Overcoming immunosuppression using TGF-β-resistant NK cells

Abstract

FIELD: biotechnology.SUBSTANCE: invention relates to an engineered feeder cell created by modifying a feeder cell to express soluble or membrane-bound TGF-b. The engineered cell comprises an expression vector containing a nucleic acid sequence expressing soluble or membrane-bound TGF-b.EFFECT: invention is effective for creating a TGF-b-resistant NK cell.16 cl, 8 dwg, 2 ex

Description

[0001] This application claims priority to U.S. Provisional Application No. 63 / 018108, filed April 30, 2020, which is incorporated herein by reference.

[0002] I. LEVEL OF TECHNOLOGY

[0003] 1. The use of NK cells in anticancer therapy is growing. However, studies have shown that various types of cancer secrete TGF-β, which can lead to a decrease in the killing activity of NK cells. Previous efforts to reduce cell death have included the use of TGF-β inhibitors; however, the use of TGF-β inhibitors can cause systemic adverse side effects. New reagents and methods that can be used to maintain the killing activity of NK cells in the presence of TGF-β (i.e., TGF-β-resistant NK cells) are needed.

[0004] II. BRIEF DESCRIPTION OF THE INVENTION

[0005] 2. Methods and compositions related to engineered feeder cells and the use of said feeder cells for creating NK cells resistant to TGF-β are disclosed.

[0006] 3. In one aspect, the present document discloses engineered feeder cells that have been modified to express soluble or membrane-bound TGF-β. For example, TGF-β (soluble or membrane-bound) can be operably linked to a constitutive or inducible promoter, including, but not limited to, the CMV or EF1A promoter.

[0007] 4. Also disclosed herein are engineered feeder cells according to any of the previous aspects, further comprising at least one additional NK cell effector agent on the cell surface thereof, wherein the at least one additional NK cell effector agent is a cytokine, an adhesion molecule, or an NK cell activating agent (such as, for example, NK cell effector agents including but not limited to 4-1BBL, IL-2, IL-12, IL-15, IL-18, IL-21, MICA, LFA-1, 2 B4, CCR7, OX40L, UBLP2, BCM1 / SLAMF2, NKG2D agonists, CD155, CD112, Jagged1, Jagged2, delta-1, Pref-1, DNER, Jedi, SOM-11, wingless, CCN3, MAGP2, MAGP1, TSP2, YB-1, EGFL7, CCR7, DAP 12 and DAP10, Notch ligands, NKp46 agonists, NKp44 agonists, NKp30 agonists, other NCR agonists, CD16 agonists).In one aspect, the at least one additional NK cell effector agent is IL-21, 4-1 BBL, IL-15, IL-21 and 4-1BBL, IL-21 and IL-15, or IL-15 and 4-1 BBL.

[0008] 5. In one aspect, disclosed herein are engineered feeder cells according to any of the previous aspects, wherein the feeder cells are PBMCs, RPMI8866, HFWT, K562, EBV-LCL cells, NK cells (including but not limited to PBMCs, RPMI8866, NK-92, NK-92MI, NK-YTS, NK, NKL, KIL, KILC.2, NK 3.3, NK-YS, HFWT, K562 cells) transfected with membrane-bound IL-21, NK cells (including but not limited to PBMCs, RPMI8866, NK-92, NK-92MI, NK-YTS, NK, NKL, KIL, KILC.2, NK3.3, NK-YS, HFWT, K562 cells) transfected with membrane-bound 4-1BBL, NK cells (including but not limited to PBMCs, RPMI8866, NK-92, NK-92MI, NK-YTS, NK, NKL, KIL, KIL C.2, NK3.3, NK-YS, HFWT, K562 cells) transfected with membrane-bound IL-15 and 4-1BBL, or NK cells (including but not limited to PBMCs, RPMI8866, NK-92, NK-92MI, NK-YTS, NK, NKL, KIL, KILC.2, NK3.3, NK-YS, HFWT, K562 cells) transfected with membrane-bound IL-21 and 4-1BBL.

[0009] 6. Also disclosed herein are cytoplasmic membrane particles or exosomes obtained from an engineered feeder cell according to any of the preceding aspects. In some cases, the particles or exosomes may be obtained using nitrogen cavitation.

[0010] 7. In one aspect, the document discloses methods for generating TGF-β-resistant NK cells, comprising incubating NK cells in the presence of engineered feeder cells, cytoplasmic membrane particles, or exosomes according to any of the preceding claims. For example, disclosed herein are methods for generating TGF-β-resistant NK cells comprising incubating NK cells in the presence of feeder cells (including, but not limited to, PBMCs, RPMI8866, NK-92, NK-92MI, NK-YTS, NK, NKL, KIL, KIL C.2, NK 3.3, NK-YS, HFWT, K562, EBV-LCL, NK cells transfected with membrane-bound IL-21, NK cells transfected with membrane-bound 4-1 BBL, NK cells transfected with membrane-bound IL-15 and 4-1 BBL, or NK cells transfected with membrane-bound IL-21 and 4-1BBL) that have been engineered to express TGF-β, or incubating NK cells in the presence of cytoplasmic membrane particles or exosomes obtained from the indicated feeder cells.

[0011] 8. Also disclosed herein are methods for generating TGF-β resistant NK cells according to any of the previous aspects, 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 activating agent (such as, for example, NK cell effector agents including but not limited to 4-1 BBL, IL-2, IL-12, IL-15, IL-18, IL-21, MICA, LFA-1, 2 B4, CCR7, OX40L, UBLP2, BCM1 / SLAMF2, NKG2D agonists, CD155, CD112, Jagged1, Jagged2, delta-1, Pref-1, DNER, Jedi, SOM-11, wingless, CCN3, MAGP2, MAGP1, TSP2, YB-1, EGFL7, CCR7, DAP 12 and DAP10, Notch ligands, NKp46 agonists, NKp44 agonists, NKp30 agonists, other NCR agonists, CD16 agonists).In one aspect, the at least one additional NK cell effector agent is IL-21, 4-1 BBL, IL-15, IL-21 and 4-1 BBL, IL-21 and IL-15, or IL-15 and 4-1 BBL.

[0012] 9. In one aspect, this document discloses methods for generating TGF-β resistant NK cells according to any of the previous aspects, wherein the NK cells are memory cell-like NK cells such as NKG2C + , CD56 bright NK cells, CD56 dim NK cells, peripheral NK cells, NK T cells, or tumor-infiltrating NK cells (including, but not limited to, NK cells derived from cell lines or obtained from a donor source (such as an autologous donor, an allogeneic donor, or a syngeneic donor).

[0013] 10. This document also discloses methods for generating TGF-β-resistant NK cells according to any of the previous aspects, wherein the NK cells are incubated in the presence of engineered feeder cells, cytoplasmic 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.

[0014] 11. In one aspect, the present document discloses TGF-β-resistant NK cells obtained by the method of any of the previous aspects.

[0015] 12. Also disclosed herein are methods for treating, inhibiting, reducing, attenuating, ameliorating and / or preventing cancer and / or metastasis in a subject, comprising administering to the subject a TGF-β-resistant NK cell according to any of the previous aspects.For example, disclosed herein are methods for treating, inhibiting, reducing, attenuating, ameliorating, and / or preventing cancer and / or metastasis (e.g., a solid tumor) in a subject using a TGF-β-resistant NK cell, comprising obtaining the NK cell; culturing an NK cell in the presence of feeder cells (including, but not limited to, PBMCs, RPMI8866 cells, HFWT cells, K562 cells, EBV-LCL cells, 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) engineered to express TGF-β, thereby generating a TGF-β-resistant NK cell, or in the presence of cytoplasmic membrane particles or exosomes derived from said feeder cells; and administering the TGF-β-resistant NK cell to the subject.Also disclosed herein is a TGF-β resistant NK cell of the present invention for use as a medicament, preferably for use in a method of treating cancer in a subject. For example, disclosed herein is a TGF-β resistant NK cell for use in a method of treating, inhibiting, reducing, attenuating, ameliorating and / or preventing cancer and / or metastasis or a solid tumor in a subject; wherein said TGF-β resistant NK cell can be obtained by a method of creating a TGF-β resistant NK cell of the present invention, preferably a method comprising obtaining an NK cell; culturing the NK cell in the presence of feeder cells (including but not limited to PBMC, RPMI8866, NK-92, NK-92MI, NK-YTS, NK, NKL, KIL, KIL C.2, NK 3 cells.3, NK-YS, HFWT, K562, EBV-LCL, or NK cells) engineered to express TGF-β, thereby generating a TGF-β-resistant NK cell, or culturing the NK cell in the presence of cytoplasmic membrane particles or exosomes obtained from said feeder cells, thereby generating a TGF-β-resistant NK cell. Preferably, the feeder cells (including, but not limited to, PBMCs, RPMI8866, NK-92, NK-92MI, NK-YTS, NK, NKL, KIL, KIL C.2, NK 3.3, NK-YS, HFWT, K562, EBV-LCL, or NK cells) are (i) transfected with membrane-bound IL-21, (ii) transfected with membrane-bound 4-1 BBL, (iii) transfected with membrane-bound IL-15 and 4-1 BBL, or (iv) transfected with membrane-bound IL-21 and 4-1 BBL. Also disclosed herein is the use of a TGF-β-resistant NK cell of the present invention for the production of a medicament for the treatment of cancer in a subject.For example, disclosed herein is the use of a TGF-β-resistant NK cell for the production of a medicament for treating, inhibiting, reducing, attenuating, ameliorating and / or preventing cancer and / or metastasis or a solid tumor in a subject; wherein said TGF-β-resistant NK cell can be obtained by a method for creating a TGF-β-resistant NK cell according to the present invention, preferably a method comprising the steps of obtaining an NK cell; culturing the NK cell in the presence of feeder cells (including but not limited to PBMCs, RPMI8866, NK-92, NK-92MI, NK-YTS, NK, NKL, KIL, KIL C.2, NK 3.3, NK-YS, HFWT, K562, EBV-LCL cells, or NK cells) engineered to express TGF-β, thereby generating a TGF-β-resistant NK cell, or culturing the NK cell in the presence of cytoplasmic membrane particles or exosomes obtained from said feeder cells, thereby generating a TGF-β-resistant NK cell.Preferably, the feeder cells (including but not limited to PBMCs, RPMI8866 cells, NK-92, NK-92MI, NK-YTS, NK, NKL, KIL, KIL C.2, NK 3.3, NK-YS, HFWT, K562, EBV-LCL, or NK cells) are (i) transfected with membrane-bound IL-21, (ii) transfected with membrane-bound 4-1 BBL, (iii) transfected with membrane-bound IL-15 and 4-1 BBL, or (iv) transfected with membrane-bound IL-21 and 4-1 BBL.

