Transforming growth factor beta superfamily-imprinted natural killer cells for cancer immunotherapy

Abstract

The present disclosure relates to combination anti-cancer therapies using transforming growth factor beta (TGF-β) superfamily imprinted natural killer (TGF-βi NK) cells and CD38 targeting agents and methods of use thereof.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This PCT application claims priority to and benefit of U.S. Provisional Patent Application No. 63 / 449,419, entitled "Transforming Growth Factor Beta Superfamily Imprinted Natural Killer Cells for Cancer Immunotherapy," filed March 2, 2023, which is incorporated herein by reference in its entirety.

[0002] The present invention relates to combination anti-cancer therapies using transforming growth factor beta (TGF-β) superfamily imprinted natural killer (TGF-βi NK) cells and CD38 targeting agents and methods of use thereof. [Background technology]

[0003] Daratumumab (DARA) is a monoclonal antibody that binds to CD38 and has been successfully tested in patients with CD38+ multiple myeloma (MM) and is currently being studied in CD38+ T-ALL. DARA targets CD38+ cancer cells through several mechanisms, including antibody-induced cytotoxicity (ADCC), thereby activating NK cells and inducing them to secrete cytotoxic granules and death ligands against cancer targets. However, DARA also depletes NK cells that highly express CD38. This phenomenon, known as "fratricide," reduces the antitumor efficacy of DARA. What is needed are new reagents and methods that can be used to maintain the killing activity of NK cells in the presence of anti-CD38 antibodies. The therapies and methods disclosed herein address these and other needs. Summary of the Invention

[0004] Disclosed are methods and compositions relating to reducing CD38 on NK cells by imprinting the NK cells with TGF-β, and their use in combination with CD38 inhibitors for therapy.

[0005] In one aspect, disclosed herein are methods for treating, inhibiting, alleviating, reducing, ameliorating, and / or preventing cancer (e.g., leukemia, lymphoma, or myeloma) or an infectious disease in a subject in need thereof, the methods comprising administering to the subject a therapeutically effective number of transforming growth factor beta (TGF-β) superfamily-imprinted natural killer (TGF-βi NK) cells and a CD38-targeting agent (e.g., an anti-CD38 inhibitor such as an anti-CD38 antibody, including but not limited to, daratumumab or isatuximab). In some aspects, the TGF-βi NK cells have reduced expression of CD38. In some embodiments, the TGF-βi NK cells exhibit increased resistance to TGF-β superfamily cytokines (e.g., TGF-β). In some embodiments, the TGF-βi NK cells produced increased amounts of one or more of IFN-γ, TNF-α, and GM-CSF, hi some embodiments, the TGF-βi NK cells exhibit decreased levels of SMAD3 and / or TGFBR3 protein.

[0006] Also disclosed are methods for treating, inhibiting, alleviating, reducing, ameliorating, and / or preventing cancer or infectious disease of any preceding embodiment, wherein said TGF-βi NK cells are prepared by incubating NK cells (e.g., in vitro or ex vivo) in the presence of TGF-β. In some embodiments, the TGF-βi NK cells are prepared by incubating NK cells in the presence of feeder cells engineered to express TGF-β, or by incubating NK cells in the presence of cell membrane particles or exosomes derived from said feeder cells. In some embodiments, the feeder cells are selected from the group consisting of PBMC, RPMI8866, HFWT, K562 cells, EBV-LCL, NK cells transfected with membrane-bound IL-21 (including but not limited to PBMC, RPMI8866, NK-92, NK-92MI, NK-YTS, NK, NKL, KIL, KIL C.2, NK 3.3, NK-YS, HFWT, K562 cells), NK cells transfected with membrane-bound 4-1BBL (including but not limited to PBMC, RPMI8866, NK-92, NK-92MI, NK-YTS, NK, NKL, KIL, KIL C.2, NK 3.3, NK-YS, HFWT, K562 cells), NK cells transfected with membrane-bound IL-15 and 4-1BBL (including but not limited to PBMC, RPMI8866, NK-92, NK-92MI, NK-YTS, NK, NKL, KIL, KIL C.2, NK 3.3, NK-YS, HFWT, K562 cells), or NK cells transfected with membrane-bound IL-21 and / or 4-1BBL (including but not limited to PBMC, RPMI8866, NK-92, NK-92MI, NK-YTS, NK, NKL, KIL, KIL C.2, NK 3.3, NK-YS, HFWT, K562 cells). In one embodiment, the NK cells are incubated in the presence of engineered feeder cells, cell membrane particles, or exosomes for at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 45, or 60 days.

[0007] In one aspect, a method is disclosed for treating, inhibiting, alleviating, reducing, ameliorating, and / or preventing cancer or an infectious disease of any preceding aspect, wherein the feeder cells further comprise at least one additional NK cell effector agent on their cell surface, wherein said at least one additional NK cell effector agent is a cytokine, adhesion molecule, or NK cell activator, and is selected from the group consisting of 4-1BBL, IL-2, IL-12, IL-15, IL-18, IL-21, MICA, LFA-1, 2B4, CCR7, OX40L, UBLP2, B Examples of additional NK cell effector agents include, but are not limited to, CM1 / SLAMF2, NKG2D agonists, CD155, CD112, Jagged1, Jagged2, Delta-1, Pref-1, DNER, Jedi, SOM-11, wingless, CCN3, MAGP2, MAGP1, TSP2, YB-1, EGFL7, CCR7, DAP12, and DAP10, Notch ligands, NKp46 agonists, NKp44 agonists, NKp30 agonists, other NCR agonists, and CD16 agonists. In one embodiment, the at least one additional NK cell effector agent comprises IL-21, 4-1BBL, IL-15, IL-21 and 4-1BBL, IL-21 and IL-15, or IL-15 and 4-1BBL.

[0008] Also disclosed are methods for treating, inhibiting, alleviating, reducing, ameliorating, and / or preventing cancer or an infectious disease of any preceding aspect, wherein the NK cells are NKG2C + , CD56 bright NK cells, CD56 dim The NK cells include NK cells, peripheral NK cells, memory-like NK cells such as NK T cells, or tumor-infiltrating NK cells (including, but not limited to, NK cells obtained from a cell line or NK cells obtained from a donor source). In some embodiments, the NK cells are human NK cells or canine NK cells. In some embodiments, the NK cells are primary NK cells (e.g., NI cells obtained from a donor subject, including, but not limited to, an autologous donor, an allogeneic donor, or a syngeneic donor) or an NK cell line. In some embodiments, the NK cells are activated in vitro or ex vivo.

[0009] In one aspect, disclosed herein is a method for treating, inhibiting, alleviating, reducing, ameliorating, and / or preventing cancer or an infectious disease of any preceding aspect, wherein the subject comprises tumor cells having an increased level of CD38 compared to a reference control.

[0010] Also disclosed herein are anti-cancer therapies comprising transforming growth factor beta (TGF-β) superfamily imprinted natural killer (TGF-βi NK) cells and a CD38 targeting agent (e.g., an anti-CD38 inhibitor such as an anti-CD38 antibody, including but not limited to, daratumumab or isatuximab).

[0011] In one aspect, the anti-cancer therapy of any preceding aspect is disclosed herein, wherein the TGF-βi NK cells are prepared by incubating NK cells (e.g., in vitro or ex vivo) in the presence of TGF-β. In some aspects, the TGF-βi NK cells are prepared by incubating NK cells in the presence of feeder cells engineered to express TGF-β, or by incubating NK cells in the presence of cell membrane particles or exosomes derived from the feeder cells. In some embodiments, the feeder cells are selected from the group consisting of PBMC, RPMI8866, HFWT, K562 cells, EBV-LCL, NK cells transfected with membrane-bound IL-21 (including but not limited to PBMC, RPMI8866, NK-92, NK-92MI, NK-YTS, NK, NKL, KIL, KIL C.2, NK 3.3, NK-YS, HFWT, K562 cells), NK cells transfected with membrane-bound 4-1BBL (including but not limited to PBMC, RPMI8866, NK-92, NK-92MI, NK-YTS, NK, NKL, KIL, KIL C.2, NK 3.3, NK-YS, HFWT, K562 cells), NK cells transfected with membrane-bound IL-15 and 4-1BBL (including but not limited to PBMC, RPMI8866, NK-92, NK-92MI, NK-YTS, NK, NKL, KIL, KIL C.2, NK 3.3, NK-YS, HFWT, K562 cells), or NK cells transfected with membrane-bound IL-21 and / or 4-1BBL (including but not limited to PBMC, RPMI8866, NK-92, NK-92MI, NK-YTS, NK, NKL, KIL, KIL C.2, NK 3.3, NK-YS, HFWT, K562 cells). In one embodiment, the NK cells are incubated in the presence of engineered feeder cells, cell membrane particles, or exosomes for at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 45, or 60 days.

[0012] Also disclosed herein is the anti-cancer therapy of any preceding embodiment, wherein the feeder cells further comprise at least one additional NK cell effector agent on their cell surface, wherein the at least one additional NK cell effector agent is a cytokine, adhesion molecule, or NK cell activator, and is selected from the group consisting of 4-1BBL, IL-2, IL-12, IL-15, IL-18, IL-21, MICA, LFA-1, 2B4, CCR7, OX40L, UBLP2, BCM1 / SLAMF2, NKG2D, IL-1B, IL-1C, IL-1D, IL-1E, IL-1F, IL-1G, IL-1H, IL-1H, IL-1I ... agonists, CD155, CD112, Jagged1, Jagged2, Delta-1, Pref-1, DNER, Jedi, SOM-11, wingless, CCN3, MAGP2, MAGP1, TSP2, YB-1, EGFL7, CCR7, DAP12, and DAP10, Notch ligands, NKp46 agonists, NKp44 agonists, NKp30 agonists, other NCR agonists, and CD16 agonists. In one embodiment, the at least one additional NK cell effector agent comprises IL-21, 4-1BBL, IL-15, IL-21 and 4-1BBL, IL-21 and IL-15, or IL-15 and 4-1BBL.

[0013] In one aspect, the anti-cancer therapy of any preceding aspect is disclosed herein, wherein the NK cells are selected from the group consisting of NKG2C + , CD56 bright NK cells, CD56 dim The NK cells include NK cells, peripheral NK cells, memory-like NK cells such as NK T cells, or tumor-infiltrating NK cells (including, but not limited to, NK cells obtained from a cell line or NK cells obtained from a donor source). In some embodiments, the NK cells are human NK cells or canine NK cells. In some embodiments, the NK cells are primary NK cells (e.g., NI cells obtained from a donor subject, including, but not limited to, an autologous donor, an allogeneic donor, or a syngeneic donor) or an NK cell line. In some embodiments, the NK cells are activated in vitro or ex vivo.

[0014] In one aspect, disclosed herein are engineered natural killer (NK) cells, wherein the engineered NK cells are cultured and / or prepared (e.g., in vitro or ex vivo) for at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 45, or 60 days in the presence of a TGF-β superfamily cytokine and feeder cells or cell membrane particles or exosomes derived from feeder cells engineered to express TGF-β.

[0015] In some embodiments, the modified NK cells are selected from the group consisting of PBMC, RPMI8866, HFWT, K562 cells, EBV-LCL, NK cells transfected with membrane-bound IL-21 (including but not limited to PBMC, RPMI8866, NK-92, NK-92MI, NK-YTS, NK, NKL, KIL, KIL C.2, NK 3.3, NK-YS, HFWT, K562 cells), NK cells transfected with membrane-bound 4-1BBL (including but not limited to PBMC, RPMI8866, NK-92, NK-92MI, NK-YTS, NK, NKL, KIL, KIL C.2, NK In some embodiments, the NK cells are prepared in the presence of feeder cells comprising NK cells transfected with membrane-bound IL-15 and 4-1BBL (including but not limited to PBMC, RPMI8866, NK-92, NK-92MI, NK-YTS, NK, NKL, KIL, KIL C.2, NK 3.3, NK-YS, HFWT, K562 cells), NK cells transfected with membrane-bound IL-21 and / or 4-1BBL (including but not limited to PBMC, RPMI8866, NK-92, NK-92MI, NK-YTS, NK, NKL, KIL, KIL C.2, NK 3.3, NK-YS, HFWT, K562 cells). In some embodiments, the NK cells have increased resistance to TGF-β compared to naturally occurring NK cells.

