in antibody-dependent cellular cytotoxicity and will exhibit higher antibody-coupled tumor target killing activity.

Example 4: Desired receptor expression

[106] NK cells were expanded from PBMCs obtained from two L43 (●) or L44 (■) donors using either the CSTX002 or CSTX002-Fc cell lines as feeder cells as described in Example 1. The resulting NK cells were then analyzed. Figure 8 is a series of six (6) graphs, each showing comparative receptor expression by NK cells expanded with CSTX002 feeder cells with or without membrane-bound Fc. NK cells were expanded from PBMCs obtained from two L43 (●) or L44 (■) donors using either the CSTX002 or CSTX002-Fc cell lines as feeder cells. The expanded NK cells were analyzed for the expression of receptors considered important for cytotoxic function or homing. NK cells expanded with CSTX002-Fc have higher expression of CD16, NKp46 and CD62L compared to cells expanded with CSTX002.

Example 5: Fc-linked plasma membrane particles

[107] Engineered K562 cells, a cell line expressing 41BBL and membrane-bound IL-21, were processed as described in U.S. Patent No. 9,623,082 to produce PM-mb21-41BBL plasma membrane vesicles or “CSTX002” particles or PM21 particles. Briefly, K562 were cultured in RPMI medium supplemented with 10% FBS and the culture was scaled up to 1 L. Cells were harvested by centrifugation at 1000×g, washed with cold PBS with 10 mM EDTA and resuspended in lysis buffer (50 mM HEPES, pH 7.4, protease inhibitor cocktail). Cells are disrupted and the lysate solution is centrifuged at 300×g for 15 min to remove any remaining intact cells. Crude plasma membranes are separated from cytosolic components by centrifugation for 30 min at 4°C. Crude membranes are resuspended and further purified using a sucrose density gradient to obtain pure plasma membrane vesicles termed PM-mb21-41BBL.

[108] A separate sample of K562 cells was transfected for Fc expression to produce Fc-linked K562, which was then processed as described above to produce Fc-linked PM-mb21-41BBL plasma membrane vesicles or “CSTX002-Fc” particles.

[109] Peripheral blood mononuclear cells (PBMCs) were isolated from a single donor and divided into samples. NK cell expansion from PBMCs was tested in the presence of CSTX002 membrane particles or CSTX002-Fc membrane particles. The amount of each membrane particle was 200 μg of membrane protein per 1 ml of culture. PBMCs isolated from blood using a Ficoll-Paque density gradient were grown in SCGM CellGro medium supplemented with 10% FBS and 100 U/ml IL-2. Cells were cultured at 37°C in a humidified incubator with 5% CO2. Starting from day 5, the culture media were changed every other day by replacing half of the medium with fresh medium, and the amounts of membrane removed were replaced by replacing the medium. Cells were counted every other day, and the culture status was checked on days 7, 10, and 14.

[110] Cytotoxicity assays are performed as described in Example 1. NK cells expanded with CSTX002-Fc will have enhanced cytotoxicity to SKOV3 cells.

SEQUENCES

SEQ ID NO:29

ATGAATCCAAATCAGAAAATAACAACCATTGGATCAATCTGTCTGGTAGTCGGACTAATTAGCCTAATATTGCAAATAGGGAATATAATCTCAATATGGATTAGCCATTCAATTCAAACTGGAAGTCAAA ACCATACTGGAATATGCAACCAAAACATCATTACCTATAAAAATAGCACCTGGGTAAAGGACACAACTTCAGTGATATTAACCGGCAATTCATCTCTTTGTCCCATCCGTGGGTGGGCTATATACAGCAA AGACAATAGCATAAGAATTGGTTCCAAAGGAGACGTTTTTGTCATAAGAGAGCCCTTTATTTCATGTTCTCACTTGGAATGCAGGACCTTTTTTCTGACCGACAAAACTCACACATGCCCACCGTGCCCA GCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCCTGCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCA AGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCCGGAGGAGCAGTACAACAGCACGCTGCGTGTGGTCAGCATTCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAA GGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAG AACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCTGGTGCTGGACTCCGACGGCTCCT TCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCACGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA

SEQ ID NO:30

ATGAATCCAAATCAGAAAATAACAACCATTGGATCAATCTGTCTGGTAGTCGGACTAATTAGCCTAATATTGCAAATAGGGAATATAATCTCAATATGGATTAGCCATTCAATTCAAACTGGAAGTCAAA ACCATACTGGAATATGCAACCAAAACATCATTACCTATAAAAATAGCACCTGGGTAAAGGACACAACTTCAGTGATATTAACCGGCAATTCATCTCTTTGTCCCATCCGTGGGTGGGCTATATACAGCAAA GACAATAGCATAAGAATTGGTTCCAAAGGAGACGTTTTTGTCATAAGAGAGCCCTTTATTTCATGTTCTCACTTGGAATGCAGGACCTTTTTTCTGACCGACAAAACTCACACATGCCCACCGTGCCCAGC ACCTGAACTCCTGGGGGGACCGGATGTCTTTCCTTCTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGT TCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAG TACAAGTGCAAGGTCTCCAACAAAGCCCTCCCACTGCCCGAAGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGGAGGAGATGACCAAGAACCA GGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCC TCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCACGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATAATAA

SEQ ID NO:31

ATGAATCCAAATCAGAAAATAACAACCATTGGATCAATCTGTCTGGTAGTCGGACTAATTAGCCTAATATTGCAAATAGGGAATATAATCTCAATATGGATTAGCCATTCAATTCAAACTGGAAGTCAAAACCATACTGGAATATGCAACCAAAACATCATTACCTATAAAAATAGCACCTGGGTAAAGGACACAACTTCAGTGATATTAACCGG CAATTCATCTCTTTGTCCCATCCGTGGGTGGGCTATATACAGCAAAGACAATAGCATAAGAATTGGTTCCAAAGGAGACGTTTTTGTCATAAGAGAGCCCTTTATTTCATGTTCTCACTTGGAATGCAGGACCTTTTTTCTGACCGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCC CCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAAT GGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCG GAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCACGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAAGACAAAACTCACACATGCCCACCGTGCCCAGCACC TGAACTCCTGGGGGGACCGATGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCA GCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCACTGCCCGAAGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGC GACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCACGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA

--->

SEQUENCE LIST

<110> University of Central Florida Research Foundation, Inc.

