Overcoming immunosuppression in TGF-β resistant NK cells

Abstract

This invention provides novel reagents and methods that can be used to maintain the killing effect of NK cells in the presence of TGF-β. [Solution] Disclosed are engineered feeder cells containing soluble or membrane-bound TGF-β, methods of using them in the production of TGF-β resistant NK cells, and the use of the aforementioned produced TGF-β resistant NK cells for the treatment of cancer.

Description

[Technical Field]

[0001] This application is a U.S. Provisional Patent Application No. 63 / 018,10, filed on April 30, 2020. The interests of Article 8 are asserted and incorporated herein by reference in their entirety. [Background technology]

[0002] The use of NK cells in cancer therapy is increasing. However, research shows that cancer It has been shown that it secretes TGF-β, which can reduce the killing effect of NK cells. Previous efforts to address the decrease in killings included the use of TGF-β inhibitors. However, the use of TGF-β inhibitors can cause harmful systemic side effects. It is possible. What is needed is to maintain the killing effect of NK cells in the presence of TGF-β. These are novel reagents and methods that can be used for (i.e., TGF-β resistant NK cells). [Overview of the Initiative]

[0003] Disclosed are methods and compositions related to manipulated feeder cells, as well as TGF -β-resistant NK cells are produced using the aforementioned feeder cells.

[0004] In one embodiment, the herein discloses a method for expressing soluble or membrane-bound TGF-β. These are modified and engineered feeder cells. For example, TGF-β (soluble or membrane-bound). The combined form includes, but is not limited to, CMV or EF1A promoters. It can be operably linked to a constitutive or inductive promoter.

[0005] Also disclosed herein is at least one additional NK cell on its cell surface. It further contains effector agents, and at least one additional NK cell effector agent is used at the site Cain, adhesion molecules, or NK cell activators (e.g., 4-1BBL, IL-2, IL-1) 2, 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, DN ER, Jedi, SOM-11, wingless, CCN3, MAGP2, MAGP1 , TSP2, YB-1, EGFL7, CCR7, DAP12, and DAP10, Notc h ligand, NKp46 agonist, NKp44 agonist, NKp30 agonist, etc. NK cell effects including, but not limited to, NCR agonists and CD16 agonists. The agent is a modified feeder cell of any prior embodiment. At least one additional NK cell effector agent is IL-21, 4-1BBL, IL -15, IL-21 and 4-1BBL, IL-21 and IL-15, or IL-15 and Includes 4-1BBL.

[0006] In one embodiment, the feeder cells disclosed herein are PBMCs, RPMI88 66, HFWT, K562 cells, EBV-LCL, membrane-bound IL-21 transfect NK cells (PBMC, RPMI8866, NK-92, NK-92MI, NK- YTS, NK, NKL, KIL, KIL C.2, NK 3.3, NK-YS, HFWT (including, but not limited to, K562 cells), membrane-bound 4-1BBL transfer NK cells (PBMC, RPMI8866, NK-92, NK-92MI, NK) -YTS, NK, NKL, KIL, KIL C.2, NK 3.3, NK-YS, HFW T, K562 cells (including but not limited to these), membrane-bound IL-15 and 4-1BB NK cells (PBMC, RPMI8866, NK-92, NK -92MI, NK-YTS, NK, NKL, KIL, KIL C.2, NK 3.3, N K-YS, HFWT, K562 cells (including but not limited to these), or membrane-bound IL -21 and 4-1BBL transfected NK cells (PBMC, RPMI886 6, NK-92, NK-92MI, NK-YTS, NK, NKL, KIL, KIL C. 2, NK 3.3, NK-YS, HFWT, K562 cells (including but not limited to these ), which are engineered feeder cells of any preceding aspect.

[0007] Also disclosed herein are plasma membrane particles or exosomes derived from engineered feeder cells of any preceding aspect. In some cases, the particles or exosomes can be obtained by nitrogen cavitation.

[0008] In one aspect, disclosed herein is a method for generating TGF-β-resistant NK cells, which includes incubating NK cells in the presence of engineered feeder cells, plasma membrane particles, or exosomes of any preceding claim. For example, disclosed herein are feeder cells (PBMC, RPMI 8866, NK-92, NK-92MI, NK-YTS, NK, NKL, KIL, KIL engineered to express TGF-β, 8866, NK-92, NK-92MI, NK-YTS, NK, NKL, KIL, KIL C.2, NK 3.3, NK-YS, HFWT, K562 cells, EBV-LCL, membrane-bound NK cells transfected with synthetic 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 (including, but not limited to), incubating NK cells in the presence of, or incubating NK cells in the presence of plasma membrane particles or exosomes derived from the aforementioned feeder cells, a method for generating TGF-β-resistant NK cells.

[0009] Also disclosed herein is that the feeder cell further comprises at least one additional NK cell effector agent on its cell surface, and at least one additional NK cell effector agent is a cytokine, an adhesion molecule, or an NK cell activator (e.g., 4-1BBL, IL-2, IL-12, IL-15, IL-18, IL-21, MICA, LFA-1, 2B4, CCR7, OX40L, UBLP2, BCM1 / SLAMF2, NKG2D agonist, CD155, CD112, Jagged1, Jagged2, Delta-1, Pref-1, DNER, Jedi, SOM-11, wingless, CCN3, MAGP2, MAGP1, TSP2, YB-1, EGFL7, CCR7, DAP12, and DAP10, Notch ligands, NKp46 agonist, NKp44 agonist, NKp 30 agonist, other NCR agonists, CD16 agonist, etc.), a method for generating TGF-β-resistant NK cells of any of the preceding aspects. In one aspect, at least one additional NK cell effector agent are IL-21, 4-1BBL, IL-15, IL-21 and 4-1BBL, IL-21 and IL-15, or IL-15 and 4-1BBL.

[0010] In one embodiment, the herein discloses NK cells as NKG2C + CD56 bri ght NK cells, CD56 dim NK cells, peripheral NK cells, NK T cells, or tumors Infiltrating NK cells (NK cells obtained from cell lines, or donor sources (e.g., autologous donors, allogeneic donors)) This includes, but is not limited to, NK cells obtained from allogeneic or syngeneic donors. Any preceding embodiment of TGF-β resistant NK cells, including memory-like NK cells such as those mentioned above. This is a method for generating [something].

[0011] Furthermore, disclosed herein are NK cells, engineered feeder cells, plasma membrane granules. In the presence of children or exosomes, at least 7, 8, 9, 10, 11, 12, 13, 1 4, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 Any preceding state that is incubated for 28, 29, 30, 45, or 60 days This is a method for generating TGF-β resistant NK cells.

[0012] In one embodiment, disclosed herein are those produced by any prior embodiment. These are TGF-β resistant NK cells.

[0013] Furthermore, disclosed herein are any prior embodiments of TGF-β-resistant NK cells Including administration to elephants, the treatment, inhibition, reduction, or decrease of cancer and / or metastasis in the subject. Methods for improvement and / or prevention. For example, disclosed herein are methods for TGF-β Resistant NK cells can treat or inhibit cancer and / or metastases (e.g., solid tumors) in the target. Methods for reducing, decreasing, improving, and / or preventing NK cells, and for obtaining NK cells Feeder cells (PBMC, RPMI8866) modified to express TGF-β Transfected with HFWT, K562 cells, EBV-LCL, and membrane-bound IL-21. NK cells, NK cells transfected with membrane-bound 4-1BBL, membrane-bound IL- NK cells transfected with 15 and 4-1BBL, or membrane-bound IL-21 and Existence of NK cells transfected with 4-1BBL (including, but not limited to, NK cells) To culture under the conditions described above and thereby produce TGF-β resistant NK cells, or to produce TGF-β resistant NK cells, or the aforementioned fee Culturing in the presence of plasma membrane particles or exosomes derived from Dah cells, and T The method involves administering a substance to GF-β-resistant NK cells. What is to be developed is, preferably, a method for treating cancer in a target, for use as a drug. The present invention relates to TGF-β resistant NK cells for use in the following contexts. For example, in the following contexts To treat, inhibit, reduce, decrease, improve, and / or prevent cancer and / or metastases or solid tumors. TGF-β resistant NK cells for use in the method are disclosed herein, and the aforementioned TG F-β resistant NK cells are produced by the method for generating TGF-β resistant NK cells according to the present invention, preferably, Obtaining NK cells, feeding NK cells (PBMC, RPMI8866, NK- 92, NK-92MI, NK-YTS, NK, NKL, KIL, KIL C.2, NK 3.3, including NK-YS, HFWT, K562 cells, EBV-LCL, or NK cells, Culturing in the presence of (but not limited to) these cells, thereby generating TGF-β resistant NK cells. to do so, or to use NK cells to obtain plasma membrane particles or exosols derived from the aforementioned feeder cells. A method comprising culturing in the presence of a fumes to generate TGF-β resistant NK cells. Obtained by: Preferably, feeder cells (PBMC, RPMI8866, NK- 92, NK-92MI, NK-YTS, NK, NKL, KIL, KIL C.2, NK 3.3, including NK-YS, HFWT, K562 cells, EBV-LCL, or NK cells, (Not limited to these) are (i) transfected with membrane-bound IL-21, or ( ii) Transfected with membrane-bound 4-1BBL, or (iii) membrane-bound IL-1 Transfected with 5 and 4-1BBL, or (iv) membrane-bound IL-21 and It is transfected with 4-1BBL. Also disclosed herein is the subject The use of TGF-β resistant NK cells of the present invention for the manufacture of drugs for the treatment of cancer For example, to treat, inhibit, reduce, or decrease cancer and / or metastases or solid tumors in the subject. The use of TGF-β resistant NK cells for the manufacture of drugs for improvement and / or prevention is The TGF-β resistant NK cells disclosed herein and described above are the TGF-β resistant NK cells of the present invention. A method for generating cells, preferably a step of obtaining NK cells, and feeding the NK cells ( PBMC, RPMI8866, NK-92, NK-92MI, NK-YTS, NK, NK L, KIL, KIL C.2, NK 3.3, NK-YS, HFWT, K562 cells, E Culture in the presence of BV-LCL or NK cells (including, but not limited to, these), and Therefore, the step of generating TGF-β resistant NK cells, or NK cells, is performed using the aforementioned feeder cells. The cells were cultured in the presence of plasma membrane particles or exosomes derived from cytoplasm, thereby enabling TGF- They are obtained by a method that includes the step of generating β-resistant NK cells. Preferably, a feeder - Cells (PBMC, RPMI8866, NK-92, NK-92MI, NK-YTS, N K, NKL, KIL, KIL C.2, NK 3.3, NK-YS, HFWT, K562 Cells, including but not limited to EBV-LCL or NK cells, are (i) membrane-bound (ii) Transfected with type IL-21, or (ii) Transfected with membrane-bound 4-1BBL (iii) Transfected with membrane-bound IL-15 and 4-1BBL (iv) It is either (iv) transfected with membrane-bound IL-21 and 4-1BBL.

[0014] In one embodiment, the herein discloses NK cells as NKG2C + CD56 bri ght NK cells, CD56 dim NK cells, peripheral NK cells, NK T cells, or tumors Infiltrating NK cells (NK cells obtained from cell lines, or donor sources (e.g., autologous donors, allogeneic donors)) This includes, but is not limited to, NK cells obtained from allogeneic or syngeneic donors. Memory-like NK cells, such as those mentioned above, can cure any preceding form of cancer and / or metastasis. A method of treating, inhibiting, reducing, decreasing, improving, and / or preventing.