[0016] Also disclosed herein is the modified NK cell of any preceding embodiment, wherein the feeder cells further comprise at least one additional NK cell effector agent on their cell surface, wherein the at least one additional NK cell effector agent is a cytokine, adhesion molecule, or NK cell activator, and is selected from the group consisting of 4-1BBL, IL-2, IL-12, IL-15, IL-18, IL-21, MICA, LFA-1, 2B4, CCR7, OX40L, UBLP2, BCM1 / SLAMF2, NKG2D, and the like. agonists, CD155, CD112, Jagged1, Jagged2, Delta-1, Pref-1, DNER, Jedi, SOM-11, wingless, CCN3, MAGP2, MAGP1, TSP2, YB-1, EGFL7, CCR7, DAP12, and DAP10, Notch ligands, NKp46 agonists, NKp44 agonists, NKp30 agonists, other NCR agonists, and CD16 agonists. In one embodiment, the at least one additional NK cell effector agent comprises IL-21, 4-1BBL, IL-15, IL-21 and 4-1BBL, IL-21 and IL-15, or IL-15 and 4-1BBL.

[0017] In some embodiments, the modified NK cells of any preceding aspect are activated in vitro or ex vivo. In some embodiments, the modified NK cells of any preceding aspect are human NK cells or canine NK cells. In some embodiments, the modified NK cells of any preceding aspect are primary NK cells or NK cell lines. In some embodiments, the modified NK cells of any preceding aspect are NKG2C + CD56 bright NK cells, CD56 dim These include NK cells, peripheral NK cells, and memory-like NK cells such as NK T cells, or tumor-infiltrating NK cells.

[0018] In some embodiments, the modified NK cells of any preceding aspect are obtained from a donor subject, including but not limited to, an autologous donor, an allogeneic donor, and / or a syngeneic donor.

[0019] The accompanying figures, which are incorporated in and constitute a part of this specification, illustrate several aspects described below. [Brief explanation of the drawings]

[0020] [Figure 1] Figures 1A-1D show real-time efficacy assessment of WT and TGF-βi NK cells killing suspended target cells. Figure 1A shows the time course data of CML cytolysis rates. Figures 1B and 1D show graphical analyses at different time points. Figure 1C shows the time course data of AML cytolysis rates. All impedance experiments were performed using an xCELLigence RTCA MP instrument (Agilent Technologies). To adapt the xCELLigence E-Plate for analysis of suspended target cell killing, wells of the E-Plate were incubated with the tethering reagent CD29 for Kassumi (AML cell line) or CD71 for K562 (CML cell line) at a concentration of 2 μg / mL for 3 hours at 37°C. After gently washing the wells with PBS, tumor cells were seeded at a density of 60,000 cells per well. The cytotoxic activity of STD (standard) NK cells and TGF-βi NK cells was evaluated based on the viability of tumor cells attached to the E-plate surface, as reflected by the Cell Index value. [Figure 2] Figures 2A and 2B show the real-time efficacy of wild-type and TGF-βi NK cells in killing adherent target cells. An xCELLigence RTCA MP instrument (Agilent Technologies) was used for all impedance experiments. Tumor cells were seeded at a density of 20,000 cells per well. The cytotoxic activity of STD NK cells and TGF-βi NK cells was evaluated based on the viability of tumor cells attached to the E-Plate surface, as reflected by the Cell Index value. Time course data for the cytolysis rate of MEL-LM33 cells (Figures 2A and 2B represent E:T ratios of 2:1 and 0.5:1, respectively) were also shown. [Figure 3]Figures 3A-C show that TGF-β imprinting downregulates CD38 and STAT1 signaling. Figure 3A shows CD38 expression on STD NK cells and TGF-βi NK cells. CD38 expression was assessed by flow cytometry. Data are presented as percentages and MFI. Figure 3B shows RNA-seq analysis. Figure 3C shows STAT1 expression and comparison between STD NK cells and TGF-βi NK cells. [Figure 4] Figures 4A-4F show favorable metabolic reprogramming of TGF-βi NK cells. Figures 4B and 4C show mitochondrial and glycolytic stress. Summary data (n = 3, mean ± SD) from metabolic analysis of paired STD and TGF-βi NK cells. Figure 4D shows a graphical analysis of maximal and spare respiratory capacity derived from Figure 4B. Figure 4E shows glycolysis, glycolytic capacity, and glycolytic reserve derived from Figure 4C. Figure 4F shows MitoTracker staining of STD and TGF-βi NK cells. Increased MitoTracker staining is associated with a metabolic shift from glycolysis to OXPHOS. The ratio of OCR / ECAR is shown. All experiments were performed using quinuplicated samples. FCCP, carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone; ROT / AA, rotenone; and antimycin A. [Figure 5] Figures 5A-5C show the resistance of TGF-βi NK cells to DARA-induced fratricide. Figure 5A shows live cells (n=1). Figure 5B shows dead cells (n=1). Figure 5C shows representative data on the change in DARA-induced lysis of paired STD NK cells and TGF-βi NK cells against JURKAT, MOLT4, and RPMI8226 (T-ALL and multiple myeloma cell lines, respectively). Data on the viability of DARA-treated STD NK cells and TGF-βi NK cells compared with the viability of controls. [Figure 6]Figures 6A-6C show the expression of CD38 on expanded NK cells. Figure 6A shows a representative FACS analysis of the percentage of CD38+ NK cells in CD38 WT, TGF-βi, or CD38 KO NK cells that express CD38. Figure 6B shows a graph representing the FACS analysis. Figure 6C shows RNA-seq analysis. Comparison of naive (non-expanded NK cells), expanded NK cells, and TGF-βi (TGF-β imprinted) NK cells. [Figure 7] Figures 7A-7E show the effects of TGF-β imprinting. Figure 7A shows increased production of pro-inflammatory cytokines. Figure 7B shows loss of sensitivity to TGF-β immunosuppression. Figure 7C shows downregulation of canonical SMAD3 signaling. Figure 7D shows the profile of reprogrammed tissue-resident addressins. Figure 7E shows significantly improved serial killing. [Figure 8] Figures 8A-8C show that TGF-β imprinting alters gene expression, with 1,750 genes having a DESeqScore of greater than 1.5. [Figure 9] FIG. 9 shows that TGF-β imprinting alters addressin gene expression (similar to FIG. 7D). [Figure 10] Figures 10A and 10B show that TGF-β imprinting improves tumor control. [Figure 11] FIG. 11 shows that TGF-β imprinting protects mice from liver metastasis. DETAILED DESCRIPTION OF THE INVENTION

[0021] The following description of the disclosure is provided as an enabling teaching of the disclosure in its best currently known embodiment. In light of this, those skilled in the art will recognize and appreciate that many variations can be made to the various embodiments of the invention described herein and still obtain the beneficial results of the disclosure. It will also be apparent that some desirable advantages of the disclosure can be obtained by selecting some features of the disclosure without utilizing other features. Thus, those skilled in the art will recognize that many modifications and adaptations to the disclosure are possible and desirable in certain circumstances and are a part of the disclosure. Therefore, the following description is illustrative of the principles of the disclosure, but not limiting.

[0022] Reference will now be made in detail to certain embodiments of the invention, examples of which are illustrated in the drawings and examples, but the invention may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein.

[0023] term Unless otherwise defined, all technical and scientific terms used herein have the meanings that are commonly understood by those skilled in the art to which this invention belongs.The following references provide those skilled in the art with the general definitions of many of the terms used in this invention: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd Ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The HarperCollins Dictionary of Biology (1991).As used herein, the following terms have the meanings ascribed to them unless otherwise specified.

[0024] When introducing elements of this disclosure or preferred embodiments thereof, the articles "a," "an," "the," and "said" are intended to mean that there are one or more of the elements. The terms "comprising," "including," and "having" are intended to be inclusive and mean that there may be additional elements other than the listed elements.

[0025] Ranges can be expressed herein as from "about" one particular value and / or to "about" another particular value. When such a range is expressed, another embodiment includes from the one particular value and / or to the other particular value. Similarly, when values ​​are expressed as approximations, by use of the antecedent "about," it is understood that the particular value forms another embodiment. It will be further understood that the endpoints of each range are significant both in relation to the other endpoint, and independently of the other endpoint. Also, several values ​​are disclosed herein, and it is understood that each value is also disclosed herein as "about" that particular value in addition to the value itself. For example, if the value "10" is disclosed, "about 10" is also disclosed. As will be appreciated by those of skill in the art, when a value is disclosed "less than or equal to," it is understood that "greater than or equal to" and possible ranges therebetween are also disclosed. For example, if the value "10" is disclosed, "less than or equal to 10" as well as "greater than or equal to 10" is also disclosed. It is also understood that throughout this application, data is provided in several different formats, and that this data represents endpoints and starting points, as well as ranges for any combination of the data points. For example, if a specific data point "10" and a specific data point "15" are disclosed, it is understood that values ​​between 10 and 15, as well as values ​​greater than 10, greater than or equal to 10, less than 10, less than or equal to 10, and equal to 10, greater than 15, less than 15, less than or equal to 15, and equal to 15, are considered to be disclosed. It is also understood that each unit between two specified units is disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

[0026] As used herein, the term "optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and the description is meant to include both cases where the event or circumstance occurs and cases where it does not occur.

[0027] As used herein, the term "N-terminal" or "amino-terminal" refers to the orientation of a peptide, polypeptide, or protein and may not refer to the N-terminus. In some embodiments, when describing a chimeric or fusion peptide, polypeptide, or protein, the N-terminal side refers only to a component of the chimeric or fusion peptide, polypeptide, or protein, and not to the entire structure. For example, when an Fc domain is described and the Fc domain is described as being fused with its amino- or N-terminal side facing the intracellular space, the present specification contemplates a chimeric or fusion peptide, polypeptide, or protein in which the signal anchor is at the N-terminus of the chimeric or fusion construct and actually spans the cell membrane. Thus, in such chimeras, the transmembrane anchor is attached to the amino-terminal side of the Fc domain, and the orientation of the Fc domain has its N-terminal side facing the cell, which is reversed compared to the Fc domain on a typical B cell, which generally has a carboxy-terminus spanning the cell membrane and an amino-terminus extending to the extracellular matrix.

[0028] The terms "peptide," "polypeptide," and "protein" are used interchangeably to refer to a polymer of amino acid residues.

[0029] The term "sequence identity" as used herein quantitatively represents the degree of identity between two sequences of substantially equal length. Whether nucleic acid or amino acid, the percent identity of two sequences is calculated by dividing the number of exact matches between the two aligned sequences by the length of the shorter sequence and multiplying by 100. Approximate alignment of nucleic acid sequences is provided by the local homology algorithm of Smith and Waterman, Advances in Applied Mathematics 2:482-489 (1981). This algorithm can be applied to amino acid sequences using a scoring matrix developed by Dayhoff, Atlas of Protein Sequences and Structure, MO Dayhoff ed., 5 suppl. 3:353-358, National Biomedical Research Foundation, Washington, DC, USA, and normalized by Gribskov, Nucl. Acids Res. 14(6):6745-6763 (1986). An exemplary implementation of this algorithm for determining percent sequence identity is provided by the "BestFit" utility application from Genetics Computer Group (Madison, Wis.). Other suitable programs for calculating percent identity or similarity between sequences are generally known in the art; for example, another alignment program is BLAST, used with default parameters. For example, BLASTN and BLASTP can be used with the following default parameters: genetic code=standard; filter=none; strand=both; cutoff=60; expect=10; Matrix=BLOSUM62; Descriptions=50 sequences; sort by=HIGH SCORE; Databases=non-redundant, GenBank+EMBL+DDBJ+PDB+GenBank CDS translations+Swiss protein+Spupdate+PIR.More information about these programs can be found on the GenBank website. Generally, substitutions are conservative amino acid substitutions and are limited to exchanges within members of 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: aspartate, glutamate, asparagine, and glutamine. Preferably, the percentage sequence identity is calculated over the entire length of the sequences being compared.