<120> COMPOSITIONS AND METHODS FOR STIMULATING NATURAL KILLER CELLS

<130> 10613-070WO1

<160> 31

<170> PatentIn version 3.5

<210> 1

<211> 50

<212> PROTEIN

<213> Homo sapiens

<400> 1

Met Asn Pro Asn Gln Lys Ile Thr Thr Ile Gly Ser Ile Cys Leu Val

1 5 10 15

Val Gly Leu Ile Ser Leu Ile Leu Gln Ile Gly Asn Ile Ile Ser Ile

20 25 30

Trp Ile Ser His Ser Ile Gln Thr Gly Ser Gln Asn His Thr Gly Ile

35 40 45

Cys Asn

50

<210> 2

<211> 6

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 2

Leu Glu Gly Gly Gly Ser

1 5

<210> 3

<211> 4

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 3

Thr Gly Ser Gly

1

<210> 4

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 4

Gly Gly Ser Gly Gly Gly Ser Gly

1 5

<210> 5

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 5

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10

<210> 6

<211> 4

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 6

Gly Gly Gly Ser

1

<210> 7

<211> 6

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 7

Gly Ser Gly Gly Gly Gly

1 5

<210> 8

<211> 5

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 8

Glu Ala Ala Ala Lys

1 5

<210> 9

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 9

Ala Glu Ala Ala Ala Lys Ala

1 5

<210> 10

<211> 5

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 10

Pro Ala Pro Ala Pro

1 5

<210> 11

<211> 15

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 11

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu

1 5 10 15

<210> 12

<211> 11

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 12

Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu

1 5 10

<210> 13

<211> 279

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 13

Met Asn Pro Asn Gln Lys Ile Thr Thr Ile Gly Ser Ile Cys Leu Val

1 5 10 15

Val Gly Leu Ile Ser Leu Ile Leu Gln Ile Gly Asn Ile Ile Ser Ile

20 25 30

Trp Ile Ser His Ser Ile Gln Thr Gly Ser Gln Asn His Thr Gly Ile

35 40 45

Cys Asn Arg Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro

50 55 60

Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

65 70 75 80

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

85 90 95

Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp

100 105 110

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr

115 120 125

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

130 135 140

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu

145 150 155 160

Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

165 170 175

Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys

180 185 190

Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

195 200 205

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

210 215 220

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

225 230 235 240

Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser

245 250 255

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

260 265 270

Leu Ser Leu Ser Pro Gly Lys

275

<210> 14

<211> 212

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 14

Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu

1 5 10 15

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

20 25 30

His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu

35 40 45

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr

50 55 60

Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn

65 70 75 80

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro

85 90 95

Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

100 105 110

Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val

115 120 125

Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

130 135 140

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

145 150 155 160

Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr

165 170 175

Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val

180 185 190

Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

195 200 205

Ser Pro Gly Lys

210

<210> 15

<211> 105

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 15

Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu

1 5 10 15

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

20 25 30

His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu

35 40 45

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr

50 55 60

Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn

65 70 75 80

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro

85 90 95

Ile Glu Lys Thr Ile Ser Lys Ala Lys

100 105

<210> 16

<211> 107

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 16

Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu

1 5 10 15

Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe

20 25 30

Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu

35 40 45

Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe

50 55 60

Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly

65 70 75 80

Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr

85 90 95

Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

100 105

<210> 17

<211> 120

<212> PROTEIN

<213> Homo sapiens

<400> 17

Met Asn Pro Asn Gln Lys Ile Thr Thr Ile Gly Ser Ile Cys Leu Val

1 5 10 15

Val Gly Leu Ile Ser Leu Ile Leu Gln Ile Gly Asn Ile Ile Ser Ile

20 25 30

Trp Ile Ser His Ser Ile Gln Thr Gly Ser Gln Asn His Thr Gly Ile

35 40 45

Cys Asn Gln Asn Ile Ile Thr Tyr Lys Asn Ser Thr Trp Val Lys Asp

50 55 60

Thr Thr Ser Val Ile Leu Thr Gly Asn Ser Ser Leu Cys Pro Ile Arg

65 70 75 80

Gly Trp Ala Ile Tyr Ser Lys Asp Asn Ser Ile Arg Ile Gly Ser Lys

85 90 95

Gly Asp Val Phe Val Ile Arg Glu Pro Phe Ile Ser Cys Ser His Leu

100 105 110

Glu Cys Arg Thr Phe Phe Leu Thr

115 120

<210> 18

<211> 347

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 18

Met Asn Pro Asn Gln Lys Ile Thr Thr Ile Gly Ser Ile Cys Leu Val

1 5 10 15

Val Gly Leu Ile Ser Leu Ile Leu Gln Ile Gly Asn Ile Ile Ser Ile

20 25 30

Trp Ile Ser His Ser Ile Gln Thr Gly Ser Gln Asn His Thr Gly Ile

35 40 45

Cys Asn Gln Asn Ile Ile Thr Tyr Lys Asn Ser Thr Trp Val Lys Asp

50 55 60

Thr Thr Ser Val Ile Leu Thr Gly Asn Ser Ser Leu Cys Pro Ile Arg

65 70 75 80

Gly Trp Ala Ile Tyr Ser Lys Asp Asn Ser Ile Arg Ile Gly Ser Lys

85 90 95

Gly Asp Val Phe Val Ile Arg Glu Pro Phe Ile Ser Cys Ser His Leu

100 105 110

Glu Cys Arg Thr Phe Phe Leu Thr Asp Lys Thr His Thr Cys Pro Pro

115 120 125

Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro

130 135 140

Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr

145 150 155 160

Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn

165 170 175

Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg

180 185 190

Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val

195 200 205

Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser

210 215 220

Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys

225 230 235 240

Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu

245 250 255

Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe

260 265 270

Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu

275 280 285

Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe

290 295 300

Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly

305 310 315 320

Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr

325 330 335

Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

340 345

<210> 19

<211> 1044

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 19

atgaatccaa atcagaaaat aacaaccatt ggatcaatct gtctggtagt cggactaatt 60

agcctaatat tgcaaatagg gaatataatc tcaatatgga ttagccattc aattcaaact 120

ggaagtcaaa accatactgg aatatgcaac caaaacatca ttacctataa aaatagcacc 180

tgggtaaagg acacaacttc agtgatatta accggcaatt catctctttg tcccatccgt 240

gggtgggcta tatacagcaa agacaatagc ataagaattg gttccaaagg agacgttttt 300

gtcataagag agccctttat ttcatgttct cacttggaat gcaggacctt ttttctgacc 360

gacaaaactc acacatgccc accgtgccca gcacctgaac tcctgggggg accgtcagtc 420

ttcctcttcc ccccaaaacc caaggacacc ctcatgatct cccggacccc tgaggtcaca 480

tgcgtggtgg tggacgtgag ccacgaagac cctgaggtca agttcaactg gtacgtggac 540

ggcgtggagg tgcataatgc caagacaaag ccgcgggagg agcagtacaa cagcacgtac 600

cgtgtggtca gcgtcctcac cgtcctgcac caggactggc tgaatggcaa ggagtacaag 660

tgcaaggtct ccaacaaagc cctcccagcc cccatcgaga aaaccatctc caaagccaaa 720

gggcagcccc gagaaccaca ggtgtacacc ctgcccccat cccgggagga gatgaccaag 780

aaccaggtca gcctgacctg cctggtcaaa ggcttctatc ccagcgacat cgccgtggag 840

tggggagagca atgggcagcc ggagaacaac tacaagacca cgcctcccgt gctggactcc 900

gacggctcct tcttcctcta cagcaagctc accgtggaca agagcaggtg gcagcagggg 960

aacgtcttct catgctccgt gatgcacgag gctctgcaca accactacac gcagaagagc 1020

ctctccctgt ctccggggtaa ataa 1044

<210> 20

<211> 347

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 20

Met Asn Pro Asn Gln Lys Ile Thr Thr Ile Gly Ser Ile Cys Leu Val

1 5 10 15

Val Gly Leu Ile Ser Leu Ile Leu Gln Ile Gly Asn Ile Ile Ser Ile

20 25 30

Trp Ile Ser His Ser Ile Gln Thr Gly Ser Gln Asn His Thr Gly Ile

35 40 45

Cys Asn Gln Asn Ile Ile Thr Tyr Lys Asn Ser Thr Trp Val Lys Asp

50 55 60

Thr Thr Ser Val Ile Leu Thr Gly Asn Ser Ser Leu Cys Pro Ile Arg

65 70 75 80

Gly Trp Ala Ile Tyr Ser Lys Asp Asn Ser Ile Arg Ile Gly Ser Lys

85 90 95

Gly Asp Val Phe Val Ile Arg Glu Pro Phe Ile Ser Cys Ser His Leu

100 105 110

Glu Cys Arg Thr Phe Phe Leu Thr Asp Lys Thr His Thr Cys Pro Pro

115 120 125

Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Leu Pro

130 135 140

Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr

145 150 155 160

Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn

165 170 175

Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Pro

180 185 190

Glu Glu Gln Tyr Asn Ser Thr Leu Arg Val Val Ser Ile Leu Thr Val

195 200 205

Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser

210 215 220

Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys

225 230 235 240

Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu

245 250 255

Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe

260 265 270

Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu

275 280 285

Asn Asn Tyr Lys Thr Thr Pro Leu Val Leu Asp Ser Asp Gly Ser Phe

290 295 300

Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly

305 310 315 320

Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr

325 330 335

Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

340 345

<210> 21

<211> 211

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 21

Gly Gly Pro Asp Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu

1 5 10 15

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

20 25 30

His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu

35 40 45

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr

50 55 60

Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn

65 70 75 80

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Leu Pro

85 90 95

Glu Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

100 105 110

Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val

115 120 125

Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

130 135 140

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

145 150 155 160

Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr

165 170 175

Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val

180 185 190

Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

195 200 205

Ser Pro Gly

210

<210> 22

<211> 105

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 22

Gly Gly Pro Asp Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu

1 5 10 15

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

20 25 30

His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu

35 40 45

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr

50 55 60

Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn

65 70 75 80

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Leu Pro

85 90 95

Glu Glu Lys Thr Ile Ser Lys Ala Lys

100 105

<210> 23

<211> 347

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 23

Met Asn Pro Asn Gln Lys Ile Thr Thr Ile Gly Ser Ile Cys Leu Val

1 5 10 15

Val Gly Leu Ile Ser Leu Ile Leu Gln Ile Gly Asn Ile Ile Ser Ile

20 25 30

Trp Ile Ser His Ser Ile Gln Thr Gly Ser Gln Asn His Thr Gly Ile

35 40 45

Cys Asn Gln Asn Ile Ile Thr Tyr Lys Asn Ser Thr Trp Val Lys Asp

50 55 60

Thr Thr Ser Val Ile Leu Thr Gly Asn Ser Ser Leu Cys Pro Ile Arg

65 70 75 80

Gly Trp Ala Ile Tyr Ser Lys Asp Asn Ser Ile Arg Ile Gly Ser Lys

85 90 95

Gly Asp Val Phe Val Ile Arg Glu Pro Phe Ile Ser Cys Ser His Leu

100 105 110

Glu Cys Arg Thr Phe Phe Leu Thr Asp Lys Thr His Thr Cys Pro Pro

115 120 125

Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Asp Val Phe Leu Phe Pro

130 135 140

Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr

145 150 155 160

Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn

165 170 175

Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg

180 185 190

Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val

195 200 205

Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser

210 215 220

Asn Lys Ala Leu Pro Leu Pro Glu Glu Lys Thr Ile Ser Lys Ala Lys

225 230 235 240

Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu

245 250 255

Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe

260 265 270

Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu

275 280 285

Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe

290 295 300

Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly

305 310 315 320

Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr

325 330 335

Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

340 345

<210> 24

<211> 106

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 24

Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu

1 5 10 15

Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe

20 25 30

Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu

35 40 45

Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe

50 55 60

Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly

65 70 75 80

Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr

85 90 95

Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

100 105

<210> 25

<211> 212

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 25