[0015] Furthermore, disclosed herein is that feeder cells have at least 1 on their cell surface. It further contains two additional NK cell effector agents, and at least one additional NK cell effector The activator is a cytokine, adhesion molecule, or NK cell activator (e.g., 4-1BBL, IL-2, IL-12, IL-15, IL-18, IL-21, MICA, LFA-1, 2B4, CCR7, OX40L, UBLP2, BCM1 / SLAMF2, NKG2D Ago Nist, CD155, CD112, Jagged1, Jagged2, Delta-1, Pref-1, DNER, Jedi, SOM-11, wingless, CCN3, MA GP2, MAGP1, TSP2, YB-1, EGFL7, CCR7, DAP12, and D AP10, Notch ligand, NKp46 agonist, NKp44 agonist, NKp This includes, but is not limited to, 30 agonists, other NCR agonists, and CD16 agonists. (e.g., NK cell effector agents) any prior embodiment of cancer and / or metastasis A method of treating, inhibiting, reducing, decreasing, improving, and / or preventing. In one embodiment, at least Another additional NK cell effector is IL-21, 4-1BBL, IL-15, I L-21 and 4-1BBL, IL-21 and IL-15, or IL-15 and 4-1BB Contains L. Preferably, TGF-β is membrane-bound. Preferably, at least one The additional NK cell effector is a membrane-bound NK cell effector.

[0016] In one embodiment, disclosed herein is a modified feeder cell, shaped by NK cells. In the presence of membrane particles or exosomes, at least 7, 8, 9, 10, 11, 12, 1 3, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 Any preceding period of incubation for 27, 28, 29, 30, 45, or 60 days. To treat, inhibit, reduce, decrease, improve, and / or treat cancer and / or metastases or solid tumors in such manner. This is a method of prevention. [Brief explanation of the drawing]

[0017] The accompanying drawings incorporated herein and forming part thereof illustrate several embodiments. The disclosed compositions and methods are illustrated with descriptions.

[0018] [Figure 1] Figures 1A and 1B show schematic diagrams of lentiviral vectors expressing human transformed growth factor beta-1 (hTGFβ1). Figure 1A shows membrane-bound TGFβ (mbTGFb), in which 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. Figure 1B shows the Mcherry-TGFβ (mc-TGFB) vector, which was generated by cloning human TGFβ mRNA under the control of the EF1A promoter and expressing the mcherry protein using the CMV promoter. [Figure 2] The images show fluorescence images illustrating GFP expression in cells. Panels A-C show images of K562 cells, a clone (CXT002) infected with a lentivirus that expresses GFP 48 hours after infection, and indicate the multiplicity of infection (MOI). [Figure 3A] Figures 3A and 3B show FACS analysis indicating TGFβ expression. K562 cell clones (CSTX002) were infected with lentiviruses expressing membrane-bound TGFβ (mbTGFβ) or mcherry TGFβ (mc-TGFβ), and the degree of infection (MOI) was shown. Cells were collected 5 days after infection and stained with human anti-TGFB antibody. Figure 3A shows cells overexpressing membrane-bound TGFβ (mbTGFβ). Figure 3B shows cells overexpressing mcherry TGFβ (mc-TGFβ). [Figure 3B] The explanation is the same as in Figure 3A. [Figure 4A]Figures 4A and 4B show scatter plots illustrating the gating strategies used to sort mbTGFb or mc-TGFB-positive cells. Single-cell clones were cultured for 10–14 days. Figure 4A shows mbTGFβ cells stained with anti-TGFβ antibody. APC-positive cells were collected. Figure 4B shows mcherry TGFβ-positive cells sorted using a fluorescent marker. [Figure 4B] The explanation is the same as in Figure 4A. [Figure 5A] Figures 5A and 5B show quantitative real-time PCR (RT-PCR) results indicating TGFβ expression. RNA was isolated from single-cell clones, and TGF transcript levels were determined using RT-PCR. Results are expressed as a fold change compared to untransfected control cells. TGFβ levels in cells infected with membrane-bound TGFβ (mbTGFβ) (5A) or mcherrryTGFβ (TGFβ) (5B). [Figure 5B] The explanation is the same as in Figure 5A. [Figure 6] This report shows ELISA results indicating human TGFβ levels. Single-cell clones selected from mbTGFβ and mc-TGFβ cells (0.5 x 10^6) were cultured in 96-well plates. The supernatant was collected after 16 hours, and TGFβ1 levels were determined using a human TGFβ1 ELISA kit. [Figure 7] This immunoblot shows Smad3 expression in NK cells treated with TGFβ. NK cells isolated from normal donor leukopak were grown for 2 weeks with irradiated normal feeder cells CSTX0002 (control), soluble TGFβ (sTGFβ) (10 ng / ml), or TGFβ-overexpressing feeder cell lines (mcC10 or MB15). [Figure 8]Figures 8A and 8B show standard NK cell cytotoxicity assays against tumor cells. NK cells derived from the peripheral blood of normal donors were co-cultured with calcein-labeled tumor cells in a 5:1 effector-versus-target ratio for 4 hours. Calcein release in the supernatant was measured using a microplate reader and used to determine the mean specific lysis. NK cells co-cultured with human osteosarcoma cells (HOB) (8A) and human medulloblastoma cells (DAOY) (8B). Significance was determined by two-way ANOVA with Holm-Sidak multiple comparison tests. (*p≦0.05, **p≦0.01, ***p≦0.001, ****p≦0.0001) [Modes for carrying out the invention]

[0019] Before the compounds, compositions, articles, devices, and / or methods of the present invention are disclosed and explained Unless otherwise specified, these refer to specific synthesis methods or specific recombinant biotechnology. - Not limited to the method, or unless otherwise specified, not limited to a specific reagent, however Of course, please understand that this can change. Also, the terminology used in this specification is particularly important. This is intended solely to describe a specific embodiment, and not to limit it. I hope you understand that too.

[0020] A.Definition Unless otherwise defined, all technical and scientific terms used herein are the same as those used in this invention. It has a meaning that is generally understood by those skilled in the art of the field to which it belongs. The following references are relevant. To provide vendors with general definitions for many of the terms used in this invention: Singleton et al.,Dictionary of Microbiology and M Olecular Biology(2nd Ed.1994), The Cambri dge Dictionary of Science and Technology (Walker ed., 1988), The Glossary of Geneti cs, 5th Ed., R. Rieger et al. (eds.), Springe r Verlag (1991) and Hale & Marham, The Harper Collins Dictionary of Biology (1991). This specification. When used in this context, the following terms have the meanings associated with them unless otherwise specified. .

[0021] When describing elements of this disclosure or preferred embodiments thereof, the articles "a," "an," and "t" are used. The words "he" and "said" mean that one or more of the elements are present. As illustrated. "comprising", "including", and The term "having" is intended to be comprehensive and enumerate. This means that additional elements other than the specified element may exist.

[0022] In this specification, the range is defined as "approximately" from one specific value and / or "approximately" from another specific value. It can be expressed as such. If such a range is expressed, another embodiment is one specific value It includes from and / or up to other specific values. Similarly, the use of the antecedent "about" brings the value closer. When expressed as similar values, it will be understood that certain values ​​form other embodiments. Each endpoint of the enclosure is important both in relation to the other endpoints and independently of the other endpoints. This will be further understood. There are several values ​​disclosed herein, and each value is also In addition to the value itself, it is also understood that a particular value may be disclosed herein as "approximately". For example, if the value "10" is disclosed, then "about 10" is also disclosed. To ensure proper understanding, when a certain value is disclosed, "less than or equal to that value," "less than or equal to that value" is used. It is also understood that the possible range between "greater than or equal to" and that value will be disclosed. For example, the value "10". If disclosed, then "10 or less" and "10 or more" are also disclosed. Over the course of the journey, the data is provided in several different formats, and this data includes the endpoint and the starting point. It is also understood to represent a range of any combination of data points. For example, If a fixed data point "10" and a specific data point 15 are disclosed, then 10 and 1 Not only between 5, but also greater than 10, 10 or more, less than 10, 10 or less, and equal to 10. The following values ​​are disclosed: greater than 15, 15 or greater, less than 15, 15 or less, and equal to 15. It is understood that this is to be considered. Also, each unit between two specific units is disclosed. It is understood that, for example, if 10 and 15 are disclosed, then 11, 12, 13, and Number 14 will also be disclosed.

[0023] Where used herein, the terms “optional” or “optionally” shall have the following meanings: This means that the events or situations described above may or may not occur, and this description includes the preceding This includes cases where the aforementioned events or situations occur and cases where they do not.

[0024] As used herein, "N-terminal" or "amino-terminal" refers to peptides, polypeptides This refers to the directionality of a protein, and may not necessarily mean the N-terminus. Several aspects So, when considering chimeric or fusion peptides, polypeptides, or proteins, N The terminal end consists only of components of a chimeric or fused peptide, polypeptide, or protein. It may refer to one part, but not to the entire structure. For example, when the Fc domain is considered, F It is described as a c-domain fused with its amino-terminus or the N-terminus facing the intracellular side. In this case, what is intended herein is that a single anchor is at the N-terminus of a chimera or fusion structure. Yes, and in fact, they are chimeric or fused peptides, polypeptides, or that span the cell membrane. It is a protein. Therefore, in such a chimera, the transmembrane anchor is the Fc domain The orientation of the Fc domain, which is bound to the amino-terminal side, is such that the N-terminal side faces the cell, Typically, these have carboxyl terminals that extend across the cell membrane and amino acids that extend into the extracellular matrix. It is inverted compared to the Fc domain on a typical B cell with terminals.

[0025] The terms "peptide," "polypeptide," and "protein" refer to amino acid residues. It is used interchangeably to refer to polymers.

[0026] As used herein, the term “sequence identity” refers to two sequences of substantially equal length. This indicates a quantitative measure of the degree of identity between two sequences, regardless of whether they are nucleic acids or amino acid sequences. Identity percentage is the number of exact matches between two aligned sequences, in a shorter timeframe. This is calculated by dividing the result by the length of the sequence and multiplying it by 100. Approximate alignment of nucleic acid sequences. Smith and Waterman, Advances in Applied d Mathematics 2:482-489 (1981) Local homology algorithm Provided by [Name of company / organization]. This algorithm is based on Dayhoff's Atlas of Pr otein Sequences and Structure,MODayhof f ed.,5 suppl.3:353-358,National Biomedi. cal Research Foundation,Washington,DC, Developed in the USA, Gribskov, Nucleo Acids Res. 14 6):6745-6763(1986) Normalized scoring matrix It can be applied to amino acid sequences by using it to determine the sequence identity percentage. An exemplary implementation of this algorithm for doing so is available at Genetics Computer G "Best Fit" utility by roup (Madison, Wis.) Provided in the application. Calculates the percentage of identity or similarity between sequences. Other programs suitable for this purpose are generally known in the art, for example, another alignment The default program is BLAST, which is used with default parameters. For example, BLAST N and BLASTP can be used with the following default parameters: Genetic code = standard Semi; Filter=None; Chain=Both; Cutoff=60; Prediction=10; Matrix=BLO SUM62; Description = 50 array; Sort = High score; Database = Non-redundant, GenB ank+EMBL+DDBJ+PDB+GenBankCDS Translation+Swiss Pro tein+Spupdate+PIR. For details on these programs, see GenBank. This can be confirmed on the website. Generally, substitutions are conservative amino acid substitutions, Group 1: glycan Syn, alanine, valine, leucine, and isoleucine, Group 2: serine, cysteine, Threonine and methionine, Group 3: Proline, Group 4: Phenylalanine, Tyrosine , and tryptophan, Group 5: aspartic acid, glutamic acid, asparagine, and gu It is limited to exchanges within the members of the luthamine. Preferably, the sequence identity percentage is It is calculated over the entire length of the arrays being compared.