[0030] Techniques for determining nucleic acid and amino acid sequence identity are known in the art. Usually, such techniques involve determining the nucleotide sequence of the mRNA of a gene, and / or determining the amino acid sequence encoded thereby, and comparing these sequences with a second nucleotide sequence or amino acid sequence. Genomic sequences can also be determined and compared in this manner. Generally, "identity" refers to the exact nucleotide-to-nucleotide or amino acid-to-amino acid correspondence of two polynucleotide or polypeptide sequences, respectively. Two or more sequences (polynucleotide or amino acid) can be compared by determining their "percent identity."

[0031] Since various modifications can be made in the cells and methods described above without departing from the scope of the present invention, it is intended that all matter contained in the above description and the examples given below should be interpreted as illustrative and not in a limiting sense.

[0032] "Increase" can refer to any change that results in a greater amount of a symptom, disease, composition, condition, or activity. An increase can be a statistically significant increase in any individual, median, or mean value of a condition, symptom, activity, or composition compared to a control. Thus, an increase can be a 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% increase, as long as the increase is statistically significant.

[0033] "Reduction" can refer to any change that results in a lower amount of symptoms, diseases, compositions, conditions, or activities. A substance is also understood to reduce the genetic output of a gene when the genetic output of a gene product containing the substance is less than the output of the gene product without the substance. For example, a reduction can also be a change in the symptoms of a disorder, such that the symptoms are less than those previously observed. A reduction can be a statistically significant decrease in any individual, median, or mean value of a condition, symptom, activity, or composition compared to a control. Thus, a reduction can be a 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% reduction, as long as the reduction is statistically significant.

[0034] "Inhibit," "inhibiting," and "inhibition" refer to a decrease in an activity, response, condition, disease, or other biological parameter. This can include, but is not limited to, the complete elimination of the activity, response, condition, or disease. It can also include, for example, a 10% reduction in the activity, response, condition, or disease compared to native or control levels. Thus, the reduction can be a 10, 20, 30, 40, 50, 60, 70, 80, 90, 100%, or any amount in between, compared to native or control levels.

[0035] "Reduce" or other forms of this term, such as "reducing" or "reduction," refer to a decrease in an event or characteristic (e.g., tumor growth). This is typically relative to some standard or expected value, in other words, it is relative, but it is understood that it is not necessarily to refer to a standard or relative value. For example, "reducing tumor growth" means reducing the growth rate of a tumor compared to a standard or control.

[0036] "Prevent" or other forms of the word, such as "preventing" or "prevention," means to stop a particular event or characteristic, stabilize or slow the development or progression of a particular event or characteristic, or minimize the likelihood of a particular event or characteristic occurring. Prevention is typically more absolute than, for example, reduction and therefore does not require a comparison to a control. As used herein, something may be reduced but not prevented, although something that is reduced may be prevented. Similarly, something may be prevented but not reduced, although something that is prevented may be reduced. It is understood that where reduction or prevention is used, the use of other words is expressly disclosed unless specifically specified otherwise.

[0037] The term "subject" refers to any individual who is the target of administration or treatment. A subject can be a vertebrate, e.g., 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 an animal patient. The term "patient" refers to a subject under the care of a clinician, e.g., a physician.

[0038] The term "therapeutically effective" refers to the amount of composition used being sufficient to ameliorate one or more causes or symptoms of a disease or disorder. Such amelioration need not necessarily be elimination, but may suffice to reduce or alter.

[0039] The term "treatment" refers to the medical management of a patient with the intent to cure, ameliorate, stabilize, or prevent a disease, condition, or disorder. This term includes active treatment, i.e., treatment specifically directed at ameliorating a disease, condition, or disorder, and also includes causal treatment, i.e., treatment directed at eliminating the cause of the associated disease, condition, or disorder. In addition, this term includes palliative treatment, i.e., treatment directed at alleviating symptoms rather than curing the disease, condition, or disorder; prophylactic treatment, i.e., treatment directed at minimizing or partially or completely suppressing the onset of the associated disease, condition, or disorder; and supportive treatment, i.e., treatment used to complement another specific treatment directed at ameliorating the associated disease, condition, or disorder.

[0040] "Administration" to a subject includes any route of introducing or delivering an agent to a subject. Administration can be by any suitable route, including oral, topical, intravenous, subcutaneous, transcutaneous, transdermal, intramuscular, intraarticular, parenteral, intraarterial, intradermal, intraventricular, intracranial, intraperitoneal, intralesional, intranasal, rectal, vaginal, by inhalation, via an implanted reservoir, parenteral (e.g., subcutaneous, intravenous, intramuscular, intraarticular, intrasynovial, intrasternal, intrathecal, intraperitoneal, intrahepatic, intralesional, and intracranial injection or infusion techniques), and the like. As used herein, "concurrent administration," "administration in combination," "simultaneous administration," or "administered simultaneously" means that compounds are administered at the same time in time or essentially immediately after each other. In the latter case, the two compounds are administered sufficiently close in time that the results observed are indistinguishable from those achieved when the compounds are administered at the same time in time. "Systemic administration" refers to the introduction or delivery of an agent to a subject via a route that introduces or delivers the agent to a wide area of ​​the subject's body (e.g., more than 50% of the body), for example, through an entrance into the circulatory or lymphatic system. In contrast, "local administration" refers to the introduction or delivery of an agent to a subject via a route that introduces or delivers the agent to the area of ​​the point of administration or an area immediately adjacent thereto, and does not introduce the agent systemically in therapeutically significant amounts. For example, a locally administered agent is readily detectable in the local vicinity of the point of administration, but is undetectable or detectable in negligible amounts in distal parts of the subject's body. Administration includes self-administration and administration by another.

[0041] "Treat," "treating," "treatment," and grammatical variations thereof, as used herein, include administration of a composition intended or intended to partially or completely prevent, delay, cure, ameliorate, alleviate, relieve, alter, remedy, ameliorate, improve, stabilize, mitigate, and / or reduce the intensity or frequency of one or more diseases or conditions, symptoms of a disease or condition, or the underlying causes of a disease or condition. Treatment according to the present invention may be applied preventatively, prophylactically, palliatively, or therapeutically. Prophylactic treatment is administered to a subject before onset (e.g., before overt signs of cancer), during early onset (e.g., at the time of early signs and symptoms of cancer), or after the development of established cancer. Prophylactic administration may occur from one day (a few days) to several years before the onset of symptoms of disease or infection.

[0042] Throughout this application, various publications are referenced. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this pertains. The references disclosed are also individually and specifically incorporated by reference herein for the material contained in the reference that is discussed in the sentence in which the reference is cited.

[0043] Compositions and methods of treatment The components used to prepare the disclosed compositions, and the compositions themselves used in the methods disclosed herein are disclosed.These and other materials are disclosed herein, and when the combinations, subsets, interactions, groups, etc. of these materials are disclosed, it is understood that although specific reference to each of the various, individual and collective, combinations and permutations of these compounds may not be explicitly disclosed, each is specifically contemplated and described herein.For example, when a specific engineered feeder cell that expresses TGF-β is disclosed and discussed, and a number of modifications that can be made to a number of molecules, including the engineered feeder cell that expresses TGF-β, each and every combination and permutation of the engineered feeder cell that expresses TGF-β, and modifications that are possible unless specifically indicated to the contrary, are specifically contemplated. Thus, if classes of molecules A, B, and C, and classes D, E, and F are disclosed, and an example of a combination molecule, A-D, is disclosed, each is considered individually and collectively disclosed, even if each is not individually listed, as are combinations A-E, A-F, B-D, B-E, B-F, C-D, C-E, and C-F. Likewise, any subset or combination of these is also disclosed. Thus, for example, subgroups A-E, B-F, and C-E are considered disclosed. This concept applies to all aspects of this application, including, but not limited to, steps in methods of making and using the disclosed compositions. Thus, where there are various additional steps that may be performed, it is understood that each of these additional steps may be performed in any specific embodiment or combination of embodiments of the disclosed methods.

[0044] In one aspect, disclosed herein is a method for treating cancer or an infectious disease in a subject in need thereof, comprising administering to the subject a therapeutically effective number of transforming growth factor beta (TGF-β) superfamily-imprinted natural killer (TGF-βi NK) cells and a CD38-targeting agent.

[0045] Also disclosed herein are anti-cancer therapies that include transforming growth factor beta (TGF-β) superfamily imprinted natural killer (TGF-βi NK) cells and CD38 targeting agents (eg, CD38 inhibitors).

[0046] In some embodiments, TGF-βi NK cells are prepared by incubating NK cells (e.g., in vitro or ex vivo) in the presence of TGF-β and feeder cells engineered to express TGF-β, or cell membrane particles or exosomes derived from feeder cells.

[0047] In some embodiments, the feeder cells are selected from the group consisting of PBMC, RPMI8866, HFWT, K562 cells, EBV-LCL, NK cells transfected with membrane-bound IL-21 (including but not limited to PBMC, RPMI8866, NK-92, NK-92MI, NK-YTS, NK, NKL, KIL, KIL C.2, NK 3.3, NK-YS, HFWT, K562 cells), NK cells transfected with membrane-bound 4-1BBL (including but not limited to PBMC, RPMI8866, NK-92, NK-92MI, NK-YTS, NK, NKL, KIL, KIL C.2, NK 3.3, NK-YS, HFWT, K562 cells), NK cells transfected with membrane-bound IL-15 and 4-1BBL (including but not limited to PBMC, RPMI8866, NK-92, NK-92MI, NK-YTS, NK, NKL, KIL, KIL C.2, NK 3.3, NK-YS, HFWT, K562 cells), or NK cells transfected with membrane-bound IL-21 and / or 4-1BBL (including but not limited to PBMC, RPMI8866, NK-92, NK-92MI, NK-YTS, NK, NKL, KIL, KIL C.2, NK 3.3, NK-YS, HFWT, K562 cells). In one embodiment, the NK cells are incubated in the presence of engineered feeder cells, cell membrane particles, or exosomes for at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 45, or 60 days.

[0048] The NK cells incubated by the methods disclosed herein can be primary NK cells or NK cell lines. In some embodiments, the NK cells are NKG2C + , CD56 bright NK cells, CD56 dim These include NK cells, peripheral NK cells, memory-like NK cells such as NK T cells, or tumor-infiltrating NK cells (including, but not limited to, NK cells obtained from a cell line or from a donor source (e.g., autologous, allogeneic, or syngeneic donor)).

[0049] In some aspects, disclosed herein are methods and anti-cancer therapies for treating, inhibiting, alleviating, reducing, ameliorating, and / or preventing cancer or an infectious disease in a subject in need thereof, wherein the TGF-βi NK cells exhibit increased resistance to a TGF-β superfamily cytokine (e.g., TGF-β). In some embodiments, the TGF-βi NK cells produced increased amounts of one or more of IFN-γ, TNF-α, and GM-CSF. In some embodiments, the TGF-βi NK cells exhibit decreased levels of SMAD3 protein and / or TGFBR3 protein.

[0050] In some embodiments, the NK cells are human NK cells or canine NK cells.

[0051] In some embodiments, the NK cells are primary NK cells (e.g., NI cells obtained from a donor subject, including but not limited to, an autologous donor, an allogeneic donor, or a syngeneic donor) or an NK cell line.

[0052] In some embodiments, the NK cells are activated in vitro or ex vivo.

[0053] Also disclosed herein are anti-cancer therapies, wherein the feeder cells further comprise at least one additional NK cell effector agent on their cell surface, wherein the at least one additional NK cell effector agent is a cytokine, adhesion molecule, or NK cell activator, including, but not limited to, 4-1BBL, IL-2, IL-12, IL-15, IL-18, IL-21, MICA, LFA-1, 2B4, CCR7, OX40L, UBLP2, BCM1 / SLAMF2, NK These include, but are not limited to, G2D agonists, CD155, CD112, Jagged1, Jagged2, Delta-1, Pref-1, DNER, Jedi, SOM-11, wigless, CCN3, MAGP2, MAGP1, TSP2, YB-1, EGFL7, CCR7, DAP12, and DAP10, Notch ligands, NKp46 agonists, NKp44 agonists, NKp30 agonists, other NCR agonists, CD16 agonists. In one embodiment, the at least one additional NK cell effector agent comprises IL-21, 4-1BBL, IL-15, IL-21 and 4-1BBL, IL-21 and IL-15, or IL-15 and 4-1BBL.