[0016] 13. In one aspect, the document discloses methods for treating, inhibiting, reducing, ameliorating, alleviating and / or preventing cancer and / or metastasis according to any of the previous aspects, wherein the NK cells are memory-like NK cells such as NKG2C+, CD56 bright NK cells, CD56 dimNK cells, peripheral NK cells, NK T cells, or tumor-infiltrating NK cells (including, but not limited to, NK cells derived from cell lines or derived from a donor source (such as an autologous donor, an allogeneic donor, or a syngeneic donor).

[0017] 14. Also disclosed herein are methods for treating, inhibiting, reducing, attenuating, ameliorating, and / or preventing cancer and / or metastasis according to any of the previous aspects, 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 activating agent (such as, for example, NK cell effector agents including but not limited to 4-1 BBL, IL-2, IL-12, IL-15, IL-18, IL-21, MICA, LFA-1, 2 B4, CCR7, OX40L, UBLP2, BCM1 / SLAMF2, NKG2D agonists, CD155, CD112, Jagged1, Jagged2, delta-1, Pref-1, DNER, Jedi, SOM-11, wingless, CCN3, MAGP2, MAGP1, TSP2, YB-1, EGFL7, CCR7, DAP 12 and DAP10, Notch ligands, NKp46 agonists, NKp44 agonists, NKp30 agonists, other NCR agonists, CD16 agonists).In one aspect, the at least one additional NK cell effector agent is IL-21, 4-1 BBL, IL-15, IL-21 and 4-1 BBL, IL-21 and IL-15, or IL-15 and 4-1 BBL. Preferably, TGF-β is membrane-bound. Preferably, the at least one additional NK cell effector agent is a membrane-bound NK cell effector agent.

[0018] 15. In one aspect, the document discloses methods for treating, inhibiting, reducing, attenuating, ameliorating and / or preventing cancer and / or metastasis or a solid tumor according to any of the previous aspects, wherein NK cells are incubated in the presence of engineered feeder cells, cytoplasmic 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.

[0019] III. BRIEF DESCRIPTION OF GRAPHIC MATERIALS

[0020] 16. The accompanying drawings, which are incorporated into and constitute a part of this description, illustrate several embodiments and, together with this description, illustrate the disclosed compositions and methods.

[0021] 17. Fig. 1A and 1B show schematic diagrams of lentiviral vectors expressing human transforming growth factor beta-1 (hTGFβ). Fig. 1A shows membrane-bound TGFβ (mbTGFb), human TGFβ mRNA (1170 bp) was fused to the CD4 transmembrane region and IgG4 domain under the control of the CMV promoter and cloned into a lentiviral vector. Fig. 1B shows that the Mcherry-TGFβ (mc-TGFB) vector was generated by cloning human TGFβ mRNA under the control of the EF1A promoter and expressing the mcherry protein using the CMV promoter.

[0022] 18. Fig. 2 shows fluorescence images showing GFP expression in cells. Panels A–C show images of K562 cells, clone (CXT002), infected with a GFP-expressing lentivirus at 48 h post-infection at the indicated multiplicity of infection (MOI).

[0023] 19. Figures 3A and 3B show FACS analysis showing TGFβ expression. A K562 cell clone (CSTX002) was infected with a lentivirus expressing membrane-bound TGFβ (mbTGFβ) or mcherry TGFβ (mc-TGFβ) at the indicated multiplicity of infection (MOI). Cells were harvested 5 days after infection and stained with human anti-TGFβ antibody. Figure 3A shows cells overexpressing membrane-bound TGFβ (mbTGFβ). Figure 3B shows cells overexpressing mcherry TGFβ (mc-TGFβ).

[0024] 20. Figures 4A and 4B depict scatter plots showing the gating strategy used to sort mbTGFβ or mc-TGFβ-positive cells. Single cell clones were cultured for 10-14 days. Figure 4A shows that mbTGFβ cells were stained with an anti-TGFβ antibody. APC-positive cells were collected. Figure 4B shows that mcherry TGFβ-positive cells were sorted using a fluorescent marker.

[0025] 21. Fig. 5A and 5B show the result of quantitative real-time PCR (RT-PCR) showing the expression of TGFβ. RNA was isolated from a single-cell clone, and the TGF transcript levels were determined by RT-PCR. The results are presented as fold change compared to untransfected control cells. TGFβ levels in cells infected with membrane-bound TGFβ (mb TGFβ) (5A) or TGFβ mcherrry (TGFβ) (5B).

[0026] 22. Fig. 6 shows the ELISA result showing human TGFβ levels. A single-cell clone sorted from mbTGFβ and mc-TGFβ cells (0.5×10^6) was cultured in a 96-well plate. Supernatants were collected after 16 hours, and TGFβ1 levels were determined using a human TGFβ1 ELISA kit.

[0027] 23. Fig. 7 shows an immunoblot showing the expression of Smad3 in NK cells treated with TGFβ. NK cells isolated from the leukopaque of a healthy donor were expanded with irradiated normal feeder cells CSTX0002 (control), soluble TGFβ (s TGFβ) (10 ng / ml), or TGFβ-overexpressing feeder cell lines (mc10 or MB15) for two weeks.

[0028] 24. Figures 8A and 8B show standard NK cell cytotoxicity assays against tumor cells. NK cells from the peripheral blood of healthy donors were cocultured with calcein-labeled tumors at an effector-to-target (E:T) ratio of 5:1 for 4 hours. Calcein release into the supernatant was measured using a microplate reader and used to determine the mean specific lysis. NK cells cocultured with human osteosarcoma HOV cells (8A). Human medulloblastoma DAOY cells (8B). Significance was determined by two-way ANOVA with the Holm-Sidak multiple comparison test (*p≤0.05, **p≤0.01, ***p≤0.001, ****p≤0.0001).

[0029] IV. DETAILED DESCRIPTION OF THE ESSENCE OF THE INVENTION

[0030] 25. Before disclosing and describing the compounds, compositions, articles, devices, and / or methods of the present invention, it should be understood that they are not limited to specific synthetic methods or specific recombinant biotechnology methods, unless otherwise indicated, or to specific reagents, unless otherwise indicated, since these can, of course, vary. It should also be understood that the terminology used herein is for the purpose of describing specific embodiments only and is not intended to be limiting.

[0031] A. Definitions

[0032] 26. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The following references provide one of ordinary skill in the art with a general definition 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 Harper Collins Dictionary of Biology (1991). As used herein, the following terms have the meanings ascribed to them unless otherwise defined.

[0033] 27. When presenting elements of the present invention or its preferred embodiments, reference to certain terms, such as the term "said," is intended to mean the presence of one or more elements. The terms "comprising," "including," and "having" are intended to be inclusive and mean that there may be additional elements other than the listed elements.

[0034] 28. In this document, ranges may be expressed as from "about" one specific value and / or to "about" another specific value. When such a range is expressed, another embodiment includes from one specific value and / or to another specific value. Similarly, when values ​​are indicated as approximations using the preceding word "about," it should be understood that the specific value forms another embodiment. It will also be understood that the endpoints of each range are significant both in relation to the other endpoint and independently of the other endpoint. It is also understood that there are a number of values ​​disclosed in this document, and that each value is also disclosed herein as "about" that specific value in addition to the value itself. For example, if the value "10" is disclosed, then "about 10" is also disclosed.It is also understood that when a value is disclosed that is "less than or equal to a value," then "greater than or equal to a value" and possible ranges between values ​​are also disclosed, as appropriately understood by one of skill in the art. For example, if the value "10" is disclosed, then "less than or equal to 10" is also disclosed, as well as "greater than or equal to 10." It should also be understood that throughout this application, data is provided in various formats and that this data represents endpoints, startpoints, and ranges for any combination of data points. For example, if a specific data point of "10" and a specific data point of 15 are disclosed, it is understood that a data point that is greater than, greater than or equal to, less than, less than or equal to, and equal to 10 and 15, as well as one that is between 10 and 15, is disclosed. It should also be understood that each unit between two specific units is also disclosed.For example, if the values ​​10 and 15 are revealed, then the values ​​11, 12, 13, and 14 are also revealed.

[0035] 29. As used in this document, the terms "optional" or "optionally" mean that the phenomenon or circumstance described below may or may not occur, and that the description includes cases in which the stated phenomenon or circumstance occurs and cases in which it does not occur.

[0036] 30. As used herein, the term "N-terminal side" or "amino-terminal end" refers to the directionality of a peptide, polypeptide, or protein and may not refer to the N-terminus. In some aspects, when discussing a chimeric or fusion peptide, polypeptide, or protein, the N-terminal side may refer only to a component of the chimeric or fusion peptide, polypeptide, or protein, rather than the entire structure. For example, when discussing an Fc domain and the Fc domain is described as fused to its amino-terminal end or N-terminal side facing the interior of the cell, this document refers to chimeric or fusion peptides, polypeptides, or proteins in which the signal anchor is located at the N-terminus of the chimeric or fusion construct and actually spans the cell membrane.Thus, in such a chimera, the transmembrane anchor is attached to the amino-terminal side of the Fc domain, with the Fc domain having its N-terminal side facing the cell, which is inverted relative to the Fc domain on a typical B cell, which typically has the carboxyl terminus spanning the cell membrane and the amino terminus extending into the extracellular matrix.