[0027] Techniques for determining nucleic acid and amino acid sequence identity are known in the relevant field. In terms of type, such techniques involve determining the nucleotide sequence of a gene's mRNA, and / or determining the amino acid sequence encoded thereby, and these sequences as a second This includes comparing with nucleotide or amino acid sequences. The genome sequence is determined in this manner and They can also be compared. Generally, the identity is determined by the number of polynucleotides or polynucleotides. This refers to the precise nucleotide-to-nucleotide or amino acid-to-amino acid correspondence in a peptide sequence. Two or more sequences (polynucleotides or amino acids) determine their percentage of identity. They can be compared by defining them.

[0028] Without departing from the scope of the present invention, various modifications may be made to the above cells and methods. Therefore, all matters contained in the above description and the examples given below are illustrative and It is intended to be interpreted in a certain way, and not in a restrictive sense.

[0029] "Increase" means any more amount of symptoms, disease, composition, condition, or activity that results from It can refer to a change. An increase means a statistically significant amount of a state, symptom, activity, or composition compared to a control. It can be an increase in any individual value, median, or mean of something. Therefore, an increase is that As long as the increase is statistically significant, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 2 0, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85 It could be an increase of 90%, 95%, or 100%.

[0030] "Reduction" means any amount of symptom, disease, composition, condition, or activity that results in a smaller amount of the disease. It can refer to a change. A substance may also have a genetic output in the gene product containing that substance. When the output of a gene product that does not contain is lower, it reduces the genetic output of the gene. It is understood that, for example, a decrease is when the symptoms are less frequent than previously observed. This could be a change in the symptoms of the disorder. A decrease is a statistically significant amount of the condition compared to the control. This could be a decrease in any individual value, median, or mean of the state, activity, or composition. Therefore, A decrease is defined as a decrease that is statistically significant, as long as it is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 7 The decrease could be 5, 80, 85, 90, 95, or 100%.

[0031] "Inhibit," "the act of inhibiting," and "inhibition" refer to activity, response, state, disease, or This means reducing other biological parameters, including activity, response, state, and This may include, but is not limited to, the complete elimination of the disease. This includes, for example, natural or This may also include a 10% reduction in activity, response, condition, or disease compared to control levels. Therefore, the reduction is 10, 20, 30, 40, 50, 60 compared to natural or control levels. This could be a reduction of 70%, 80%, 90%, 100%, or any amount in between.

[0032] "To reduce" or other forms of the word, such as "to reduce" or "reduction," means an event. Alternatively, it means a decrease in a characteristic (e.g., tumor growth). This is typically due to several standard values. Or, in terms of expected value, in other words, it is relative, but a standard value or relative value is referred to. It is understood that this is not always necessary. For example, "reducing tumor growth" means, This means reducing the tumor's growth rate compared to the standard or control.

[0033] "To prevent" or other forms of the word, such as "the act of preventing" or "prevention," To halt a specific event or characteristic, or to stabilize the development or progression of a specific event or characteristic. To standardize or delay, or to reduce the likelihood of a particular event or characteristic occurring. It means minimizing. Prevention is typically more absolute than reduction, for example. Therefore, comparison with a control is not necessary. When used herein, it reduces something. It is possible, but sometimes it is not possible to prevent something, although it is possible to prevent something that can be reduced. In some cases, this may be the case. Similarly, there are cases where something can be prevented but not reduced. However, there are cases where something that is prevented can be reduced. Reduction or prevention is used. In such cases, unless otherwise specifically designated, the use of other words will also be explicitly disclosed. I want to.

[0034] The term "target" refers to any individual that is the target of administration or treatment. It could be an object, for example, a mammal. In one aspect, the object could be a human, a non-human primate, a cow, or a cow. It may be a man, a pig, a dog, or a cat. The subject may also be a guinea pig, a rat, a hamster, It could be a rabbit, mouse, or mole. Therefore, the subject is a human or veterinary patient. That's possible. The term "patient" refers to a person under the treatment of a clinician, for example, a physician.

[0035] The term "therapeutably effective" means that the amount of the composition used is sufficient to treat one or more diseases or disorders. This refers to being sufficient to alleviate the cause or symptoms of a condition. Such alleviation is reduction or It only requires modification; it does not need to be eliminated.

[0036] The term "treatment" means curing, alleviating, or easing a disease, pathological condition, or disorder. This term refers to the medical management of a patient with the intention of stabilizing or preventing the condition. Treatment, that is, treatment specifically aimed at improving a disease, pathological condition, or disorder, Causal treatment, that is, treatment aimed at eliminating the cause of the associated disease, pathological condition, or disorder. This also includes palliative care, i.e., treatment of disease, pathological condition, or disability. Treatments designed not for healing, but for symptom relief, preventative treatments, i.e., treatments for related diseases. , to minimize or partially or completely inhibit the onset of a pathological condition or disorder Treatment directed towards, and supportive treatment for, namely, related diseases, pathological conditions, or disabilities. This includes treatments used to supplement other specific therapies aimed at improving harm.

[0037] "Administration" to a subject includes any route through which the drug is introduced or delivered to the subject. Oral, topical, intravenous, subcutaneous, transcutaneous, transcutaneous (dermal), intramuscular, intra-articular, parenteral, intra-arterial, intradermal, ventricular, intracranial, intraperitoneal, Intralesional, intranasal, rectal, vaginal, by inhalation, via transplanted reservoir, parenteral (e.g., subcutaneous), Intravenous, intramuscular, intraarticular, synovial fluid, intrasternal, intramedullary, intraperitoneal, intrahepatic, intralesional, and cranial This can be carried out by any suitable route, including internal injection or infusion techniques. The term "concurrent administration" as used in the specification. n, administration in combination, simultan eous administration, or administered simul (taneously) means that the compounds are administered at the same time in time, or are essentially mutually exclusive. This means that it is administered immediately after. In the latter case, the two compounds, the observed results a sufficiently close time to the point where the results are indistinguishable from those obtained when administered at the same time. It is administered to the target. "Systemic administration" means, for example, through an entry point into the circulatory or lymphatic system. via routes that introduce or deliver drugs to a wide area of ​​the body (e.g., more than 50% of the body) This refers to introducing or delivering a drug to a target. In contrast, "local administration" refers to the area of ​​the administration site. Alternatively, the drug may be introduced or delivered to an area directly adjacent to it, and a therapeutically significant amount of the drug may be administered systemically. This refers to introducing or delivering a drug to a target via a route other than local administration. For example, local administration. The drug being administered can be easily detected locally near the administration site, but not in the distal part of the target body. It is either undetectable or detectable in negligible amounts. Administration includes self-administration and other This includes administration by the person concerned.

[0038] "to treat", "the act of treating", "treatment (tr When used herein, one or more of the following are used: "eatment)" and their grammatical variations. The severity or frequency of the disease or condition, the symptoms of the disease or condition, or the underlying cause of the disease or condition To partially or completely prevent, delay, cure, alleviate, or reduce the cause. , change, treatment (remedying), improvement (ameliorating), improvement (im To provide, stabilize, mitigate, and / or reduce , including administration of the composition intended or intended. The treatment according to the present invention is preventive, prophylactic, It can be applied palliatively or therapeutically. Preventive treatment is applied before the onset of disease (e.g., before obvious signs of cancer). Before symptoms appear, during early-stage cancer (for example, when there are early signs and symptoms of cancer), or in established cancer It is administered to the target after development. Prophylactic administration is given one day (or several days) before the onset of symptoms of the disease or infection. This could take place over several years.

[0039] Throughout this application, various publications are referenced. The disclosures in these publications are... The entirety of this application is incorporated by reference to this application in order to more fully explain the state of the art relating to this application. The disclosed references are also discussed in the texts that refer to them and are included therein. The materials described herein are incorporated by reference individually and specifically.

[0040] B. Composition Components used to prepare the disclosed compositions, and methods disclosed herein. The compositions used within are disclosed. These and other materials are disclosed herein. When combinations, subsets, interactions, groups, etc. of these materials are disclosed, Specific references to various individual and collective combinations and permutations of each compound are explicitly disclosed. While this may not always be the case, it is understood that each is specifically intended and described herein. For example, engineered feeder cells expressing a specific TGF-β have been disclosed and discussed. This was performed on several molecules, including engineered feeder cells expressing TGF-β. When considering the modifications that can be obtained, engineered feeder cells expressing TGF-β Each and every combination and permutation of them, and all of them, are possible unless the opposite is specifically shown. The following modifications are specifically intended. Therefore, classes A, B, and C of molecules, and divisions If classes D, E, and F are disclosed, and an example of a combined molecule, AD, is disclosed, Next, even if each is not listed individually, each is intended individually and collectively, The combinations AE, AF, BD, BE, BF, CD, CE, and CF are open. It means that it is considered to be shown. Similarly, any subset or combination of these This will also be disclosed. Therefore, for example, the subgroups AE, BF, and CE are, This will be deemed to be disclosed. This concept applies to those who prepare and use the disclosed composition. This applies to all aspects of this application, including but not limited to the steps in the law. Therefore, if there are various additional steps that can be implemented, these additional steps Each of these may be implemented in any particular embodiment or combination of embodiments of the disclosed method. It is understood that one can obtain something.

[0041] The use of NK cells in antitumor therapy is becoming increasingly common. However, However, research has shown that cancer secretes TGF-β, which protects against the killing action of NK cells. It has been shown that TGF-β secretion leads to a decrease in killing capacity. Previous efforts included the use of TGF-β inhibitors, however TGF-β The use of inhibitors can cause harmful systemic side effects. Alternatively, clinically gray TGF-β can be used during NK cell proliferation, but excessive amounts are already expensive. Because the reagent (i.e., TGF-β) is required to observe any effect, clinical The use of TGF-β in RADE is time-consuming and extremely expensive. TGF-β resistance To address the need for NK cells, TGF-β inhibitors or soluble TGF-β culture supplements are used. To avoid a deficiency in either of these, the organism was modified to express soluble or membrane-bound TGF-β. A modified feeder cell is disclosed herein.

[0042] Also disclosed herein is at least one additional NK cell on its cell surface. It further contains effector agents, and at least one additional NK cell effector agent is used at the site Cain, adhesion molecules, or NK cell activators (e.g., 4-1BBL, IL-2, IL-1) 2, 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, DN ER, Jedi, SOM-11, wingless, CCN3, MAGP2, MAGP1 , TSP2, YB-1, EGFL7, CCR7, DAP12, and DAP10, Notc h ligand, NKp46 agonist, NKp44 agonist, NKp30 agonist, etc. NK cell effects including, but not limited to, NCR agonists and CD16 agonists. These are engineered feeder cells (such as those containing a stimulant). In one embodiment, at least one The additional NK cell effector agents are IL-21, 4-1BBL, IL-15, and IL-2 Includes 1 and 4-1BBL, IL-21 and IL-15, or IL-15 and 4-1BBL. Preferably, at least one additional NK cell effector agent is membrane-bound NK cell It is an effects agent.

