Compositions and methods for stimulating natural killer cells

Abstract

The invention relates to compositions and methods for stimulating natural killer cells. Compositions and methods for stimulating expansion and cytotoxicity of natural killer (NK) cells are described. Therapeutic compositions and methods of using the expanded and stimulated NK cells are also described.

Description

[0001] This invention application is a divisional application of the invention patent application filed on January 24, 2020, with application number 202080011416.7 (international application number PCT / US2020 / 015021) entitled "Composition and method for stimulating natural killer cells".

[0002] This application claims the benefit of U.S. Provisional Application No. 62 / 796,575, filed January 24, 2019, which is incorporated herein by reference in its entirety. Technical Field

[0003] This disclosure relates to compositions and methods for stimulating natural killer (NK) cells. Background Technology

[0004] Natural killer (NK) cell therapy is emerging as a treatment for cancer and a potential treatment for other diseases. The challenges of fully realizing the clinical potential of NK cell therapy include obtaining large quantities of robust, healthy NK cells exhibiting high tumor cytotoxicity; the ability of NK cells to target disease targets; and, once introduced into the patient, ensuring their sustained presence in the body to achieve therapeutic effects. This challenge is partly due to the fact that NK cell activity is tightly regulated by a balance between activating and inhibiting receptors, including immune checkpoints. For example, ligands that activate NK cell receptors are expressed only on stressed, transformed, or virus-infected cells, so that the cytotoxic activity of NK cells targets these cells without harming normal, healthy tissue. The cytotoxic activity of NK cells is further limited by inhibitory ligands expressed on “self” cells. Simultaneously, the inhibitory regulatory mechanisms controlling NK cell cytotoxicity can be attack pathways of tumor cells, which utilize various immunosuppressive interactions to prevent immune attack. One example of how NK cells resist tumor immunosuppression is the binding of target cells labeled with antibodies that induce antibody-dependent cytotoxicity (ADCC) in NK cells. Therefore, the efficacy of many newer anti-tumor antibodies depends on the presence of a large number of healthy NK cells in the patient to support anti-tumor activity. In conclusion, the field of NK cell therapy still requires methods to obtain large numbers of healthy NK cells and to stimulate NK cells to achieve higher cytotoxicity and / or better ADCC function. Summary of the Invention

[0005] In various aspects of this disclosure, there are feeder cell compositions comprising at least one feeder cell, said feeder cell comprising a fragment crystallizable (Fc) domain that binds to the outer surface of the feeder cell. In some aspects, the at least one feeder cell further comprises one or more NK cell effectors. In some aspects, the at least one feeder cell comprises at least one NK cell effector, wherein the NK cell effector is IL-21. In another aspect, the at least one feeder cell further comprises at least two NK cell effectors, wherein one of the at least two NK cell effectors is IL-21.

[0006] This document also discloses NK cell expansion compositions without feeder cells, the compositions comprising engineered particles containing an Fc domain that binds to the outer surface of the engineered particle in any of the foregoing aspects. In some aspects, the engineered particles further comprise one or more NK cell effectors. In some aspects, the engineered particles further comprise at least one NK cell effector, wherein the NK cell effector is IL-21. In another aspect, the engineered particles further comprise at least two NK cell effectors, wherein one of the at least two NK cell effectors is IL-21.

[0007] In one aspect of this disclosure, there is a therapeutic dose of a combination of NK cells and an NK cell expansion composition comprising a plurality of in vitro expanded NK cells, said composition being free of feeder cells and comprising at least one engineered particle, said at least one engineered particle comprising an Fc domain that binds to the outer surface of the engineered particle. In some aspects, the engineered particle further comprises one or more NK cell effectors. In some aspects, the engineered particle further comprises at least one NK cell effector, wherein the NK cell effector is IL-21. In another aspect, the engineered particle further comprises at least two NK cell effectors, wherein one of the at least two NK cell effectors is IL-21.

[0008] In one aspect of this disclosure, there is an expanded population of NK cells exposed in vitro to an NK cell expansion composition, said composition being free of feeder cells and comprising at least one engineered cell membrane (PM) particle disclosed herein. In another aspect of this disclosure, there is an expanded population of NK cells exposed in vitro to an NK cell expansion composition, said composition comprising at least one feeder cell containing an Fc domain that binds to the outer surface of the feeder cell disclosed herein. The method optionally further comprises exposing the NK cells to one or more NK cell effectors. The one or more effectors may be in a cell culture medium containing a solution and / or bound to the surface of the Fc-binding feeder cell or engineered PM particle disclosed herein.

[0009] This document also discloses methods for treating, improving, alleviating, and / or inhibiting cancer or cancer metastasis or infectious diseases, said methods comprising administering to a subject in need an effective amount of any of the disclosed NK cell expansion compositions or any of the NK cell expansion infusion formulations described above. In one aspect, the NK cell expansion composition or NK cell expansion infusion formulation may be combined with or administered simultaneously with a therapeutic agent, such as an anticancer agent, an antiviral agent, or an antibiotic.

[0010] One aspect of this disclosure includes a method for preventing, mitigating, alleviating, and / or inhibiting cancer recurrence or metastasis before or after stem cell transplantation, the method comprising administering to a subject in need an effective amount of any of the disclosed expanded NK cell populations, said expanded NK cell populations having been exposed in vitro to an NK cell expansion composition or an NK cell stimulation composition or any of the disclosed NK cell stimulation or expansion infusion formulations. Any of the disclosed NK cell stimulation or expansion compositions or formulations may be administered in combination with or separately from stem cell transplantation.

[0011] One aspect of this disclosure includes a method for regulating a T-cell pool, the method comprising administering to a subject in need an effective amount of any of the disclosed expanded NK cell populations that have been in vitro exposed to an NK cell expansion composition or any of the disclosed NK cell expansion infusion formulations.

[0012] One aspect of this disclosure includes a method for preventing, inhibiting, mitigating, or alleviating acute or chronic graft-versus-host disease, the method comprising administering to a subject in need an effective amount of any of the disclosed expanded NK cell populations that have been in vitro exposed to an NK cell expansion composition, or an NK cell expansion composition, or any of the disclosed NK cell expansion infusion formulations.

[0013] Specifically, the present invention includes, but is not limited to, the following:

[0014] 1. A fusion protein comprising a transmembrane domain linked to the amino terminus of an Fc domain.

[0015] 2. The fusion protein according to claim 1, wherein the transmembrane domain comprises a signal anchoring sequence selected from the following: a transmembrane domain of a neuraminidase, a signal anchor from a parainfluenza virus hemagglutinin-neuraminidase, a signal anchor from a transferrin receptor, a signal anchor from a class II MHC invariant strand, a signal anchor from a P glycoprotein, a signal anchor from a desialyl glycoprotein receptor, and a signal anchor from a neutral endopeptidase.

[0016] 3. The fusion protein according to claim 1, wherein the transmembrane domain comprises a parainfluenza virus hemagglutinin-neuraminidase (NA) peptide sequence.

[0017] 4. The fusion protein according to claim 3, wherein the parainfluenza virus hemagglutinin-neuraminidase (NA) peptide sequence comprises a sequence having at least about 81% sequence identity with SEQ ID NO:1 or SEQ ID NO:17.

[0018] 5. The fusion protein according to claim 4, wherein the parainfluenza virus hemagglutinin-neuraminidase (NA) peptide sequence comprises a sequence having at least about 95% sequence identity with SEQ ID NO:1 or SEQ ID NO:17.

[0019] 6. The fusion protein according to claim 1, wherein the Fc domain comprises an immunoglobulin Fc domain selected from IgG1, IgG2, IgG3, IgG4, IgA and IgE.

[0020] 7. The fusion protein according to claim 1, further comprising a peptide linker between the transmembrane domain and the Fc domain.

[0021] 8. A nucleic acid encoding a fusion protein according to any one of items 1 to 7.

[0022] 9. A vector comprising the nucleic acid according to item 8.

[0023] 10. A cell comprising the carrier according to item 9.

[0024] 11. An engineered cell membrane (PM) particle or exosome comprising a fusion protein according to any one of claims 1 to 7.

[0025] 12. An NK cell expansion composition comprising a membrane-bound reverse Fc domain that binds to the outer surface of a feeder cell, engineered PM particle, or exosome.

[0026] 13. The NK cell expansion composition according to item 12, comprising engineered PM particles or engineered exosomes that are substantially free of feeder cells.

[0027] 14. The NK cell expansion composition according to claim 13, wherein the engineered particles further comprise at least one NK cell effector.

[0028] 15. The NK cell expansion composition according to claim 12, further comprising at least one NK cell effector.

[0029] 16. The NK cell expansion composition according to item 14 or 15, wherein the at least one NK cell effector is IL-21 or IL-15.

[0030] 17. The NK cell expansion composition according to claim 16, further comprising a second NK cell effector, wherein the second NK cell effector is 41BBL.

[0031] 18. The NK cell expansion composition according to claim 12, wherein the engineered PM particles comprise cytoplasmic membrane vesicles purified from NK cell feeder cells transfected or transduced with the fusion protein and the at least one NK cell effector, the fusion protein comprising a transmembrane domain linked to an Fc domain.

[0032] 19. The NK cell expansion composition according to claim 12, wherein the engineered PM particles comprise exosomes derived from NK cell feeder cells transfected with the fusion protein and the at least one NK cell effector, the fusion protein comprising a transmembrane domain linked to an Fc domain.

[0033] 20. The NK cell expansion composition according to claim 17, further comprising at least one additional NK cell effector, wherein the at least one additional NK cell effector is a cytokine, adhesion molecule, or NK cell activator; wherein the at least one additional NK cell effector is selected from IL-15, IL-2, IL-12, IL-18, IL-21, MICA, UBLP, 2sB4, LFA-1, Notch ligand, NKp46 ligand, or BCM1 / SLAMF2, TLR ligand, and NKG2D ligand.

[0034] 21. The NK cell expansion composition according to claim 12, wherein the engineered particles are cytoplasmic membrane particles comprising a cytoplasmic membrane, and the composition further comprises a solid surface, wherein the cytoplasmic membrane covers at least a portion of the solid surface.

[0035] 22. The NK cell expansion composition according to claim 21, wherein the solid surface comprises at least one of magnetic microparticles, silica beads, polystyrene beads, latex beads, microstructures, contrast agents, and / or cancer therapeutic agents.

[0036] 23. An NK cell expansion infusion formulation comprising an NK cell expansion composition according to any one of claims 12 to 22 and a pharmaceutically acceptable carrier.

[0037] 24. The NK cell expansion infusion formulation according to claim 23, wherein the formulation is selected from parenteral infusions, arterial infusions, intravenous infusions, catheter-mediated infusions, intravenous injections, intraperitoneal injections, subcutaneous injections, oral formulations, and topical formulations.

[0038] 25. The NK cell expansion infusion formulation according to item 24 is infused into a subject who requires in vivo NK cell expansion.

[0039] 26. An NK cell composition comprising an in vitro population of NK cells in contact with the NK cell expansion composition according to claim 12.

[0040] 27. The NK cell composition according to claim 26, further comprising at least one NK cell effector.

[0041] 28. The NK cell composition according to claim 27, wherein the at least one NK cell effector is IL-21 or IL-15.

[0042] 29. The NK cell composition according to claim 27, wherein the at least one NK cell effector is soluble.

[0043] 30. An expanded population of NK cells exposed in vitro to an NK cell expansion composition according to claim 26, the composition comprising feeder cells or comprising at least one engineered particle and not containing feeder cells, wherein the feeder cells or engineered particles comprise an Fc domain that binds to their outer surface.

[0044] 31. The expanded NK cell population according to claim 32, wherein the NK cells have increased cytotoxicity compared to non-expanded NK cells.

[0045] 32. The expanded NK cell population according to claim 30, wherein the cytotoxicity of the expanded NK cells is at least about twice that of the cytotoxicity of the non-expanded NK cells.

[0046] 33. The expanded NK cell population according to item 30, wherein the cytotoxicity of the expanded NK cells is at least about 5 times that of the cytotoxicity of the non-expanded NK cells.