[0037] 31. The terms "peptide", "polypeptide", and "protein" are used interchangeably to refer to a polymer of amino acid residues.

[0038] 32. As used herein, the term "sequence identity" means a quantitative measure of the degree of identity between two sequences of substantially equal length. The percentage identity of two sequences, whether nucleic acid sequences or amino acid sequences, is the number of exact matches between the two aligned sequences divided by the length of the shorter sequence and multiplied by 100. Approximate alignment of nucleic acid sequences is provided by the local homology algorithm of Smith and Waterman, Advances in Applied Mathematics 2:482–489 (1981). This algorithm can be applied to amino acid sequences using the scoring matrix developed by Dayhoff, Atlas of Protein Sequences and Structure, M.O. Dayhoff ed., 5 suppl.3:353–358, National Biomedical Research Foundation, Washington, DC, USA, and normalized by Gribskov, Nucl. Acids Res. 14(6):6745–6763 (1986). An illustrative implementation of this algorithm for determining percent sequence identity is provided by the Genetics Computer Group (Madison, WI) in the utility model application "BestFit". Other suitable programs for calculating percent identity or similarity between sequences are widely 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; band=both; cutoff=60; expectation=10; matrix=BLOSUM62; descriptions=50 sequences; sort by=HIGH SCORE; databases=non-redundant, GenBank+EMBL+DDBJ+PDB+GenBank CDS translations+Swiss protein+Spupdate+PIR.Detailed information about these programs can be found on the GenBank website. In general, the substitutions are conservative amino acid substitutions: limited to exchanges within members of group 1: glycine, alanine, valine, leucine, and isoleucine; group 2: serine, cysteine, threonine, and methionine; group 3: proline; group 4: phenylalanine, tyrosine, and tryptophan; group 5: aspartate, glutamate, asparagine, and glutamine. Preferably, percent sequence identity is calculated over the full length of the sequences to be compared.

[0039] 33. Techniques for determining the identity of nucleic acid and amino acid sequences are known in the art. Typically, such techniques involve determining the nucleotide sequence of mRNA for a gene and / or determining the amino acid sequence it encodes and comparing these sequences to a second nucleotide or amino acid sequence. Genomic sequences can be determined and compared in a similar manner. In general, identity refers to 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.

[0040] 34. Since various changes can be made in the above-described cells and methods without departing from the scope of the present invention, it is intended that all matter contained in the above description and in the examples below should be interpreted as illustrative and not as limiting.

[0041] 35. "Increase" may refer to any change that results in a greater degree of a symptom, disease, constituent, condition, or activity. An increase may be any individual, median, or mean increase in a condition, symptom, activity, constituent by a statistically significant amount compared to the control. Thus, an increase may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% increase, as long as the increase is statistically significant.

[0042] 36. "Reduction" may refer to any change that results in a lesser degree of a symptom, disease, composition, condition, or activity. A substance is also considered to result in a decrease in the genetic output of a gene when the genetic output of the gene product with the substance is less than the genetic output of the gene product without the substance. Also, for example, a decrease may represent a change in the symptoms of a disorder such that the symptoms become less pronounced than previously observed. A decrease may represent any individual, median, or average decrease in a condition, symptom, activity, composition by a statistically significant amount compared to the control. Thus, the reduction can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% reduction if the reduction is statistically significant.

[0043] 37. “Inhibit,” “inhibiting,” and “inhibition” mean a reduction in an activity, response, condition, disease, or other biological parameter. This may include, without limitation, complete elimination of the activity, response, condition, or disease. This may also include, for example, a 10% reduction in the activity, response, condition, or disease compared to the native or control level. Thus, the reduction may be 10, 20, 30, 40, 50, 60, 70, 80, 90, 100%, or any intermediate value compared to native or control levels.

[0044] 38. By "reduce," or other forms of the word such as "effect of reduction" or "reduction," is meant a decrease in a phenomenon or characteristic (e.g., tumor growth). It should be understood that this usually refers to some standard or expected value; in other words, it is relative, but it is not always necessary to refer to a standard or relative value. For example, "reduces tumor growth" means a decrease in the rate of tumor growth compared to a standard or control.

[0045] 39. By "prevent," or other forms of this word, such as "implementing a prevention" or "prevention," is meant the stopping of a specific phenomenon or characteristic, stabilizing or delaying the development or progression of a specific phenomenon or characteristic, or minimizing the likelihood that a specific phenomenon or characteristic will occur. The term "prevent" does not require comparison with control, since it is generally more absolute than, for example, "mitigate." As used herein, something can be mitigated but not prevented, but something that is mitigated can also be prevented. Similarly, something could be prevented but not mitigated, but what was prevented could also be mitigated. It should be understood that if "mitigate" or "prevent" is used, unless specifically stated otherwise, the use of the other word is also expressly disclosed.

[0046] 40. The term "subject" refers to any individual that is the target of administration or treatment. A subject may be a vertebrate, such as a mammal. In one aspect, a subject may be a human, a non-human primate, a bovine, a horse, a pig, a dog, or a cat. A subject may also be a guinea pig, a rat, a hamster, a rabbit, a mouse, or a mole. Thus, a subject may be a human patient or an animal patient. The term "patient" refers to a subject undergoing treatment by a clinician, such as a physician.

[0047] 41. The term "therapeutically effective" refers to an amount of the composition used that is sufficient to alleviate one or more causes or symptoms of the disease or disorder. Such alleviation requires only a reduction or modification, but not necessarily elimination.

[0048] 42. The term "treatment" refers to the medical management of a patient with the goal of curing, alleviating, stabilizing, or preventing a disease, pathological condition, or disorder. This term includes active treatment, that is, treatment specifically aimed at ameliorating the disease, pathological condition, or disorder, and also includes causal therapy, that is, treatment aimed at eliminating the cause of an associated disease, pathological condition, or disorder.In addition, the term includes palliative treatment, that is, treatment aimed at relieving symptoms rather than curing a disease, condition, or disorder; prophylactic treatment, that is, treatment aimed at minimizing or partially or completely suppressing the progression of an associated disease, condition, or disorder; and maintenance treatment, that is, treatment used in addition to other specific therapy aimed at reducing the symptoms of an associated disease, condition, or disorder.

[0049] 43. "Administration" to a subject includes any route of administration or delivery of a drug to the subject. Administration may be by any suitable route, including oral, topical, intravenous, subcutaneous, transdermal, intramuscular, intra-articular, parenteral, intra-arterial, intradermal, intraventricular, intracranial, intraperitoneal, intralesional, intranasal, rectal, vaginal, by inhalation, via an implanted reservoir, parenteral (e.g., subcutaneous, intravenous, intramuscular, intra-articular, intrasynovial, intrasternal, intrathecal, intraperitoneal, intrahepatic, intralesional, and intracranial injections or infusion techniques), etc.As used herein, the terms "concomitant administration," "administration in combination," "simultaneous administration," or "simultaneous administration" mean that the compounds are administered at the same time or substantially immediately after one another. In the latter case, the two compounds are administered at a sufficiently close time that the observed results are indistinguishable from the results achieved when the compounds are administered at the same time. "Systemic administration" refers to the administration or delivery of a drug to a subject via a route that delivers the drug to large areas of the subject's body (e.g., more than 50% of the body), such as through the circulatory or lymphatic systems.In contrast, local administration refers to the administration or delivery of a drug to a subject via a route in which the drug is administered or delivered to a site or region immediately adjacent to the site of administration, and the drug is not administered systemically in a therapeutically significant amount. For example, locally administered drugs are readily detectable in the immediate vicinity of the administration site but are not detectable, or detectable in negligible amounts, in distal parts of the subject's body. Administration includes self-administration and administration by another person.

[0050] 44. "Treat," "treating," "treatment," and their grammatical variations as used herein include administering a composition with the intent or purpose of partially or completely preventing, delaying, curing, treating, alleviating, mitigating, modifying, correcting, improving, normalizing, stabilizing, mitigating, and / or reducing the intensity or frequency of one or more diseases or conditions, a symptom of a disease or condition, or the underlying cause of a disease or condition. Treatment according to the present invention can be administered in a preventive, prophylactic, palliative, or curative manner. Prophylactic treatments are administered to a subject before the onset of a disease (e.g., before overt signs of cancer), during the early onset of a disease (e.g., at the initial signs and symptoms of cancer), or after the confirmed development of cancer. Prophylactic administration can occur from day(s) to several years before the onset of symptoms of a disease or infection.

[0051] 45. Throughout this application, various publications are referenced. The disclosures of these publications in their entirety are incorporated by reference into this application for a more complete description of the state of the art to which this application pertains. The disclosed references are also individually and specifically incorporated herein by reference for the material contained therein that is discussed in the sentence to which the reference refers.

[0052] B. Compositions

[0053] 46. Disclosed are the components used to prepare the disclosed compositions, as well as the compositions themselves used in the disclosed methods disclosed herein. These and other materials are disclosed herein, and it is understood that when disclosing combinations, subsets, interactions, groups, etc. of these materials, 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, if a particular TGF-β-expressing engineered feeder cell is disclosed and discussed, and a number of modifications that can be made to a number of molecules, including a TGF-β-expressing engineered feeder cell, are discussed, each combination and permutation of the TGF-β-expressing engineered feeder cell and possible modifications are specifically contemplated, unless otherwise specifically stated.Thus, if a class of molecules A, B and C is disclosed, as well as a class of molecules D, E and F, and an example of a combination molecule AD is disclosed, then even if each of them is not mentioned individually, each of them is considered individually and together denoting combinations A-E, A-F, BD, B-E, BF, CD, C-E and CF are considered to be disclosed. Similarly, any subset or combination of them is also disclosed. Thus, for example, a subgroup A-E, BF and C-E will be considered to be disclosed. This concept applies to all aspects of the present application, including, without limitation, the steps of the methods for making and using the disclosed compositions. Thus, if there are a plurality of additional steps that can be performed, it is understood that each of these additional steps can be performed with any particular embodiment or combination of embodiments of the disclosed methods.