[0054] In one aspect, disclosed herein are engineered natural killer (NK) cells, wherein the engineered NK cells are cultured and / or prepared (e.g., in vitro or ex vivo) for at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 45, or 60 days in the presence of a TGF-β superfamily cytokine and feeder cells or cell membrane particles or exosomes derived from feeder cells engineered to express TGF-β.

[0055] In some embodiments, the modified NK cells are selected from the group consisting of PBMC, RPMI8866, HFWT, K562 cells, EBV-LCL, NK cells transfected with membrane-bound IL-21 (including but not limited to PBMC, RPMI8866, NK-92, NK-92MI, NK-YTS, NK, NKL, KIL, KIL C.2, NK 3.3, NK-YS, HFWT, K562 cells), NK cells transfected with membrane-bound 4-1BBL (including but not limited to PBMC, RPMI8866, NK-92, NK-92MI, NK-YTS, NK, NKL, KIL, KIL C.2, NK In some embodiments, the NK cells are prepared in the presence of feeder cells comprising NK cells transfected with membrane-bound IL-15 and 4-1BBL (including but not limited to PBMC, RPMI8866, NK-92, NK-92MI, NK-YTS, NK, NKL, KIL, KIL C.2, NK 3.3, NK-YS, HFWT, K562 cells), NK cells transfected with membrane-bound IL-15 and 4-1BBL (including but not limited to PBMC, RPMI8866, NK-92, NK-92MI, NK-YTS, NK, NKL, KIL, KIL C.2, NK 3.3, NK-YS, HFWT, K562 cells), or NK cells transfected with membrane-bound IL-21 and / or 4-1BBL (including but not limited to PBMC, RPMI8866, NK-92, NK-92MI, NK-YTS, NK, NKL, KIL, KIL C.2, NK 3.3, NK-YS, HFWT, K562 cells). In some embodiments, the NK cells have increased resistance to TGF-β compared to naturally occurring NK cells.

[0056] Also disclosed herein are modified NK cells, wherein the feeder cells further comprise at least one additional NK cell effector agent on their cell surface, wherein the at least one additional NK cell effector agent is a cytokine, adhesion molecule, or NK cell activator, including 4-1BBL, IL-2, IL-12, IL-15, IL-18, IL-21, MICA, LFA-1, 2B4, CCR7, OX40L, UBLP2, BCM1 / SLAMF2, NKG2D agonist, , CD155, CD112, Jagged1, Jagged2, Delta-1, Pref-1, DNER, Jedi, SOM-11, wingless, CCN3, MAGP2, MAGP1, TSP2, YB-1, EGFL7, CCR7, DAP12, and DAP10, Notch ligands, NKp46 agonists, NKp44 agonists, NKp30 agonists, other NCR agonists, and CD16 agonists. In one embodiment, the at least one additional NK cell effector agent comprises IL-21, 4-1BBL, IL-15, IL-21 and 4-1BBL, IL-21 and IL-15, or IL-15 and 4-1BBL.

[0057] In some embodiments, the modified NK cells are activated in vitro or ex vivo.

[0058] In some embodiments, the modified NK cells are human NK cells or canine NK cells. In some embodiments, the modified NK cells of any preceding aspect are primary NK cells or NK cell lines.

[0059] In some embodiments, the engineered NK cells are NKG2C + CD56 bright NK cells, CD56 dim These include NK cells, peripheral NK cells, memory-like NK cells such as NK T cells, or tumor-infiltrating NK cells.

[0060] In some embodiments, the modified NK cells of any preceding aspect are obtained from a donor subject, including but not limited to, an autologous donor, an allogeneic donor, and / or a syngeneic donor.

[0061] In some embodiments, the CD38-targeting agent used in the disclosed anti-cancer therapies and methods for treating, inhibiting, alleviating, reducing, ameliorating, and / or preventing cancer or infectious diseases is an anti-CD38 antibody. The term "antibody" is used broadly herein to include both polyclonal and monoclonal antibodies. In addition to intact immunoglobulin molecules, the term "antibody" also includes fragments or polymers of these immunoglobulin molecules, as well as human or humanized versions of immunoglobulin molecules or fragments thereof, so long as they are selected based on their ability to interact with CD38. Antibodies can be tested for their desired activity using in vitro assays or similar methods described herein, and then their in vivo therapeutic and / or prophylactic activity is tested according to known clinical trial methods. There are five major classes of human immunoglobulins: IgA, IgD, IgE, IgG, and IgM, some of which are further divided into subclasses (isotypes), such as IgG-1, IgG-2, IgG-3, and IgG-4; IgA-1 and IgA-2. Those skilled in the art will recognize the murine analogue class. The heavy-chain constant domains that correspond to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively.

[0062] The term "monoclonal antibody," as used herein, refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies within the population are identical except for naturally occurring mutations that may be present in a small subset of antibody molecules. Monoclonal antibodies herein specifically include "chimeric" antibodies in which a portion of the heavy and / or light chain is identical to or homologous to corresponding sequences in antibodies from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical to or homologous to corresponding sequences in antibodies from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, provided that they exhibit the desired antagonistic activity.

[0063] The disclosed monoclonal antibodies can be made using any procedure that produces monoclonal antibodies. For example, the disclosed monoclonal antibodies can be prepared using hybridoma methods, such as those described in Kohler and Milstein, Nature, 256:495 (1975). In the hybridoma method, typically, a mouse or other suitable host animal is immunized with an immunizing agent to induce lymphocytes that produce or are capable of producing antibodies that specifically bind to the immunizing agent. Alternatively, lymphocytes may be immunized in vitro.

[0064] Monoclonal antibodies may be produced by recombinant DNA methods. DNA encoding the disclosed monoclonal antibodies can be readily isolated and sequenced using conventional procedures (e.g., using oligonucleotide probes capable of specifically binding to genes encoding the heavy and light chains of murine antibodies). Libraries of antibodies or active antibody fragments can also be generated and screened using phage display techniques, for example, as described in U.S. Patent No. 5,804,440 to Burton et al. and U.S. Patent No. 6,096,441 to Barbas et al.

[0065] In vitro methods are suitable for preparing monovalent antibodies. Digestion of antibodies to generate fragments, particularly Fab fragments, can be achieved using conventional techniques known in the art. For example, digestion can be performed using papain. Examples of papain digestion are described in WO94 / 29348 published December 22, 1994, and U.S. Pat. No. 4,342,566. Papain digestion of antibodies typically produces two identical antigen-binding fragments, called Fab fragments, each with a single antigen-binding site, and a residual Fc fragment. Pepsin treatment produces a fragment with two antigen-binding sites and the ability to cross-link antigens.

[0066] As used herein, the term "antibody or fragment thereof" includes fragments such as F(ab')2, Fab', Fab, Fv, sFv, and scFv, including chimeric and hybrid antibodies with dual or multiple antigen or epitope specificities, as well as hybrid fragments. Thus, antibody fragments that retain the ability to bind to a specific antigen are provided. For example, antibody fragments that retain CD38-binding activity are included within the meaning of the term "antibody or fragment thereof." Such antibodies and fragments can be produced by techniques known in the art and screened for specificity and activity according to methods described in the Examples and general methods for producing and screening antibodies for specificity and activity (see Harlow and Lane, Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, New York, (1988)). Thus, in some embodiments, anti-CD38 antibodies contain a fragment crystallizable region (Fc region) that binds to an Fc receptor, or lack an Fc region.

[0067] The term "antibody or fragment thereof" also includes conjugates of antibody fragments and antigen-binding proteins (single chain antibodies).

[0068] Fragments, whether or not associated with other sequences, can include insertions, deletions, substitutions, or other selected modifications of specific regions or specific amino acid residues, so long as the activity of the antibody or antibody fragment is not significantly altered or impaired compared to the unmodified antibody or antibody fragment. These modifications can confer additional properties, such as removing / adding amino acids capable of disulfide bonding, extending biological lifespan, or altering secretion characteristics. In either case, the antibody or antibody fragment must retain biologically active properties, such as specific binding to its cognate antigen. Functional or active regions of an antibody or antibody fragment can be identified by mutagenesis of specific regions of the protein, followed by expression and testing of the expressed polypeptide. Such methods are readily apparent to those skilled in the art and may include site-directed mutagenesis of nucleic acids encoding the antibody or antibody fragment. (Zoller, M.J. Curr. Opin. Biotechnol. 3:348-354, 1992).

[0069] As used herein, the term "antibody" or "antibodies" can also refer to human antibodies and / or humanized antibodies. Many non-human antibodies (e.g., those derived from mice, rats, or rabbits) exhibit natural antigenicity in humans and may induce undesirable immune responses when administered to humans. Therefore, the use of human or humanized antibodies in the methods of the present invention can reduce the likelihood that antibodies administered to humans will induce undesirable immune responses.

[0070] In some embodiments, the anti-CD38 antibody is daratumumab or isatuximab. The TGF-βi NK cells can be administered prior to, concurrently with, and / or after administration of a CD38 targeting agent (e.g., a CD38 inhibitor).

[0071] a) TGF-β imprinted natural killer cells In some aspects, disclosed herein are natural killer (NK) cells or NK cell lines cultured in the presence of TGF-β superfamily cytokines (referred to herein as TGF-βi NK cells). This includes NK cells or cell lines produced by the methods described herein, and compositions comprising the NK cells provided herein. In certain aspects, the composition is a pharmaceutical composition comprising one or more of the NK cells or cell lines provided herein. In some embodiments, the TGF-βi NK cells exhibit increased resistance to TGF-β.

[0072] The TGF-βi NK cells may be allogeneic or autologous. In some aspects, the NK cells are mammalian NK cells. Examples of "mammals" or "mammals" include primates (e.g., humans), canines, felines, rodents, porcines, ruminants, and the like. Specific examples include humans, dogs, cats, horses, cows, sheep, goats, rabbits, guinea pigs, rats, and mice. In certain embodiments, the mammalian NK cells are human NK cells.

[0073] TGF-βi NK cells exhibit many properties that distinguish them from naturally occurring NK cells. In some embodiments, the NK cells or cell lines exhibit increased resistance to TGF-β. In other embodiments, the NK cells produce increased amounts of interferon gamma (IFN-γ), tumor necrosis factor alpha (TNF-α), and / or granulocyte-macrophage colony-stimulating factor (GM-CSF). In further embodiments, the NK cells have reduced levels of SMAD family member 3 (SMAD3) protein and / or transforming growth factor beta receptor III (TGFBR3) protein. SMAD proteins, named as a contraction of the names C. elegans Sma and Drosophila Mad (Derynck et al., Cell, 95(6), p737-740, 1998), are transcriptional activators of the TGF-β response.

[0074] TGF-βi NK cells exhibit many properties that differ from naturally occurring NK cells. In some embodiments, the NK cells have a gene expression profile substantially similar to that shown in FIG. 7D. A substantially similar gene expression profile is one in which gene expression is within 10% of that shown. In some embodiments, TGF-βi NK cells produce increased amounts of one or more of IFN-γ, TNF-α, and GM-CSF proteins. In some embodiments, the NK cells or cell lines exhibit increased expression of SCUBE1, MYO7A, KLF3, WIPF3, and EPHA1.

[0075] TGF-βi NK cells exhibit many properties that distinguish them from naturally occurring NK cells. In some embodiments, TGF-βi NK cells exhibit reduced levels of SMAD3 protein and / or TGFBR3 protein and / or CD38 protein. In some embodiments, NK cells or cell lines exhibit reduced expression of CD300A, SGSM1, SMAD3, TBX21, and GZMK, TGFBR3, and GZMA.

[0076] In one aspect, disclosed herein is a method for treating, inhibiting, alleviating, reducing, ameliorating, and / or preventing cancer and / or metastasis, wherein NK cells are incubated in the presence of engineered feeder cells, cell membrane particles, or exosomes for at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 45, or 60 days.