[0047] 34. The expanded NK cell population according to claim 30, wherein the cytotoxicity of the expanded NK cells is at least about 10 times that of the non-expanded NK cells.

[0048] 35. A composition comprising a therapeutic dose of NK cells and a pharmaceutically acceptable carrier, said NK cells comprising an expanded NK cell population according to any one of claims 30 to 34.

[0049] 36. The expanded NK cell population according to claim 30 or the composition according to claim 35, further comprising at least one NK cell effector.

[0050] 37. The composition according to claim 36, wherein the at least one NK cell effector is selected from IL-15, IL-2, IL-12, IL-18, IL-21, MICA, UBLP, 2sB4, LFA-1, Notch ligand, NKp46 ligand, or BCM1 / SLAMF2, TLR ligand, and NKG2D ligand.

[0051] 38. The composition according to claim 37, wherein the at least one NK cell effector is soluble.

[0052] 39. The composition according to claim 37, further comprising a second NK cell effector, wherein the second NK cell effector is 41BBL.

[0053] 40. A method for treating, improving, alleviating, and / or inhibiting cancer or cancer metastasis or infectious disease in a subject, comprising administering to the subject in need an effective amount of a composition or expanded NK cell population optionally in contact with an NK cell population.

[0054] 41. The method according to item 40, wherein the cancer is selected from the group consisting of: tumors, hematologic cancers, lymphomas, leukemia, acute myeloid leukemia, myelodysplastic syndrome, chronic myeloid leukemia, acute lymphoblastic leukemia, myelofibrosis, multiple myeloma, colorectal cancer, colon cancer, lung cancer, head and neck cancer, ovarian cancer, pancreatic cancer, liver cancer, skin cancer, prostate cancer, kidney cancer, intraperitoneal cancer, and breast cancer.

[0055] 42. A method for inhibiting, mitigating, and / or preventing cancer or cancer metastasis recurrence before or after stem cell transplantation, comprising administering to a subject in need an effective amount of the composition or expanded NK cell population according to any one of claims 12 to 39.

[0056] 43. The method according to any one of claims 40 to 42, further comprising administering to the subject at least one cancer therapeutic agent and the effective amount of the composition or expanded NK cell population.

[0057] 44. The method according to claim 43, wherein the at least one cancer therapeutic agent is selected from chemotherapeutic agents, drug-based preparation methods, or combinations thereof.

[0058] 45. The method according to item 44, wherein the chemotherapeutic agent is selected from CHOP, FLAG, 7+3, and the drug preparation method is selected from Cy-Flu, Bu-Flu, and Flu-Mel.

[0059] 46. ​​A method for regulating a T-cell pool during or after stem cell transplantation, comprising administering to a subject in need an effective amount of the composition or expanded NK cell population according to any one of claims 12 to 39.

[0060] 47. A method for preventing, inhibiting, reducing or alleviating acute or chronic graft-versus-host disease, comprising administering to a subject in need an effective amount of the composition or expanded NK cell population according to any one of claims 12 to 39.

[0061] 48. The method of preventing, inhibiting, reducing or alleviating acute or chronic graft-versus-host disease according to claim 47, further comprising administering to the subject a GvHD prophylactic agent and the effective amount of the composition or expanded NK cell population.

[0062] 49. A method for preventing, inhibiting, mitigating or alleviating viral reactivation, comprising administering to a subject in need an effective amount of the composition or expanded NK cell population according to any one of claims 12 to 39.

[0063] 50. The method for preventing, inhibiting, mitigating or alleviating viral reactivation according to item 49, wherein the viral infection includes 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 BK virus.

[0064] 51. A method for preventing, inhibiting, reducing or alleviating opportunistic infections, comprising administering to a subject in need an effective amount of the composition or expanded NK cell population according to any one of claims 12 to 39.

[0065] 52. An NK cell expansion culture medium formulation comprising the NK cell expansion composition according to claim 12 and at least one culture medium component.

[0066] 53. The NK cell expansion culture medium formulation according to claim 52, further comprising at least one additional component selected from cytokines, IL-2, IL-12, IL-18, NAM, reducing agents, human platelets, human platelet lysate, insulin, and ascorbate.

[0067] 54. The expanded NK cell population according to items 30 to 34, wherein the expanded NK cells exhibit increased secretion of antitumor cytokines compared with non-expanded NK cells.

[0068] 55. The expanded NK cell population according to items 30 to 34, wherein the expanded NK cells exhibit increased expression of NKG2D, NKp46, and CD16 compared to non-expanded NK cells.

[0069] 56. A cryopreserved therapeutic dose of an expanded NK cell population according to any one of claims 30 to 34, wherein the expanded NK cells remain viable after thawing.

[0070] 57. A method for increasing the cytotoxicity of NK cells, comprising exposing an initial NK cell population to an NK cell expansion composition, the composition comprising at least one feeder cell or engineered particle and an Fc domain binding to the outer surface of the feeder cell or engineered particle, the at least one feeder cell or engineered particle comprising at least two NK cell effectors, one of said at least two NK cell effectors being IL-21.

[0071] 58. The method according to claim 57, wherein the NK cell expansion composition comprises at least one feeder cell having a membrane-bound Fc domain.

[0072] 59. The method of claim 57, wherein the NK cell expansion composition comprises engineered particles selected from PM particles having a membrane-binding Fc domain and exosomes having a membrane-binding Fc domain.

[0073] 60. The method according to claim 57, further comprising obtaining an expanded NK cell population with increased cytotoxicity compared to the initial NK cell population.

[0074] 61. The method according to item 57, wherein the cytotoxicity of the expanded NK cells is at least about twice that of the cytotoxicity of the initial NK cell population.

[0075] 62. The method according to claim 57, wherein the cytotoxicity of the expanded NK cells is at least about 5 times that of the initial NK cell population.

[0076] 63. The method according to item 57, wherein the cytotoxicity of the expanded NK cells is at least about 10 times that of the initial NK cell population.

[0077] 64. The method according to any one of claims 40 to 51 or 57 to 63, wherein the source of the NK cells to be expanded or stimulated may include peripheral blood (PBMC, apheresis, leukopaks, erythrocyte sedimentation rate), iPSC-derived NK cells, ESC-derived NK cells, NK cells with polymorphisms of high-affinity Fc receptors of Phe or Val at position 158, and genetically modified NK cells.

[0078] Other aspects and features of this disclosure are detailed below. Attached Figure Description

[0079] Figure 1A and Figure 1B The construction of membrane-bound immune cell targeting ligands containing unly cleaved signal anchors is shown. Figure 1A The structures of type I and type II integrated membrane proteins are shown, which differ in orientation in terms of their N-terminus and C-terminus. Figure 1B The structure of a NA-Fc chimeric protein used as a membrane-bound immune cell targeting ligand is shown. The NA-Fc chimeric protein consists of a neuraminidase transmembrane domain acting as a membrane anchor, a stem region, and a human IgG'Fc region.

[0080] Figure 2 An alternative construction of a membrane-bound immune cell targeting ligand is shown, the ligand comprising an Fc domain containing a neuraminidase (NA) signaling anchor and an increased NA stem length.

[0081] Figure 3 An example of a membrane-bound immune cell targeting ligand sequence (SEQ ID NO:13) is shown, in which the NA signal anchor is fused to the IgG Fc domain via an RS linker.

[0082] Figure 4 This is a schematic diagram of Fc stimulation of NK cells.

[0083] Figure 5 CD16 conjugation via the Fc region was shown to enhance proliferation after day 14. NK cells were expanded from PBMCs derived from two donors [L54 (circle) or L44 (square)] using either CSTX002 (open sign) or CSTX002-Fc cell lines (closed sign) as feeder cells. CD16 conjugation allowed for increased NK cell proliferation. NK cells from both donors expanded at the same rate until day 14 after stimulation with IL-21 alone (CSTX2) or IL-21 and Fc (CSTX2-Fc), at which point Fc-stimulated cultures divided at an increased rate compared to IL-21 alone.

[0084] Figure 6Two graphs are presented, each showing the cytotoxicity of NK cells expanded from initial PBMC populations obtained from different donors. NK cells were expanded from PBMCs using either CSTX002(●) or CSTX002-Fc(■) cell lines as feeder cells. Increased cytotoxicity against SKOV3 cells was found in NK cells expanded from both different donors using CSTX002-Fc.

[0085] Figure 7 NK cells expanded from poorly responding donors using Fc-binding feeder cells were shown to exhibit increased cytotoxicity against tumor targets expressing membrane-bound Fc to mimic antibody-coated tumor cells that would engage antibody-dependent cytotoxicity (ADCC). NK cells were expanded from PBMCs using CSTX002(●) or CSTX002-Fc(■) cell lines as feeder cells. NK cells expanded using CSTX002-Fc were found to have increased cytotoxicity against SKOV3-Fc cells.

[0086] Figure 8 This is a series of six (6) figures, each showing comparative receptor expression via NK cells expanded using CSTX002 feeder cells with (CSTX002-Fc) and without (CSTX-002) membrane-binding 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. Receptor expression in the expanded NK cells was analyzed, and these receptors are considered essential for cytotoxic function and homing. NK cells expanded with CSTX002-Fc showed higher expression of CD16, NKp46, and CD62L. Detailed Implementation

[0087] This disclosure provides compositions and formulations comprising NK cell stimulants, and provides methods of use thereof relating to NK cell stimulation and various treatments described in further detail below.

[0088] Binding to CD16 receptors (FcγRIIIa receptor (CD16a) and FcγRIIIb receptor (CD16b)) on NK cells is potentially a very potent NK cell stimulation mechanism. The Fc (fragment crystallizable region) domain of an antibody is recognized by CD16, and binding of the Fc domain to CD16 induces antibody-dependent cytotoxicity (ADCC). This disclosure describes engineered stimulation of NK cells via CD16 binding for improving NK cell expansion and enhancing NK cell cytotoxicity. Alternatively, this disclosure contemplates stimulating NK cells using the Fc domain of an antibody, wherein the Fc domain is competent to activate CD16 on NK cells and present it to NK cells, wherein the Fc domain binds to feeder cells, plasma membrane (PM) granules, exosomes (EX), or solid carriers. Fc-bound feeder cells, PM particles, exosomes, and solid carriers may further contain other NK cell stimulating factors in various forms, along with corresponding signaling pathways or combinations thereof, such as membrane-bound or soluble IL-15, IL-21, 4-1BBL, other cytokines, or other chemical motifs simultaneously conjugated to other stimulatory or inhibitory receptors. NK cells expanded according to the methods disclosed herein and using the compositions disclosed herein can exhibit higher cytotoxicity, higher CD16 expression, and / or improved ADCC function. These NK cells are suitable for therapeutic compositions and methods for treating human diseases and conditions, including various types of cancer.

[0089] definition

[0090] Unless otherwise defined, all technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains. The following references provide general definitions for many of the terms used herein: Singleton et al., *Dictionary of Microbiology and Molecular Biology* (2nd edition, 1994); *The Cambridge Dictionary of Science and Technology* (Walker, ed., 1988); *The Glossary of Genetics*, 5th edition, R. Rieger et al., Springer Verlag (1991); and Hale and Marham, *The Harper Collins Dictionary of Biology* (1991). Unless otherwise specified, the following terms as used herein have their assigned meanings.

[0091] When describing elements of this disclosure or one or more preferred embodiments thereof, the articles “a / an” and “the / said” are intended to mean the presence of one or more of the stated elements. The terms “comprising,” “including,” and “having” are intended to be inclusive and mean that additional elements may exist in addition to those listed.

[0092] A range herein may be expressed as starting “about” of a particular value and / or ending “about” of another particular value. When representing such a range, another embodiment includes starting from a particular value and / or ending at another particular value. Similarly, when a value is expressed as an approximation, the use of the antecedent “about” should be understood as the particular value forming another embodiment. It should be further understood that the endpoint values ​​of each range are substantially related to or substantially unrelated to another endpoint value. It should also be understood that multiple values ​​are disclosed herein, and each value is also disclosed herein as “about” the particular value, in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed. It should also be understood that, as will be properly understood by those skilled in the art, when a value is disclosed, “less than or equal to” the value, “greater than or equal to” the value, and possible ranges between values ​​are also disclosed. For example, if the value “10” is disclosed, then “less than or equal to 10” and “greater than or equal to 10” are also disclosed. It should also be understood that throughout the application, data is provided in a variety of different formats, and this data represents a range of endpoint values ​​and start-point values, as well as any combination of data points. For example, if specific data point "10" and specific data point 15 are disclosed, it should be understood that greater than, greater than or equal to, less than, less than or equal to, and equal to 10 and 15, as well as between 10 and 15, are disclosed. It should also be understood that each unit between two specific units is also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

[0093] As used herein, the terms “optional” or “optionally” mean that the event or situation described below may or may not occur, and the description includes both scenarios in which the event or situation occurs and scenarios in which it does not occur.