[0043] Feeder cells are feeder cells that can proliferate and / or activate NK cells. It is understood and intended herein that it may originate from any source for the purpose of The feeder cells were PBMC, RPMI8866, HFWT, K562 cells, and EBV-LC. L, NK cells transfected with membrane-bound IL-21 (PBMC, RPMI886) 6, NK-92, NK-92MI, NK-YTS, NK, NKL, KIL, KIL C. This includes, but is not limited to, 2, NK 3.3, NK-YS, HFWT, and K562 cells. ), NK cells (PBMC, RPMI88) transfected with membrane-bound 4-1BBL 66, NK-92, NK-92MI, NK-YTS, NK, NKL, KIL, KIL C This includes, but is not limited to, .2, NK 3.3, NK-YS, HFWT, and K562 cells. i) NK cells (PBM) transfected with membrane-bound IL-15 and 4-1BBL C, RPMI8866, NK-92, NK-92MI, NK-YTS, NK, NKL, K This includes IL, KIL C.2, NK 3.3, NK-YS, HFWT, and K562 cells. (and not limited to these) or transfected with membrane-bound IL-21 and 4-1BBL NK cells (PBMC, RPMI8866, NK-92, NK-92MI, NK-YT) S, NK, NKL, KIL, KIL C.2, NK 3.3, NK-YS, HFWT, K Includes 562 cells (but not limited to these).

[0044] In addition, also disclosed herein are the operated feeders disclosed herein. - Derived from any of the cells or prepared by the methods disclosed herein. These are plasma membrane particles or exosomes.

[0045] 1. Delivery of the composition to cells It can be used to deliver nucleic acids to cells either in vitro or in vivo. There are several compositions and methods that can be used. These methods and compositions are virus-based. They can be broadly classified into two classes: virus-based delivery systems and non-virus-based delivery systems. Nucleic acids are processed by electroporation, lipofection, calcium phosphate precipitation, and plus Cosmids, viral vectors, viral nucleic acids, phage nucleic acids, phages, cosmids, etc. via a few direct delivery systems, or through cells or carriers such as cationic liposomes, genetic information is transmitted. It can be delivered via the transfer of substances. Viral vectors, chemical transfectors This includes physical and mechanical methods such as electroporation and direct diffusion of DNA. Appropriate means for transfection include, for example, Wolff, JA, et al. al., Science, 247, 1465-1468, (1990), and Wolff ,JANature,352,815-818,(1991) Such methods are well known in the art and, together with the compositions and methods described herein, It is easily adaptable for use with large DNA molecules. In certain cases, this method can be used with large DNA molecules. It will be modified to function specifically in [the case]. Furthermore, these methods will be used to [the carrier] By utilizing targeting properties, it is possible to target specific diseases and cell populations. .

[0046] Using endonucleazeriboprotein complexes (e.g., Cas9 / RNP) This involves reprogramming (i.e., manipulating or modifying) the feeder cells.

[0047] Endonucleases / RNPs (e.g., Cas9 / RNP) are located at the CRISPR locus. A three-component recombinant endonuclease protein complexed with (e.g., Cas9 endonuclease) It consists of endonucleases. Endonucleases complexed with the CRISPR gene locus are This can be called a CRISPR / Cas guide RNA. The CRISPR locus is the target sequence. Complementary repetitive RNA (crRNA) and trans complementary repetitive RNA (tra) complexed with A synthetic single guide RNA (gRNA) consisting of RNA that can hybridize to crRNA. It includes. Therefore, CRISPR / Cas guide RNA is a target within the cell's genomic DNA. Hybridizes into a specific array. In some cases, Class 2 CRISPR / Cas end The nuclease is a type II CRISPR / Cas endonuclease. In some cases Class 2 CRISPR / Cas endonucleases are Cas9 polypeptides. Therefore, the corresponding CRISPR / Cas guide RNA is the Cas9 guide RNA. Cas9 / RNPs are delivered as a functional complex to foreign DNA. Compared to existing approaches, it can cleave genome targets with higher efficiency. Therefore, rapid clearance of Cas9 / RNP from cells is important for inducing apoptosis and other processes. The targeting effect can be reduced. Therefore, in one embodiment, as disclosed herein What is being described is a method for genetically modifying NK cells, specifically targeting the DNA sequence of NK cells. a) obtaining a suitable guide RNA (gRNA), and targeting within the genomic DNA of feeder cells. The corresponding CRISPR / Cas guide RNA that hybridizes to the sequence is complexed with the CRISPR / Cas guide RNA. Last 2 CRISPR / Cas endonuclease (Cas9) ribonucleoprotein Transduction of the (RNP) complex into target NK cells (e.g., electroporation) This method includes (introducing through) and

[0048] a) Nucleic acid-based delivery systems A transvector is used to deliver a gene into a cell (for example, a plasmid). ), or as part of a general strategy for delivering genes, for example, recombinant retrovials It may be any nucleotide construct used as part of Rus or adenovirus. Ram et al. Cancer Res. 53:83-88, (1993)).

[0049] When used herein, plasmids or viral vectors are open-source proteins such as TGF-β. The nucleic acids are transported into the cell without degradation, and the genes are expressed within the cell to which they are delivered. It is a drug containing a promoter that brings about the effect. A viral vector is, for example, adenovirus. Rus, adeno-associated virus, herpesvirus, vaccinia virus, poliovirus, AIDS virus, neurotrophic virus, Syndbis virus, and this HIV skeleton These are other RNA viruses, including these viruses. Preferably, the characteristics of these viruses are Any shared virus family that is suitable for use as a vector It is also a family of viruses. As a retrovirus, it is the mouse Maloney's leukemia virus (MM). Examples include retroviruses that express desired characteristics of LV and MMLV as vectors. Retroviral vectors have a larger gene payload than other viral vectors. In other words, it can carry an introduced gene or a marker gene, and for this reason, it is commonly used These are the vectors used. However, they are not useful in non-proliferating cells. Adenovirus vectors are relatively stable, easy to handle, and have high titers. It is delivered as an aerosol formulation and can transfect non-dividing cells. The virus vector is large, has several sites for inserting genes, and is heat-stable. It has the properties to be stored at room temperature. A preferred embodiment is one in which the virus antigen is triggered. This is a viral vector that has been engineered to suppress the immune response of the host organism. Preferred vectors of this type carry the coding region of interleukin 8 or 10.

[0050] Viral vectors are more effective than chemical or physical methods for introducing genes into cells. It can have transaction (gene transfer) capabilities. Typically, viruses The vector consists of an unstructured early gene, a structural late gene, and RNA polymerase III transmutation. Photoproducts, inverted terminal repeats necessary for replication and capsid formation, and transcription of the viral genome. It contains a promoter that controls replication. When used as a vector, the virus Typically, one or more of the initial genes are removed, and the removed viral DNA is replaced by In addition, a gene or gene / promoter cassette is inserted into the viral genome. The construct can carry foreign genetic material up to approximately 8kb. The necessary function of the child is typically to manipulate the gene product of the initial gene to be expressed in trans. It is supplied by the created cell line.

[0051] (1) Retroviral vector Retroviruses are animal viruses belonging to the Retroviridae family, and are not limited to any particular type. Includes type, subfamily, genus, or orientation.

[0052] A retrovirus is essentially a package containing nucleic acids packed into a nucleic acid cargo. Cargo has a packaging signal, which indicates that the replicated daughter molecule is packaged. Ensure that it is efficiently packaged within the package. In addition to the package signal, Several molecules required in cis are present for the replication and packaging of replicated viruses. They exist. Typically, retroviral genomes are involved in the production of protein coats. It contains the g, pol, and env genes. It is typically introduced into target cells as an exogenous agent. These are the gag, pol, and env genes that are replaced by DNA. Retrovirus A vector is typically a packaging signal to be incorporated into the package coat. The sequence that signals the initiation of the gag transcription unit, and the sequence that binds to the tRNA primer for reverse transcription. Elements necessary for reverse transcription, including primer binding sites, and switches of the RNA strand during DNA synthesis. A guiding terminal repeat sequence, serving as a priming site for the synthesis of the second strand of DNA. A purine-rich sequence 5' for the functional 3'LTR, and a ray for insertion into the host genome. It contains a specific sequence near the end of the LTR that allows for the insertion of the trovirus's DNA state. By removing the ag, pol, and env genes, approximately 8kb of foreign sequences are removed from the virus. The process involves inserting the retroviral into the genome, reverse transcribing it, and packaging it into a new retroviral particle during replication. This allows for the delivery of one nucleic acid to many genes, depending on the size of each transcript. It is sufficient to do so. Within the insertion, along with other genes, there is a choice between positive or negative. It is preferable to include a marker.

[0053] The replication mechanism and packaging proteins in most retroviral vectors are Because they have been removed (gag, pol, and env), the vectors typically do not It is produced by placing it in a packaging cell line. Packaging cell lines are multiple It includes a manufacturing and packaging mechanism, but lacks any packaging signal, retro These are cell lines that have been transfected or transformed with a virus. They carry selected DNA. When the vector is transfected into these cell lines, helper cells cis The provided mechanism replicates the vector containing the target gene, and a new retrograde It is packaged into an Ilus particle. Lacking the necessary signal, the genome of the mechanism , not packaged. In one embodiment, the nucleic acid encoding TGF-b is lenticular It can be delivered via a ruth vector.

[0054] (2) Adenovirus vector The construction of a replication-defective adenovirus has been described (Berkner et al., J. Virology 61:1213-1220 (1987), Massie et al. 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 identity on of recombinant adenovirus by liposome -mediated transfection and PCR analysis” BioTechniques 15:868-872(1993). These viruses The advantage of using them as vectors is that they can replicate within the initially infected cells, Because they cannot form new infectious virus particles, they cannot spread to other cell types. This means that it is limited to the extent that it can be controlled. Recombinant adenoviruses affect the airway epithelium and liver cells. High after direct in vivo delivery to cells, vascular endothelium, CNS parenchyma, and several other tissue sites. It has been shown that efficient gene transfer can be achieved (Morsy, J. Clin. Inve st.92:1580-1586(1993), Kirshenbaum, J.Clin .Invest.92:381-387(1993), Roessler, J.Clin .Invest.92:1085-1092(1993), Moullier, Natu. re Genetics 4:154-159(1993), La Salle, Sci. ence 259:988-990 (1993), Gomez-Foix, J. Biol. .Chem.267:25129-25134(1992), Rich, Human G. ene Therapy 4:461-476(1993), Zabner,Natur. e Genetics 6:75-83(1994), Guzman,Circulat. ion Research 73:1201-1207(1993), Bout, Hum. an Gene Therapy 5:3-10(1994), Zabner,Cell 75:207-216 (1993), Caillaud, Eur. J. Neurosc. ience 5:1287-1291 (1993), and Ragot, J. Gen. Vi rology 74:501-507(1993). Recombinant adenoviruses are specific By binding to cell surface receptors, the virus achieves gene transfer, and subsequently, the virus becomes wild. By receptor-mediated endocytosis, in the same manner as with type or replication-defective adenoviruses. Internalized (Chardonnet and Dales, Virology 40: 462-477(1970), Brown and Burlingham, J. Vir. 12:386-396 (1973), Svensson and Pers. son, J. Virology 55:442-449 (1985), Seth, et al. al., J. Virol. 51:650-655 (1984), Seth, et al. ,Mol.Cell.Biol.4:1528-1533(1984), Varga e t al., J. Virology 65:6061-6070 (1991), Wick Ham et al., Cell 73:309-319(1993)).