[0054] 47. The use of NK cells in anticancer therapy is becoming increasingly popular. However, studies have shown that various cancer types secrete TGF-β to protect against the killing action of NK cells. Secretion of TGF-β leads to decreased killing. Previous efforts to reduce killing have included the use of TGF-β inhibitors; however, the use of TGF-β inhibitors can cause systemic adverse side effects. Alternatively, TGF-β can be used during NK cell expansion, but the use of clinical-grade TGF-β is labor-intensive and prohibitively expensive due to the excessively large quantities of an already expensive reagent (i.e., TGF-β) required to observe any effect.To address the need for TGF-β-resistant NK cells and to overcome the lack of either TGF-β inhibition or soluble TGF-β culture supplements, engineered feeder cells that have been modified to express soluble or membrane-bound TGF-β are disclosed herein.

[0055] 48. Also disclosed herein are engineered feeder cells further comprising at least one additional NK cell effector agent on the cell surface thereof, wherein the at least one additional NK cell effector agent is a cytokine, an adhesion molecule, or an NK cell activating agent (such as, for example, NK cell effector agents including but not limited to 4-1 BBL, IL-2, IL-12, IL-15, IL-18, IL-21, MICA, LFA-1, 2 B4, CCR7, OX40L, UBLP2, BCM1 / SLAMF2, NKG2D agonists, CD155, CD112, Jagged1, Jagged2, delta-1, Pref-1, DNER, Jedi, SOM-11, wingless, CCN3, MAGP2, MAGP1, TSP2, YB-1, EGFL7, CCR7, DAP 12 and DAP10, Notch ligands, NKp46 agonists, NKp44 agonists, NKp30 agonists, other NCR agonists, CD16 agonists). In one aspect, at least one additional NK cell effector agent is IL-21, 4-1 BBL, IL-15, IL-21 and 4-1 BBL, IL-21 and IL-15, or IL-15 and 4-1 BBL.Preferably, the at least one additional NK cell effector agent is a membrane-bound NK cell effector agent.

[0056] 49. It is understood and is implied in this document that feeder cells may be obtained from any source of feeder cells that can proliferate and / or activate NK cells. For example, feeder cells are PBMCs, RPMI8866, HFWT, K562, EBV-LCL cells, NK cells (including but not limited to PBMCs, RPMI8866, NK-92, NK-92MI, NK-YTS, NK, NKL, KIL, KIL C.2, NK3.3, NK-YS, HFWT, K562 cells) transfected with membrane-bound IL-21, NK cells (including but not limited to PBMCs, RPMI8866, NK-92, NK-92MI, NK-YTS, NK, NKL, KIL, KILC.2, NK3.3, NK-YS, HFWT, K562 cells) transfected with membrane-bound 4-1 BBL, NK cells (including but not limited to PBMCs, RPMI8866, NK-92, NK-92MI, NK-YTS, NK, NKL, KIL, KIL C.2, NK3.3, NK-YS, HFWT, K562 cells) transfected with membrane-bound IL-15 and 4-1 BBL, or NK cells (including, but not limited to, PBMCs, RPMI8866, NK-92, NK-92MI, NK-YTS, NK, NKL, KIL, KILC.2, NK3 cells.3, NK-YS, HFWT, K562), transfected with membrane-bound IL-21 and 4-1 BBL.

[0057] 50. Furthermore, this document also discloses cytoplasmic membrane particles or exosomes obtained from any of the engineered feeder cells described herein or obtained using the methods disclosed herein.

[0058] 1. Delivery of compositions to cells

[0059] 51. A number of compositions and methods exist that can be used to deliver nucleic acids into cells either in vitro or in vivo. These methods and compositions can be generally divided into two classes: viral-based delivery systems and non-viral-based delivery systems. For example, nucleic acids can be delivered using a number of direct delivery systems, such as electroporation, lipofection, calcium phosphate precipitation, plasmids, viral vectors, viral nucleic acids, phage nucleic acids, phages, cosmids, or by transferring genetic material in cells or carriers such as cationic liposomes. Suitable means for transfection, including viral vectors, chemical transfectants, or physicomechanical methods such as electroporation and direct DNA diffusion, are described, for example, by Wolff, J. A., et al., Science, 247, 1465-1468, (1990); and Wolff, J. A. Nature, 352, 815-818, (1991).Such methods are well known in the art and are readily adapted for use with the compositions and methods described herein. In some cases, the methods will be modified to work with larger DNA molecules. Furthermore, these methods can be used to target specific diseases and cell populations using the targeting properties of the carrier.

[0060] 52. The use of endonuclease ribonucleoprotein complexes (such as Cas9 / RNP) for reprogramming (i.e., constructing or modifying) feeder cells occurs.

[0061] 53. Endonuclease / RNP complexes (e.g., Cas9 / RNP) consist of three components: a recombinant endonuclease protein (e.g., Cas9 endonuclease) in complex with CRISPR loci. The endonuclease complexed with CRISPR loci may be referred to as CRISPR / Cas guide RNA. CRISPR loci contain a synthetic single guide RNA (gRNA), consisting of RNA that can hybridize to a target sequence to form a complex with complementary repetitive RNA (crRNA) and trans-complementary repetitive RNA (tracrRNA). Accordingly, the CRISPR / Cas guide RNA hybridizes to the target sequence in the cell's genomic DNA. In some cases, class 2 CRISPR / Cas endonuclease is type II CRISPR / Cas endonuclease. In some cases, the class 2 CRISPR / Cas endonuclease is a Cas9 polypeptide and the corresponding CRISPR / Cas guide RNA is a Cas9 guide RNA.These Cas9 / RNPs are capable of cleaving genomic targets with greater efficiency compared to foreign DNA-dependent approaches due to their delivery as functional complexes. Furthermore, rapid clearance of Cas9 / RNPs from cells may result in reduced off-target effects, such as apoptosis induction. Accordingly, in one aspect, disclosed herein are methods for genetically modifying an NK cell, comprising (a) producing a guide RNA (gRNA) specific for a target DNA sequence in an NK cell; and (b) transducing (e.g., introducing by electroporation) into the target NK cell a ribonucleoprotein (RNP) complex comprising a class 2 CRISPR / Cas endonuclease (Cas9) in complex with an appropriate CRISPR / Cas guide RNA that hybridizes to a target sequence in the genomic DNA of the feeder cell.

[0062] a) Nucleic acid-based delivery systems

[0063] 54. Transfer vectors can be any nucleotide construct used to deliver genes into cells (e.g., a plasmid), or as part of a general gene delivery strategy, such as part of a recombinant retrovirus or adenovirus (Ram et al. Cancer Res. 53:83-88, (1993)).

[0064] 55. Plasmid or viral vectors as used herein are vehicles that transfer exposed nucleic acids, such as TGF-β, into a cell without cleavage and include a promoter that provides for gene expression in the cells into which it is delivered. Viral vectors include, for example, adenovirus, adeno-associated virus, herpes virus, vaccinia virus, poliovirus, AIDS virus, neuronal trophic virus, Sindbis virus, and other RNA viruses, including those viruses with the HIV backbone. Also preferred are any viral families that possess the properties of these viruses, making them suitable for use as vectors. Retroviruses include Maloney murine leukemia virus, MMLV, and retroviruses that exhibit the desired properties of MMLV as a vector. Retroviral vectors are capable of carrying a larger genetic payload, i.e.A transgene or marker gene is carried by adenovirus vectors more easily than by other viral vectors, making them widely used. However, they are less applicable to non-proliferating cells. Adenovirus vectors are relatively stable, easy to handle, have high titers, can be delivered by aerosol, and can transfect non-dividing cells. Smallpox virus vectors are large and have multiple gene insertion sites. They are thermostable and can be stored at room temperature. A preferred embodiment is a viral vector engineered to suppress the host immune response elicited by viral antigens. Preferred vectors of this type will carry regions encoding interleukin 8 or 10.

[0065] 56. Viral vectors can have higher translocation capacities (the ability to introduce genes) than chemical or physical methods of introducing genes into cells. Typically, viral vectors contain nonstructural early genes, structural late genes, an RNA polymerase III transcript, inverted terminal repeats required for replication and encapsidation, and promoters to control transcription and replication of the viral genome. When engineered as vectors, viruses typically have one or more early genes deleted, and a gene or gene / promoter cassette is inserted into the viral genome in place of the deleted viral DNA. Constructs of this type can carry up to 8 kb of foreign genetic material. The necessary functions of the deleted early genes are usually provided by cell lines engineered to express the gene products of the early genes in trans.

[0066] (1) Retroviral vectors

[0067] 57. A retrovirus is an animal virus belonging to the virus family Retroviridae, including any phylum, subfamilies, genera, or tropisms.

[0068] 58. A retrovirus is essentially a package containing a nucleic acid payload. The nucleic acid payload carries a packaging signal, which ensures that the replicated progeny molecules are efficiently packaged within the package shell. In addition to the packaging signal, there are a number of molecules required in cis for replication and packaging of the replicated virus. Typically, the retroviral genome contains the gag, pol, and env genes, which are involved in the formation of the protein coat. These genes, gag, pol, and env, are typically replaced by foreign DNA to be transferred to the target cell.Retroviral vectors typically contain a packaging signal for inclusion in the packaging envelope, a sequence that signals the start of the gag transcription unit, elements necessary for reverse transcription, including a primer binding site for binding the tRNA primer for reverse transcription, terminal repeat sequences that direct RNA strand switching during DNA synthesis, a purine-rich 5'-3' LTR sequence that serves as a priming site for second-strand DNA synthesis, and specific sequences near the ends of the LTR that allow insertion of the retroviral DNA state for insertion into the host genome. Removal of the gag, pol, and env genes allows approximately 8 kb of foreign sequence to be inserted into the viral genome, reverse transcribed, and, after replication, packaged into a new retroviral particle. This amount of nucleic acid is sufficient to deliver from one to many genes, depending on the size of each transcript.It is preferable to include either positive or negative selectable markers along with other genes in the insert.