[0094] As used herein, “N-terminal side” or “amino terminus” refers to the orientation of a peptide, polypeptide, or protein and may not refer to the N-terminus. In some aspects, when discussing chimeric or fused peptides, polypeptides, or proteins, the N-terminal side may refer only to a component of the chimeric or fused peptide, polypeptide, or protein and not the entire structure. For example, when discussing an Fc domain and describing the Fc domain as fused to its intracellularly facing N-terminus or N-terminal side, this text considers chimeric or fused peptides, polypeptides, or proteins where the signal anchor is located at the N-terminus of the chimeric or fused construct and effectively crosses the cell membrane. Thus, in such chimeras, the transmembrane anchor is attached to the N-terminal side of the Fc domain, where the orientation of the Fc domain causes the N-terminal side to face the cell, and the N-terminal side is inverted relative to the Fc domain on a typical B cell, which typically has a carboxyl terminus crossing the cell membrane and an N-terminus extending into the extracellular matrix.

[0095] The terms “peptide,” “polypeptide,” and “protein” are used interchangeably to refer to polymers of amino acid residues.

[0096] As used herein, the term "sequence identity" refers to a quantitative measure of the degree of identity between two sequences of substantially equal length. The percentage of identity between 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. The local homology algorithm of Smith and Waterman, Advances in Applied Mathematics 2:482-489 (1981), provides an approximate alignment of nucleic acid sequences. This algorithm can be applied to amino acid sequences using the following developed scoring matrix: Dayhoff, Atlas of Protein Sequences and Structure, MO Dayhoff ed., 5 Supplement 3:353-358, National Biomedical Research Foundation, Washington, DC, USA, and normalized by: Gribskov, Nucleic Acids Res. 14(6):6745-6763 (1986). The Genetics Computer Group (Madison, Wisconsin) provides an exemplary implementation of this algorithm for determining the percentage of sequence similarity in the "BestFit" utility application. Other suitable procedures for calculating the percentage of similarity or identity between sequences are well known in the art; for example, another alignment procedure is BLAST, all of which are used with default parameters. For example, BLASTN and BLASTP can be used with the following default parameters: Genetic Code = Standard; Filter = None; Chain = Both; Cutoff = 60; Expected Value = 10; Matrix = BLOSUM62; Description = 50 sequences; Sort by = High Score; Database = Non-redundant, GenBank+EMBL+DDBJ+PDB+GenBank CDS Translation+Swiss Protein+Spupdate+PIR. Details of these procedures can be found on the GenBank website. Generally, substitutions are conserved amino acid substitutions: limited to exchanges within the following members: Group 1: glycine, alanine, valine, leucine, and isoleucine; Group 2: serine, cysteine, threonine, and methionine; Group 3: proline; Group 4: phenylalanine, tyrosine, and tryptophan; Group 5: aspartic acid, glutamic acid, asparagine, and glutamine.

[0097] Techniques for determining the identity of nucleic acid and amino acid sequences are known in the art. Typically, these techniques involve determining the mRNA nucleotide sequence of a gene and / or the amino acid sequence it encodes, and comparing these sequences with second nucleotide or amino acid sequences. Genomic sequences can also be determined and compared in this manner. Generally, identity refers to the precise nucleotide-to-nucleotide or amino acid-to-amino acid correspondence between two polynucleotide or polypeptide sequences, respectively. Comparisons can be made by determining the percentage of identity between two or more sequences (polynucleotides or amino acids).

[0098] Since various changes can be made to the cells and methods described above without departing from the scope of the invention, all content contained in the above description and the examples given below should be interpreted as illustrative and not restrictive.

[0099] An "increase" can refer to any change that results in a greater quantity of symptoms, disease, composition, condition, or activity. An increase can be any individual, median, or average increase in condition, symptoms, activity, or composition by a statistically significant amount. Therefore, an increase can be 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%, as long as the increase is statistically significant.

[0100] "Reduction" can refer to any change that results in a smaller amount of symptoms, disease, composition, condition, or activity. When the genetic output of a gene product containing a substance is lower than the output of a gene product not containing said substance, that substance is also understood to reduce the genetic output of the gene. Furthermore, for example, reduction can be a change in the symptoms of a disease so that the symptoms are less than previously observed. Reduction can be any individual, median, or average reduction in the condition, symptoms, activity, or composition by a statistically significant amount. Therefore, a reduction can be 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.

[0101] "Inhibition" refers to a reduction in activity, response, condition, disease, or other biological parameters. This can include, but is not limited to, the complete elimination of activity, response, condition, or disease. It can also include, for example, a 10% reduction in activity, response, condition, or disease compared to natural or control levels. Therefore, the reduction compared to natural or control levels can be 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any amount between these values.

[0102] "Reduce" or other forms of the term, such as "reducing / reduction," means a reduction in an event or characteristic (e.g., tumor growth). It should be understood that this is usually related to a standard or expected value; in other words, it is relative, but not always requires reference to a standard or relative value. For example, "reducing tumor growth" means a reduction in the rate of tumor growth relative to a standard or control.

[0103] The word “prevent” or other forms of the term such as “preventing / prevention” are intended to terminate a particular event or characteristic, stabilize or delay the development or progression of a particular event or characteristic, or minimize the probability of a particular event or characteristic occurring. Prevention does not require a comparison with a control, as it is generally more absolute than, for example, reduction. As used herein, some things can be reduced but not prevented, but some things that are reduced can also be prevented. Similarly, some things can be prevented but not reduced, but some things that are prevented can also be reduced. It should be understood that in the use of reduction or prevention, the use of other words is also explicitly disclosed unless otherwise specifically indicated.

[0104] The term "subject" refers to any individual who is the target of administration or treatment. A subject can be a vertebrate, such as a mammal. In one aspect, a subject can be a human, a non-human primate, a cow, a horse, a pig, a dog, or a cat. A subject can also be a guinea pig, a rat, a hamster, a rabbit, a mouse, or a mole. Thus, a subject can be a human or mammalian patient. The term "patient" refers to a subject under the treatment of a clinician, such as a physician.

[0105] The term "therapeuticly effective" means that the amount of the composition used is sufficient to improve one or more causes or symptoms of the disease or condition. Such improvement requires only reduction or alteration, not necessarily elimination.

[0106] The term "treatment" refers to the medical management of a patient aimed at curing, improving, stabilizing, or preventing a disease, pathological condition, or symptom. This term includes active treatment, which is treatment specifically designed to improve a disease, pathological condition, or symptom, and also includes etiological treatment, which is treatment aimed at eliminating the cause of the related disease, pathological condition, or symptom. Additionally, this term includes palliative treatment, which is treatment designed to relieve symptoms rather than cure a disease, pathological condition, or symptom; preventive treatment, which is treatment aimed at minimizing or partially or completely suppressing the development of a related disease, pathological condition, or symptom; and supportive treatment, which is treatment used to complement another specific therapy aimed at improving a related disease, pathological condition, or symptom.

[0107] "Administration" to a subject includes any route by which a drug is introduced or delivered to the subject. Administration can be performed via any suitable route, including oral, local, intravenous, subcutaneous, transcutaneous / transdermal, intramuscular, intra-articular, parenteral, intra-arterial, intradermal, intravenous, intracranial, intraperitoneal, intralesional, intranasal, rectal, vaginal, by inhalation, by implantable cartridges, parenteral (e.g., subcutaneous, intravenous, intramuscular, intra-articular, intrasynovial, intrasternal, intrasheath, intraperitoneal, intrahepatic, intralesional, and intracranial injection or infusion techniques), and so on. As used herein, "concurrent administration," "combined administration," or "simultaneous administration / administered simultaneously" means that the compounds are administered at the same time point or substantially immediately following each other. In the latter case, the administration of the two compounds is close enough that the observed results are indistinguishable from those achieved when the compounds are administered at the same time point. "Systemic administration" refers to a route that introduces or delivers a drug to a wide area (e.g., more than 50% of the body) of the subject, such as through an inlet into the circulatory or lymphatic system. In contrast, "local administration" refers to a route that introduces or delivers a drug to the area or immediately adjacent to the application site without introducing the drug systemically in a therapeutically significant amount. For example, a locally administered drug may be readily detectable in the vicinity of the application site, but may be undetectable or detectable in negligible amounts in distal parts of the subject's body. Administration includes self-administration and administration by another person.

[0108] As used herein, “treat / treating / treatment” and its grammatical variations include the application of a composition with the intent or purpose of partially or completely preventing, delaying, curing, alleviating, relieving, altering, remedying, improving, stabilizing, weakening and / or reducing the intensity or frequency of one or more diseases or conditions, symptoms of diseases or conditions, or the underlying cause of diseases or conditions. The treatments of the present invention can be applied preventively, preventively, palliatively, or remedially. Preventive treatments are administered to a subject before onset (e.g., before signs of cancer become apparent), during early onset (e.g., after initial signs and symptoms of cancer), or after cancer development is determined. Preventive application can be performed days to years before symptoms of disease or infection appear.

[0109] (I) Fc fusion peptide

[0110] In one aspect, this article discloses engineered feeder cells, engineered plasma membrane (PM) particles, engineered exosomes, engineered platelets (including but not limited to Fc-binding platelets), and engineered lymphocytes (e.g., lymphocytes engineered to express an Fc domain to stimulate NK cells (e.g., T cells)), as well as solid carriers containing membrane-bound Fc fusion peptides (referred to herein as Fc-binding feeder cells, Fc-binding PM particles, Fc-binding exosomes, Fc-binding platelets, and Fc-binding lymphocytes, respectively), wherein the Fc fusion peptide contains a transmembrane peptide domain linked to the amino terminus of the Fc domain. In one aspect, the transmembrane domain of the Fc fusion peptide may contain cleaved or uncleaved signal anchoring sequences, such as the transmembrane domain of a neuraminidase, a signal anchor from parainfluenza virus hemagglutinin-neuraminidase, a signal anchor from a transferrin receptor, a signal anchor from the invariant strand of class II MHC, a signal anchor from a P glycoprotein, a signal anchor from a desialylate glycoprotein receptor, or a signal anchor from a neutral endopeptidase. In one example, the transmembrane domain contains a parainfluenza virus hemagglutinin-neuraminidase (NA) peptide sequence. As schematically shown in Figure 1, the transmembrane neuraminidase (NA) peptide domain is used to couple or bind the Fc domain to the outer surface of a feeder cell. In other respects, the transmembrane neuraminidase (NA) peptide domain is used to couple or bind the Fc domain to the outer surface of PM nanoparticles, exosomes, or solid carriers. The NA peptide domain consists of an N-terminal cytoplasmic tail, an uncleaved signal anchor acting as the transmembrane domain, and a stem region extending from the cytoplasmic membrane. It should be understood that the length of the stem region can vary.

[0111] As used herein, “NA peptide domain” means a peptide sequence comprising at least fifty (50) amino acid sequences of SEQ ID NO:1MNPNQKITTIGSICLVVGLISLILQIGNIISIWISHSIQTGSQNHTGICN, 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 with SEQ ID NO:1.

[0112] The Fc domain is a ligand that binds to the NK cell surface receptor CD16 (FcγRIII). CD16 is one of the major 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), this activates NK cell antibody-dependent cell-mediated cytotoxicity (ADCC). In another aspect, the Fc domain (IgG1, IgG2, IgG3, and / or IgG4) can also bind to the CD16 receptor on other immune cells such as mast cells, macrophages, and γ-δ T cells; thereby similarly stimulating the proliferation of said cells and enhancing their cytotoxicity. In yet another aspect, other cell types can be engineered to be Fc-binding. Therefore, this disclosure also covers, for example, Fc-binding engineered platelets, Fc-binding primary tumor samples that can be used for tumor vaccines, and Fc-expressing engineered iPSCs.