[0055] The viral vector may be based on an adenovirus from which the E1 gene has been removed. These byrons are generated in cell lines such as the human 293 cell line. Another preferred fruit In this application, both the E1 and E3 genes are removed from the adenovirus genome.

[0056] (3) Adeno-associated virus vector Another type of viral vector is based on adeno-associated virus (AAV). Some parvoviruses can infect many cell types and are nonpathogenic to humans. Therefore, it is a preferred vector. AAV type vectors transport approximately 4-5 kb. This can occur, and wild-type AAV is known to stably insert into chromosome 19 (for example). (e.g., AAV integration site 1 (AAVS1)). This site contains site-specific integration characteristics. Vectors are preferred. The AAVs used are AAC1, AAV2, AAV3, AAV4. AAV5, AAV6, AAV7, AAV8, AAV9, and, for example, AAV-Rh74 Which recombinant (rAAV) and / or synthetic AAV (e.g., AAV-DJ, Anc80) AAV serotypes can be derived from any AAV serotype, including but not limited to these. The composition and method disclosed may be selected based on cell or tissue tropism. AAV vectors for use in this context are single-stranded (SS) or self-complementary (SC) vectors. It is possible.

[0057] In other types of AAV viruses, AAVs are cellularly specific, linked to heterologous genes. A pair adjacent to at least one cassette containing a promoter that directs heterologous expression It contains inverted terminal repeats (ITRs). In this context, heterogeneous refers to AAV or B19. This refers to any nucleotide sequence or gene that is not native to rubovirus.

[0058] Typically, the AAV and B19 coding regions are deleted, resulting in a safe, cytotoxic vector. AAV ITR, or its modifications, confer infectivity and site-specific integration. However, it does not confer cytotoxicity, and the promoter directs cell-specific expression.

[0059] Therefore, the disclosed vectors can be incorporated into mammalian chromosomes without substantial toxicity. It provides a DNA molecule that can do this.

[0060] Inserted genes in viruses and retroviruses typically enhance the expression of the desired gene product. It contains promoters and / or enhancers that help control the process. Generally, DNA functions when it is in a position fixed relative to the transcription start site. This is a sequence. The promoter is essential for the fundamental interaction between RNA polymerase and transcription factors. It may contain essential core elements, as well as upstream and response elements.

[0061] It is understood and intended herein that the packaging capacity of AAVs is limited. One way to overcome the load capacity of AV vectors is through the use of two vectors. The transgene is split between two plasmids, forming a 3' splice donor and a 5' splice donor. IS acceptors are used to ligate two sections of an introduced gene into a single full-length introduced gene. It is used. Alternatively, the two transgenes can be made by substantial duplication, homologous. Recombination involves binding two segments to a full-length transcript.

[0062] 2. Expression System Nucleic acids delivered to cells typically contain expression regulatory systems. For example, viruses and Inserted genes in retroviral systems typically control the expression of a desired gene product. It contains useful promoters and / or enhancers. Promoters are generally... This is a DNA sequence that functions when it is in a relatively fixed position relative to the start site. Promoters are core elements necessary for the fundamental interaction between RNA polymerase and transcription factors. It contains and may contain upstream elements and response elements. TGF-β (soluble or membrane-bound type) The promoter used to produce disclosed feeder cells expressing any of the following is It can be constitutive or inductive.

[0063] a) Virus promoters and enhancers Preferred promoters that control transcription from vectors within mammalian host cells are various Sources include, for example, polyoma, Simian virus 40 (SV40), adenovirus, and viruses such as trophovirus, hepatitis B virus, and most preferably cytomegalovirus From the Rus genome, or from a different mammalian promoter, such as the β-actin promoter. It can be obtained from. The early and late promoters of the SV40 virus This can be conveniently obtained as an SV40 restriction fragment that also contains the replication origin (Fiers et a l., Nature, 273:113 (1978). The first human cytomegalovirus The period promoter can be conveniently obtained as a HindIIIE restriction fragment (Greenwa y, PJet al., Gene 18:355-360 (1982). Of course. Promoters derived from host cells or related species are also useful herein. In one embodiment, TG Fb mRNA is expressed under the control of either the EF1A promoter or the CMV promoter. obtain.

[0064] Enhancers generally function even if they are not at a certain distance from the transcription start site, and they affect the transcription unit. and 5'(Laimins,L.et al.,Proc.Natl.Acad.Sci .78:993(1981)) or 3'(Lusky, ML, et al., Mol The DNA sequence that could be any of the following (Cell Bio.3:1108(1983)) It points to. Furthermore, enhancers are located within introns (Banerji, J. Let al., Cell 33:729(1983)), and within the code array itself (Osborne, T. F., et al., Mol. Cell Bio. 4:1293 (1984) These are also good. These are typically 10-300 bp in length and function in cis. Enhancers are It works to increase transcription from nearby promoters. Enhancers also Often, it contains response elements that mediate the regulation of transcription. Promoters mediate the regulation of transcription. It may also contain response elements. Enhancers often determine the regulation of gene expression. Many enhancer sequences are currently used in mammalian genes (globin, elastase, albumin). Although known from methyl, fetoprotein, and insulin, typically, common Currently, we use virus-derived enhancers from eukaryotic cells. A preferred example is the origin of replication. The late-stage SV40 enhancer (100-270 bp), early stage cytomegalovirus Promoter enhancer, late-stage polyoma enhancer of the origin of replication, and ad It is a novirus enhancer.

[0065] Promoters and / or enhancers are activated by light or specific chemicals that trigger their functions. It can be specifically activated by any of the following events. The system is tetracycline and dexa It can be adjusted using reagents such as methazone. Furthermore, it can be used for irradiation such as gamma ray irradiation. Exposure to or alkylating chemotherapeutic agents enhances gene expression in viral vectors. There are other methods as well.

[0066] In certain embodiments, the promoter and / or enhancer region is the transcription unit to be transcribed. Designed as a constitutive promoter and / or enhancer to maximize the expression of the region at a specific position. It can be used. In certain constructs, the promoter and / or enhancer regions are Even if it is expressed only in specific types of cells at specific times, all eukaryotic cell types It is active in this context. A preferred promoter of this type is the CMV promoter (650 (Base). Other preferred promoters include the SV40 promoter and the Cytomegalovirus. This is the LTR (full-length promoter) of the retroviral vector.

[0067] All specific regulatory elements have been cloned and are present in certain cell types, such as melanoma cells. It has been shown that it can be used to construct selectively expressed expression vectors. The glial fibrillary protein (GFAP) promoter is a gene that selects genes in glial-derived cells. It has been used for selective expression.

[0068] Furthermore, it can be used in eukaryotic host cells (yeast, fungi, insects, plants, animals, humans, or nucleated cells). The expression vector used contains sequences necessary for transcription termination that can affect mRNA expression. These regions may be included. These regions are the untranslated parts of mRNA encoding tissue factor proteins. At this point, it is transcribed as a polyadenylated segment. The 3' untranslated region contains the transcription terminus. The binding site is also included. The transcription unit preferably also contains a polyadenylated region. One of the advantages of this region is that the transcription units can be processed and transported like mRNA. The goal is to increase it. The identification and use of polyadenylation signals in expression constructs is It is well established. Homologous polyadenylation signals are used in the transgene construct. It is preferable that the polyadenylated region in a specific transcription unit is the SV40 initial poly It originates from a riadenylation signal and consists of approximately 400 bases. Furthermore, the transcription unit can be used alone or... This is a combination of the above sequences with other standard sequences that improve expression or stability from the construct. It is also preferable that it contains this ingredient.

[0069] b) Marker Viral vectors can contain nucleic acid sequences that encode marker products. Using the maker product, we can determine whether a gene is delivered to a cell and, once delivered, expressed. This determines the preferred marker gene, E.Coli lacZ gene (β-galact). It encodes sidase and is also known as green fluorescent protein.

[0070] In some embodiments, the marker may be a selectable marker. Mammalian cells Examples of suitable selectable markers include dihydrofolate reductase (DHFR) and thymidine. Kinase, neomycin, neomycin analog G418, hydromycin, and Puro Mycin is a selectable marker that has been successfully translocated to mammalian host cells. When transformed mammalian host cells are subjected to selective pressure, they can survive. Yes. There are two different categories of selective regimes that are widely used. The first is The category involves the use of mutant cell lines that lack cellular metabolism and the ability to grow independently of supplemental media. Based on this, two examples are CHO DHFR cells and mouse LTK cells. These cells have the ability to grow without the addition of nutrients such as thymidine or hypoxanthine. They lack. These cells lack specific genes necessary for the complete nucleotide synthesis pathway. Therefore, unless the missing nucleotide is provided in the supplement medium, it cannot survive. No. An alternative method for supplementing the culture medium is to add intact DHFR or to the cells lacking each gene. This involves altering those growth requirements by introducing the TK gene. DHFR or Individual cells that were not transformed with the TK gene could survive in the unsupplemented medium. It probably won't happen.

[0071] The second category refers to selection schemes used for any cell type, and the use of mutant cell lines. This is a form of dominant selection that does not require [something]. These schemes typically stop the growth of host cells. Drugs are used to stop it. Those cells with novel genes transmit drug resistance. It will express the protein and survive through selection. An example of such dominant selection is the drug Neo. Mycin, (Southern P. and Berg, P., J. Molec. App l.Genet. 1:327 (1982), mycophenolic acid, (Mulligan, RC and Berg, P. Science 209:1422 (1980)) or Hygromycin (Sugden, B. et al., Mol. Cell. Biol.) Use 5:410-413 (1985). Three examples show the controlled development of eukaryotic cells. Using bacterial genes, the appropriate drug G418 or neomycin (geneticin) is used. It transmits resistance to XGPT (mycophenolic acid) or hygromycin. Other examples include... It contains omycin analog G418 and puramycin.

[0072] 3. Delivery of pharmaceutical carriers / medicines As described above, the composition can also be administered in vivo in a pharmaceutically acceptable carrier. Yes, it is possible. "Pharmacologically acceptable" means that it does not cause any undesirable biological effects. without interacting in a deleterious manner with any of the other components of the pharmaceutical composition or coming into contact therewith a material that is not biologically or otherwise undesirable, i.e., a nucleic acid or a material that can be administered to a subject together with a vector. Carriers are well known to those skilled in the art and can necessarily be selected so as to minimize any degradation of the active ingredient and minimize any adverse side effects in the subject when used.