[0077] The disclosed compositions can be used to treat any disease in which uncontrolled cell proliferation occurs, such as cancer. A representative, but non-limiting, list of cancers that can be treated using the disclosed compositions is as follows: lymphoma, B-cell lymphoma, T-cell lymphoma, mycosis fungoides, Hodgkin's disease, myeloid leukemia, bladder cancer, brain cancer, nervous system cancer, head and neck cancer, squamous cell carcinoma of the head and neck, lung cancer such as small cell lung cancer and non-small cell lung cancer, neuroblastoma / glioblastoma, ovarian cancer, skin cancer, liver cancer, melanoma, cancer of the mouth, pharynx, larynx, and squamous cell carcinoma of the lung, cervical cancer, cervical carcinoma, breast cancer, and epithelial cancer, kidney cancer, genitourinary cancer, lung cancer, esophageal cancer, head and neck cancer, colorectal cancer, hematopoietic cancer, testicular cancer, colon cancer, rectal cancer, prostate cancer, or pancreatic cancer.

[0078] In some embodiments, disclosed herein is a method, composition, use, and / or anti-cancer therapy of any preceding embodiment, wherein the cancer cells have an increased level of CD38 compared to a reference level.

[0079] b) Pharmaceutical Carriers / Delivery of Pharmaceutical Products As mentioned above, the composition can also be administered in vivo in a pharmaceutically acceptable carrier. "Pharmaceutically acceptable" means a material that is not biologically or otherwise undesirable, i.e., a material that can be administered to a subject together with a nucleic acid or vector, without causing any undesirable biological effects or interacting in a harmful manner with any of the other components of the pharmaceutical composition that come into contact with it. As is well known to those skilled in the art, the carrier can necessarily be selected to minimize any degradation of the active ingredient and minimize any adverse side effects in the subject.

[0080] The compositions may be administered orally, parenterally (e.g., intravenously), by intramuscular injection, intraperitoneal injection, transdermally, extracorporeally, topically, etc. (including local intranasal administration or administration by inhalation). As used herein, "local intranasal administration" refers to delivery of a composition to the nose and nasal passages through one or both nostrils and can include delivery by a spray or droplet mechanism, or by aerosolization of the nucleic acid or vector. Administration of a composition by inhalation can be through the nose or mouth via delivery by a spray or droplet mechanism. Delivery can also be directly to any region of the respiratory system (e.g., the lungs) via intubation. The exact amount of composition required will vary from subject to subject, depending on the species, age, weight, and general condition of the subject, the severity of the allergic disorder being treated, the particular nucleic acid or vector used, its method of administration, etc. Therefore, it is not possible to specify an exact amount for every composition. However, appropriate amounts can be determined by one of ordinary skill in the art using only routine experimentation given the teachings herein.

[0081] Parenteral administration of compositions when used is generally characterized by injection.Injectables can be prepared in conventional form, either as liquid solution or suspension, solid form suitable for solution in liquid suspension before injection, or emulsion.Recently revised parenteral administration approach involves the use of slow release or sustained release to maintain a constant dosage.For example, see U.S. Patent No. 3,610,795, which is incorporated herein by reference.

[0082] The materials may be in solution, suspension (e.g., incorporated into microparticles, liposomes, or cells), and may be targeted to specific cell types via antibodies, receptors, or receptor ligands. The following references are examples of using this technology to target specific proteins to tumor tissue (Senter, et al., Bioconjugate Chem., 2:447-451, (1991); Bagshawe, KD, Br. J. Cancer, 60:275-281, (1989); Bagshawe, et al., Br. J. Cancer, 58:700-703, (1988); Senter, et al., Bioconjugate Chem., 4:3-9, (1993); Battelli, et al., Cancer Immunol. Immunother., 35:421-425, (1992); Pietersz and McKenzie, Immunolog. Reviews, 129:57-80, (1992); and Roffler, et al. al., Biochem. Pharmacol, 42:2062-2065, (1991)). Vehicles such as "stealth" and other antibody-conjugated liposomes (including lipid-mediated drugs targeting colon cancer), receptor-mediated targeting of DNA via cell-specific ligands, lymphocyte-specific tumor targeting, and highly specific therapeutic retroviral targeting of mouse glioma cells in vivo. The following references are examples of the use of this technology to target specific proteins to tumor tissue (Hughes et al., Cancer Research, 49:6214-6220, (1989), and Litzinger and Huang, Biochimica et Biophysica Acta, 1104:179-187, (1992)). Generally, receptors are involved in either constitutive or ligand-induced endocytic pathways. These receptors cluster in clathrin-coated pits, enter the cell via clathrin-coated vesicles, pass through acidified endosomes where the receptors are sorted, and then are either recycled to the cell surface, stored intracellularly, or degraded in lysosomes.Internalization pathways perform a variety of functions, including nutrient uptake, removal of activated proteins, clearance of macromolecules, opportunistic entry of viruses and toxins, ligand dissociation and degradation, and regulation of receptor levels. Many receptors follow more than one intracellular pathway, depending on the cell type, receptor concentration, ligand type, ligand valency, and ligand concentration. The molecular and cellular mechanisms of receptor-mediated endocytosis have been reviewed (Brown and Greene, DNA and Cell Biology 10:6, 399-409 (1991)).

[0083] Pharmaceutically acceptable carrier The present compositions comprising antibodies can be used therapeutically in combination with a pharmaceutically acceptable carrier.

[0084] Suitable carriers and their formulations are described in Remington: The Science and Practice of Pharmacy (19th ed.) ed. A.R. Gennaro, Mack Publishing Company, Easton, PA 1995. Typically, an appropriate amount of a pharmaceutically acceptable salt is used in the formulation to render the formulation isotonic. Examples of pharmaceutically acceptable carriers include, but are not limited to, saline, Ringer's solution, and dextrose solution. The pH of the solution is preferably about 5 to about 8, more preferably about 7 to about 7.5. Additional carriers include sustained-release preparations such as semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, liposomes, or microparticles. It will be apparent to those skilled in the art that certain carriers may be more preferable depending, for example, on the route of administration and concentration of the administered composition.

[0085] Pharmaceutical carriers are known to those skilled in the art.They are most typically the standard carriers for human drug administration, including sterile water, physiological saline, and buffer solution at physiological pH.These compositions can be administered intramuscularly or subcutaneously.Other compounds will be administered according to the standard procedures used by those skilled in the art.

[0086] Pharmaceutical compositions may include, in addition to the molecule of choice, carriers, thickeners, diluents, buffers, preservatives, surface active agents, etc. Pharmaceutical compositions may also include one or more active ingredients, e.g., antibacterial agents, anti-inflammatory agents, anesthetics, etc.

[0087] The pharmaceutical composition can be administered in several ways, depending on whether local or systemic treatment is desired and the area to be treated. Administration can be topical (ophthalmic, vaginal, rectal, intranasal, etc.), oral, inhalation, or parenteral, for example, by intravenous drip, subcutaneous, intraperitoneal, or intramuscular injection. The disclosed antibodies can be administered intravenously, intraperitoneally, intramuscularly, subcutaneously, intracavity, or transdermally.

[0088] Preparations 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 such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic / aqueous solutions, emulsions, or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's solution, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives, such as antibacterial agents, antioxidants, chelating agents, and inert gases, may also be present.

[0089] Formulations for topical administration may include ointments, lotions, creams, gels, drops, suppositories, sprays, liquids, and powders. Conventional pharmaceutical carriers, aqueous, powder, or oily bases, thickeners, and the like may be necessary or desirable.

[0090] Compositions for oral administration include powders or granules, suspensions or solutions in water or non-aqueous media, capsules, sachets, or tablets. Thickeners, flavorings, diluents, emulsifiers, dispersing aids, or binders may be desirable.

[0091] Some of the present compositions may be capable of being 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, thiocyanic acid, sulfuric acid, and phosphoric acid, and organic acids such as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid, maleic acid, and fumaric acid, or by reaction with inorganic bases such as sodium hydroxide, ammonium hydroxide, potassium hydroxide, and organic bases such as mono-, di-, tri-alkyl, and aryl amines, and substituted ethanol amines.

[0092] therapeutic use Effective dosages and schedules for administering the composition can be determined empirically, and making such determinations is within the skill of one of ordinary skill in the art. The dosage range for administering the composition is large enough to produce the desired effect of affecting the symptoms of the disorder. The dosage should not be so large as to cause adverse side effects, such as undesirable cross-reactions, anaphylactic reactions, etc. Generally, dosages will vary depending on the patient's age, condition, sex, and extent of disease, the route of administration, or whether other drugs are included in the regimen, which can be determined by one of ordinary skill in the art. Dosages can be adjusted by individual physicians in the event of any contraindications. Dosages may vary and can be administered in one or more doses per day for one or several days. Guidance can be found in the literature regarding appropriate dosages for a given class of pharmaceuticals. For example, guidance for selecting an appropriate dose of an antibody can be found in literature on the therapeutic use of antibodies, e.g., Handbook of Monoclonal Antibodies, Ferrone et al., eds., Noges Publications, Park Ridge, NJ, (1985) ch. 22 and pp. 303-357; Smith et al., Antibodies in Human Diagnosis and Therapy, Haber et al., eds., Raven Press, New York (1977) pp. 365-389. A typical daily dosage of an antibody used alone can range from about 1 μg / kg body weight up to 100 mg / kg body weight, or more, depending on the factors mentioned above.

[0093] c) Methods for generating TGF-β imprinted NK cells As noted throughout, the primary purpose of TGF-β imprinting is to generate NK cells with reduced expression of CD38. Accordingly, in one aspect, disclosed herein is a method for generating TGF-β imprinted NK cells, the method comprising incubating NK cells in the presence of engineered feeder cells, cell membrane particles, or exosomes disclosed herein. For example, disclosed herein is a method for generating TGF-β-imprinted NK cells, the method comprising: generating TGF-β-imprinted NK cells from feeder cells (PBMC, RPMI8866, HFWT, K562 cells, EBV-LCL, NK cells transfected with membrane-bound IL-21 (including, but not limited to, NK-92, NK-92MI, NK-YTS, NK, NKL, KIL, KIL C.2, NK 3.3, NK-YS), NK cells transfected with membrane-bound 4-1BBL (including, but not limited to, NK-92, NK-92MI, NK-YTS, NK, NKL, KIL, KIL C.2, NK 3.3, NK-YS), NK cells transfected with membrane-bound IL-15 and 4-1BBL (including, but not limited to, NK-92, NK-92MI, NK-YTS, NK, NKL, KIL, KIL C.2, NK The method includes incubating NK cells in the presence of TGF-β expressed by feeder cells (including but not limited to NK cells transfected with membrane-bound IL-21 and 4-1BBL (including but not limited to NK-92, NK-92MI, NK-YTS, NK, NKL, KIL, KIL C.2, NK 3.3, NK-YS), or ... incubating NK cells in the presence of cell membrane particles or exosomes derived from feeder cells. Preferably, the TGF-β expressed by the feeder cells is membrane-bound.

[0094] In one aspect, disclosed herein is a method for generating TGF-β imprinted NK cells, wherein the feeder cells comprise at least one additional NK cell effector agent on their cell surface, wherein the at least one additional NK cell effector agent is a cytokine, adhesion molecule, or NK cell activator (e.g., 4-1BBL, IL-2, IL-12, IL-15, IL-18, IL-21, MICA, LFA-1, 2B4, CCR7, OX40L, UBLP2, BCM1 / SLAMF2, NKG2D antigen, etc.). The at least one additional NK cell effector agent is a NK cell effector agent, including, but not limited to, NK cell agonists, CD155, CD112, Jagged1, Jagged2, Delta-1, Pref-1, DNER, Jedi, SOM-11, wingless, CCN3, MAGP2, MAGP1, TSP2, YB-1, EGFL7, CCR7, DAP12, and DAP10, Notch ligands, NKp46 agonists, NKp44 agonists, NKp30 agonists, other NCR agonists, and CD16 agonists. In one embodiment, the at least one additional NK cell effector agent comprises IL-21, 4-1BBL, IL-15, IL-21, and 4-1BBL, IL-21 and IL-15, or IL-15 and 4-1BBL. Preferably, the at least one additional NK cell effector agent is a membrane-bound NK cell effector agent.

[0095] It is understood and contemplated herein that the methods for generating TGF-β imprinted NK cells disclosed herein can be used for any NK cells (exogenous or endogenous) for which TGF-β resistance is desired. Thus, methods for generating TGF-β imprinted NK cells are disclosed herein, which NK cells are NKG2C + , CD56 bright NK cells, CD56 dim These include NK cells, peripheral NK cells, memory-like NK cells such as NK T cells, or tumor-infiltrating NK cells (including, but not limited to, NK cells obtained from a cell line or from a donor source (e.g., autologous, allogeneic, or syngeneic donor)).