[0113] In another aspect, other Fc immunoglobulin isotypes besides IgG (IgA, IgE, IgM) can be used to stimulate their respective corresponding Fc receptors to stimulate other immune cell types. For example, the domain FcαRI (CD89) specifically binds to IgA on macrophages, neutrophils, and eosinophils; FcRI (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 thus stimulates them. Therefore, fusion peptides, Fc-bound feeder cells (FC), Fc-bound lymphocytes, Fc-bound engineered cell membrane (PM) granules, Fc-bound engineered exosomes, and compositions containing the like can also be used to substantially amplify mast cells and / or macrophages according to the methods for amplifying NK cells described herein.

[0114] In one aspect, this document discloses fusion peptides comprising immunoglobulin Fc domains (e.g., IgG1, IgG2, IgG3, IgG4, IgA, and / or IgE Fc domains) fused with transmembrane domains such as NA peptide domains as described above. One or more Fc domains may be presented as monomeric, dimer, or multimeric constructs. In one aspect, one or more Fc domains may be further modified to optimize or enhance antibody-mediated killing, NK cell recognition, and control the amplification of activated Fc receptors. For example, one or more Fc domains may be modified to increase affinity for CD16. Thus, for example, one or more Fc domains may contain one or more mutations, such as T256A, K290A, S298A, E333A, K334A, L235V, F243L, R292P, Y300L, and / or P396L. Similarly, one or more Fc domains can be further modified to increase selectivity for binding to activating (IIIa) receptors relative to binding to repressive Fc (IIb) receptors. Thus, for example, one or more Fc domains can contain one, two, three, four, five, six, seven, eight, or more mutant or alternative forms such as S239D, I332E, A330L, F243L, R292P, V305I, and / or P396L. For example, in one aspect, the Fc domain can be modified to contain R292L, Y300L, V305I, and P396L. In another instance, the Fc domain can be modified to contain S239D, I332E, and A330L. In yet another aspect, engineered mutants of the Fc domain with lower affinity can be used to induce higher expression of CD16 on NK cells.

[0115] Transmembrane domains, such as NA peptide domains, can be directly linked to Fc domains via chemical bonds or indirectly linked to Fc domains via linkers. Direct chemical bonds can be covalent (e.g., peptide bonds, ester bonds, etc.) or non-covalent (e.g., ionic, electrostatic, hydrogen, hydrophobic, Van der Waal interaction, or π-effect). Indirect linkages can be achieved using linkers, which are chemical groups connected to one or more other chemical groups via at least one covalent bond. Suitable linkers include amino acids, peptides, nucleotides, nucleic acids, dimer hinge Fc molecules, organic linker molecules (e.g., maleimide derivatives, N-ethoxybenzylimidazole, biphenyl-3,4',5-tricarboxylic acid, p-aminobenzyloxycarbonyl, etc.), disulfide linkers, and polymer linkers (e.g., PEG). Linkers can include one or more spacer groups, including but not limited to alkylene, alkenylene, alkyneene, alkyl, alkenyl, alkyne, alkoxy, aryl, heteroaryl, aralkyl, areneyl, arynyl, etc. Linkers can be neutral or positively or negatively charged. Additionally, the linker can be cleavable so that the linker covalent bond connecting the linker to another chemical group can be broken or cleaved under certain conditions, including pH, temperature, salt concentration, light, catalyst, or enzyme. In one aspect, the NA peptide domain can be NA4-Fc Siadel (S239D / I332E / A330L).

[0116] In one aspect, the linker can be a peptide linker. Examples of suitable peptide linkers are well known in the art, and procedures for designing linkers are readily available (see, for example, Crasto et al., *Protein Engineering*, 2000, 13(5):309-312). Peptide linkers can be restriction site linkers, such as short-sequence RS linkers, or flexible amino acid linkers (e.g., containing small, nonpolar, 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), and (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. The linker includes (EAAAK). 1-4 (SEQ ID NO:8), A(EAAAK) 2-5 A (SEQ ID NO:9), PAPAP (SEQ ID NO:10), and (AP) 6-8The Fc domain can be linked to the N-terminus, C-terminus, and / or internal sites of the NA peptide.

[0117] In some aspects, the Fc fusion peptide has an amino acid sequence that is 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 identical to SEQ ID NO:13. For targeting the placement of the Fc domain on the cytoplasmic membrane, a membrane-targeting domain from a well-characterized influenza virus neuraminidase protein (NA) can be used, the membrane-targeting domain consisting of an N-terminal cytoplasmic tail, an uncleaved signal anchor acting as a transmembrane domain, and a stem region extending from the cytoplasmic membrane. Figure 1A and 1B This is a schematic diagram showing the construction of a membrane-bound immune cell targeting ligand containing an unlyzed signal anchoring sequence. Figure 1A The structures of type I and type II integrated membrane proteins and the signal anchors for each protein are shown. Figure 1B The structure of an unlysevered signal anchor from a type II integrated membrane protein used in membrane-bound immune cell targeting ligands is shown. Figure 1B As shown, the exemplary but non-limiting construct of this disclosure includes an NA-Fc chimera, wherein the Fc domain (IgG1) is connected to the unlysed NA stem region via a short connector.

[0118] It is noteworthy that NA-Fc chimeras can be inserted into recombinant P / V / F viruses to generate novel oncolytic viruses that are specific to tumor cells relative to normal cells (attributed to P / V mutations) and can enhance ADCC via NK cells. Figure 2 An alternative construction of NA-Fc chimeras with gradually increasing NA stem lengths is shown.

[0119] Figure 3 An exemplary sequence of an NA-Fc chimera is shown, wherein the Fc domain (IgG1) is linked to a 50-amino acid NA sequence via a short RS linker sequence to produce a chimera with the structure described below and shown in Figure 1. Figure 3Non-limiting examples of NA-Fc constructs with 279 amino acid sequences: MNPNQKITTIGSICLVVGLISLILQIGNIISIWISHSIQTGSQNHTGICNRSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:13).

[0120] like Figure 2The document indicates that the NA-Fc structure may include an NA-targeted structural domain (SEQ ID NO:1), a connector (e.g., an RS connector), a hinge region DKTHTCPPCPAPELL (SEQ ID NO:11) or TCPPCPAPELL (SEQ ID NO:12), and an Fc region GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:1) NO:14), the Fc region contains the CH2 domain GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRV VSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK (SEQ ID NO:15) and the CH3 domain GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFF LYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:16) or the domain SEQ ID NO:14. NO:14, 15 or 16 have a sequence with 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.It should be understood and considered herein that NA-Fc chimeras may comprise membrane-targeting domains of any length from a well-characterized influenza virus neuraminidase protein (NA), said membrane-targeting domains comprising MNPNQKITTIGSICLVVGLISLILQIGNIISIWISHSIQTGSQNHTGICNQNIITYKNSTWVKDT TSVILTGNSSLCPIRGWAIYSKDNSIRIGSKGDVFVIREPFISCSHLECRTFFLT (SEQ ID NO:17) or sequences 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 with SEQ ID NO:17. In one aspect, the NA-Fc fusion may comprise...

[0121]

[0122] As noted above, the Fc region can contain one or more mutations, such as L234Y, L235V, L235Q, G236W, S239D, S239M, F243L, T256A, K290A, R292P, N297Q, S298A, Y300L, V305I, A330L, I332E, E333A, K334A and / or P396L. Therefore, this paper specifically discloses an Fc region containing the following: leucine (L) or tyrosine (Y) at residue 234; leucine (L), glutamine or valine (V) at residue 235; glutamine (G) or tryptophan (W) at residue 236; serine (S), methionine (M) or aspartic acid (D) at residue 239; phenylalanine (F) or leucine (L) at residue 243; threonine (T) or alanine (A) at residue 256; histidine (H) or aspartic acid (D) at residue 268; aspartic acid (D) or glutamic acid (E) at residue 270; lysine (K) or alanine (A) at residue 290; and arginine at residue 292. (R) or proline (P), serine (S) or alanine (A) at residue 298, asparagine or glutamine at residue 297, tyrosine (Y) or leucine (L) at residue 300, valine (V) or isoleucine (I) at residue 305, lysine (K) or aspartic acid (D) at residue 326, alanine (A), methionine (M) or leucine (L) at residue 330, isoleucine (I) or glutamic acid (E) at residue 332, glutamic acid (E) or alanine (A) at residue 333, lysine (K), glutamic acid (E) or alanine (A) at residue 334, and / or proline (P) or leucine (L) at residue 396. It should be understood specifically that the Fc region may be devoid of substitutions or contain any one or two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, or seventeen combinations of substitutions mentioned herein.Therefore, in one aspect disclosed herein, there exists a fusion protein comprising substitutions in the Fc region at F243L, R292P, Y300L, V305I, and P396L, wherein the Na4-Fc sequence comprises MNPNQKITTIGSICLVVGLISLILQIGNI ISIWISHSIQTGSQNHTGICNQNIITYKNSTWVKDTTSVILTGNSSLCPIRGWAIYSKDNSIRIGSKGDVFVIREPFISCSHLECRTFFLTDKTHTCPPCPAPELLGGPSVFLLPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTK PPEEQYNSTLRVVSILTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPLVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:20); the substitution includes the sequence GGPSVFLLPPKPKDTLMISRTPEVTCVVVDVSHEDPEV The sequence KFNWYVDGVEVHNAKTKPPEEQYNSTLRVVSILTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKA KGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPLVLDSDGSFF LYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:25) contains an Fc domain, and the sequence comprises a CH2 domain having the sequence GGPSVFLLPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPPEEQYNSTLRVVSILTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK (SEQ ID NO:26) and a CH2 domain having the sequence GQPREPQVYTLPPSREEMTKNQVSLTC The CH3 domain of LVKGFYPSDIAVEWESNGQPENNYKTTPLVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL HNHYTQKSLSLSPGK (SEQ ID NO:27).

[0123] In one aspect, the Na4-Fc fusion comprises S239D, I332E, and A330L substitutions, said substitutions having an Fc domain having the sequence GGPDVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPLPEEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO:21), said sequence comprising an Fc domain having the sequence GGPDVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF The CH2 domain of NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPLPEEKTISKAK (SEQ ID NO:22) and the CH2 domain of GQPREPQVYTLPPSREEMTKNQVSLTC LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL HNHYTQKSLSLSPG (SEQ ID NO:24) and MNPNQKITTIGSICLVVG LISLILQIGNIISIWISHSIQTGSQNHTGICNQNIITYKNSTWVKDTTSVILTGNSSLCPIRGWAIYSKDNSIRIGSKGDVFVIREPFISCSHLECRTFFLTDKTHTCPPCPAPELLGGPDVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEV HNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPLPEEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:23).

[0124] In one aspect, the Na4-Fc fusion protein may contain two Fc domains connected by a hinge region. For example, the Na-Fc fusion protein may contain the sequence (SEQ ID NO:28).

[0125] In another aspect, the Fc domain can be an asymmetric variant; for example, one heavy chain Fc domain may contain L234Y / L235Q / G236W / S239M / H268D / D270E / S298A, while another Fc domain may contain D270E / K326D / A330M / K334E.

[0126] Generally, any amino acid substitutions are conserved: that is, they are limited to exchanges within the following groups: Group 1: glycine, alanine, valine, leucine, and isoleucine; Group 2: serine, cysteine, threonine, and methionine; Group 3: proline; Group 4: phenylalanine, tyrosine, and tryptophan; and Group 5: aspartic acid, glutamic acid, asparagine, and glutamine.

[0127] This disclosure also contemplates nucleic acids encoding any fusion protein disclosed herein, for example, SEQ ID NO:19 encoding the Na-Fc fusion protein described in SEQ ID NO:18; SEQ ID NO:29 encoding the Na-Fc fusion protein described in SEQ ID NO:20; SEQ ID NO:30 encoding the Na-Fc fusion protein described in SEQ ID NO:23; and SEQ ID NO:31 encoding the NA-2xFc fusion protein described in SEQ ID NO:28. This disclosure further contemplates vectors comprising such nucleic acids according to the present invention and cells comprising such vectors. Vectors and cells containing said vectors can be prepared using methods known in the art.