[0069] 59. Since the replication machinery and packaging proteins (gag, pol, and env) have been removed from most retroviral vectors, vectors are typically created by placing them in a packaging cell line. A packaging cell line is a cell line that has been transfected or transformed with a retrovirus that contains the replication and packaging machinery but lacks any packaging signal. When a vector carrying the selected DNA is transfected into these cell lines, the vector containing the gene of interest is replicated and packaged into new retroviral particles via machinery provided in cis by the helper cell. The genomes for the machinery are not packaged because they lack the necessary signals. In one aspect, nucleic acid encoding TGF-β can be delivered using a lentiviral vector.

[0070] (2) Adenoviral vectors

[0071] 60. The construction of replication-defective adenoviruses has been described (Berkner et al., J. Virology 61:1213–1220 (1987); Massie et al., Mol. Cell. Biol. 6:2872–2883 (1986); Haj-Ahmad et al., J. Virology 57:267–274 (1986); Davidson et al., J. Virology 61:1226–1239 (1987); Zhang, "Generation and identification of recombinant adenovirus by liposome-mediated transfection and PCR analysis," BioTechniques 15:868–872 (1993)). The advantage of using these viruses as vectors is that they are limited in the extent to which they can spread to other cell types, as they can replicate within the original infected cell but cannot produce new infectious viral particles. Recombinant adenoviruses have been shown to provide high gene transfer efficiency after direct in vivo delivery to respiratory epithelium, hepatocytes, vascular endothelium, CNS parenchyma, and a number of other tissue sites (Morsy, J. Clin. Invest.92:1580-1586 (1993); Kirshenbaum, J. Clin. Invest. 92:381-387 (1993); Roessler, J. Clin. Invest. 92:1085-1092 (1993); Moullier, Nature Genetics 4:154-159 (1993); La Salle, Science 259:988-990 (1993); Gomez-Foix, J. Biol. Chem. 267:25129-25134 (1992); Rich, Human Gene Therapy 4:461-476 (1993); Zabner, Nature Genetics 6:75-83 (1994); Guzman, Circulation Research 73:1201-1207 (1993); Bout, Human Gene Therapy 5:3–10 (1994); Zabner, Cell 75:207–216 (1993); Caillaud, Eur. J. Neuroscience 5:1287–1291 (1993); and Ragot, J. Gen. Virology 74:501–507 (1993)). Recombinant adenoviruses carry out gene transduction by binding to specific cell surface receptors, after which the virus is internalized by receptor-mediated endocytosis in the same manner as wild-type or replication-defective adenoviruses (Chardonnet and Dales, Virology 40:462–477 (1970); Brown and Burlingham, J. Virology 12:386–396 (1973); Svensson and Persson, J.Virology 55:442-449 (1985); Seth, et al., J. Virol. 51:650-655 (1984); Seth, et al., Mol. Cell. Biol. 4:1528-1533 (1984); Varga et al., J. Virology 65:6061-6070 (1991); Wickham et al., Cell 73:309-319 (1993)).

[0072] 61. The viral vector may be an adenovirus-based vector in which the E1 gene has been deleted, and these viruses are produced in a cell line such as the human 293 cell line. In another preferred embodiment, both the E1 gene and the E3 gene are deleted from the adenovirus genome.

[0073] (3) Adeno-associated viral vectors

[0074] 62. Another type of viral vector is based on the adeno-associated virus (AAV). This defective parvovirus is the preferred vector because it can infect many cell types and is nonpathogenic to humans. AAV-type vectors can transport 4 to 5 kb, and wild-type AAVs are known to stably integrate into chromosome 19 (e.g., at AAV integration site 1 (AAVS1)). Vectors that contain this site-specific integration feature are preferred. The AAVs used can be derived from any AAV serotype, including but not limited to AAC1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, and recombinant (rAAV), such as, for example, AAV-Rh74, and / or synthetic AAV (such as, for example, AAV-DJ, Anc80). AAV serotypes can be selected based on cell or tissue tropism. AAV-based vectors for use in the disclosed compositions and methods can be single-chain (SS) or self-complementary (SC).

[0075] 63. In another type of AAV virus, the AAV contains a pair of inverted terminal repeats (ITRs) flanking at least one cassette containing a promoter that directs cell-specific expression, operably linked to a heterologous gene. Heterologous in this context refers to any nucleotide sequence or gene that is not native to the AAV or B19 parvovirus.

[0076] 64. Typically, the coding regions of AAV and B19 are deleted, resulting in a safe, non-cytotoxic vector. AAV ITRs or their modifications confer infectivity and site-specific integration, but not cytotoxicity, and the promoter directs cell-specific expression.

[0077] 65. Thus, the disclosed vectors provide DNA molecules that are capable of integrating into the mammalian chromosome without significant toxicity.

[0078] 66. Integrated genes in viruses and retroviruses typically contain promoters and / or enhancers that facilitate control of expression of the desired gene product. A promoter is typically a DNA sequence or sequences that function when in a relatively fixed position relative to the transcription start site. The promoter contains upstream elements necessary for the primary interaction of RNA polymerase and transcription factors and may contain upstream and response elements.

[0079] 67. It should be understood, and is assumed in this document, that the packaging capacity of AAV is limited. One way to overcome the loading capacity of the AAV vector is to use two vectors, with the transgene divided between two plasmids and a 3' splice donor and 5' splice acceptor used to join the two transgene regions into a single full-length transgene. Alternatively, two transgenes can be produced with significant overlap, and homologous recombination will result in the joining of the two segments into a full-length transcript.

[0080] 2. Expression systems

[0081] 68. Nucleic acids delivered to cells typically contain expression control systems. For example, inserted genes in viral and retroviral systems typically contain promoters and / or enhancers that facilitate control of expression of the desired gene product. A promoter is typically a DNA sequence or sequences that function when in a relatively fixed position relative to the transcription start site. The promoter contains upstream elements necessary for the basic interaction of RNA polymerase and transcription factors and may contain upstream and response elements. Promoters used to generate open feeder cells expressing TGF-β (either soluble or membrane-bound) can be constitutive or inducible.

[0082] a) Viral promoters and enhancers

[0083] 69. Preferred promoters controlling transcription from vectors in mammalian host cells can be obtained from various sources, for example, from the genomes of viruses such as: polyoma virus, simian virus 40 (SV40), adenovirus, retroviruses, hepatitis B virus and most preferably cytomegalovirus, or from heterologous mammalian promoters, for example, the beta-actin promoter. The early and late promoters of SV40 virus are conveniently obtained as a restriction fragment of SV40, which also contains the origin of replication of SV40 virus (Fiers et al., Nature, 273: 113 (1978)). The immediate-early promoter of human cytomegalovirus is conveniently obtained as a restriction fragment of Hind III E (Greenway, PJ et al., Gene 18: 355-360 (1982)). Of course, promoters from the host cell or related species may also be applicable herein. In one aspect, TGF-β mRNA can be expressed under the control of the EF1A promoter or the CMV promoter.

[0084] 70. An enhancer generally refers to a DNA sequence that functions at any fixed distance from the transcription start site and can be either 5' (Laimins, L. et al., Proc. Natl. Acad. Sci. 78: 993 (1981)) or 3' (Lusky, ML, et al., Mot. Cell Bio. 3: 1108 (1983)) with respect to the transcription unit. Additionally, enhancers can be located within an intron (Banerji, JL et al., Ceil 33: 729 (1983)) as well as within the coding sequence itself (Osborne, TF, et al., Mol. Cell Bio. 4: 1293 (1984)). They are typically 10 to 300 bp in length and function in cis. Enhancers enhance transcription from nearby promoters. Enhancers also often contain response elements that mediate transcriptional regulation. Promoters may also contain response elements that mediate transcriptional regulation. Enhancers often determine the regulation of gene expression.Although many enhancer sequences are currently known from mammalian genes (globin, elastase, albumin, α-fetoprotein, and insulin), an enhancer from a eukaryotic virus is typically used for general expression. Preferred examples include the SV40 enhancer on the late side of the replication origin (bp 100-270), the cytomegalovirus early promoter enhancer, the polyoma virus enhancer on the late side of the replication origin, and adenovirus enhancers.

[0085] 71. The promoter and / or enhancer can be specifically activated either by light or by specific chemical events that trigger their function. These systems can be regulated using reagents such as tetracycline and dexamethasone. There are also ways to enhance viral vector gene expression by exposure to radiation, such as gamma irradiation, or alkylating chemotherapeutic drugs.

[0086] 72. In certain embodiments, the promoter and / or enhancer region may act as a constitutive promoter and / or enhancer to maximize expression of the region of the transcription unit to be transcribed. In certain constructs, the promoter and / or enhancer region is active in all eukaryotic cell types, even if it is expressed only in a specific cell type at a specific time. A preferred promoter of this type is the CMV promoter (650 bases). Other preferred promoters include the SV40 promoter, the cytomegalovirus promoter (full-length promoter), and the LTR of a retroviral vector.

[0087] 73. It was shown that all specific regulatory elements can be cloned and used to construct expression vectors that are selectively expressed in specific cell types, such as melanoma cells. The glial fibrillary acidic protein (GFAP) promoter was used for selective gene expression in glial-derived cells.