[0073] The composition can be administered locally, including by oral, parenteral (e.g., intravenous), intramuscular injection, intraperitoneal injection, transdermal, ex vivo local nasal administration or inhalation. As used herein, "local nasal administration" means delivering the composition to the nose and nasal cavity through one or both nostrils and can include delivery by a nebulizer or droplet mechanism or by aerosolization of the nucleic acid or vector. Administration of the composition by inhalation can be effected through the nose or mouth via delivery by a nebulizer or droplet mechanism and can also include direct delivery to any region of the respiratory system (e.g., the lungs) via an endotracheal tube. The exact amount of the composition required varies for each subject depending on the species, age, weight, and general condition of the subject, the severity of the allergic disorder being treated the particular nucleic acid or vector used, its mode of administration, etc. Thus, it is not possible to specify an exact amount for all compositions However, an appropriate amount can be determined by one skilled in the art using only routine experimentation providing the teachings of this specification . Parenteral administration of the composition, when used, generally features injection. Injectables are liquid solutions or suspensions, solid forms suitable for solution of the suspension in a liquid prior to injection, or emulsions

[0074] ​ It can be prepared in the conventional form as one of the following. The recently revised parenteral approach This involves the use of sustained-release or prolonged-release formulations to maintain a constant dosage. For example, see See U.S. Patent No. 3,610,795 incorporated herein by reference.

[0075] The material is in a solution or suspension (e.g., incorporated into microparticles, liposomes, or cells). These may also be antibodies, receptors, or receptor ligands that target specific cell types. This can be targeted. The following references are books on targeting specific proteins in tumor tissue. This is an example of the use of the technology (Senter, et al., Bioconjugate Che m.,2:447-451,(1991),Bagshawe,KD,Br.JC ancer,60:275-281,(1989),Bagshawe,et al., Br. J. Cancer, 58:700-703, (1988), Senter, et al. al.,Bioconjugate Chem.,4:3-9,(1993), Batt elli,et al.,Cancer Immunol.Immunother.,3 5:421-425, (1992), Pietersz and McKenzie,I. mmunolog.Reviews,129:57-80,(1992), and Roff ler,et al.,Biochem.Pharmacol,42:2062-206 5, (1991)). Vehicles, e.g., "Stealth" and other antibody-conjugated lipo Somesomes (including lipid-mediated drugs that target colon cancer), D through cell-specific ligands Receptor-mediated targeting of NA, lymphocyte-specific tumor targeting, and in vivo mouse neural Highly specific therapeutic retroviral targeting of glioma cells. The following references are for tumor tissue. This is an example of using this technology to target specific proteins (Hughes et al.) ., Cancer Research, 49:6214-6220, (1989), and Litzinger and Huang, Biochimica et Biophy sica Acta, 1104:179-187, (1992). Generally, receptors are, They are involved in either constitutive or ligand-induced endocytosis pathways. The cells cluster within clathrin-coated pits and then travel through clathrin-coated vesicles into cells. It enters the cell, passes through acidified endosomes where receptors are selected, and is then recycled to the cell surface. It is either processed, stored within the cell, or degraded in lysosomes. Internal transport pathways include nutrient uptake, activated protein removal, and macromolecule clearance. opportunistic entry of viruses and toxins, dissociation and degradation of ligands, and regulation of receptor levels. They perform various functions such as cell type, receptor concentration, ligand type, etc. Depending on the Gand titer and ligand concentration, it follows two or more intracellular pathways. Receptor-mediated enzyme The molecular and cellular mechanisms of docytosis are outlined (Brown and Green). e,DNA and Cell Biology 10:6,399-409(1991 ))

[0076] a) A pharmaceutically acceptable carrier This antibody-containing composition can be used therapeutically in combination with a pharmaceutically acceptable carrier. can.

[0077] Suitable carriers and their formulations are described in Remington: The Science and d Practice of Pharmacy(19th ed.)ed.A.R.G ennaro,Mack Publishing Company,Easton,PA It is described in 1995. Typically, to make the formulation isotonic, an appropriate amount of pharmaceutically acceptable salt is used in the formulation. Examples of pharmaceutically acceptable carriers include, but are not limited to, physiological saline, Ringer's solution, and dextrose solution. The pH of the solution is preferably about 5 to about 8, more preferably about 7 to about 7.5. Further carriers include sustained-release preparations such as semipermeable matrices of solid hydrophobic polymers containing antibodies, and this matrix is in the form of a shaped article, such as a film, liposome, or microparticle. It will be apparent to those skilled in the art that a particular carrier may be more preferred depending, for example, on the route of administration and the concentration of the composition being administered.

[0078] Pharmaceutical carriers are known to those skilled in the art. These are most typically standard carriers for drug administration to humans, including solutions such as sterile water at physiological pH, physiological saline, and buffer solutions. These compositions can be administered intramuscularly or subcutaneously. Other compounds will be administered according to standard procedures used by those skilled in the art.

[0079]

[0080] The pharmaceutical composition may also include carriers, thickeners, diluents, buffers, preservatives, surfactants, etc. in addition to the selected molecule. The pharmaceutical composition may also include one or more active ingredients such as antimicrobial agents, anti-inflammatory agents, anesthetics, etc. This pharmaceutical composition depends on whether local or systemic treatment is desired and the area to be treated. Depending on the method, it may be administered in several ways. Administration may be local (including in the eyes, vagina, rectum, and nasal cavity). , by oral, inhalation, or parenteral administration, for example, by intravenous infusion, subcutaneous, intraperitoneal, or intramuscular injection. The procedure may be carried out intravenously, intraperitoneally, intramuscularly, subcutaneously, intracavitarially, or percutaneously. The disclosed antibodies may be administered intravenously, intraperitoneally, intramuscularly, subcutaneously, intracavitarially, or percutaneously. It can be administered as a target.

[0081] Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. This includes non-aqueous solvents such as propylene glycol, polyethylene glycol, and olive oil. It is a vegetable oil such as cereal oil, and an injectable organic ester such as ethyl oleate. The carrier contains physiological saline and a buffer medium, as well as water, alcoholic / aqueous solutions, and emulsions. , or suspension. Parenteral vehicles include sodium chloride solution, Ringer's dextrin. It contains rose, dextrose, sodium chloride, lactated Ringer's solution, or a fixative oil. Intravenous vehicles include fluids and nutritional supplements, and electrolyte supplements (such as Ringer's dextrose). This includes preservatives and other additives, such as antimicrobial agents and antioxidants. Chelating agents and inert gases may also be present.

[0082] Preparations for topical administration include ointments, lotions, creams, gels, intravenous infusions, suppositories, and sprays. Aerosols, liquids, and powders may be included. Conventional pharmaceutical carriers, aqueous, powder, or oily bases. A thickening agent or similar may be necessary or desirable.

[0083] Compositions for oral administration include powders or granules, suspensions or solutions in water or a non-aqueous medium, Contains capsules, sachets, or tablets. Includes thickeners, flavorings, diluents, emulsifiers, and dispersing agents. Alternatively, a binder may be preferred.

[0084] Some of these compositions contain hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, and sulfuric acid. Acids, and inorganic acids such as phosphoric acid, as well as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, Organic acids such as pyruvic acid, oxalic acid, malonic acid, succinic acid, maleic acid, and fumaric acid By the reaction of, or by the aerosol of sodium hydroxide, ammonium hydroxide, potassium hydroxide, etc. Organic bases, as well as mono, di, trialkyl, and arylamines, and substituted ethanolamines. As a pharmaceutically acceptable acid or base addition salt formed by reaction with organic bases such as n. It may be administered as such.

[0085] b) Therapeutic use The effective dosage and schedule for administering this composition can be determined empirically. Therefore, making such a decision falls within the scope of the art of those skilled in the art. For the administration of this composition The dosage range is large enough to produce the desired effect that affects the symptoms of the disorder. Yes. The dosage may affect adverse side effects, such as undesirable cross-reactions and anaphylactic reactions. It should not be so large as to cause such problems. Generally, the dosage should be adjusted according to the patient's age. , condition, sex, and severity of the disease, route of administration, or whether other drugs are included in the regimen The dosage will vary depending on the factors, which can be determined by a person skilled in the art. In the event of any contraindications, the dosage may be adjusted by the individual physician. It may be administered in doses once or twice daily for one or several days. Guidelines for appropriate dosages of these medications can be found in the literature. For example, Guidance on selecting the appropriate dosage for antibodies can be found in the literature on the therapeutic use of antibodies. For example, Handbook of Monoclonal Antibodies, Fe rrone et al., eds., Noges Publications, Par. k Ridge, NJ, (1985) ch.22 and pp.303-357, Smith et al., Antibodies in Human Diagnos is and Therapy, Haber et al., eds., Raven P. This can be found in Ress, New York (1977), pp. 365–389. The typical daily dose of an antibody used alone is approximately 1 per day, depending on the factors mentioned above. The range may be from μg / kg body weight to a maximum of 100 mg / kg body weight or more.

[0086] Method for generating C.TGF-β resistant NK cells As stated throughout, the primary purpose of the disclosed feeder cells is to provide TGF-β to The objective is to create resistant NK cells. Therefore, in one embodiment, as disclosed herein, This includes the manipulated feeder cells, plasma membrane particles, or exotypes disclosed herein. TGF-β-resistant NK cells, including incubation of NK cells in the presence of TGF-β. This is a method for generating a TGF-β expression. For example, disclosed herein is a method for generating a TGF-β expression Manipulated feeder cells (PBMC, RPMI8866, HFWT, K562 cells, EBV-LCL, NK cells (NK-92, ) transfected with membrane-bound IL-21 NK-92MI, NK-YTS, NK, NKL, KIL, KIL C.2, NK 3.3 (including but not limited to NK-YS), transfected with membrane-bound 4-1BBL NK cells (NK-92, NK-92MI, NK-YTS, NK, NKL, KIL) (including, but not limited to, KIL C.2, NK 3.3, and NK-YS), membrane binding NK cells transfected with type IL-15 and 4-1BBL (NK-92, NK- 92MI, NK-YTS, NK, NKL, KIL, KIL C.2, NK 3.3, NK -YS (including but not limited to these), or membrane-bound IL-21 and 4-1BBL Specified NK cells (NK-92, NK-92MI, NK-YTS, NK, N This includes, but is not limited to, KL, KIL, KIL C.2, NK 3.3, and NK-YS. Incubating NK cells in the presence of (i) (including, but not limited to) , or in the presence of plasma membrane particles or exosomes derived from the aforementioned feeder cells, NK A method for generating TGF-β resistant NK cells, which includes incubating cells. Preferably, the TGF-β expressed by feeder cells is membrane-bound.

[0087] In one embodiment, disclosed herein is a feeder cell having a small amount on its cell surface. Each contains one additional NK cell effector agent, and at least one additional NK cell effector The inhibitor agent is a cytokine, adhesion molecule, or NK cell activator (e.g., 4-1BBL). , IL-2, IL-12, IL-15, IL-18, IL-21, MICA, LFA-1 , 2B4, CCR7, OX40L, UBLP2, BCM1 / SLAMF2, NKG2D Gonist, CD155, CD112, Jagged1, Jagged2, Delta-1 , Pref-1, DNER, Jedi, SOM-11, wingless, CCN3, M AGP2, MAGP1, TSP2, YB-1, EGFL7, CCR7, DAP12, and DAP10, Notch ligand, NKp46 agonist, NKp44 agonist, NK p30 agonist, other NCR agonists, CD16 agonist, and other NK cell effector agents (including, but not limited to, etc.), a method for generating TGF-β-resistant NK cells is provided. In one embodiment, at least one additional NK cell effector agent is IL-21, 4 -1BBL, IL-15, IL-21 and 4-1BBL, IL-21 and IL-15, or IL-15 and 4-1BBL. Preferably, at least one additional NK cell e ffector agent is a membrane-bound NK cell effector agent.