[0096] To generate TGF-β-imprinted NK cells, NK cells must be exposed to TGF-β-expressing feeder cells, exosomes, or cell membrane particles (on membrane or soluble), or in the presence of soluble TGF-β, for a period of time to confer tolerance. Thus, in one aspect, a method for generating TGF-β-imprinted NK cells is disclosed herein, in which the NK cells are incubated in the presence of TGF-β and engineered feeder cells, cell membrane particles, or exosomes for at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 45, or 60 days. It is further understood and contemplated herein that NK cells can be cultured for an additional period of time after exposure to engineered feeder cells or feeder cell-derived cell membrane particles or exosomes. In one embodiment, NK cells can be contacted with engineered feeder cells or cell membrane particles or exosomes derived from the feeder cells for 7 to 21 days, preferably 7 to 14 days.

[0097] As noted throughout, the disclosed methods generate TGF-β-imprinted NK cells. Thus, in one aspect, disclosed herein are TGF-β-imprinted NK cells produced by the methods for generating TGF-β-imprinted NK cells disclosed herein.

[0098] In some cases, plasma membrane particles or exosomes derived from engineered feeder cells are obtained by nitrogen cavitation.

[0099] Generally, cells are maintained under conditions suitable for cell growth and / or maintenance.Suitable cell culture conditions are well known in the art, and are described, for example, in Santiago et al., Proc.Natl.Acad.Sci.USA, 2008,105:5809-5814; Moehle et al., Proc.Natl.Acad.Sci.USA, 2007,104:3055-3060; Urnov et al., Nature, 2005,435:646-651; and Lombardo et al., Nat.Biotechnol., 2007,25:1298-1306.Those skilled in the art will understand that methods for culturing cells are known in the art and may vary depending on cell type.In any case, routine optimization may be used to determine the optimal method for a particular cell type.

[0100] d) Dosage and Administration TGF-βi NK cells should be administered and dosed in accordance with good medical practice, taking into account the site and method of administration, the administration schedule, the patient's age, sex, and weight, the nature and severity of the disease being treated or prevented, and other factors known to medical professionals. The cells may be administered in a single dose or in divided doses. Therefore, a pharmaceutically "effective amount" for purposes herein is determined by considerations known in the art. The amount should be effective to achieve improvement, including, but not limited to, improved survival or more rapid recovery, or improvement or elimination of symptoms, and other indicators selected as appropriate by those skilled in the art.

[0101] Typically, the dose is about 10 x 10 6 Cells / kg of subject weight or less, approximately 9 x 10 6 Cells / kg or less, approximately 8×10 6 Cells / kg or less, approximately 7×10 6 Cells / kg or less, approximately 6×10 6 Cells / kg or less, approximately 5×10 6 In other embodiments, the dose is about 0.25 x 10 cells / kg or less. 6 cells / kg ~ approx. 5×106 cells / kg, more preferably about 1 x 10 6 cells / kg ~ approx. 5×10 6 Thus, in yet another embodiment, the dose may be about 0.25×10 cells / kg. 6 cells / kg, 0.5×10 6 cells / kg, 0.6×10 6 cells / kg, 0.7×10 6 cells / kg, 0.8×10 6 cells / kg, 0.9×10 6 cells / kg, 1.1×10 6 cells / kg, 1.2×10 6 cells / kg, 1.3×10 6 cells / kg, 1.4×10 6 cells / kg, 1.5×10 6 cells / kg, 1.6×10 6 cells / kg, 1.7×10 6 cells / kg, 1.8×10 6 cells / kg, 1.9×10 6 cells / kg or 2 x 10 6 In other embodiments, the dose may be 100,000 to 1 million cells / kg, or 1 million to 2 million cells / kg, or 2 million to 3 million cells / kg, or 3 million to 4 million cells / kg, or 4 million to 5 million cells / kg, or 5 million to 6 million cells / kg, or 6 million to 7 million cells / kg, or 7 million to 8 million cells / kg, or 8 million to 9 million cells / kg, or 9 million to 10 million cells / kg.

[0102] The cancer can be selected from, but is not limited to, blood cancer, lymphoma, colorectal cancer, colon cancer, lung cancer, head and neck cancer, ovarian cancer, prostate cancer, testicular cancer, kidney cancer, skin cancer, cervical cancer, pancreatic cancer, and breast cancer. In one aspect, the cancer comprises a solid tumor. In another aspect, the cancer is selected from acute myeloid leukemia, myelodysplastic syndrome, chronic myeloid leukemia, acute lymphoblastic leukemia, myelofibrosis, and multiple myeloma. In another aspect, the cancer is selected from leukemia, lymphoma, sarcoma, and carcinoma, and can occur in the bone marrow, brain, lung, breast, pancreas, liver, head and neck, skin, reproductive tract, prostate, colon, liver, kidney, intraperitoneal cavity, bone, joint, and eye.