[0128] (II) Engineered feeder cells, engineered cell membrane granules, and engineered exosomes containing membrane-bound Fc

[0129] The compositions disclosed herein include compositions comprising Fc-binding feeder cells (FC), compositions comprising Fc-binding engineered cell membrane (PM) particles, and compositions comprising Fc-binding engineered exosomes. The Fc-binding engineered PM particles comprise PM nanoparticles derived from Fc-binding feeder cells. The Fc-binding engineered exosomes comprise exosomes or other extracellular vesicles derived from Fc-binding feeder cells, as further described below. Alternatively, exosomes may be derived from other sources such as platelets and megakaryocytes.

[0130] As used herein, the term "Fc-binding" should be understood to mean that the Fc domain is coupled to the outer surface of a feeder cell or engineered particle in an inverted direction (i.e., the intracellular facing N-terminus) via a transmembrane peptide. This can be achieved using the Fc fusion peptides disclosed herein. Therefore, one aspect of this disclosure provides a feeder cell composition comprising at least one Fc-binding feeder cell, i.e., a feeder cell comprising an Fc domain that binds to the outer surface of the feeder cell, as described in further detail below. For example, the feeder cell may be genetically modified to express an Fc domain that binds to the outer surface of the feeder cell, i.e., to express the Fc fusion peptide described in further detail below. Another aspect of this disclosure provides a feeder cell-free NK cell expansion composition comprising at least one Fc-binding engineered particle, i.e., an engineered particle comprising an Fc domain that binds to the outer surface of the feeder cell in an inverted direction. In some aspects, the feeder cell may be engineered to express a ligand that can be labeled with a humanized antibody (e.g., CD20).

[0131] In the feeder cell composition, at least one Fc-binding feeder cell optionally comprises at least one NK cell effector. In one example, the Fc-binding feeder cell comprises one NK cell effector, namely IL-15 or IL-21. The Fc-binding feeder cell may comprise at least two or more different NK cell effectors.

[0132] In NK cell expansion compositions that do not contain feeder cells, Fc-bound engineered PM particles optionally comprise at least one NK cell effector. In one example, the Fc-bound engineered particle comprises one NK cell effector, namely IL-15 or IL-21. The Fc-bound engineered PM particle may comprise at least two or more different NK cell effectors.

[0133] In the feeder cell composition or the composition without feeder cells, at least two NK cell effectors are present, and the second NK cell effector may be, for example, 41BBL. In the feeder cell composition or the NK cell expansion composition without feeder cells, the feeder cells or engineered PM particles contain one or more NK cell effectors, which may be selected from 41BBL, IL-15, IL-2, IL-12, IL-18, IL-21, MICA, UBLP, 2sB4, LFA-1, Notch ligand, NKp46 ligand or BCM1 / SLAMF2, TLR ligand, and NKG2D ligand or cytokines. In the exemplary compositions, at least one additional NK cell effector is IL-15 or IL-21.

[0134] (a) Fc binding to feeder cells

[0135] This disclosure provides feeder cells comprising the Fc fusion peptide as detailed above. The NK cell feeder cells used in the methods disclosed herein and for manufacturing the PM particles and exosomes disclosed herein can be irradiated autologous or allogeneic peripheral blood mononuclear cells (PBMCs) or unirradiated autologous or allogeneic PBMCs, RPMI8866, HFWT, 721.221, or K562 cells, as well as EBV-LCL, other non-HLA or low-HLA expressing cell lines, or patient-derived primary tumors that can be used as tumor vaccines. Fc-bound feeder cells can be prepared by transfecting or transducing feeder cells with any of the Fc fusion peptides described herein using standard transduction or transfection techniques well known in the art. For example, a cDNA vector of the Fc fusion peptide disclosed herein can be ligated into an expression plasmid, allowing expression in bacterial (Escherichia coli), insect, or mammalian cells. The cDNA vector may be tagged with a FLAG or HIS tag. Suitable transfection methods include nuclear transfection (or electroporation), calcium phosphate-mediated transfection, cationic polymer transfection (e.g., DEAE-dextran or polyethyleneimine), viral transduction, virion transfection, viral particle transfection, liposome transfection, cationic liposome transfection, immunoliposome transfection, non-liposomal lipid transfection, dendritic polymer transfection, heat shock transfection, magnetic transfection, lipid transfection, gene gun delivery, puncture transfection, acoustic perforation, optical transfection, and nucleic acid uptake enhanced by proprietary reagents. Transfection methods are well-known in their respective fields (see, for example, *Current Protocols in Molecular Biology*, Ausubel et al., John Wiley & Sons, New York, 2003; or *Molecular Cloning: A Laboratory Manual*, Sambrook and Russell, Cold Spring Harbor Press, Cold Spring Harbor, NY, 3rd ed., 2001). Alternatively, molecules can be introduced into cells via microinjection. For example, molecules can be injected into the cytoplasm or nucleus of the cell of interest. The amount of each molecule introduced into the cell can vary, but those skilled in the art are familiar with the methods used to determine the appropriate amount.

[0136] It should be understood that various molecules can be introduced into cells simultaneously or sequentially. For example, an Fc fusion peptide and one or more membrane-bound NK cell effectors can be introduced simultaneously into feeder cells. Alternatively, one molecule can be introduced first, followed by one or more other molecules. For example, feeder cells transfected or transduced with an Fc fusion peptide can be further transfected and / or infected with membrane-bound NK cell effectors such as IL-15 and / or IL-21 and / or 41BBL and / or EBV-LCL and / or one or more other NK cell effectors. Alternatively, feeder cells can be simultaneously transfected or transduced with an Fc fusion peptide and membrane-bound NK cell effectors such as IL-15 and / or IL-21 and / or 41BBL and / or EBV-LCL and / or one or more other NK cell effectors. Alternatively, feeder cells previously transfected or transduced to express membrane-bound NK cell effectors such as IL-15 and / or IL-21 and / or 41BBL and / or infected with EBV-LCL and / or one or more other NK cell effectors can be transfected or transduced with the Fc fusion peptide. It should also be understood that membrane-bound Fc can be obtained using other means, such as chemical conjugation methods known in the art.

[0137] Generally, cells are maintained under conditions suitable for cell growth and / or maintenance. Suitable cell culture conditions are well-known in the field and described, for example, in Santiago et al., *Proceedings of the National Academy of Sciences of the United States of America*, 2008, 105:5809-5814; Moehle et al., *Proceedings of the National Academy of Sciences of the United States of America*, 2007, 104:3055-3060; Urnov et al., *Nature*, 2005, 435:646-651; and Lombardo et al., *Nature Biotechnology*, 2007, 25:1298-1306. Those skilled in the art will understand that the methods used for culturing cells are known in the field and can and will vary depending on the cell type. In all cases, routine optimization can be used to determine the optimal technique for a particular cell type.

[0138] Fc-bound feeder cells can be used in cell culture to directly stimulate NK cells, or to prepare PM particles or exosomes derived from feeder cells.

[0139] (b) Fc binds to PM particles

[0140] Fc-bound engineered PM particles include Fc-bound PM particles prepared from Fc-bound NK cell feeder cells using well-known methods. PM particles are vesicles (i.e., liposomes) made from cell membranes or artificially manufactured. PM particles may contain a lipid bilayer or only a lipid monolayer. PM particles may be prepared in thin-layer, multi-layer, or inverted forms. PM particles can be prepared from Fc-bound feeder cells as described herein using known cell membrane preparation 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 average diameter of PM particles as disclosed herein ranges from about 170 nm to about 300 nm.

[0141] (c)Fc-binding exosomes

[0142] Fc-binding exosomes disclosed herein can be prepared from cells that secrete exosomes, which can be prepared from Fc-binding NK cell feeder cells using well-known methods, wherein the exosomes are extracellular products of the cells that secrete exosomes, as described in U.S. Patent Application Publication No. 20170333479, the entire disclosure of which is incorporated herein by reference. Exosomes contain lipids and proteins, and the identity of the proteins found in a particular exosome depends on one or more of the cells that produce it. Exosomes disclosed herein contain the Fc fusion peptide (i.e., Fc-binding) as disclosed herein, and optionally contain one or more stimulating peptides (NK cell effectors) present in the exosome membrane. Exosomes can be produced, for example, from cell lines engineered to improve the formation or release of exosomes. Such cell lines include, but are not limited to, the Fc-binding cell lines described above in Part II(a). Non-restricted cell lines are Fc-binding K562-mb15-41BBL and Fc-binding K562. In some respects, as disclosed herein, the average diameter of exosomes ranges from about 30 nm to about 100 nm or up to about 160 nm. In one respect, the average diameter of exosomes is about 60-80 nm. The ability of exosomes to achieve a smaller particle size than readily available PM particles means that exosomes can be more easily adapted for use in applications where smaller sizes are preferred. For example, exosomes may be preferred in applications requiring diffusion across physiological barriers, enhanced biodistribution through tissue compartments, or intravenous injection.

[0143] (III) Composition

[0144] This disclosure provides various NK cell expansion compositions comprising Fc-binding feeder cells as disclosed above, and in other aspects provides NK cell expansion compositions without feeder cells, the compositions comprising one or more engineered Fc-binding particles, such as PM particles or exosomes as disclosed above. Any Fc-binding feeder cells or Fc-binding engineered PM particles used in the compositions optionally further comprise at least one, two or more different NK cell effectors. In one aspect, one NK cell effector is IL-21, and in some aspects, one NK cell effector is IL-21, while a second NK cell effector is 41BBL. The Fc-binding feeder cells or Fc-binding engineered PM particles optionally comprise one or more additional NK cell effectors as disclosed above.

[0145] NK cell expansion compositions comprising PM particles containing a cytoplasmic membrane may further comprise a plurality of microparticles / nanoparticles, wherein the cytoplasmic membrane coats a plurality of microparticles and / or nanoparticles. The microparticles / nanoparticles may comprise magnetic microparticles, silica beads, polystyrene beads, latex beads, particulate contrast agents, particulate cancer therapeutic agents, or any combination thereof.

[0146] This disclosure also considers NK cell expansion infusion formulations comprising any NK cell expansion compositions disclosed herein and pharmaceutically acceptable carriers.

[0147] Therapeutic pharmaceutical compositions can be prepared by combining Fc-bound feeder cells or engineered PM particles with pharmaceutically acceptable carriers known in the art, such as those described in Remington: The Science and Practice of Pharmacy (19th edition), edited by ARGennaro, Mack Publishing Company, Easton, PA, 1995. Examples of pharmaceutically acceptable carriers include, but are not limited to, sterile water, saline, Ringer's solution, glucose solution, and buffer 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.

[0148] In addition to the selected molecules, the pharmaceutical composition may also include a carrier, thickener, diluent, buffer, preservative, surfactant, etc. The pharmaceutical composition may also include one or more active ingredients, such as antimicrobial agents, anti-inflammatory agents, anesthetics, etc.

[0149] 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 applied. Depending on whether local or systemic treatment is desired and the area to be treated, pharmaceutical compositions can be suitably prepared for administration to mammals, particularly humans, via any of a variety of known routes of administration. Administration can be local (including ocular, vaginal, rectal, and intranasal), oral, by inhalation or parenteral administration, such as by intravenous infusion or injection, or by subcutaneous, intraperitoneal, intramuscular, intracavitary, or percutaneous injection.

[0150] Preparations intended for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils (e.g., olive oil), and injectable organic esters (e.g., ethyl oleate). Aqueous carriers include water, alcoholic solutions / aqueous solutions, emulsions, or suspensions, including saline and buffer media. Parenteral carriers include sodium chloride solutions, Ringer's glucose, glucose and sodium chloride, lactated Ringer's solution, or fixed oils. Intravenous carriers include fluids and nutritional supplements, electrolyte supplements (e.g., Ringer's glucose-based electrolyte supplements), and so on. Preservatives and other additives may also be present, such as antimicrobial agents, antioxidants, chelating agents, and inert gases, etc.