[0088] 74. Expression vectors for use in eukaryotic host cells (yeast, fungi, insect, plant, animal, human, or nucleated cells) may also contain sequences required for transcription termination, which can influence mRNA expression. These regions are transcribed as polyadenylated segments in the untranslated portion of mRNA encoding the tissue factor protein. The 3'-untranslated regions also include transcription termination sites. Preferably, the transcription unit also contains a polyadenylation region. One advantage of this region is that it increases the likelihood that the transcribed unit will be processed and transported similarly to mRNA. The identification and use of polyadenylation signals in expression constructs is well known. Preferably, homologous polyadenylation signals are used in transgene constructs.In some transcription units, the polyadenylation region is derived from the SV40 early polyadenylation signal and consists of approximately 400 bases. It is also preferred that the transcribed units contain other standard sequences, either alone or in combination with the above sequences, that improve expression or construct stability.

[0089] b) Markers

[0090] 75. Viral vectors may include a nucleic acid sequence encoding a marker product. This marker product is used to determine whether a gene has been delivered to a cell and whether it is expressed after delivery. Preferred marker genes are the E. coli lacZ gene, which encodes β-galactosidase, and green fluorescent protein.

[0091] 76. In some embodiments, the marker may be a selectable marker. Examples of suitable selectable markers for mammalian cells include dihydrofolate reductase (DHFR), thymidine kinase, neomycin, neomycin analog G418, hygromycin, and puromycin. When such selectable markers are successfully transferred into a mammalian host cell, the transformed mammalian host cell can survive under selective pressure. There are two widely used distinct categories of selection schemes. The first category is based on cellular metabolism and the use of a mutant cell line that is unable to grow independently of supplemented medium. Two examples are DHFR-CHO cells and LTK- mouse cells. These cells are unable to grow without the addition of nutrients such as thymidine or hypoxanthine.Because these cells lack certain genes required for the complete nucleotide synthesis pathway, they cannot survive unless the missing nucleotides are provided in the supplemented medium. An alternative to supplementing the medium is to introduce the intact DHFR or TK gene into cells lacking the corresponding genes, which alters their growth requirements. Individual cells that are not transformed with the DHFR or TK gene will not survive in the unsupplemented medium.

[0092] 77. The second category is dominant selection, which refers to a selection scheme used for any cell type and does not require the use of a mutant cell line. These schemes typically use a drug to arrest the growth of the host cell. Those cells that have the new gene will express the drug resistance protein and survive the selection. Examples of such dominant selection include the drugs neomycin (Southern P. and Berg, P., J. Molec. Appl. Genet. 1: 327 (1982)), mycophenolic acid (Mulligan, RC and Berg, P. Science 209: 1422 (1980)) or hygromycin (Sugden, B. et al., Moi. Cell. Biol. 5: 410-413 (1985)). Three examples use bacterial genes under eukaryotic control to confer resistance to the respective drug G418 or neomycin (geneticin), xgpt (mycophenolic acid), or hygromycin, respectively.Others include neomycin analogue G418 and puramycin.

[0093] 3. Pharmaceutical carriers / Delivery of pharmaceuticals

[0094] 78. As described above, the compositions can also be administered in vivo in a pharmaceutically acceptable carrier. By "pharmaceutically acceptable" is meant a material that is not biologically or otherwise undesirable, i.e., the material can be administered to a subject along with the nucleic acid or vector without causing any undesirable biological effects or harmful interactions with any of the other components of the pharmaceutical composition in which it is contained. The carrier, of course, should be selected to minimize any degradation of the active ingredient and minimize any adverse side effects in the subject, as is well known to those skilled in the art.

[0095] 79. The compositions can be administered orally, parenterally (e.g., intravenously), intramuscularly, intraperitoneally, transdermally, extracorporeally, topically, and the like, including topical intranasal administration or administration via an inhaler. The term "topical intranasal administration" as used herein means delivery of the compositions to the nasal cavity and nasal passages through one or both nostrils and may include delivery via a spray or dropper mechanism or by aerosolization of nucleic acid or vector. Administration of the compositions via an inhaler can be accomplished through the nasal cavity or oral cavity using a spray or dropper mechanism. Delivery can also be accomplished directly to any area 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 health of the subject, the severity of the allergic disorder being treated, the specific nucleic acid or vector used, the route of administration, and the like. Therefore, it is impossible to specify a precise amount for each composition. However, the appropriate amount can be determined by one skilled in the art using only routine experimentation, taking into account the teachings of this document.

[0096] 80. Parenteral administration of the composition, if used, is typically by injection. Injectable preparations can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for reconstitution of the suspension in liquid prior to injection, or as emulsions. A recently revised approach to parenteral administration involves the use of slow-release or extended-release systems so that a constant dose is maintained. See, for example, U.S. Patent No. 3,610,795, which is incorporated herein by reference.

[0097] 81. Materials may be in solution or suspension (e.g., as microparticles, liposomes, or cells). They can be targeted to a specific cell type via antibodies, receptors, or receptor ligands. The following references are examples of the application of 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., Biochem. Pharmacol, 42:2062-2065, (1991)).Carriers such as stealth and other antibody-conjugated liposomes (including a lipid-mediated drug targeting colon carcinoma), receptor-mediated DNA targeting via cell-specific ligands, lymphocyte-directed tumor targeting, and highly specific therapeutic retroviral targeting of mouse glioma cells in vivo. The following references are examples of the application 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)). In general, receptors are involved in endocytosis pathways, either constitutive or ligand-induced.These receptors are clustered in clathrin-coated pits, enter the cell through clathrin-coated vesicles, pass through an acidified endosome where the receptors are sorted, and then either return to the cell surface, accumulate intracellularly, or are degraded in lysosomes. Internalization pathways serve a variety of functions, such as nutrient uptake, removal of activated proteins, clearance of macromolecules, opportunistic entry of viruses and toxins, ligand dissociation and degradation, and receptor-level regulation. Many receptors follow more than one intracellular pathway, depending on the cell type, receptor concentration, ligand type, ligand valence, and ligand concentration. The molecular and cellular mechanisms of receptor-mediated endocytosis are reviewed (Brown and Greene, DNA and Cell Biology 10:6, 399–409 (1991)).

[0098] a) Pharmaceutically acceptable carriers

[0099] 82. Compositions containing antibodies can be used therapeutically in combination with a pharmaceutically acceptable carrier.

[0100] 83. 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 a pharmaceutically acceptable carrier include, but are not limited to, saline, Ringer's solution, and dextrose solution. The pH of the solution is preferably from about 5 to about 8, and more preferably from about 7 to about 7.5. Additional carriers include sustained-release preparations, such as semipermeable matrices of solid hydrophobic polymers containing the antibody, wherein the matrices are in the form of shaped articles, for example, films, liposomes, or microparticles.It will be apparent to those skilled in the art that certain carriers may be preferred depending on, for example, the route of administration and the concentration of the composition being administered.

[0101] 84. Pharmaceutical carriers are familiar to those skilled in the art. These will most commonly be standard carriers for administering drugs to humans, including solutions such as sterile water, saline, and buffered solutions at physiological pH. The compositions can be administered intramuscularly or subcutaneously. Other compounds will be administered according to standard procedures used by those skilled in the art.

[0102] 85. Pharmaceutical compositions may include carriers, thickeners, diluents, buffers, preservatives, surfactants, and the like in addition to the selected molecule. Pharmaceutical compositions may also include one or more active ingredients, such as antimicrobials, anti-inflammatory agents, anesthetics, and the like.

[0103] 86. The pharmaceutical composition can be administered in several ways, depending on whether local or systemic treatment is required and the area to be treated. Administration can be carried out locally (including ophthalmologically, vaginally, rectally, intranasally), orally, by inhalation, or parenterally, for example, by intravenous drip, subcutaneous, intraperitoneal, or intramuscular injection. The disclosed antibodies can be administered intravenously, intraperitoneally, intramuscularly, subcutaneously, intracavitarily, or transdermally.

[0104] 87. Parenteral preparations include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic / aqueous solutions, emulsions, or suspensions, including saline and buffered media. Parenteral carrier media include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, Ringer's lactate, or fixed oils. Intravenous carrier media include fluid and nutrient replenishers, electrolyte replenishers (e.g., Ringer's dextrose), etc. Preservatives and other additives may also be present, such as antimicrobial agents, antioxidants, chelating agents, inert gases, etc.

[0105] 88. Formulations for topical use may include ointments, lotions, creams, gels, drops, suppositories, sprays, liquids, and powders. Conventional pharmaceutical carriers, aqueous, powder, or oily bases, thickening agents, and the like may be necessary or desirable.

[0106] 89. Compositions for oral administration include powders or granules, suspensions or solutions in aqueous or non-aqueous media, capsules, sachets, or tablets. Thickening agents, flavoring agents, diluents, emulsifiers, dispersing agents, or binders may be desirable.

[0107] 90. Some of the compositions can potentially be administered in the form of a pharmaceutically acceptable acid or base addition salt formed by reaction with inorganic acids such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid and phosphoric acid, and organic acids such as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid, maleic acid and fumaric acid, or by reaction with an inorganic base such as sodium hydroxide, ammonium hydroxide, potassium hydroxide and organic bases such as mono-, di-, trialkyl- and arylamines and substituted ethanolamines.

[0108] b) Therapeutic uses

[0109] 91. Effective dosages and administration regimens for the compositions can be determined empirically, and such determinations are within the skill of the art. Dose ranges for administering the compositions are sufficiently wide to achieve the desired effect at which the symptoms of the disorder are manifested. The dosage should not be so high as to cause adverse side effects, such as unwanted cross-reactions, anaphylactic reactions, etc. Typically, the dosage will vary depending on the age, health condition, gender, and severity of the patient's disease, the route of administration, or whether other drugs are included in the regimen, and can be determined by a person skilled in the art. The dosage can be adjusted by the individual's physician in the event of any contraindications. The dosage can vary and can be administered as one or more doses daily for one or more days.Recommendations on appropriate dosages for these classes of pharmaceutical products can be found in the literature. For example, guidance on selecting appropriate dosages of antibodies can be found in the literature on therapeutic uses of antibodies, such as 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 approximately 1 μg / kg to 100 mg / kg body weight or more per day, depending on the factors mentioned above.