[0103] As used herein, the disclosed methods for inhibiting, reducing, and / or preventing cancer metastasis and / or recurrence may include, but are not limited to, the administration of any anticancer agent known in the art, including, but not limited to, abemaciclib, abiraterone acetate, avitrexate (methotrexate), Abraxane (paclitaxel albumin-stabilized nanoparticle formulation), ABVD, ABVE, ABVE-PC, AC, AC-T, Adcetris (Brentuximab), and the like. ciimab vedotin), ADE, adotrastuzumab emtansine, Adriamycin (doxorubicin hydrochloride), afatinib dimaleate, Afinitor (everolimus), Aquinzeo (netupitant and palonosetron hydrochloride), Aldara (imiquimod), aldesleukin, Alecensa (alectinib), alectinib, alemtuzumab, Alimta (pemetrexed disodium), Alicopa (copanlisib hydrochloride), Alkeran injection (melphalan hydrochloride), Alkeran tablets (melphalan), Aloxi (palonosetron hydrochloride), Alunbrig (brigatinib), Ambochlorin (chlorambucil), Ambochlorin (chlorambucil), Amifostine, Aminolevulinic acid, Anastrozole, Aprepitant, Aredia (pamidronate disodium), Arimidex (anastrozole), Aromasin (exemestane), Alanon (nelarabine), Arsenic trioxide, Alze La (ofatumumab), asparaginase Erwinia chrysanthemi, atezolizumab, Avastin (bevacizumab), avelumab, axitinib, azacitidine, Bavencio (avelumab), BEACOPP, Besenam (carmustine), Beleodac (belinostat), belinstat, bendamustine hydrochloride, BEP, Besponsa (inotuzumab ozogamicin), bevacizumab, bexarotene, Bexar (tositumomab and iodine I 131 tositumomab), bicalutamide, BiCNU (carmustine), bleomycin, blinatumomab, Blincyto (blinatumomab), bortezomib, Bosurif (bosutinib), bosutinib, brentuximab vedotin, brigatinib, BuMel, busulfan, Busulfex (busulfan), cabazitaxel, Cabometyx (cabozantinib-S-malate), cabozantinib-S-malate, CAF,Campath (alemtuzumab), Camptosar (irinotecan hydrochloride), capecitabine, CAPOX, Carlac (topical fluorouracil), carboplatin, carboplatin-taxol, carfilzomib, Carmbris (carmustine), carmustine, carmustine implant, Casodex (bicalutamide), CEM, ceritinib, Cerbidine (daunorubicin hydrochloride), Cervarix (recombinant HPV bivalent vaccine), cetuximab, CEV, chlorambucil, chlorambucil-prednisone, CHOP, cisplatin, cladribine, Rafen (cyclophosphamide), clofarabine, Clofarex (clofarabine), Chlor (clofarabine), CMF, cobimetinib, Cometriq (cabozantinib-S-malate), copanlisib hydrochloride, COPDAC, COPP, COPP-ABV, Cosmegen (dactinomycin), Cotellic (cobimetinib), crizotinib, CVP, cyclophosphamide, Cyphos (ifosfamide), Cyramza (ramucirumab), cytarabine, cytarabine liposome, Cytosar U (cytarabine), Cytoxan (cyclophosphamide), Dabrafenib, dacarbazine, Dacogen (decitabine), dactinomycin, daratumumab, Darzalex (daratumumab), dasatinib, daunorubicin hydrochloride, daunorubicin hydrochloride and cytarabine liposome, decitabine, defibrotide sodium, Defitelio (defibrotide sodium), degarelix, denileukin diftitox, denosumab, Depocyte (cytarabine liposome), dexamethasone, dexrazoxane hydrochloride, dinutuximab, docetaxel, Doxil (doxorubicin hydrochloride liposome), doxorubicin hydrochloride Salt, doxorubicin hydrochloride liposome, Dox-SL (doxorubicin hydrochloride liposome), DTIC-Dome (dacarbazine), durvalumab, Efudex (fluorouracil - topical), ERYTECH (rasburicase), Elence (epirubicin hydrochloride), elotuzumab, Eloxatin (oxaliplatin), eltrombopag olamine, Emend (aprepitant), Empliciti (elotuzumab), enasidenib mesylate, enzalutamide, epirubicin hydrochloride, EPOCH, Erbitux (cetuximab), eribulin mesylate,Erivage (vismodegib), erlotinib hydrochloride, Erwinase (asparaginase erwinia chrysanthemi), Ethiol (amifostine), Etopofos (etoposide phosphate), etoposide, etoposide phosphate, Everset (doxorubicin hydrochloride liposome), everolimus, Evista (raloxifene hydrochloride), Evomela (melphalan hydrochloride), exemestane, 5-FU (fluorouracil injection), 5-FU (fluorouracil topical), Fairston (toremifene), Farydak (panobinostat), Fasolodex (fulves) Trant), FEC, Femara (letrozole), filgrastim, Fludara (fludarabine phosphate), fludarabine phosphate, Fluoroplex (fluorouracil - topical), fluorouracil injection, fluorouracil - topical, flutamide, Folex (methotrexate), Folex PFS (methotrexate), Forfiri, forfiri-bevacizumab, forfiri-cetuximab, Forfirinox, Forfox, Folotyn (pralatrexate), FU-LV, fulvestrant, Gardasil (recombinant HPV 4-valent vaccine), Gardasil 9 (recombinant HPV 9-valent vaccine), Gazyva (obinutuzumab), gefitinib, gemcitabine hydrochloride, cisplatin gemcitabine, oxaliplatin gemcitabine, gemtuzumab ozogamicin, Gemzar (gemcitabine hydrochloride), Gilotrif (afatinib dimaleate), Glivec (imatinib mesylate), Gliadel (carmustine implant), Gliadel wafer (carmustine implant), glucarpidase, goserelin acetate, Halaven (eribulin mesylate), Hemangeol (propranolol) hydrochloride), Herceptin (trastuzumab), HPV bivalent vaccine (recombinant), HPV nonavalent vaccine (recombinant), HPV quadrivalent vaccine (recombinant), Hycamtin (topotecan hydrochloride), Hydrea (hydroxyurea), hydroxyurea, Hyper CVAD, Ibrance (palbociclib), ibritumomab tiuxetan, ibrutinib, ICE, Iclusig (ponatinib hydrochloride), Idamycin (idarubicin hydrochloride), idarubicin hydrochloride, idelalisib, Idifa (enasidenib mesylate), Ifex (ifosfamide), ifosfamide,Ifosfamidam (ifosfamide), IL-2 (aldesleukin), imatinib mesylate, ibrutinib (ibrutinib), Imfinzi (durvalumab), imiquimod, Imlijik (talimogene laherparepvec), Inlyta (axitinib), inotuzumab ozogamicin, interferon alfa-2b (recombinant), interleukin-2 (aldesleukin), Intron A (recombinant interferon alfa-2b), iodine I 131 Tositumomab and tositumomab, ipilimumab, Iressa (gefitinib), irinotecan hydrochloride, irinotecan hydrochloride liposomal, Istodax (romidepsin), ixabepilone, ixazomib citrate, Ixempra (ixabepilone), Jakafi (ruxolitinib), JEB, Jevtana (cabazitaxel), Kadcyla (adotrastuzumab emtansine), Keoxifen (raloxifene hydrochloride), Kepiba ance (palifermin), Keytruda (pembrolizumab), Kisqali (ribociclib), Kymriah (tisagenlecleucel), Kyprolis (carfilzomib), lanreotide acetate, lapatinib ditosylate, Raltruvo (olaratumab), lenalidomide, lenvatinib mesylate, Lenvima (lenvatinib mesylate), letrozole, leucovorin calcium, Leukelan (chlorambucil), leuprorethamine acetate, Leukelan Statins (cladribine), Levran (aminolevulinic acid), Linfolidine (chlorambucil), Lipodox (doxorubicin hydrochloride liposome), lomustine, Lonsurf (trifluridine and tipiracil hydrochloride), Lupron (leuproreductamine acetate), Lupron Depot (leuproreductamine acetate), Lupron Depot Ped (leuproreductamine acetate), Lynparza (olaparib), Marchibo (vincristine sulfate liposome), Matulane ( Procarbazine hydrochloride), mechlorethamine hydrochloride, megestrol acetate, Mekinist (trametinib), melphalan, melphalan hydrochloride, mercaptopurine, mesna, Mesnex (mesna), metazolastone (temozolomide), methotrexate, methotrexate LPF (methotrexate), methylnaltrexone bromide, mexate (methotrexate), mexate AQ (methotrexate), midostaurin, mitomycin C,Mitoxantrone hydrochloride, Mitozitrex (mitomycin C), MOPP, Mozobil (plelixafor), Mustagen (mechlorethamine hydrochloride), Mutamycin (mitomycin C), Myleran (busulfan), Mylocer (azacitidine), Mylotarg (gemtuzumab ozogamicin), nanoparticle paclitaxel (paclitaxel albumin-stabilized nanoparticle formulation), Navelbine (vinorelbine tartrate), necitumumab, nelarabine, Neocer (cyclophosphamide), neratinib maleate, Nerlinx (neratinib maleate) , Netpitant and Palonosetron Hydrochloride, Neulasta (Pegfilgrastim), Neupogen (Filgrastim), Nexavar (Sorafenib Tosylate), Nilandrone (Nilutamide), Nilotinib, Nilutamide, Ninlaro (Ixazomib Citrate), Niraparib Tosylate Monohydrate, Nivolumab, Nolvadex (Tamoxifen Citrate), Nplate (Romiplostim), Obinutuzumab, Odomzo (Sonidegib), OEPA, Ofatumumab, OFF, Olaparib, Olaratumab, Omacetaxine Mepescofate, Oncaspar (pegaspargase), ondansetron hydrochloride, Onivyde (irinotecan hydrochloride liposome), Ontak (denileukin diftitox), Opdivo (nivolumab), OPPA, osimertinib, oxaliplatin, paclitaxel, paclitaxel albumin-stabilized nanoparticle formulation, PAD, palbociclib, palifermin, palonosetron hydrochloride, palonosetron hydrochloride and netupitant, pamidronate disodium, panitumumab, panobinostat, Paraplat (carboplatin), Paraplatin (carboplatin), pazopanib hydrochloride , PCV, PEB, pegaspargase, pegfilgrastim, peginterferon alfa-2b, pegIntron (peginterferon alfa-2b), pembrolizumab, pemetrexed disodium, Perjeta (pertuzumab), pertuzumab, Platinol (cisplatin), Platinol-AQ (cisplatin), plerixafor, pomalidomide, Pomalyst (pomalidomide), ponatinib hydrochloride, Portrazza (necitumumab), pralatrexate, prednisone, procarbazine hydrochloride, Proleukin (aldesleukin),Prolia (denosumab), Promacta (eltrombopag olamine), propranolol hydrochloride, Provenzi (sipuleucel-T), Purinetol (mercaptopurine), Prixan (mercaptopurine), radium 223 dichloride, raloxifene hydrochloride, ramucirumab, rasburicase, R-CHOP, R, -CVP, recombinant human papillomavirus (HPV) bivalent vaccine, recombinant human papillomavirus (HPV) nonavalent vaccine, recombinant human papillomavirus (HPV) quadrivalent vaccine, recombinant interferon alfa-2b, regorafenib, Ristol (methylnaltrexone bromide), R-EPOCH, Revlimid (lenalidomide), Rheumatrex (methotrexate), ribociclib, R-ICE, Rituxan (rituximab), Rituxan Hycera (rituximab and hyaluronidase human), rituximab, rituximab and hyaluronidase human Human, rolapitant hydrochloride, romidepsin, romiplostim, rubidomycin (daunorubicin hydrochloride), Rubraca (rucaparib camsylate), rucaparib camsylate, ruxolitinib phosphate, Ridapt (midostaurin), Sclerosol intrapleural aerosol (talc), siltuximab, sipuleucel-T, Somatuline Depot (lanreotide acetate), sonidegib, sorafenib tosylate, Sprycel (dasatinib), Stanford V, sterile talc powder (talc), Steritalc (talc), Stivarga (regorafenib), sunitinib malate, Sutent (sunitinib malate), Silatron (peginterferon alfa-2b), Sylvant (siltuximab), Synribo (omacetaxel) Pescoquinate), Tabloid (thioguanine), TAC, Tafinlar (dabrafenib), Tagrisso (osimertinib), talc, talimogene laherparepvec, tamoxifen citrate, Tarabin PFS (cytarabine), Tarceva (erlotinib hydrochloride), Targretin (bexarotene), Tasigna (nilotinib), Taxol (paclitaxel) , Taxotere (docetaxel), Tecentriq, (atezolizumab), Temodar (temozolomide), temozolomide, temsirolimus, thalidomide, Thalomid (thalidomide), thioguanine, thiotepa, tisagenlecleucel, Trac (fluorouracil - topical), topotecan hydrochloride, toremifene, Tolicel (temsirolimus), tositumomab, iodine I 131 tositumomab, Totect (dexrazoxane hydrochloride), TPF, trabectedin, trametinib, trastuzumab, Treanda (bendamustine hydrochloride), trifluridine tipiracil hydrochloride,Trisenox (arsenic trioxide), Tykerb (lapatinib ditosylate), Unituxin (dinutuximab), uridine triacetate, VAC, vandetanib, VAMP, Barbi (rolapitant hydrochloride), Vectibix (panitumumab), VeIP, Velban (vinblastine sulfate), Velcade (bortezomib), Belsar (vinblastine sulfate), vemurafenib, Venclexta (venetoclax), venetoclax s, Verzenio (abemaciclib), Viadur (leuprolide acetate), Vidaza (azacitidine), vinblastine sulfate, Vincasar PFS (vincristine sulfate), vincristine sulfate, vincristine sulfate liposomal, vinorelbine tartrate, VIP, vismodegib, Vistogard (uridine triacetate), voraxaze (glucarpidase), vorinostat, Votrient (pazopanib hydrochloride), Viqios (Dow Norubicin hydrochloride and cytarabine liposome), Wellcovorin (leucovorin calcium), Xalkori (crizotinib), Xeloda (capecitabine), Zeliri, Xerox, Zegeva (denosumab), Zofigo (radium-223 chloride), Xtandi (enzalutamide), Yervoy (ipilimumab), Yondelis (trabectedin), Zaltrap (ziflibercept), Zarxio (filgrastim), Zejula (niraparib) tosylate monohydrate), Zelboraf (vemurafenib), Zevalin (ibritumomab tiuxetan), Zincard (dexrazoxane hydrochloride), dibuaflibercept, Zofran (ondansetron) hydrochloride, Zoladex (goserelin acetate), zoledronic acid, Zolinza (vorinostat), Zometa (zoledronic acid), Zydelig (idelalisib), Zykadia (ceritinib), and / or Zytiga (abiraterone acetate). Also contemplated herein are chemotherapeutic agents that are PD1 / PDL1 blockade inhibitors (e.g., lambrolizumab, nivolumab, pembrolizumab, pidilizumab, BMS-936559, atezolizumab, durvalumab, or avelumab). Also provided herein are the disclosed uses of the disclosed compositions and / or engineered NK cell populations to inhibit, reduce, and / or prevent cancer metastasis and / or recurrence, including:It is also contemplated that the use of any anti-cancer agent known in the art, including but not limited to those listed above, may be included in combination.

[0104] Although several embodiments of the present disclosure have been described, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims.

[0105] By way of non-limiting example, examples of specific embodiments of the present disclosure are provided below.

[0106] Example The following examples are intended to illustrate compositions, devices, methods, and results in accordance with the disclosed subject matter. These examples are not intended to be inclusive of all aspects of the subject matter disclosed herein, but rather to illustrate representative methods and results. These examples are not intended to exclude equivalents and variations of the present invention that would be apparent to one skilled in the art.

[0107] Example 1: CD38 for cancer immunotherapy Low Natural killer (NK) cells Daratumumab (DARA), a monoclonal antibody that binds to CD38, has been successfully tested in patients with CD38+ multiple myeloma (MM) and is currently being studied in patients with CD38+ T-ALL. DARA targets CD38+ cancer cells through several mechanisms, including antibody-induced cytotoxicity (ADCC), thereby activating NK cells and inducing them to secrete cytotoxic granules and death ligands against cancer targets. However, DARA also depletes NK cells that highly express CD38. This is known as "fratricide," reducing the antitumor efficacy of DARA.

[0108] showed that the CRISPR / Cas9 system was highly effective in deleting CD38 in ex vivo expanded peripheral blood NK cells. These CD38KO NK cells were completely resistant to DARA-induced fratricide, exhibited superior persistence in DARA-pretreated immunodeficient mice, and showed enhanced ADCC activity against CD38-expressing MM cell lines and primary MM cells.

[0109] Although CRISPR-based gene modification is difficult to translate into clinical practice, TGFβ imprinting may be a viable non-genetic alternative if it can produce comparable or even better results, thereby leading to the development of a new cell therapy product, TGF-βi-CD38, for treating MM. low This may support the development of clinical trials of the combination of NK cells and DARA for patients with MM.

[0110] Example 2: TGF-βi imprinting of primary NK cells reduces CD38-Nadase, promotes a tissue-resident addressin profile, and improves cytotoxicity and metabolism.

[0111] Transforming growth factor beta (TGFβ) is a potent immunosuppressive cytokine that inhibits the antitumor responses of NK cells and T cells. However, stimulation of natural killer (NK) cells with proinflammatory cytokines reduces the sensitivity of NK cells to TGFβ. Previously, we demonstrated that TGFβ imprinting (TGFβi) during IL-21-induced expansion further reduces NK cell sensitivity to TGFβ through SMAD3 suppression, enhancing a proinflammatory phenotype accompanied by excessive secretion of IFN-γ, TNF-α, and GM-CSF.

[0112] To evaluate the sequential killing capacity of TGFβi-NK cells, real-time cell analysis was performed. TGFβi-NK cells exhibited faster and more efficient cytotoxicity against both liquid cancer cell lines (K562 and Kasumi) and solid cancer cell lines (MEL33 and MEL41) compared with standard expanded NK cells. Furthermore, TGF-βi-NK cells exhibited significantly superior killing potency than standard expanded NK cells at a low E:T ratio (0.5:1). To gain a deeper understanding of the impact of TGFβ-imprinting on gene expression in NK cells, we performed RNA-seq. At the mRNA level, TGF-βi-imprinting altered the expression of chemokine receptors, decreasing CCR2, CXCR1, CXCR6, and CX3CR1 and increasing CCR4 and 7. Furthermore, TGF-β-imprinted NK cells showed increased expression of integrins such as ITGA1, ITGB1, and ITGAE, indicating that TGF-β imprinting can induce a reprogrammed tissue-resident addressin profile similar to that of ILC1s. Next, we evaluated the efficacy of TGF-βi NK cells in a disseminated tumor model and found that while standard and TGF-βi NK cells had similar abilities to control primary tumors, TGF-βi NK cells were superior in protecting mice from liver metastases (Figure 7).