[0151] Formulations intended for topical application may include ointments, lotions, creams, gels, drops, suppositories, sprays, liquids, and powders. Conventional drug carriers, aqueous solutions, powders or oily bases, thickeners, etc., may be necessary or desired.

[0152] Compositions intended for oral administration include powders or granules, suspensions or solutions in aqueous or non-aqueous media, capsules, sachets or tablets. Thickeners, flavoring agents, diluents, emulsifiers, dispersants or binders may be desired.

[0153] Some compositions can potentially be administered as pharmaceutically acceptable acid or base addition salts, formed by reaction with inorganic acids such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanate, 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 inorganic bases such as sodium hydroxide, ammonium hydroxide, potassium hydroxide, and organic bases such as mono-, di-, and trialkyl and arylamines and substituted ethanolamines.

[0154] Therefore, NK cell expansion infusion formulations can be formulated for parenteral infusion, arterial infusion, intravenous infusion, catheter-mediated infusion, intravenous injection, intraperitoneal injection, subcutaneous injection, oral delivery, or local delivery.

[0155] In one aspect, this disclosure covers any NK cell expansion composition prepared in vitro or ex vivo as disclosed herein, administered or injected into a subject requiring NK cell expansion. It should be understood and contemplated herein that infusion may be performed in vitro with commercially available NK cells or ex vivo from a donor source (e.g., an allogeneic donor or an autologous donor (i.e., the recipient subject receiving the expanded NK cells)).

[0156] In another aspect, this disclosure contemplates NK cell compositions comprising an in vitro population of NK cells in contact with either an Fc-binding feeder cell composition as disclosed herein or an Fc-binding NK cell expansion composition without feeder cells as disclosed herein.

[0157] In another aspect, this disclosure considers sources of NK cells, including but not limited to peripheral blood, iPSC-derived NK cells, ESC-derived NK cells, NK cells with high-affinity Fc receptor Phe or Val polymorphisms at position 158, and genetically modified NK cells.

[0158] In another aspect, this disclosure contemplates an expanded NK cell population exposed in vitro to an NK cell expansion composition, said composition being free of feeder cells and comprising at least one Fc-binding engineered particle as disclosed herein, said Fc-binding engineered particle comprising at least two NK cell effectors, wherein one of the at least two NK cell effectors is IL-21 or IL-15. The expanded NK cell population can exhibit increased cytotoxicity compared to unexpanded NK cells. In various aspects, the expanded NK cell population can exhibit at least about 2, 5, or 10 times the cytotoxicity of unexpanded NK cells.

[0159] In another aspect, this disclosure provides compositions comprising a therapeutic dose of NK cells and optionally a pharmaceutically acceptable carrier, said NK cells comprising an expanded NK cell population as disclosed herein. The expanded NK cell population may exhibit higher CD16 levels and other advantageous properties such as enhanced cytotoxicity and ADCC function. As will be appreciated by those skilled in the art, the amount of NK cells providing a therapeutic dose will vary depending on many factors, discussed, for example, in U.S. Patent No. 9,623,082. These factors include the age, sex, and diagnosis of the subject, as well as the route of administration, which may be, but is not limited to, oral, oral, mucosal, and intravenous routes. For example, the therapeutic dose may be 1 × 10⁻⁶ per dose. 4 / kg to 1×10 8 The dose can be between / kg and may be included in a single dose or divided into multiple doses. It should be understood that the equivalent of the therapeutic dose described above may also be expressed as a quantity per total body surface area.

[0160] In another aspect, this disclosure also provides NK cell expansion culture medium formulations comprising any NK cell expansion composition as disclosed herein and NK cell expansion culture medium solutions comprising at least one soluble culture medium component such as cytokines, IL-2, IL-12, IL-15, IL-18, IL-21, NAM, ascorbate, or any combination thereof.

[0161] (IV) Method

[0162] (a) Methods for increasing the cytotoxicity of NK cells

[0163] In one aspect, this disclosure provides a method for increasing the cytotoxicity of NK cells by expanding an initial NK cell population using an NK cell expansion composition or formulation as disclosed herein. The disclosed method provides a simple expansion platform that avoids complex alternative expansion processes, including, for example, coating a solid carrier with a monoclonal antibody and using one or more soluble cytokines in solution. Instead, in the method disclosed herein, an initial NK cell population is obtained from a donor and exposed to an NK cell expansion composition as disclosed herein. Exposure can be performed in vitro or in vivo. Figure 4 This is a schematic diagram of Fc stimulation of NK cells according to the present disclosure. NK cells contact one or more Fc-binding feeder cells, Fc-binding PM particles, or Fc-binding exosomes, or any combination thereof. The exposed Fc domains bind to CD16 on the surface of NK cells, thereby stimulating NK cells to expand more rapidly and / or more effectively and produce NK cells with higher antitumor toxicity and a more favorable overall phenotype.

[0164] like Figure 4As indicated herein, the composition for contacting NK cells may comprise any Fc-binding feeder cells, Fc-binding engineered PM particles, or Fc-binding engineered exosomes disclosed herein. The engineered PM particles may be Fc-binding PM particles. In one aspect, the optionally present NK cell effector is IL-21 or IL-15. An optionally present second NK cell effector may be selected from 41BBL, IL-2, IL-12, IL-15, IL-18, IL-21, MICA, UBLP, 2B4, LFA-1, Notch ligand, NKp46 ligand, or BCM1 / SLAMF2, TLR ligand, and NKG2D ligand. In one aspect, the second NK cell effector is 41BBL. The composition may further comprise at least one additional (i.e., a third, fourth, fifth, etc.) NK cell effector selected from IL-2, IL-12, IL-15, IL-18, IL-21, MICA, UBLP, 2sB4, LFA-1, Notch ligand, NKp46 ligand or BCM1 / SLAMF2, TLR ligand, and NKG2D ligand. NK cell expansion performed in this manner can achieve far more than a few-fold (approximately 3-4 times) within 10 days. More specifically, NK cell expansion according to the method of the present invention can achieve 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 to about 2000-fold cell number increases within 16 days, or even greater multiple increases over a longer period. Therefore, the disclosed method is suitable for scaling up the production of NK cells. NK cells can be derived from peripheral blood, spleen NK cells, lymphocyte preparations (e.g., erythrocyte sedimentation rate (ESR) amber layer, 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 receptors such as Phe or Val at position 158, as known in the art and described in, for example, *Blood* (1997) 90:1109-14 and *J Clin Invest.* (1997) 100:1059–70)). The genetically modified NK cell sources can be engineered using methods known in the art. Alternatively, NK cells can be derived from a cell donor carrying the desired polymorphism, and the supplied cells serve as the initial NK cell population expanded by the methods described herein and using the compositions described herein. Thus, in this context, "genetically modified" encompasses naturally occurring NK cells carrying the polymorphism.The method can be applied to NK cells derived from human sources or other animals.

[0165] Furthermore, the disclosed method has the additional benefit of providing cells with higher cytotoxicity and ADCC function. An initial NK cell population expanded according to the disclosed method produces an expanded NK cell population exhibiting at least about 2 times the cytotoxicity of the initial NK cell population, at least about 4 times the cytotoxicity of the initial NK cell population, at least about 5 times the cytotoxicity of the initial NK cell population, at least about 8 times the cytotoxicity of the initial NK cell population, or at least about 10 times the cytotoxicity of the initial NK cell population. Moreover, NK cells expanded according to the disclosed method exhibit higher cytotoxicity against targets with ADCC capability. Higher expression of ADCC-related proteins, such as CD16 in non-limiting examples, or other NK cell ligands, such as NKG2D, NKp46, and CD62L in non-limiting examples, 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 cell activation. Combining markers can provide signals of increased cytotoxicity, even when cytotoxicity cannot be directly assessed. For example, compared to non-expanded NK cells, expanded NK cell populations, as disclosed herein, may exhibit increased killing of tumor targets or secretion of higher levels of anti-tumor cytokines (IFN, TNF). In another aspect, compared to non-expanded NK cells, expanded NK cell populations, as disclosed herein, may exhibit increased expression of NKG2D, NKp46, and CD16. Various methods for detecting the amount of specific proteins to assess the activation status of NK cells are known in the art and can be used, including spectroscopic methods such as flow cytometry or immunoassays such as Western blotting, enzyme-linked immunosorbent assay (ELISA), protein immunoprecipitation; immunoelectrophoresis or immunostaining.

[0166] In addition, the expanded NK cell populations disclosed herein can exhibit improved tolerance to cryopreservation, viability and cytotoxicity, and post-freezing and post-thawing capabilities.

[0167] NK cell compositions expanded from Fc-expressing feeder cells exhibited higher cytotoxicity against SKOV3 ovarian cancer target cells, such as... Figure 2 and Figure 3As shown in the diagram, the NK cell composition expanded using feeder cells expressing the Fc domain possesses an enhanced phenotype with increased CD16, NKp46, and CD62L. These NK cells with the enhanced phenotype can have enhanced therapeutic efficacy. Increased CD16 allows for enhanced binding to antibody-coated target cells. Increased NKp46 can have a stronger ability to bind activation ligands. Increased CD62L, as an L-selectin ligand, can enhance NK cell transport to lymphatic or bone marrow compartments.

[0168] (b) Therapeutic methods

[0169] The compositions and methods disclosed herein can be used in a variety of therapeutic, diagnostic, industrial, and research applications. In some aspects, this disclosure can be used to treat cancer. Thus, in one aspect, this document discloses a method for treating, inhibiting, alleviating, and / or preventing cancer, cancer recurrence or metastasis, or infectious diseases such as viral or bacterial infections in a subject, said method comprising administering to a subject in need an effective amount of the composition as described herein or an expanded population of NK cells.

[0170] Cancer can be selected from, but is not limited to, hematologic cancers, lymphomas, 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, cancer includes solid tumors. In another aspect, cancer is selected from acute myeloid leukemia, myelodysplastic syndrome, chronic myeloid leukemia, acute lymphoblastic leukemia, myelofibrosis, and multiple myeloma. In yet another aspect, cancer is selected from leukemia, lymphoma, sarcoma, and carcinoma, and can originate from the bone marrow, brain, lungs, breast, pancreas, liver, head and neck, skin, reproductive tract, prostate, colon, liver, kidneys, peritoneum, bone, joints, and eyes.

[0171] In another aspect, treatment methods include: methods for preventing, inhibiting, mitigating, or reducing cancer recurrence or metastasis after stem cell transplantation; methods for regulating the T-cell repertoire after stem cell transplantation; general methods for regulating the immune repertoire; methods for preventing, inhibiting, mitigating, or reducing acute or chronic graft-versus-host disease; and methods for preventing, inhibiting, mitigating, or reducing 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 an effective amount of the composition or expanded NK cell population described herein to a subject in need.