[0110] C. Methods for generating TGF-β-resistant NK cells

[0111] 92. As noted, the primary purpose of the disclosed feeder cells is to generate NK cells that are resistant to TGF-β. Accordingly, in one aspect, this document discloses methods for generating TGF-β-resistant NK cells, comprising incubating NK cells in the presence of engineered feeder cells, cytoplasmic membrane particles, or exosomes disclosed herein. For example, this document discloses methods for creating TGF-β-resistant NK cells comprising incubating NK cells in the presence of feeder cells (including but not limited to PBMCs, RPMI8866, HFWT, K562, EBV-LCL cells, NK cells (including but not limited to NK-92, NK-92MI, NK-YTS, NK, NKL, KIL, KIL C.2, NK 3.3, NK-YS) transfected with membrane-bound IL-21, NK cells (including but not limited to NK-92, NK-92MI, NK-YTS, NK, NKL, KIL, KIL C.2, NK 3.3, NK-YS) transfected with membrane-bound 4-1BBL cells, NK cells (including but not limited to NK-92, NK-92MI, NK-YTS, NK, NKL, KIL, KIL C.2, NK 3.3, NK-YS) transfected with membrane-bound IL-15 and 4-1BBL, or NK cells (including but not limited to NK-92, NK-92MI, NK-YTS, NK, NKL, KIL, KIL C.2, NK 3.3, NK-YS) transfected with membrane-bound IL-21 and 4-1 BBL) that have been engineered to express TGF-β or incubate NK cells in the presence of cytoplasmic membrane particles or exosomes derived from said feeder cells. Preferably, TGF-β expressed by feeder cells is membrane-bound.

[0112] 93. In one aspect, disclosed herein are methods for generating TGF-β-resistant 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, an adhesion molecule, or an NK cell activating agent (such as, for example, NK cell effector agents including but not limited to 4-1 BBL, IL-2, IL-12, IL-15, IL-18, IL-21, MICA, LFA-1, 2 B4, CCR7, OX40L, UBLP2, BCM1 / SLAMF2, NKG2D agonists, CD155, CD112, Jagged1, Jagged2, delta-1, Pref-1, DNER, Jedi, SOM-11, wingless, CCN3, MAGP2, MAGP1, TSP2, YB-1, EGFL7, CCR7, DAP 12 and DAP10, Notch ligands, NKp46 agonists, NKp44 agonists, NKp30 agonists, other NCR agonists, CD16 agonists).In one aspect, the at least one additional NK cell effector agent is IL-21, 4-1 BBL, IL-15, IL-21 and 4-1 BBL, IL-21 and IL-15, or IL-15 and 4-1 BBL. Preferably, the at least one additional NK cell effector agent is a membrane-bound NK cell effector agent.

[0113] 94. It should be understood and is contemplated herein that the methods for generating TGF-β-resistant NK cells disclosed herein can be applied to any NK cell (exogenous or endogenous) where TGF-β resistance is desired. Accordingly, methods for generating TGF-β-resistant NK cells are disclosed herein, wherein the NK cells are memory-like NK cells such as NKG2C + , CD56 bright NK cells, CD56 dimNK cells, peripheral NK cells, NK T cells, or tumor-infiltrating NK cells (including, but not limited to, NK cells derived from cell lines or derived from a donor source (such as an autologous donor, an allogeneic donor, or a syngeneic donor).

[0114] 95. To generate TGF-β-resistant NK cells, NK cells must be exposed to a feeder cell, exosome, or cytoplasmic membrane particle expressing TGF-β (on its membrane or in soluble form) for a certain period of time to confer resistance. Thus, in one aspect, the present document also discloses methods for generating TGF-β-resistant NK cells, wherein the NK cells are incubated in the presence of engineered feeder cells, cytoplasmic 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. Furthermore, it should be understood and is contemplated herein that the NK cells can be cultured for additional periods of time after exposure to the engineered feeder cells or cytoplasmic membrane particles or exosomes obtained from said feeder cells.In one aspect, NK cells may be contacted with engineered feeder cells or cytoplasmic membrane particles or exosomes obtained from said feeder cells for 7 to 21 days, preferably 7 to 14 days.

[0115] 96. As noted, the disclosed methods create NK cells that are resistant to TGF-β. Thus, in one aspect, this document discloses TGF-β-resistant NK cells created using the method for creating TGF-β-resistant NK cells disclosed herein.

[0116] 97. In some cases, cytoplasmic membrane particles or exosomes derived from engineered feeder cells can be obtained through nitrogen cavitation.

[0117] 98. Typically, the cell is maintained under conditions suitable for growth and / or maintenance of the cell. Suitable cell culture conditions are well known in the art and are described, for example, in Santiago et al., Proc. Natl. Acad. Sci. USA, 2008, 105:5809-5814; Moehle et al. Proc. Natl. Acad. Sci. USA, 2007, 104:3055-3060; Urnov et al., Nature, 2005, 435:646-651; and Lombardo et al., Nat. Biotechnol., 2007, 25:1298-1306. Those skilled in the art will appreciate that cell culture methods are known in the art and may vary depending on the cell type. Routine optimization can be used in all cases to determine the best methods for a particular cell type.

[0118] D. Method for treating cancer

[0119] 99. The disclosed compositions can be used to treat any disease in which uncontrolled cellular proliferation occurs, such as cancer. Accordingly, disclosed herein are methods of treating, inhibiting, reducing, attenuating, ameliorating, and / or preventing cancer and / or metastasis in a subject, comprising administering to the subject a TGF-β-resistant NK cell disclosed herein. For example, disclosed herein are methods of treating, inhibiting, reducing, attenuating, ameliorating, and / or preventing cancer and / or metastasis (such as, for example, a solid tumor) in a subject with a TGF-β-resistant NK cell, comprising obtaining an NK cell; cell cultivation in the presence of feeder cells (including but not limited to PBMC, RPMI8866, HFWT, K562, EBV-LCL cells, NK cells (including but not limited to NK-92, NK-92MI, NK-YTS, NK, NKL, KIL, KIL C.2, NK 3.3, NK-YS), transfected with membrane-bound IL-21, NK cells (including but not limited to NK-92, NK-92MI, NK-YTS, NK, NKL, KIL, KIL C.2, NK3.3, NK-YS), transfected with membrane-bound 4-1 BBL, NK cells (including but not limited to NK-92, NK-92MI, NK-YTS, NK, NKL, KIL, KIL C.2, NK 3.3, NK-YS), transfected with membrane-bound IL-15 and 4-1 BBL, or NK cells (including but not limited to NK-92, NK-92MI, NK-YTS, NK, NKL, KIL, KIL C.2, NK 3.3, NK-YS, transfected membrane-bound IL-21 and 4-1 BBL) engineered to express TGF-β, thereby generating a TGF-β-resistant NK cell, or in the presence of cytoplasmic membrane particles or exosomes obtained from said feeder cells; and administering the TGF-β-resistant NK cells to the subject. Preferably, the TGF-β expressed by the feeder cell is membrane-bound.

[0120] 100. It is to be understood and contemplated herein that feeder cells can be used in the disclosed treatment methods not only to confer resistance to TGF-β, but also to activate and / or expand populations of exogenous NK cells (such as, for example, memory-like NK cells such as NKG2C + , CD56 bright NK cells, CD56 dim NK cells, peripheral NK cells, NK T cells, or tumor-infiltrating NK cells), but they can be used to render endogenous NK cell populations resistant to TGF-β and / or to activate and / or expand endogenous NK cell populations (such as, for example, memory-like NK cells such as NKG2C + , CD56 bright NK cells, CD56 dimNK cells, peripheral NK cells, NK T cells, or tumor-infiltrating NK cells). NK cells that can be used in cancer treatment methods can be any donor NK cell or NK cell line. In one aspect, disclosed herein are methods for treating, inhibiting, reducing, attenuating, ameliorating, and / or preventing cancer and / or metastasis, wherein the NK cells are memory-like NK cells such as NKG2C+, CD56 bright NK cells, CD56 dimNK cells, peripheral NK cells, NK T cells, or tumor-infiltrating NK cells (including, but not limited to, NK cells derived from cell lines or obtained from a donor source (such as, for example, an autologous donor, an allogeneic donor, or a syngeneic donor). In one aspect, the NK cell may be an endogenous NK cell that is rendered stable in vivo by exposure to modified feeder cells and / or cytoplasmic membrane particles or exosomes derived from said feeder cells, as disclosed herein.

[0121] 101. Feeder cells, cytoplasmic membrane particles and / or exosomes used in the disclosed treatment methods may further comprise additional effector agents for the expansion and / or activation of NK cells.Thus, in one aspect, disclosed herein are methods for treating, inhibiting, reducing, attenuating, ameliorating, and / or preventing cancer and / or metastasis, 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 activating agent (such as, for example, NK cell effector agents including but not limited to 4-1 BBL, IL-2, IL-12, IL-15, IL-18, IL-21, MICA, LFA-1, 2 B4, CCR7, OX40L, UBLP2, BCM1 / 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, CD16 agonists).In one aspect, at least one additional NK cell effector agent is IL-21, 4-1 BBL, IL-15, IL-21 and 4-1 BBL, IL-21 and IL-15, or IL-15 and 4-1 BBL. Preferably, said cytokine, adhesion molecule, or NK cell activating agent is a human cytokine, adhesion molecule, or NK cell activating agent. Preferably, at least one additional NK cell effector agent is a membrane-bound NK cell effector agent.

[0122] 102. In one aspect, the document discloses methods for treating, inhibiting, reducing, attenuating, ameliorating and / or preventing cancer and / or metastasis, wherein NK cells are incubated in the presence of engineered feeder cells, cytoplasmic 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.