[0113] RNA sequencing also revealed that TGF-β imprinting significantly suppressed CD38 expression in NK cells by flow cytometry. Because CD38 is an extracellular enzyme that regulates NAD+, a key component of OXPHOS in both T and NK cells, the effect of TGF-βi on NK cell metabolism was examined. Compared to standard NK cells, TGF-βi NK cells exhibited a higher oxygen consumption rate (OCR) accompanied by a higher OCR / ECAR ratio. Because the level of surface CD38 expression was significantly reduced, their ability to resist daratumumab (DARA)-induced fratricide was also assessed. It was found that TGF-βi NK cells were resistant to DARA-induced fratricide and exhibited higher cytotoxicity against CD38+ cell lines in the presence of DARA. (Figure 7)

[0114] Here, it has been shown that TGF-βi suppresses CD38, leading to improved OXPHOS and elimination of fratricide, increasing NK cell potency, tissue homing, and serial killing. TGF-βi NK cells combined with CD38 targeting are considered for CD38+ malignancies.

[0115] It will be apparent to those skilled in the art that various modifications and variations can be made in the present disclosure without departing from the scope or spirit of the invention. Other embodiments of the present disclosure will be apparent to those skilled in the art from consideration of the specification and practice of the methods disclosed herein. It is intended that the specification and examples be considered as exemplary only, with the true scope and spirit of the invention being indicated by the following claims.

Claims

1. 1. A method for treating cancer or an infectious disease in a subject in need thereof, comprising administering to the subject a therapeutically effective number of transforming growth factor beta (TGF-β) superfamily imprinted natural killer (TGF-βi NK) cells and a CD38 targeting agent.

2. The method of claim 1, wherein the TGF-βi NK cells have reduced expression of CD38.

3. The method of claim 1, wherein the TGF-βi NK cells have increased resistance to TGF-β superfamily cytokines.

4. The method of any one of claims 1 to 3, wherein the TGF-βi NK cells have increased resistance to TGF-β.

5. The method of any one of claims 1 to 4, wherein the TGF-βi NK cells produced increased amounts of one or more of IFN-γ, TNF-α, and GM-CSF.

6. The method of any one of claims 1 to 5, wherein the TGF-βi NK cells have reduced levels of SMAD3 and / or TGFBR3 proteins.

7. The method of any one of claims 1 to 6, wherein the TGF-βi NK cells are prepared by incubating NK cells in the presence of TGF-β.

8. The method of any one of claims 1 to 7, wherein the TGF-βi NK cells are prepared by incubating NK cells in the presence of feeder cells engineered to express TGF-β, or by incubating NK cells in the presence of cell membrane particles or exosomes derived from the feeder cells.

9. The method of claim 8, wherein the feeder cells comprise PBMC, RPMI8866, HFWT, K562 cells, EBV-LCL, NK cells transfected with membrane-bound IL-21, NK cells transfected with membrane-bound 4-1BBL, NK cells transfected with membrane-bound IL-15 and 4-1BBL, or NK cells transfected with membrane-bound IL-21 and 4-1BBL.

10. 10. The method of claim 8 or 9, wherein the feeder cells further comprise at least one additional NK cell effector agent on their cell surface, wherein the at least one additional NK cell effector agent is a cytokine, an adhesion molecule, or an NK cell activator.

11. The at least one additional NK cell effector agent is selected from the group consisting of 4-1BBL, IL-2, IL-12, IL-15, IL-18, IL-21, MICA, LFA-1, 2B4, CCR7, OX40L, UBLP2, BCM1 / SLAMF2, NKG2D agonist, CD155, CD112, Jagged1, Jagged2, Delta-1, Pref-1, DNER , Jedi, SOM-11, wingless, CCN3, MAGP2, MAGP1, TSP2, YB-1, EGFL7, CCR7, DAP12, and DAP10, Notch ligands, NKp46 agonists, NKp44 agonists, NKp30 agonists, other NCR agonists, and CD16 agonists.

12. 11. The method of claim 10, wherein the at least one additional NK cell effector agent comprises IL-21, 4-1BBL, IL-15, IL-21 and 4-1BBL, IL-21 and IL-15, or IL-15 and 4-1BBL.

13. The method of any one of claims 7 to 12, wherein the NK cells are activated in vitro or ex vivo.

14. The method according to any one of claims 7 to 13, wherein the NK cells are human NK cells or canine NK cells.

15. The method according to any one of claims 7 to 14, wherein the NK cells are primary NK cells or an NK cell line.

16. The NK cells are NKG2C ＋ CD56 ｂｒｉｇｈｔ NK cells, CD56 ｄｉｍ The method of any one of claims 7 to 15, comprising NK cells, peripheral NK cells, and memory-like NK cells such as NK T cells, and tumor-infiltrating NK cells.

17. The method of any one of claims 7 to 16, wherein the NK cells are obtained from a donor subject.

18. The method of any one of claims 7 to 17, wherein the NK cells are obtained from an autologous donor, an allogeneic donor, or a syngeneic donor.

19. 19. The method of any one of claims 7 to 18, wherein the NK cells are incubated in the presence of the engineered feeder cells, the cell membrane particles, or the exosomes for at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 45, or 60 days.

20. The method of any one of claims 1 to 19, wherein the CD38 targeting agent is a CD38 inhibitor.

21. The method of any one of claims 1 to 20, wherein the CD38 targeting agent is an anti-CD38 antibody.

22. 22. The method of claim 21, wherein the anti-CD38 antibody comprises a fragment crystallizable region (Fc region) that binds to an Fc receptor or lacks an Fc region.

23. 23. The method of claim 21 or 22, wherein the anti-CD38 antibody is daratumumab or isatuximab.

24. The method of any one of claims 1 to 23, wherein the TGF-βi NK cells are administered before, simultaneously with, and / or after administration of the CD38 inhibitor.

25. The method of any one of claims 1 to 24, wherein the subject comprises tumor cells that have elevated levels of CD38 compared to a reference control.

26. The method of any one of claims 1 to 25, wherein the cancer is leukemia, lymphoma, or myeloma.

27. An anti-cancer therapy comprising transforming growth factor beta (TGF-β) superfamily imprinted natural killer (TGF-βi NK) cells and a CD38-targeting agent.

28. 28. The anticancer therapy of claim 27, wherein the TGF-βi NK cells are generated by incubating NK cells in the presence of feeder cells engineered to express TGF-β, or by incubating NK cells in the presence of cell membrane particles or exosomes derived from the feeder cells.

29. The anticancer therapy of claim 28, wherein the feeder cells comprise PBMC, RPMI8866, HFWT, K562 cells, EBV-LCL, NK cells transfected with membrane-bound IL-21, NK cells transfected with membrane-bound 4-1BBL, NK cells transfected with membrane-bound IL-15 and 4-1BBL, or NK cells transfected with membrane-bound IL-21 and 4-1BBL.

30. 30. The anticancer therapy of claim 28 or 29, wherein the feeder cells further comprise at least one additional NK cell effector agent on their cell surface, and the at least one additional NK cell effector agent is a cytokine, an adhesion molecule, or an NK cell activator.

31. The at least one additional NK cell effector agent may be 4-1BBL, IL-2, IL-12, IL-15, IL-18, IL-21, MICA, LFA-1, 2B4, CCR7, OX40L, UBLP2, BCM1 / SLAMF2, NKG2D agonist, CD155, CD112, Jagged1, Jagged2, Delta-1, Pref-1, DNER, 31. The anti-cancer therapy of claim 30, wherein the agonist is selected from Jedi, SOM-11, wingless, CCN3, MAGP2, MAGP1, TSP2, YB-1, EGFL7, CCR7, DAP12, and DAP10, Notch ligands, NKp46 agonists, NKp44 agonists, NKp30 agonists, other NCR agonists, and CD16 agonists.

32. 31. The anti-cancer therapy of claim 30, wherein the at least one additional NK cell effector agent comprises IL-21, 4-1BBL, IL-15, IL-21 and 4-1BBL, IL-21 and IL-15, or IL-15 and 4-1BBL.

33. The anti-cancer therapy of any one of claims 28 to 32, wherein the NK cells are activated in vitro or ex vivo.

34. The anti-cancer therapy according to any one of claims 28 to 33, wherein the NK cells are human NK cells or canine NK cells.

35. The anti-cancer therapy of any one of claims 28 to 34, wherein the NK cells are primary NK cells or an NK cell line.

36. The NK cells are NKG2C ＋ CD56 ｂｒｉｇｈｔ NK cells, CD56 ｄｉｍ The anti-cancer therapy of any one of claims 26 to 33, comprising NK cells, peripheral NK cells, and memory-like NK cells such as NK T cells, and tumor-infiltrating NK cells.

37. The anti-cancer therapy of any one of claims 28 to 36, wherein the NK cells are obtained from a donor subject.

38. The anti-cancer therapy of any one of claims 28 to 37, wherein the NK cells are obtained from an autologous donor, an allogeneic donor, or a syngeneic donor.

39. The anti-cancer therapy of any one of claims 27 to 38, wherein the CD38 targeting agent is a CD38 inhibitor.

40. The anti-cancer therapy of any one of claims 27 to 39, wherein the CD38 targeting agent is an anti-CD38 antibody.

41. 41. The anti-cancer therapy of claim 40, wherein the anti-CD38 antibody comprises a fragment crystallizable region (Fc region) that binds to an Fc receptor or lacks an Fc region.

42. 42. The anti-cancer therapy of claim 40 or 41, wherein the anti-CD38 antibody is daratumumab or isatuximab.

43. 1. A modified natural killer (NK) cell, prepared by culturing a first NK cell in the presence of a TGF-β superfamily cytokine and incubating the first NK cell in the presence of a feeder cell engineered to express TGF-β, or incubating the first NK cell in the presence of a cell membrane particle or exosome derived from the feeder cell.

44. 44. The modified NK cell of claim 43, wherein the first NK cell has improved resistance to TGF-β compared to a naturally occurring NK cell.

45. The modified NK cell of claim 43 or 44, wherein the feeder cells comprise PBMC, RPMI8866, HFWT, K562 cells, EBV-LCL, NK cells transfected with membrane-bound IL-21, NK cells transfected with membrane-bound 4-1BBL, NK cells transfected with membrane-bound IL-15 and 4-1BBL, or NK cells transfected with membrane-bound IL-21 and 4-1BBL.

46. 46. ​​The modified NK cell of any one of claims 43 to 45, wherein the feeder cell further comprises at least one additional NK cell effector agent on its cell surface, wherein the at least one additional NK cell effector agent is a cytokine, an adhesion molecule, or an NK cell activator.

47. The at least one additional NK cell effector agent may be 4-1BBL, IL-2, IL-12, IL-15, IL-18, IL-21, MICA, LFA-1, 2B4, CCR7, OX40L, UBLP2, BCM1 / SLAMF2, NKG2D agonist, CD155, CD112, Jagged1, Jagged2, Delta-1, Pref-1, DNER, J 47. The modified NK cell of claim 46, wherein the NK cell is selected from edi, SOM-11, wingless, CCN3, MAGP2, MAGP1, TSP2, YB-1, EGFL7, CCR7, DAP12, and DAP10, a Notch ligand, an NKp46 agonist, an NKp44 agonist, an NKp30 agonist, other NCR agonists, and a CD16 agonist.

48. 47. The modified NK cell of claim 46, wherein the at least one additional NK cell effector agent comprises IL-21, 4-1BBL, IL-15, IL-21 and 4-1BBL, IL-21 and IL-15, or IL-15 and 4-1BBL.

49. The modified NK cell of any one of claims 43 to 48, wherein the modified NK cell is activated in vitro or ex vivo.

50. 50. The modified NK cell of any one of claims 43 to 49, wherein the first NK cell is a human NK cell or a canine NK cell.

51. The modified NK cell of any one of claims 43 to 50, wherein the first NK cell is a primary NK cell or an NK cell line.

52. The first NK cell is NKG2C ＋ CD56 ｂｒｉｇｈｔ NK cells, CD56 ｄｉｍ 52. The modified NK cells of any one of claims 43 to 51, comprising NK cells, peripheral NK cells, and memory-like NK cells such as NK T cells, or tumor-infiltrating NK cells.

53. 53. The modified NK cell of any one of claims 43 to 52, wherein the first NK cell is obtained from a donor subject.

54. 54. The modified NK cell of any one of claims 43 to 53, wherein the first NK cell is obtained from an autologous donor, an allogeneic donor, or a syngeneic donor.

55. 55. The modified NK cell of any one of claims 43-54, wherein the first NK cell is incubated in the presence of the engineered feeder cells, the cell membrane particles, or the exosomes for at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 45, or 60 days.

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