[0172] Any disclosed treatment method may further comprise administration to the subject (simultaneously or as a single agent) of an additional therapeutic agent or regimen and an effective amount of the composition or expanded NK cell population as described herein. The additional therapeutic agent may be a pharmaceutical preparation regimen such as Cy-Flu, Bu-Flu, Flu-Mel, or similar to dose or administration adjustments. Alternatively, the additional therapeutic agent may be a graft-versus-host disease (GvHD) agent, such as, but not limited to, cyclophosphamide. Alternatively, the additional therapeutic agent or regimen may be selected from chemotherapeutic agents and chemotherapy regimens, in non-limiting instances such as those known by the acronym CHOP, FLAG (including FLAG-Ida or FLAG-IDA or IDA-FLAG or Ida-FLAG; and FLAG-Mito or FLAG-MITO or Mito-FLAG or MITO-FLAG or FLANG), IA or IAC or 7+3. For example, this document anticipates that the disclosed methods for inhibiting, mitigating, and / or preventing cancer metastasis and / or recurrence may include the administration of any anticancer agent known in the art, including but not limited to abemaciclib, abiraterone acetate, abitrexate (methotrexate), abraxane (paclitaxel albumin-stabilized nanoparticle formulation), ABVD, ABVE, ABVE-PC, AC, AC-T, Adcetris (brentuximab vedotin), ADE, ado-trastuzumab emtansine, adriamycin (doxorubicin hydrochloride), and afatinib dimaleate. Dimaleate, Afinitor (everolimus), Akynzeo (netupitant hydrochloride and palonosetron hydrochloride), Aldara (imiquimod), Aldesleukin, Alecensa (alectinib), Alemtuzumab, Alimta (pemetrexed disodium), Aliqopa (copanlisib)Hydrochloride), Alkeran (Melphalan Hydrochloride for Injection), Alkeran Tablets (Melphalan), Aloxi (Palonosetron Hydrochloride), Alunbrig (Brigatinib), Ambochlorin (Chloramic Acid), Amifostine, Aminolevulinic Acid, Anastrozole, Aprepitant, Aredia (Pamidronate Disodium) Disodium), Arimidex (anastrozole), Aromasin (exemestane), Arranon (nelarabine), arsenic trioxide, Arzerra (ofatumumab), Asparaginase Erwinia Chrysanthemi), Atezolizumab, Avastin (Bevacizumab), Avelumab, Axitinib, Azacitidine, Bavencio (Avelumab), BEACOPP, Becenum (Carmustine), Belinostat (Belinostat), Bendamustine Hydrochloride, BEP, Besponsa (Inotuzumab Ozogamicin), Bevacizumab, Bexarotene, Bexxar (Tositumomab and Iodine I)131 Tocetosumab), Bicalutamide, BiCNU (carmustine), Bleomycin, Blinatumomab, Blincyto (Blinatumomab), Bortezomib, Bosulif (Bosutinib), Bosutinib, Belentosumab-Vidotine, Brigatinib, BuMel, Busulex (Busulfan), Cabazitaxel, Cabometyx (Cabozantinib-S-Malate) Cabozantinib S-malate, CAF, Camppath (alemumab), Camptosar, Irinotecan Hydrochloride, Capecitabine, CAPOX, Carac (topical fluorouracil), Carboplatin, Carboplatin-paclitaxel, Carfilzomib, Carmubris (carmustine), Carmustine, Carmustine implant, Casodex (bicalutamide), CEM, Ceritinib, Cerubidine (daunorubicin hydrochloride)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), Cobancoxide Hydrochloride, COPDAC, COPP, COPP-ABV, Cosmegen (actinomycin D), Cotellic (cobimetinib), Crizotinib Crizotinib, CVP, cyclophosphamide, Cyfos (ifosfamide), Cyramza (ramucirumab), cytarabine, cytarabine liposomes, Cytosar-U (cytarabine), Cytoxan / Cyclophosphamide, Dabrafenib, Dacarbazine, Dacogen (decitabine), Actinomycin D, Daratumumab, Darzalex (daratumumab), Dasatinib, daunorubicin hydrochloride, daunorubicin hydrochloride and cytarabine liposomes, decitabine, sodium defibrinoside, sodium defibrinoside, Degarelix, Denileukin Diftitox, Denosumab, DepoCyt (cytarabine liposome), Dexamethasone, Dexrazoxane Hydrochloride, Dinutuximab, Docetaxel, Doxil (doxorubicin hydrochloride liposome), Doxorubicin Hydrochloride, Dox-SL (doxorubicin hydrochloride liposome), DTIC-Dome (dacarbazine), Durvalumab, Efudex (topical fluorouracil), Elitek (rasburicase), Ellence (epirorubicin hydrochloride)Hydrochloride, Elotuzumab, Eloxatin (oxaliplatin), Eltrombopag Olamine, Emend (aprepitant), Empliciti (Elotuzumab), Enasidenib Mesylate Mesylate, Enzalutamide, Epirubicin Hydrochloride, EPOCH, Erbitux (Cetuximab), Eribulin Mesylate, Erivedge (Vismodegib), Erlotinib Hydrochloride, Erwinaze (Erwinia asparaginase), Ethyol (Amifostine), Etopophos (Etoposide Phosphate), Etoposide, Etoposide Phosphate, Evacet (Liposome Doxorubicin Hydrochloride), Everolimus, Evista (Raloxifene Hydrochloride) Hydrochloride), Evomela (melphalan hydrochloride), Exemestane, 5-FU (fluorouracil injection), 5-FU (topical fluorouracil), Fareston (toremifene), Farydak (panobinostat), Faslodex (fullvestrant), FEC, Femara (letrozole), Filgrastim, Fludara (fludarabine phosphate)Phosphate, fludarabine phosphate, Fluoroplex cream (topical fluorouracil), Fluoroplex injection (topical fluorouracil), Flutamide, Phosphite (methotrexate), Phosphite PFS (methotrexate), FOLFIRI, FOLFIRI-bevacizumab, FOLFIRI-cetuximab, FOLFIRINOX, FOLFOX, Folotyn (Pralatrexate), FU-LV, Fulvestrant, Gardasil (Gardasil) rdasil (recombinant quadrivalent HPV vaccine), Gardasil 9 (recombinant nine-valent HPV vaccine), Gazyva (Obinutuzumab), Gefitinib, Gemcitabine hydrochloride, Gemcitabine-cisplatin, Gemcitabine-oxaliplatin, Gemcitabine-Ozomicin, Gemzar (Gemcitabine hydrochloride), Gilotrif (Afatinib dimaleate), Gleevec (Imatinib mesylate) Mesylate), Gliadel (carmustine implant), Gliadel chip (carmustine implant), Glucarpidase, Goserelin Acetate, Halaven (eribulin mesylate), Hemangeol (propranolol hydrochloride), Herceptin (trastuzumab), recombinant bivalent HPV vaccine, recombinant nine-valent 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), Idelalisib, Idhifa (Enasidenib Mesylate)Mesylate), Ifex (ifosphosphatamide), ifosphosphatamide, Ifosfamidum (ifosphosphatamide), IL-2 (aldesleukin), Imatinib Mesylate, Imbruvica (ibrutinib), Imfinzi (durvalumab), imiquimod, Imlygic (talimogene Laherparepvec), Inlyta (axitinib), Inotuzumab Ozogamicin, recombinant interferon α-2b, interleukin-2 (aldesleukin), intron A (recombinant interferon α-2b), iodine I 131. Tocetosumab and Ipilimumab, Iressa (Gefitinib), Irinotecan Hydrochloride, Irinotecan Liposome Hydrochloride, Istodax (Romidepsin), Ixabepilone, Ixazomib Citrate, Ixempra (Ixabepilone), Jakafi (Ruxolitinib Phosphate), JEB, Jevtana (Cabazitaxel), Kadcyla (Adoxifene-Trastuzumab-Mestane), Keoxifene (Raloxifene Hydrochloride) Hydrochloride, Kepivance (Palifermin), Keytruda (Pembrolizumab), Kisqali (Ribociclib), Kymriah (Tisagenlecleucel), Kyprolis (Carfilzomib), Lanreotide Acetate, Lapatinib Ditosylate, Lartruvo (Olaratumab), Lenalidomide, Lenvatinib MesylateMesylate, Lenvima (lenvatinib mesylate), Letrozole, Leucovorin Calcium, Leukeran (chlorambucil), Leuprolide Acetate, Leustatin (cladribine), Levulan (aminolevulinic acid), Linfolizin (chlorambucil), LipoDox (liposome doxorubicin hydrochloride), Lomustine, Lonsurf (trifluorothymidine and tipiracil hydrochloride), Lupron (leuprolide), LupronDepot (leuprolide) Depot-Ped (Leuprorelin Acetate), Lynparza (Olaparib), Marqibo (Vincristine Sulfate Liposome), Matulane (Procarbazine Hydrochloride), Methionine Hydrochloride, Medroxyprogesterone Acetate, Mekinist (Trametinib), Melphalan, Melphalan Hydrochloride, Mercaptopurine, Mesna, Mesnex, Methazolastone (Temozolomide), Methotrexate, Methotrexate LPF, Methylnaltrexone Bromide, Methotrexate Sodium (Methotrexate), Methotrexate Sodium-AQ (Methotrexate), Midostaurin, Mitomycin C, Mitoxantrone Hydrochloride Hydrochloride, Mitozytrex (mitomycin C), MOPP, Mozobil (Plerixafor), Nitrogen Mustard (methylchloroethylamine hydrochloride), Mutamycin (mitomycin C), Myleran (Busulfan), Mylosar (azacitidine), Mylotarg (gefitinumab ozomicin), Paclitaxel nanoparticles (paclitaxel albumin-stabilized nanoparticle formulation), Navelbine (vinorelbine tartrate), Necitumumab, Nelarabine, Neosar (cyclophosphamide), Neratinib maleateMaleate, Nerlynx (lenatinib maleate), Netupitant, and palonosetron hydrochloride, Neulasta (polyethylene glycol filgrastim), Neupogen (filgrastim), Nexavar (sorafenib tosylate), Nilandron (nilutamide), Nilotinib, Ninlaro (ixazomib citrate), and Niraparib tosylate monohydrate Monohydrate, Nivolumab, Nolvadex (tamoxifen citrate), Nplate (Romiplostim), Obituzumab, Odomzo (Sonidegib), OEPA, Ofamumab, OFF, Olaparib, Olamab, Omacetaxine Mepesuccinate, Oncaspar (pegasparase), Ondansetron Hydrochloride, Onivyde (irinotecan liposome hydrochloride), Ontak (denileukin) Diftitox, Opdivo (nivolumab), OPPA, Osimertinib, Oxaliplatin, Paclitaxel, Paclitaxel albumin-stabilized nanoparticle formulation, PAD, Palbociclib, Palifermin, Palonosetron hydrochlorideHydrochloride, palonosetron hydrochloride and netupitant, pamidronate disodium, panitumumab, panobinostat, paraplatin (carboplatin), paraplatin (carboplatin), pazopanib hydrochloride, PCV, PEB, pegaspargase, PEG-filgrastim, PEG-interferon α-2b, PEG-intron (PEG-interferon α-2b), pembrolizumab, pemetrexed disodium, Perjeta (pertuzumab) Monoclonal antibodies, pertuzumab, cisplatin, cisplatin-AQ, praxavir, pomalidomide, pomalyst, ponatinib hydrochloride, Portrazza (nesetuzumab), pralatrexate, prednisone, procarbazine hydrochloride, interleukin (alder interleukin), Prolia (denosumab), Promacta (eltrombopag Olamine), propranolol hydrochloride Hydrochloride, Provenge (Sipuleucel-T), purine (mercaptopurine), Purixan (mercaptopurine), radium-223 dichloride, raloxifene hydrochloride, ramucirumab, rasburicase, R-CHOP, R-CVP, recombinant human papillomavirus (HPV) bivalent vaccine, recombinant human papillomavirus (HPV) nine Bivalent vaccines, recombinant human papillomavirus (HPV) quadrivalent vaccine, recombinant interferon alpha-2b, regorafenib, Relistor (methylnaltrexone bromide), R-EPOCH, Revlimid (lenalidomide), Rheumatrex (methotrexate), ribociclib, R-ICE, rituximab / rituximab, rituximab-hyaluronidase (Rituxan Hycela) (human rituximab and hyaluronidase)Human), rituximab, human rituximab and hyaluronidase, Rolapitant Hydrochloride, Romidepsin, Romiplostim, Rubidomycin (daunorubicin hydrochloride), Rubraca (rucaparib camsylate), ruxotinib phosphate, Rydapt (midotutolin), Sclerosol intrapleural aerosol (talc), Siltuximab, Siprulusel-T, Somatuline Depot (lanreotide acetate), Sonidegib, Sorafenib tosylate Tosylate, Sprycel (Dasatinib), STANFORDV, sterile talc, Stivarga (Regorafenib), Sunitinib malate, Sutent (Sunitinib malate), Sylatron (Pegylated interferon alpha-2b), Sylvant (Stutuximab), Synribo (Homoharringtonine), Tabloid (Thioguanine), TAC, Tafinlar (Dabrafenib), Tagrisso (Osimertinib), Talc, Latamoxetine, Tamoxifen citrate, Tarabine PFSPFS (cytarabine), Tarceva (erlotinib hydrochloride), Targretin (bexarotene), Tasigna (nilotinib), Taxol / Paclitaxel, Taxotere (docetaxel), Tecentriq (atezumab), Temodar (temozolomide), Temozolomide, Temsirolimus, Thalidomide (thalidomide / thalidomide), Thioguanine, Thiotepa, Tesalex, Tolak (topotecan hydrochloride), Toremifene, Torisel (temsirolimus), Tocetumab, and Iodine I 131. Totect (dexrazoxan hydrochloride), TPF, Trabectedin, Trametinib, Trastuzumab, Treanda (bendamustine hydrochloride), Trifluorothymidine and Tipiridine hydrochloride, Trisenox (arsenic trioxide), Tykerb (lapatinib ditosylate), Unituxin (dinutuximab), Uric acid triacetate, VAC, Vandetanib, VAMP, Varubi (rolapitant hydrochloride) Hydrochloride, Vectibix (panitumumab), VeIP, Velban (vincrine sulfate), Velcade (bortezomib), Velsar (vincrine sulfate), Vemurafenib, Venclexta (Venetoclax), Verzenio (Abemaciclib), Viadur (leuprolide acetate), Vidaza (azacitidine), Vincrine sulfate, Vincasar PFSPFS (Vincristine Sulfate), Vincristine Sulfate, Vincristine Sulfate Liposomes, Vinorelbine Tartrate, VIP, Vismodegib, Vistogaard (Uric acid triacetate), Voraxaze (Glucopithase), Vorinostat, Votrient (Pazopanib Hydrochloride), Vyxeos (Daunorubicin Hydrochloride and Cytarabine Liposomes), Wellcovorin (Leucovorin Calcium) Calcium), Xalkori (crizotinib), Xeloda (capecitabine), XELIRI, XELOX, Xgeva (denomab), Xofigo (radium 223 dichloride), Xtandi (enzalutamide), Yervoy (ipilimumab), Yondelis (trabectedin), Zaltrap (ziv-aflibercept), Zarxio (filgrastim), Zejula (niraparib tosylate monohydrate) Monohydrate), Zelboraf (vemurafenib), Zevalin (teimomab), Zinecard (dexrazoxan hydrochloride), Ziv-abflibercept, Zofran (ondansetron hydrochloride), Zoladex (goserelin acetate), Zoledronic acid, Zolinza (vorinostat), Zometa (zoledronic acid), Zydelig (ederaris), Zykadia (ceritinib), and / or Zytiga (abiraterone acetate). This article also considers chemotherapeutic agents as PD1 / PDL1 blocking inhibitors (e.g., lamboruzumab, nivolumab, pembrolizumab, pidilizumab, BMS-936559, atezolizumab, durvalumab, or averulimumab).