[0123] 103. The disclosed compositions can be used to treat any disease in which uncontrolled cellular proliferation occurs, such as cancer.A representative, but non-limiting list of various cancers that the disclosed compositions can be used to treat includes the following: 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 cancer of the head and neck, various types of lung cancer such as small cell lung cancer and non-small cell lung cancer, neuroblastoma / glioblastoma, ovarian cancer, skin cancer, liver cancer, melanoma, squamous cell cancer of the mouth, throat, larynx and lungs, cervical cancer, cervical carcinoma, breast cancer and epithelial cancer, kidney cancer, genitourinary cancer, respiratory tract cancer, esophageal cancer, head and neck carcinoma, colon cancer, various types of hematopoietic cancer; testicular cancer; colon cancer, rectal cancer, prostate cancer, or pancreatic cancer.

[0124] E. Examples

[0125] 104. The following examples are presented to provide those skilled in the art with a complete disclosure and description of how the compounds, compositions, articles, devices, and / or methods claimed herein are prepared and evaluated, and are intended to be illustrative only and are not intended to limit the present invention. Efforts have been made to ensure accuracy with respect to numbers (e.g., amount, temperature, etc.), however, some errors and deviations should be taken into account. Unless otherwise noted, parts are parts by weight, temperatures are in °C or equal to ambient temperature, and pressures are equal to or near atmospheric pressure.

[0126] 1. Example 1: Generation of genetically modified feeder cell clones consisting of K562 expressing mblL21, CD137L and secreting mbTGFβ

[0127] 105. Multiple human TGFβ1-overexpressing feeder cell lines were generated by transducing K562-based feeder cell clone CSTX002 with lentiviral vectors expressing TGFβ1 as a membrane-bound mutein (mbTGFβ) or in its native secreted form. Clone CSTX002 expresses a membrane-bound mutein of the cytokine IL-21 and the costimulatory molecule 4-1 BBL (CD137). Briefly, a human mbTGFβ1 fusion construct was generated in silico using human TGFβ1 fused to the CD4 transmembrane region and the IgG4 stalk (F c) under the regulatory control of the human CMV promoter. The construct was then subjected to codon optimization and sequence homology confirmation. Similarly, a bicistronic mc-TGFβ vector was generated using human TGFβ under the control of the human elongation factor-1 (EF1A) promoter with independent expression of the mcherry fluorescent protein under the control of the CMV promoter. Detailed vector maps showing the construction of the two constructs (mbTGFβ and mc-TGFβ) are shown in Fig. 1. Both constructs were cloned into a third-generation lentiviral system, which was used to produce high-titer lentivirus.

[0128] 106. Because lentivirus transduction efficiency may vary between cell lines, and the initial transduction of CSTX002 was accomplished with green fluorescent protein (GFP)-encoding lentivirus, susceptibility to superinfection was confirmed at three different virus titers (multiplicity of infection (MOI) 5, 10, 20) using a control GFP-encoding lentivirus (Fig. 2). High GFP expression was observed in each condition 48 h post-transfection. CSTX002 infections were preceded by MOIs of 5, 10, and 20 from TGFβ-based vectors, and transduced cells were stained with anti-human TGFβ1 antibody (Biolegend, cat. #349615) 5 days post-infection (Fig. 3). Optimal TGFβ expression was observed at an MOI of 20 for cells infected with either vector (mbTGFβ or mc-TGFβ), so all subsequent experiments were performed using an MOI of 20.To generate clones overexpressing mbTGFβ or mc-TGFβ, CSTX002 was infected with two viruses as described above. After infection, the cells were cultured for 4–5 days. mbTGFβ cells were stained with an anti-TGFβ1 antibody and sorted on a BD Influx using the BD software. Positive cells were sorted directly into 96-well plates. mc-TGFβ-positive cells were sorted using a green laser (561 nm), collecting mcherry-positive cells. Single-cell clones were picked from the 96-well plates, and mbTGFβ or mc-TGFβ expression was determined using FACS (Fig. 4). In addition, mRNA expression was determined by quantitative real-time PCR (RT-PCR) using hTGFβ1 TaqMan primers. A 5- to 20-fold increase in TGFβ mRNA expression was observed in mc-TGFβ cells and a 50- to 400-fold increase in mbTGFβ cells compared to control non-transfected cells (Fig. 5).

[0129] 2. Example 2: Demonstrate that expansion of NK cells on these new feeder cells results in TGFβ-resistant NK cells and describe the characteristics of these resistant cells

[0130] 107. Next, it was determined whether new feeder cells transduced with mbTGFβ and mc-TGFβ secreted intact TGFβ. mbTGFβ and mc-TGFβ cells were seeded in a 96-well plate after 16 hours of incubation, and the supernatants were collected and stored at -80°C. The level of TGFβ1 was determined using a TGFβ1 ELISA kit (R&D, cat. #DB100B). All clones from both expression vectors were found to secrete TGFβ. The levels were ~5,000 pg / mL for mc-TGFβ cells and ranged from 10,000 to 15,000 pg / mL for mbTGFβ cells (Fig. 6).

[0131] 108. TGFβ is an immunosuppressive molecule that impairs the anti-tumor function of NK cells through the activation of downstream targets Smad2 / Smad3. Addition of soluble TGFβ1 during NK cell expansion with CSTX002 results in a marked reduction in Smad3 expression in NK cells, rendering them cytokine hypersecreting and less susceptible to TGFβ1-mediated signaling. To determine whether newly transduced clones could induce similar NK cell remodeling, NK cells from 3 healthy donors were expanded with regular CSTX002, CSTX002 with soluble TGFβ1, or TGFβ1-expressing CSTX002. Briefly, feeder cells were irradiated with 100 Gy, and NK cells isolated from PBMCs of healthy blood donors were expanded using the irradiated feeder cells for two weeks in the presence of a low dose of IL-2 (50 IU / mL).A similar decrease in Smad3 expression levels was observed in NK cells expanded with mbTGFβ and mc-TGFβ feeder cells (Fig. 7).

[0132] 109. It was then determined whether NK cells expanded with mbTGFβ and mc-TGFβ-transduced feeder cells were functionally similar to cells cultured with soluble TGFβ and CSTX002. After expansion, NK cells were cocultured with calcein-loaded HOS (osteosarcoma) or DAOY (medulloblastoma) target cells at a 5:1 ratio for 4 h. Calcein release was used to determine the average percentage of cell lysis. NK cells treated with 10 ng / mL soluble human TGFβ1 (sTGFβ) served as a positive control. As shown in Fig. 8, NK cells cultured with mbTGFβ, mc-TGFβ feeder cells, or expanded in the presence of soluble TGFβ had a significantly reduced ability to lyse HOS and DAOY target tumor cells compared to NK cells expanded with control feeder cells. This is consistent with TGFβ-imprinted NK cells.

Claims

1. An engineered feeder cell capable of generating a natural killer (NK) cell resistant to TGF-β, wherein said engineered feeder cell is created by modifying a feeder cell to express soluble or membrane-bound TGF-β, wherein said engineered cell includes an expression vector containing a nucleic acid sequence expressing soluble or membrane-bound TGF-β, wherein said NK cell is incubated in the presence of said engineered feeder cell, and wherein said soluble or membrane-bound TGF-β comes into contact with said NK cell, inducing resistance to TGF-β.

2. The engineered feeder cell according to claim 1, characterized in that said expression vector contains an inducible or constitutive promoter.

3. The engineered feeder cell according to claim 1 or 2, characterized in that said feeder cell is a K562 leukemia cell.

4. An engineered feeder cell according to any one of claims 1-3, further comprising 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 activating agent.

5. The engineered feeder cell of claim 4, wherein said at least one additional NK cell effector agent is selected from 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, DAP10, Notch ligand, NKp46 agonist, NKp44 agonist, NKp30 agonist, NCR and CD16 agonist.

6. The engineered feeder cell of claim 4, wherein said at least one additional NK cell effector agent comprises IL-21, 4-1 BBL, IL-15, IL-21 and 4-1 BBL, IL-21 and IL-15, or IL-15 and 4-1 BBL.

7. An engineered feeder cell according to any one of claims 1 to 6, wherein said feeder cells are PBMCs, RPMI8866 cells, HFWT cells, K562 cells, EBV-LCL cells, 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.

8. A method for generating a TGF-β-resistant NK cell, comprising: a) introducing into the feeder cell an expression vector containing a nucleic acid sequence expressing soluble or membrane-bound TGF-β; b) incubating the NK cell in the presence of said feeder cell engineered to express TGF-β; wherein said soluble or membrane-bound TGF-β comes into contact with said NK cell, inducing resistance to TGF-β in the NK cell.

9. The method according to claim 8, characterized in that said feeder cells are PBMCs, RPMI8866 cells, HFWT, K562, 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. The method according to claim 8 or 9, characterized in that said feeder cells additionally contain 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 activating agent.

11. The method of claim 10, wherein said at least one additional NK cell effector agent is selected from 4-1 BBL, 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, DAP10, Notch ligand, NKp46 agonist, NKp44 agonist, NKp30 agonist, NCR agonist and CD16 agonist.

12. The method according to claim 11, characterized in that at least one said additional effector agent for NK cells is IL-21, 4-1BBL, IL-15, IL-21 and 4-1 BBL, IL-21 and IL-15, or IL-15 and 4-1 BBL.

13. The method according to any one of paragraphs 8-12, characterized in that said NK cells are NK cells similar to memory cells, such as NKG2C + , CD56 bright NK cells, CD56 dim NK cells, peripheral NK cells, NK T cells, or tumor-infiltrating NK cells.

14. The method according to any one of paragraphs 8-13, characterized in that said NK cells are obtained from a donor subject.

15. The method according to any one of paragraphs 8-14, characterized in that said NK cells are obtained from an autologous donor, an allogeneic donor, or a syngeneic donor.

16. The method according to any one of claims 8-15, characterized in that said NK cells are incubated in the presence of engineered feeder cells, cytoplasmic 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.

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