Gly Gly Pro Ser Val Phe Leu Leu Pro Pro Lys Pro Lys Asp Thr Leu

1 5 10 15

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

20 25 30

His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu

35 40 45

Val His Asn Ala Lys Thr Lys Pro Pro Glu Glu Gln Tyr Asn Ser Thr

50 55 60

Leu Arg Val Val Ser Ile Leu Thr Val Leu His Gln Asp Trp Leu Asn

65 70 75 80

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro

85 90 95

Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

100 105 110

Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val

115 120 125

Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

130 135 140

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

145 150 155 160

Leu Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr

165 170 175

Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val

180 185 190

Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

195 200 205

Ser Pro Gly Lys

210

<210> 26

<211> 105

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 26

Gly Gly Pro Ser Val Phe Leu Leu Pro Pro Lys Pro Lys Asp Thr Leu

1 5 10 15

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

20 25 30

His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu

35 40 45

Val His Asn Ala Lys Thr Lys Pro Pro Glu Glu Gln Tyr Asn Ser Thr

50 55 60

Leu Arg Val Val Ser Ile Leu Thr Val Leu His Gln Asp Trp Leu Asn

65 70 75 80

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro

85 90 95

Ile Glu Lys Thr Ile Ser Lys Ala Lys

100 105

<210> 27

<211> 107

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 27

Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu

1 5 10 15

Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe

20 25 30

Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu

35 40 45

Asn Asn Tyr Lys Thr Thr Pro Leu Val Leu Asp Ser Asp Gly Ser Phe

50 55 60

Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly

65 70 75 80

Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr

85 90 95

Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

100 105

<210> 28

<211> 559

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 28

Met Asn Pro Asn Gln Lys Ile Thr Thr Ile Gly Ser Ile Cys Leu Val

1 5 10 15

Val Gly Leu Ile Ser Leu Ile Leu Gln Ile Gly Asn Ile Ile Ser Ile

20 25 30

Trp Ile Ser His Ser Ile Gln Thr Gly Ser Gln Asn His Thr Gly Ile

35 40 45

Cys Asn Gln Asn Ile Ile Thr Tyr Lys Asn Ser Thr Trp Val Lys Asp

50 55 60

Thr Thr Ser Val Ile Leu Thr Gly Asn Ser Ser Leu Cys Pro Ile Arg

65 70 75 80

Gly Trp Ala Ile Tyr Ser Lys Asp Asn Ser Ile Arg Ile Gly Ser Lys

85 90 95

Gly Asp Val Phe Val Ile Arg Glu Pro Phe Ile Ser Cys Ser His Leu

100 105 110

Glu Cys Arg Thr Phe Phe Leu Thr Asp Lys Thr His Thr Cys Pro Pro

115 120 125

Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro

130 135 140

Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr

145 150 155 160

Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn

165 170 175

Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg

180 185 190

Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val

195 200 205

Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser

210 215 220

Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys

225 230 235 240

Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu

245 250 255

Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe

260 265 270

Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu

275 280 285

Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe

290 295 300

Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly

305 310 315 320

Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr

325 330 335

Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Gly Gly Pro Ser Val

340 345 350

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

355 360 365

Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu

370 375 380

Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

385 390 395 400

Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser

405 410 415

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

420 425 430

Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile

435 440 445

Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

450 455 460

Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu

465 470 475 480

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

485 490 495

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

500 505 510

Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg

515 520 525

Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

530 535 540

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

545 550 555

<210> 29

<211> 1041

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 29