[0173] Alternatively, additional therapeutic agents may be selected from, but not limited to, 5-substituted 2-deoxyuridine analogs, nucleoside analogs, (non-nucleoside) pyrophosphate analogs, nucleoside reverse transcriptase (RT) inhibitors (NRTI), non-nucleoside reverse transcriptase inhibitors (NNRTI), protease inhibitors (PI) and integrase inhibitors, entry inhibitors and acyclic guanosine analogs, acyclic nucleoside phosphonates (ANP) analogs, hepatitis C virus (HCV) NS5A and NS5B inhibitors and influenza virus inhibitors, immunostimulants, interferons, oligonucleotides and antimitotic inhibitors. 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 isomer, tautomer, analogue, polymorph, solvate, derivative, or pharmaceutically acceptable salt thereof.

[0174] Alternatively, additional treatment agents may be antibiotics selected from, but not limited to, penicillins, tetracyclines, cephalosporins, lincomycin, macrolides, sulfonamides, glycopeptides, aminoglycosides, and carbapenems. Non-limiting examples of antiviral agents include amoxicillin, doxycycline, cephalexin, ciprofloxacin, clindamycin, metronidazole, azithromycin, sulfamethoxazole, trimethoprim, clavulanate, and levofloxacin.

[0175] (V) Kit

[0176] Another aspect of this disclosure provides a kit comprising at least one of the fusion peptides detailed above, and / or at least one of the Fc-binding feeder cells detailed above, and / or at least one of the Fc-binding engineered particles (PM particles and / or exosomes) detailed above. The fusion peptides may be provided in a suitable container along with other kit components, such as cell reagents, cell growth media, selection media, protein purification reagents, buffers, etc. The kits provided herein generally include instructions for carrying out the methods detailed below. The instructions included in the kit may be affixed to packaging material or may be included as part of the package insert. While instructions are typically written or printed material, they are not limited thereto. This disclosure contemplates any medium capable of storing and communicating the instructions to the end user. Such media include, but are not limited to, electronic storage media (e.g., disks, magnetic tapes, cassette tapes, chips), optical media (e.g., CD-ROMs), etc. As used herein, the term "instructions" may include the address of an internet site providing the instructions.

[0177] Example

[0178] Example 1 - CD16 conjugation via the Fc region enhanced proliferation rate after day 14.

[0179] The K562 cell line was obtained, which is a cell line expressing 41BBL and membrane-bound IL-21 (“CSTX-002”).

[0180] Independent samples of K562 cells were transfected with NA-Fc to generate Fc-binding K562 cells (“CSTX002-Fc”).

[0181] Peripheral blood mononuclear cells (PBMCs) were obtained from two different leukocyte sources (L43 and L44) and divided into multiple equal aliquots. NK cell expansion was tested in the PBMC sample from each donor in the presence of CSTX002, and NK cell expansion was tested in the other sample in the presence of CSTX002-Fc.

[0182] PBMCs isolated from the ESR amber layer via a Ficoll-Paque density gradient were grown in SCGM CellGro medium supplemented with 10% FBS and 100 U / mL IL-2, along with feeder cells (CSTX002 cells or CSTX002-Fc cells) treated with mitomycin C or irradiated, at a ratio of 1 feeder cell per NK cell. Cells were maintained at 37°C in a humidified atmosphere with 5% CO2. Starting from day 5, the medium was replaced every other day by replacing half of the medium with fresh medium supplemented with 100 U / mL. Cell counts were performed every other day, and culture concentration was checked periodically starting from day 7.

[0183] Figure 5 A plot showing NK cell expansion relative to culture days is presented, demonstrating that CD16 conjugation via the Fc region enhanced proliferation after day 14. NK cells were expanded from PBMCs derived from two donors' L54 (circles) or L44 (squares) cell lines using CSTX002 (open symbol) or CSTX002-Fc cell lines (closed symbol) as feeder cells. CD16 conjugation allowed for increased NK cell proliferation. NK cells from both donors expanded at the same rate until day 14 after stimulation with IL21 alone or with IL21 and Fc, at which point the Fc-stimulated culture divided at an increased rate compared to IL21 alone.

[0184] Example 2 - Cytotoxicity of Natural Killer Cells Expanded in the Presence of Fc-Binding Feeder Cells

[0185] CSTX002 cells and CSTX002-Fc cells were prepared as described in Example 1. Cytotoxicity analysis was performed as follows. The antitumor cytotoxicity of effector NK cells was measured using the ovarian cancer-derived target cell line SKOV3 transfected with green fluorescent protein (GFP) as a target. Target cells were cultured individually at 37°C in a 5% CO2 atmosphere (control wells) or at 0.5 × 10⁻⁶ cells / well. 6 Cells / mL were co-cultured with NK cells at a specified effector to target (E:T) ratio for 45 min. Cells were then centrifuged and resuspended in Annexin V-labeled buffer containing Annexin V-PacBlue antibody and incubated at 4°C for 15 min, followed by flow cytometry analysis. Analysis was based on the absolute amount of viable target cells (GFP+ / Annexin V-) remaining in each well containing the effector (VTC). E:T And refer to the average VTC in the "individual target" control well (VTC) T对照 ) Determine cytotoxicity.

[0186] Cytotoxicity E:T (%) = (VTC)E:T Average VTC T对照 )×100

[0187] Figure 6 Two graphs are presented, 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 line as feeder cells. For each of the two different donors, NK cells expanded with CSTX002-Fc were found to have increased cytotoxicity against SKOV3 cells.

[0188] Example 3 - Enhancing the cytotoxicity of natural killer cells from poorly responding donor PBMCs

[0189] PBMCs were obtained from donors previously observed to be non-cytotoxic to SKOV3 cells when expanded by stimulation with CSTX002 cells (without Fc). NK cells were expanded from previously observed poorly responding PBMCs using CSTX002(●) or CSTX002-Fc(■) cell lines as feeder cells, as described in Example 1. The cytotoxicity of the two different resulting NK cell populations was then tested, also as described in Example 1 but using SKOV3 cells transformed to express Fc. Figure 7 This is a cytotoxicity diagram of NK cells expanded from PBMCs using CSTX002(●) or CSTX002-Fc(■). Figure 7 The results showed that NK cells derived from PBMCs of donors with poor cytotoxicity towards SKOV3 cells responded well to expansion with Fc-binding feeder cells (CSTX002-Fc) that exhibited cytotoxicity against tumor targets, compared to those derived from PBMCs obtained from the same poorly responding donor and expanded with Fc-binding feeder cells (CSTX002). NK cells expanded with CSTX002-Fc exhibited better antibody-dependent cytotoxicity and higher killing activity against antibody-bound tumor targets.

[0190] Example 4 - Favorable receptor expression

[0191] NK cells were expanded from PBMCs derived from two donors, L43(●) or L44(■), using CSTX002 or CSTX002-Fc cell lines as feeder cells, as described in Example 1. The resulting NK cells were then analyzed. Figure 8This is a series of six (6) figures, each showing comparative receptor expression via NK cells expanded with CSTX002 feeder cells with or without membrane-binding Fc. NK cells were expanded from PBMCs derived from two donors, L43 (●) or L44 (■), using CSTX002 or CSTX002-Fc cell lines as feeder cells. Receptor expression in the expanded NK cells was analyzed, and these receptors are considered essential for cytotoxic function and homing. NK cells expanded with CSTX002-Fc showed higher expression of CD16, NKp46, and CD62L than NK cells expanded with CSTX002.

[0192] Example 5 - Fc binding to cell membrane granules

[0193] Engineered K562 cells, a cell line expressing 41BBL and membrane-binding IL-21, were treated as described in U.S. Patent No. 9,623,082 to obtain PM-mb21-41BBL cytoplasmic membrane vesicles, or “CSTX002” particles, or PM21 particles. Briefly, K562 cells 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 containing 10 mM EDTA, and resuspended in lysis buffer (50 mM HEPES, pH 7.4, a mixture of protease inhibitors). Cells were divided, and the lysis solution was centrifuged at 300 × g for 15 min to remove any remaining intact cells. The crude cytoplasmic membrane was separated from the cytoplasmic components by centrifugation at 4 °C for 30 min. The crude membrane was resuspended and further purified using a sucrose density gradient to produce pure cytoplasmic membrane vesicles, referred to as PM-mb21-41BBL.

[0194] Independent samples of K562 cells were transfected to express Fc to generate Fc-binding K562, and then treated as described above to obtain Fc-binding PM-mb21-41BBL cell membrane vesicles or “CSTX002-Fc” particles.

[0195] Peripheral blood mononuclear cells (PBMCs) were obtained from a single donor and aliquoted. NK cell expansion from PBMCs was tested in the presence of CSTX002 membrane particles or CSTX002-Fc membrane particles. The dosage for each membrane particle was 200 μg membrane protein / 1 mL culture. PBMCs isolated from blood were grown in SCGM CellGro medium supplemented with 10% FBS and 100 U / mL IL-2 via a Ficopac density gradient. Cells were maintained at 37°C in a humidified atmosphere with 5% CO2. Starting from day 5, the medium was replaced every other day by replacing half of the medium with fresh medium and replacing the amount of membrane removed by the medium. Cells were counted every other day, and culture content was checked on days 7, 10, and 14.

[0196] Perform cytotoxicity analysis as described in Example 1. NK cells amplified with CSTX002-Fc will show increased cytotoxicity against SKOV3 cells.

[0197] sequence

[0198] SEQ ID NO:29

[0199]

[0200] SEQ ID NO:30

[0201]

[0202] SEQ ID NO:31

[0203]

Claims

1. A fusion protein comprising a transmembrane domain linked to the amino terminus of an Fc domain.

Images