[0088] The method for generating TGF-β-resistant NK cells disclosed herein is understood to be applicable to any NK cells (exogenous or endogenous) for which resistant TGF-β is desired, and is contemplated herein. Accordingly, what is disclosed herein is that the NK cells are NKG2C , C D56 + NK cells, CD56 NK cells, peripheral NK cells, NK T bright cells, or tumor-infiltrating NK cells (including, but not limited to, NK cells obtained from cell lines or NK cells obtained from donor sources (such as autologous donors, allogeneic donors, or syngeneic donors, etc.)), a method for generating TGF-β-resistant NK cells including memory-like NK cells dim

[0089] To produce TGF-β-resistant NK cells, NK cells need to be exposed to feeder cells, exosomes, or plasma membrane particles that express TGF-β (on their membrane or soluble) for a certain period of time to confer resistance. Accordingly, in one embodiment, what is disclosed herein is NK cells, in the presence of manipulated feeder cells, plasma membrane particles, or exosomes, Even without them, 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 This is a method for generating TGF-β resistant NK cells through incubation for several days. Cells are plasma membrane particles derived from manipulated feeder cells or the aforementioned feeder cells. It is further understood that after exposure to exosomes, they may be cultured for an additional period, as specified herein. This is intended to be done. In one embodiment, NK cells are manipulated feeder cells or the aforementioned feeder - Cell-derived plasma membrane particles or exosomes, and for 7 to 21 days, preferably 7 to 1 They can be in contact for 4 days.

[0090] As described throughout, the disclosed method produces TGF-β resistant NK cells. To accomplish. Therefore, in one aspect, what is disclosed herein is the T disclosed herein. These are TGF-β resistant NK cells produced by a method for generating GF-β resistant NK cells. .

[0091] In some cases, plasma membrane particles or exoskeletons derived from manipulated feeder cells. The material can be obtained by nitrogen cavitation.

[0092] Generally, cells are maintained under conditions suitable for cell growth and / or maintenance. Suitable cells The culture conditions are well known in the relevant field, for example, Santiago et al., P roc.Natl.Acad.Sci.USA,2008,105:5809-5814 ,Moehle et al.Proc.Natl.Acad.Sci.USA,200 7,104:3055-3060, Urnov et al., Nature, 2005 435:646-651, and Lombardo et al., Nat. Biote It is described in chnol.,2007,25:1298-1306. The cell culture method is known in the relevant art, and may change depending on the cell type. I understand that deafness is a problem. In all cases, optimization of routine tasks is used, and specific This allows us to determine the best technology for each cell type.

[0093] D. Methods of treating cancer The compositions disclosed herein may be used to treat any disease in which uncontrolled cell proliferation occurs, such as cancer. Therefore, the TGF disclosed herein is - Treatment of cancer and / or metastasis in the subject, including administration to β-resistant NK cells Methods for inhibiting, reducing, decreasing, improving, and / or preventing. For example, as disclosed herein. The cells being targeted are TGF-β resistant NK cells, and they are responsible for cancer and / or metastases in the target (e.g., solid A method for treating, inhibiting, reducing, decreasing, improving, and / or preventing tumors, etc., using NK cells Obtaining cells, feeder cells (PBMCs) that have been engineered to express TGF-β. , RPMI8866, HFWT, K562 cells, EBV-LCL, membrane-bound IL-21 Transfected NK cells (NK-92, NK-92MI, NK-YTS, NK, This includes, but is not limited to, NKL, KIL, KIL C.2, NK 3.3, and NK-YS. (None), NK cells transfected with membrane-bound 4-1BBL (NK-92, NK- 92MI, NK-YTS, NK, NKL, KIL, KIL C.2, NK 3.3, NK -Including but not limited to YS, membrane-bound IL-15 and 4-1BBL Sfected NK cells (NK-92, NK-92MI, NK-YTS, NK, NKL) (Includes, but is not limited to, KIL, KIL C.2, NK 3.3, and NK-YS) , or NK cells transfected with membrane-bound IL-21 and 4-1BBL (NK- 92, NK-92MI, NK-YTS, NK, NKL, KIL, KIL C.2, NK 3.3, including but not limited to NK-YS) Culturing in the presence of TGF-β to produce TGF-β resistant NK cells, or the aforementioned process Culturing in the presence of plasma membrane particles or exosomes derived from the lead cells, and The method involves administering TGF-β-resistant NK cells as the target. Preferably, fee TGF-β expressed by Dar cells is membrane-bound.

[0094] Feeder cells develop TGF-β resistance, and also exogenous NK cell populations (e.g., NK G2C + CD56 bright NK cells, CD56 dim NK cells, peripheral NK cells Activates memory-like NK cells such as NK T cells or tumor-infiltrating NK cells and / Or, not only to increase the number of cells, but also to induce TGF-β resistance in the endogenous NK cell population, and / or Endogenous NK cell population (e.g., NKG2C) + CD56 bright NK cells, CD5 6 dim Memory of NK cells, peripheral NK cells, NK T cells, or tumor-infiltrating NK cells In order to activate and / or proliferate (such as --type NK cells), the disclosed therapeutic method It is understood and intended herein that it may be used in the treatment of cancer. The NK cells that may be used may include any NK cells derived from a donor or NK cell line. In this specification, NK cells are disclosed as NKG2C + CD56 bright NK cells, CD56 dim NK cells, peripheral NK cells, NK T cells, or tumor-infiltrating NK cells K cells (NK cells obtained from cell lines, or donor sources (e.g., autologous donors, allogeneic donors)) This includes, but is not limited to, NK cells obtained from a donor or a similar donor. Memory-like NK cells, such as those mentioned above, treat, inhibit, reduce, decrease, or improve cancer and / or metastasis. , and / or a method of prevention. In one embodiment, NK cells are engineered feeder cells and / or plasma membrane particles or exo These may be endogenous NK cells that become resistant in vivo through exposure to osomes.

[0095] Feeder cells, plasma membrane particles and / or exophytes used in the disclosed therapeutic methods The osome may further contain additional effector agents for proliferation and / or activation of NK cells. Therefore, in one embodiment, disclosed herein is a feeder cell, that cell The surface further comprises at least one additional NK cell effector agent, and at least one Additional NK cell effectors include cytokines, adhesion molecules, or NK cell activators (e.g.) For example, 4-1BBL, IL-2, IL-12, IL-15, IL-18, IL-21, M ICA, LFA-1, 2B4, CCR7, OX40L, UBLP2, BCM1 / SLAM F2, NKG2D agonist, CD155, CD112, Jagged1, Jagged 2, Delta-1, Pref-1, DNER, Jedi, SOM-11, wingle ss, CCN3, MAGP2, MAGP1, TSP2, YB-1, EGFL7, CCR7 DAP12 and DAP10, Notch ligand, NKp46 agonist, NKp4 Includes 4 agonists, NKp30 agonists, other NCR agonists, and CD16 agonists. These include NK cell effector agents, which are not limited to these, and are used to treat cancer and / or metastasis. A method of treating, inhibiting, reducing, decreasing, improving, and / or preventing. In one embodiment, at least Another additional NK cell effector is IL-21, 4-1BBL, IL-15, I L-21 and 4-1BBL, IL-21 and IL-15, or IL-15 and 4-1BB Contains L. Preferably, the aforementioned cytokine, adhesion molecule, or NK cell activator is human These are cytokines, adhesion molecules, or NK cell activators. Preferably, at least one The additional NK cell effector is a membrane-bound NK cell effector.

[0096] In one embodiment, disclosed herein is a modified feeder cell, shaped by NK cells. In the presence of membrane particles or exosomes, at least 7, 8, 9, 10, 11, 12, 1 3, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 Cancer and / or a method of treating, inhibiting, reducing, decreasing, improving, and / or preventing metastasis.

[0097] The compositions disclosed herein may be used to treat any disease in which uncontrolled cell proliferation occurs, such as cancer. It is possible to treat with the disclosed compositions, which are representative but not limited. The following is a list of typical cancers: lymphoma, B-cell lymphoma, T-cell lymphoma Mycosis fungoides, Hodgkin's disease, myeloid leukemia, bladder cancer, brain cancer, nervous system cancer, head and neck cancer Squamous cell carcinoma of the head and neck, lung cancer such as small cell lung cancer and non-small cell lung cancer, neuroblastoma Cystoma / glioblastoma, ovarian cancer, skin cancer, liver cancer, melanoma, oral, pharyngeal, laryngeal cancer, and Squamous cell carcinoma of the lung, cervical cancer, cervical cancer Cervical carcinoma, breast cancer, as well as epithelial cancer, kidney cancer, urinary carcinoma Reproductive cancer, lung cancer, esophageal cancer, head and neck cancer, colorectal cancer, hematopoietic cancer, testicular cancer, colon cancer, Rectal cancer, prostate cancer, or pancreatic cancer.

[0098] E. Examples The following examples illustrate to those skilled in the art the compounds, compositions, articles, and devices claimed herein. Provided to offer a complete disclosure and explanation of the methods for preparing and evaluating the S and / or methods. This is intended to be purely illustrative and does not limit the disclosure. No. Efforts are made to guarantee accuracy regarding numerical values ​​(for example, quantity, temperature, etc.). However, some error and deviation should be taken into consideration. Unless otherwise specified, parts ( Parts are the weight in parts, temperature is °C or ambient temperature, and pressure is atmospheric pressure or its It is nearby.

[0099] 1. Example 1: K expressing mbIL21, CD137L, and secreted or mbTGFβ This generates a genetically modified feeder cell clone consisting of 562 cells. Multiple feeder cell lines that overexpress human TGFβ1 are K562-based feeder - In cell clone CSTX002, as a membrane-bound mutant protein (mbTGFβ), This is achieved by transducing a lentiviral vector that expresses TGFβ1 in its naturally secreted form. It was generated as follows: The CSTX002 clone contains the cytokine IL-21 and the costimulatory molecule 4 -1BBL(CD137) expresses a membrane-bound mutant protein. In short, human The mbTGFβ1 fusion construct, under the regulatory control of the human CMV promoter, is found in the CD4 transmembrane region. Region and IgG4(F C Using human TGFβ1 fused to stalk, produced in silico The constructs were then subjected to codon optimization and validation for sequence homology. Similarly, human T Using GFβ, a bisistronic mc-TGFβ vector was generated, and CMV promotion Independent expression of the fluorescent protein mcherry under ointment is observed in human elongation factor-1 (E It is under the control of the F1A promoter. Two constructs (mbTGFβ and mc-TGFβ) A detailed vector map showing the design of both structures is shown in Figure 1. It was cloned into the third-generation lentiviral system used to generate Rus.