atgaatccaa atcagaaaat aacaaccatt ggatcaatct gtctggtagt cggactaatt 60

agcctaatat tgcaaatagg gaatataatc tcaatatgga ttagccattc aattcaaact 120

ggaagtcaaa accatactgg aatatgcaac caaaacatca ttacctataa aaatagcacc 180

tgggtaaagg acacaacttc agtgatatta accggcaatt catctctttg tcccatccgt 240

gggtgggcta tatacagcaa agacaatagc ataagaattg gttccaaagg agacgttttt 300

gtcataagag agccctttat ttcatgttct cacttggaat gcaggacctt ttttctgacc 360

gacaaaactc acacatgccc accgtgccca gcacctgaac tcctgggggg accgtcagtc 420

ttcctcctgc ccccaaaacc caaggacacc ctcatgatct cccggacccc tgaggtcaca 480

tgcgtggtgg tggacgtgag ccacgaagac cctgaggtca agttcaactg gtacgtggac 540

ggcgtggagg tgcataatgc caagacaaag ccgccggagg agcagtacaa cagcacgctg 600

cgtgtggtca gcattctcac cgtcctgcac caggactggc tgaatggcaa ggagtacaag 660

tgcaaggtct ccaacaaagc cctcccagcc cccatcgaga aaaccatctc caaagccaaa 720

gggcagcccc gagaaccaca ggtgtacacc ctgcccccat cccgggagga gatgaccaag 780

aaccaggtca gcctgacctg cctggtcaaa ggcttctatc ccagcgacat cgccgtggag 840

tggggagagca atgggcagcc ggagaacaac tacaagacca cgcctctggt gctggactcc 900

gacggctcct tcttcctcta cagcaagctc accgtggaca agagcaggtg gcagcagggg 960

aacgtcttct catgctccgt gatgcacgag gctctgcaca accactacac gcagaagagc 1020

ctctccctgt ctccggggtaa a 1041

<210> 30

<211> 1047

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 30

atgaatccaa atcagaaaat aacaaccatt ggatcaatct gtctggtagt cggactaatt 60

agcctaatat tgcaaatagg gaatataatc tcaatatgga ttagccattc aattcaaact 120

ggaagtcaaa accatactgg aatatgcaac caaaacatca ttacctataa aaatagcacc 180

tgggtaaagg acacaacttc agtgatatta accggcaatt catctctttg tcccatccgt 240

gggtgggcta tatacagcaa agacaatagc ataagaattg gttccaaagg agacgttttt 300

gtcataagag agccctttat ttcatgttct cacttggaat gcaggacctt ttttctgacc 360

gacaaaactc acacatgccc accgtgccca gcacctgaac tcctgggggg accggatgtc 420

ttcctcttcc ccccaaaacc caaggacacc ctcatgatct cccggacccc tgaggtcaca 480

tgcgtggtgg tggacgtgag ccacgaagac cctgaggtca agttcaactg gtacgtggac 540

ggcgtggagg tgcataatgc caagacaaag ccgcgggagg agcagtacaa cagcacgtac 600

cgtgtggtca gcgtcctcac cgtcctgcac caggactggc tgaatggcaa ggagtacaag 660

tgcaaggtct ccaacaaagc cctcccactg cccgaagaga aaaccatctc caaagccaaa 720

gggcagcccc gagaaccaca ggtgtacacc ctgcccccat cccgggagga gatgaccaag 780

aaccaggtca gcctgacctg cctggtcaaa ggcttctatc ccagcgacat cgccgtggag 840

tggggagagca atgggcagcc ggagaacaac tacaagacca cgcctcccgt gctggactcc 900

gacggctcct tcttcctcta cagcaagctc accgtggaca agagcaggtg gcagcagggg 960

aacgtcttct catgctccgt gatgcacgag gctctgcaca accactacac gcagaagagc 1020

ctctccctgt ctccggggtaa ataataa 1047

<210> 31

<211> 1722

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 31

atgaatccaa atcagaaaat aacaaccatt ggatcaatct gtctggtagt cggactaatt 60

agcctaatat tgcaaatagg gaatataatc tcaatatgga ttagccattc aattcaaact 120

ggaagtcaaa accatactgg aatatgcaac caaaacatca ttacctataa aaatagcacc 180

tgggtaaagg acacaacttc agtgatatta accggcaatt catctctttg tcccatccgt 240

gggtgggcta tatacagcaa agacaatagc ataagaattg gttccaaagg agacgttttt 300

gtcataagag agccctttat ttcatgttct cacttggaat gcaggacctt ttttctgacc 360

gacaaaactc acacatgccc accgtgccca gcacctgaac tcctgggggg accgtcagtc 420

ttcctcttcc ccccaaaacc caaggacacc ctcatgatct cccggacccc tgaggtcaca 480

tgcgtggtgg tggacgtgag ccacgaagac cctgaggtca agttcaactg gtacgtggac 540

ggcgtggagg tgcataatgc caagacaaag ccgcgggagg agcagtacaa cagcacgtac 600

cgtgtggtca gcgtcctcac cgtcctgcac caggactggc tgaatggcaa ggagtacaag 660

tgcaaggtct ccaacaaagc cctcccagcc cccatcgaga aaaccatctc caaagccaaa 720

gggcagcccc gagaaccaca ggtgtacacc ctgcccccat cccgggagga gatgaccaag 780

aaccaggtca gcctgacctg cctggtcaaa ggcttctatc ccagcgacat cgccgtggag 840

tggggagagca atgggcagcc ggagaacaac tacaagacca cgcctcccgt gctggactcc 900

gacggctcct tcttcctcta cagcaagctc accgtggaca agagcaggtg gcagcagggg 960

aacgtcttct catgctccgt gatgcacgag gctctgcaca accactacac gcagaagagc 1020

ctctccctgt ctccgggtaa agacaaaact cacacatgcc caccgtgccc agcacctgaa 1080

ctcctggggg gaccggatgt cttcctcttc cccccaaaac ccaaggacac cctcatgatc 1140

tcccggaccc ctgaggtcac atgcgtggtg gtggacgtga gccacgaaga ccctgaggtc 1200

aagttcaact ggtacgtgga cggcgtggag gtgcataatg ccaagacaaa gccgcggggag 1260

gagcagtaca acagcacgta ccgtgtggtc agcgtcctca ccgtcctgca ccaggactgg 1320

ctgaatggca aggagtacaa gtgcaaggtc tccaacaaag ccctcccact gcccgaagag 1380

aaaaccatct ccaaagccaa agggcagccc cgagaaccac aggtgtacac cctgccccca 1440

tcccgggagg agatgaccaa gaaccaggtc agcctgacct gcctggtcaa aggcttctat 1500

cccagcgaca tcgccgtgga gtgggagagc aatgggcagc cggagaacaa ctacaagacc 1560

acgcctcccg tgctggactc cgacggctcc ttcttcctct acagcaagct caccgtggac 1620

aagagcaggt ggcagcaggg gaacgtcttc tcatgctccg tgatgcacga ggctctgcac 1680

aaccactaca cgcagaagag cctctccctg tctccgggta aa 1722

<---

Claims (28)

1. A composition for increasing the number of natural killer (NK) cells, containing a) a feeder cell engineered to express a fusion protein containing a membrane-bound inverted Fc domain, b) a plasma membrane particle (PM) derived from a feeder cell engineered to express a fusion protein containing a membrane-bound inverted Fc domain, or c) an exosome derived from a feeder cell engineered to express a fusion protein containing a membrane-bound inverted Fc domain, wherein the fusion protein comprises a transmembrane domain linked to the amino terminus of a membrane-bound inverted Fc domain and is configured to link the amino terminus of the inverted Fc domain to the outer surface of an engineered feeder cell, an engineered PM particle, or an engineered exosome, and wherein the engineered feeder cell, the engineered PM particle, or the engineered exosome further comprises membrane-bound IL-21 and membrane-bound 4-1BBL. 2. A composition for increasing the number of NK cells according to claim 1, comprising an engineered PM particle or an engineered exosome substantially free of feeder cells. 3. A composition for increasing the number of NK cells according to claim 1, characterized in that the engineered PM particle is obtained from a K562 cell. 4. A composition for increasing the number of NK cells according to claim 3, characterized in that the constructed feeder cell contains a K562 cell. 5. The composition for increasing the number of NK cells according to claim 1, 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 activating agent; and wherein 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 Notch ligand, a ligand for NKp46 or BCM1/SLAMF2, a TLR ligand and an NKG2D ligand. 6. A composition for increasing the number of NK cells according to claim 1, characterized in that the designed PM particle additionally contains a solid carrier, at least part of which is covered with a plasma membrane. 7. A composition for increasing the number of NK cells according to claim 6, characterized in that the solid carrier contains at least one of magnetic microparticles, silicon dioxide granules, polystyrene granules, latex granules, microstructures, a contrast agent and/or an anticancer therapeutic agent. 8. A composition for increasing the number of NK cells according to any one of claims 1-7, formulated in combination with a pharmaceutically acceptable carrier. 9. A method for treating, reducing and/or inhibiting cancer in a subject, comprising administering to a subject in need thereof an effective amount of a population of NK cells, the number of which has been increased in vitro by contact with a composition for increasing the number of NK cells according to any one of claims 1-7. 10. The method according to claim 9, characterized in that the cancer includes ovarian cancer. 11. The method of claim 9, further comprising administering to the subject at least one therapeutic agent against cancer in combination with an effective amount of the composition. 12. The method according to claim 11, characterized in that at least one therapeutic agent against cancer is selected from a chemotherapeutic agent, a drug-based formulation, or a combination thereof. 13. The method according to claim 12, characterized in that the chemotherapeutic agent is selected from CHOP, FLAG and 7+3, and the drug-based preparative regimen is selected from Cy-Flu, Bu-Flu and Flu-Mel. 14. A method for preventing, inhibiting or reducing acute or chronic graft-versus-host disease comprising administering to a subject in need thereof an effective amount of a composition for increasing the number of NK cells according to any one of claims 1-7. 15. The method of claim 14, wherein the composition is used in combination with a prophylactic agent against graft-versus-host disease (GvHD). 16. A method for preventing, inhibiting or reducing virus reactivation, comprising administering to a subject in need thereof an effective amount of a composition for increasing the number of NK cells according to any one of claims 1-7. 17. The method according to claim 16, characterized in that the virus comprises herpes simplex virus - 1 (HSV-1), herpes simplex virus - 2 (HSV-2), cytomegalovirus (CMV), chickenpox, herpes zoster virus (VZV), Epstein-Barr virus (EBV), adenovirus, adeno-associated virus, parvovirus, JC virus and BK virus. 18. A method for preventing, inhibiting or reducing opportunistic infections, comprising administering to a subject in need thereof an effective amount of a composition for increasing the number of NK cells according to any one of claims 1-7. 19. A method for increasing the cytotoxicity of NK cells of an initial population of NK cells, comprising exposing the initial population of NK cells in vitro to a composition for increasing the number of NK cells according to any one of claims 1-7. 20. The method according to claim 19, further comprising obtaining a population of expanded NK cells having increased cytotoxicity compared to the original population of NK cells. 21. The method according to claim 19, characterized in that the cytotoxicity of the expanded NK cells is at least approximately 2 times greater than the cytotoxicity of the original population of NK cells. 22. The method according to claim 19, characterized in that the cytotoxicity of the expanded NK cells is at least approximately 5 times greater than the cytotoxicity of the original population of NK cells. 23. The method according to claim 19, characterized in that the cytotoxicity of the expanded NK cells is at least approximately 10 times greater than the cytotoxicity of the original population of NK cells. 24. The method according to any one of claims 9-23, characterized in that the sources of NK cells to be expanded may include peripheral blood (PBMC, apheresis, leukopacks, leukocytes), NK cells obtained from iPSCs, NK cells obtained from ESCs, NK cells having a high-affinity Fc receptor polymorphism having Phe or Val at 158, and genetically modified NK cells.