[0100] Lentiviral transduction efficiency may vary between cell lines, and the initial results for CSTX002 were... Transduction is induced by a lentivirus that codes for green fluorescent protein (GFP). Therefore, using a control lentivirus that codes for GFP, three different viral forces were tested. The susceptibility to co-infection was examined using titers (multiple degrees of infection (MOI) of 5, 10, and 20) (Figure 2). Good GFP expression was observed under each condition 48 hours after transfection. CS Infection with TX002 was performed prior to the TGFβ vector at MOI 5, 10, and 20. Then, five days after infection, the transduced cells were treated with human TGFβ1 antibody (Biolegen catalog number The samples were stained with (No. 349615) (Figure 3). Optimal TGFβ expression was achieved using the vector (mbTGFβ). Since it was observed at 20 MOI in cells infected with either (or mc-TGFβ), All subsequent experiments were conducted using 20 MOI. mbTGFβ or mc-TGF To generate a clone that overexpresses β, CSTX002 was subjected to two wisps as described above. The cells were infected with Russ. After infection, the cells were cultured for 4-5 days. mbTGFβ cells were cultured with TGFβ Stain with antibody 1, sort using BD sortware program, and sort with BD Influx. Positive cells were directly sorted into 96-well plates. mc-TGFβ-positive cells were used. We used a green laser (561nm) to sort and collect mcherry-positive cells. Single cell clones were collected from 96-well plates, and mbTGFβ was analyzed using FACS. Alternatively, mc-TGFβ expression was determined (Figure 4). In addition, mRNA expression was determined for hTGFβ1 Quantitative real-time PCR (RT-PCR) is performed using TaqMan primers. This was determined. Compared to untransfected cell controls, mc-TGFβ cells In cells, TGFβ mRNA expression increased 5-20 times, and in mbTGFβ cells, it increased 50- It increased 400-fold (Figure 5).

[0101] 2. Example 2: Proliferation of NK cells on these novel feeder cells is observed in TGFβ-resistant NK cells. We demonstrate the ability to produce resistant cells and describe the characteristics of these resistant cells. Next, new feeder cells transduced with mbTGFβ and mc-TGFβ were introduced. We determined whether intact TGFβ cells were secreted. mbTGFβ and mc-TGFβ cells were tested. Seeds were sown in a 96-well plate, and after 16 hours of incubation, the supernatant was collected and -8 Stored at 0°C. TGFβ1 ELISA kit (R&D catalog DB100B) was used. Then, the level of TGFβ1 was determined. All clones from both expression vectors showed T It was found that it secretes GFβ. The level was approximately 500 for mc-TGFβ cells. The concentration is 0 pg / ml, while for mbTGFβ cells it is 10,000-15,000 pg / ml. It was within the range of l (Figure 6).

[0102] TGFβ acts as an antitumor agent for NK cells by activating downstream targets Smad2 / Smad3. It is an immunosuppressive molecule that weakens function. Soluble T14 during NK cell proliferation in CSTX002 The addition of GFβ1 resulted in a significant reduction in Smad3 expression in NK cells, which indicates that These cells over-secrete cytokines, reducing sensitivity to TGFβ1 signaling. The newly transduced clones can induce similar remodeling from NK cells. To determine whether it is possible, NK cells from three healthy donors were used in normal CSTX 002, using CSTX002 having soluble TGFβ1, or novel TGFβ1 It was grown together with the current CSTX002. In short, feeder cells were divided into 100 Gray NK cells isolated from PBMCs of healthy blood donors, irradiated with (Gy), were treated with low-dose IL. Irradiated feeder cells were grown for 2 weeks in the presence of -2 (50 IU / ml). The NK cells that proliferated together with mbTGFβ and mc-TGFβ feeder cells had an odor. A similar reduction in Smad3 expression levels was observed (Figure 7).

[0103] Next, N cells proliferated together with mbTGFβ and mc-TGFβ transduced feeder cells. K cells are functionally similar to NK cells cultured with soluble TGFβ and CSTX002. It was determined whether or not this was happening. After proliferation, NK cells were treated with calcein-loaded HOS (osteosarcoma) or These cells were co-cultured with DAOY (medulloblastoma) target cells in a 5:1 ratio for 4 hours. Calcein release The mean cell lysis percentage was determined using 10 ng / mL soluble human TGF. NK cells treated with β1(sTGFβ) served as a positive control. As shown in Figure 8, Feeder cells mbTGFβ, NK cells proliferating with mc-TGFβ, or soluble T NK cells that proliferated in the presence of GFβ were compared with NK cells that proliferated together with control feeder cells. In comparison, it significantly reduced the ability to lyse target tumor cells HOS and DAOY. This matches NK cells that have been imprinted with TGFβ.

Claims

1. Modified feeder cells that express soluble or membrane-bound TGF-β. Cell.

2. The TGF-β is expressed by an inducible or constitutive promoter, according to claim 1. Manipulated feeder cells.

3. The feeder cells include leukemia K562 cells, and the operation described in claim 1 or 2 feeder cells.

4. The manipulated feeder according to claim 1 or 2, wherein the feeder cells include NK cells. -Cell.

5. The cell surface further comprises at least one additional NK cell effector agent, At the very least, one additional NK cell effector agent can stimulate cytokines, adhesion molecules, or NK cells. A modified feeder cell according to any one of claims 1 to 3, which is a cell activator.

6. The aforementioned at least one additional NK cell effector agent is 4-1BBL, IL-2, I L-12, IL-15, IL-18, IL-21, MICA, LFA-1, 2B4, CC R7, OX40L, UBLP2, BCM1 / SLAMF2, NKG2D Agonist, CD 155, CD112, Jagged1, Jagged2, Delta-1, Pref-1 , DNER, Jedi, SOM-11, wingless, CCN3, MAGP2, MA GP1, TSP2, YB-1, EGFL7, CCR7, DAP12, and DAP10, N och ligand, NKp46 agonist, NKp44 agonist, NKp30 agonist The operation according to claim 5, selected from other NCR agonists and CD16 agonists. Feeder cells that were processed.

7. The aforementioned at least one additional NK cell effector agent is IL-21, 4-1BBL, IL-15, IL-21 and 4-1BBL, IL-21 and IL-15, or IL-15 The manipulated feeder cells according to claim 5, comprising 4-1BBL.

8. The aforementioned feeder cells include PBMC, RPMI8866, HFWT, K562 cells, and EB V-LCL, NK cells transfected with membrane-bound IL-21, membrane-bound 4-1B NK cells transfected with BL, membrane-bound IL-15, and 4-1BBL Transfected NK cells, or membrane-bound IL-21 and 4-1BBL Processed feeder cells according to any one of claims 1 to 7, comprising processed NK cells Cell.

9. Plasma granules derived from manipulated feeder cells according to any one of claims 1 to 8 Child or exosome.

10. NK cells are modified feeder cells according to any one of claims 1 to 8 or Incubation in the presence of exosomes or plasma membrane particles as described in item 9 Includes a method for generating TGF-β resistant NK cells.

11. NK cells are incubated in the presence of feeder cells engineered to express TGF-β. vaping, or plasma membrane particles or exosole derived from the feeder cells This includes incubating NK cells in the presence of TGF-β to develop TGF-β resistant NK cells. How to generate it.

12. The aforementioned feeder cells include PBMC, RPMI8866, HFWT, K562 cells, and EB V-LCL, NK cells transfected with membrane-bound IL-21, membrane-bound 4-1B NK cells transfected with BL, membrane-bound IL-15, and 4-1BBL Transfected NK cells, or membrane-bound IL-21 and 4-1BBL A method for generating TGF-β resistant NK cells according to claim 11, comprising NK cells.

13. The feeder cells have at least one additional NK cell effector on their cell surface. - Further comprising an agent, the at least one additional NK cell effector agent is cytokine The TGF-β resistance according to claim 11 or 12, which is an adhesion molecule or an NK cell activator. A method for generating NK cells.

14. The aforementioned at least one additional NK cell effector agent is 4-1BBL, IL-2, I L-12, IL-15, IL-18, IL-21, MICA, LFA-1, 2B4, CC R7, OX40L, UBLP2, BCM1 / SLAMF2, NKG2D Agonist, CD 155, CD112, Jagged1, Jagged2, Delta-1, Pref-1 , DNER, Jedi, SOM-11, wingless, CCN3, MAGP2, MA GP1, TSP2, YB-1, EGFL7, CCR7, DAP12, and DAP10, N och ligand, NKp46 agonist, NKp44 agonist, NKp30 agonist T, selected from other NCR agonists, CD16 agonists, as described in claim 13. A method for generating GF-β resistant NK cells.

15. The aforementioned at least one additional NK cell effector agent is IL-21, 4-1BBL, IL-15, IL-21 and 4-1BBL, IL-21 and IL-15, or IL-15 A method for generating TGF-β resistant NK cells according to claim 14, comprising 4-1BBL.

16. The aforementioned NK cells, NKG2C ＋ CD56 ｂｒｉｇｈｔ NK cells, CD56 ｄｉｍ Memory-like NK cells such as NK cells, peripheral NK cells, NK T cells, and tumor-infiltrating NK cells. Producing TGF-β resistant NK cells according to any one of claims 11 to 15, including cells. method.

17. The NK cells are obtained from a donor subject, as described in any one of claims 11 to 16. A method for generating TGF-β resistant NK cells.

18. Claims that the NK cells are obtained from an autologous donor, an allogeneic donor, or a syngeneic donor. A method for generating TGF-β resistant NK cells as described in any one of items 11 to 16.

19. The NK cells are present in the presence of the manipulated feeder cells, plasma membrane particles, or exosomes. Under these conditions, at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 1 ８、１９、２０、２１、２２、２３、２４、２５、２６、２７、２８、２９、３０、４５ or a T according to any one of claims 11 to 18, which is incubated for 60 days. A method for generating GF-β resistant NK cells.

20. TGF-β resistant N prepared by the method described in any one of claims 10 to 19 K cells.

21. A method for treating cancer in a subject, wherein the subject is subjected to the TGF- described in claim 20. A method comprising administering β-resistant NK cells.

22. A method for treating cancer in a target with TGF-β resistant NK cells, wherein NK cells are obtained. The cells are cultured in the presence of feeder cells that have been modified to express TGF-β. Nourish the cells and thereby produce TGF-β resistant NK cells, or use the feeder cells Culturing in the presence of plasma membrane particles or exosomes, and the aforementioned target A method comprising administering the TGF-β resistant NK cells to the patient.

23. The aforementioned NK cells, NKG2C ＋ CD56 ｂｒｉｇｈｔ NK cells, CD56 ｄｉｍ Memory-like NK cells such as NK cells, peripheral NK cells, NK T cells, and tumor-infiltrating NK cells. A method for treating cancer according to claim 22, wherein the cancer is a cell.

24. The NK cells are obtained from a donor subject and are used to treat cancer according to claim 22 or 23. How to do it.

25. Claims that the NK cells are obtained from an autologous donor, an allogeneic donor, or a syngeneic donor. A method for treating any of the cancers described in items 22 to 24.

26. The aforementioned feeder cells include PBMC, RPMI8866, HFWT, K562 cells, and EB V-LCL, NK cells transfected with membrane-bound IL-21, membrane-bound 4-1B NK cells transfected with BL, membrane-bound IL-15, and 4-1BBL Transfected NK cells, or membrane-bound IL-21 and 4-1BBL A method for treating cancer according to any one of claims 22 to 25, comprising NK cells. 。

27. The feeder cells have at least one additional NK cell effector on their cell surface. - Further comprising an agent, the at least one additional NK cell effector agent is cytokine The following is a claim according to any one of claims 22 to 26, which is an adhesion molecule or an NK cell activator. How to treat it.

28. The aforementioned at least one additional NK cell effector agent is 4-1BBL, IL-2, I L-12, IL-15, IL-18, IL-21, MICA, LFA-1, 2B4, CC R7, OX40L, UBLP2, BCM1 / SLAMF2, NKG2D Agonist, CD 155, CD112, Jagged1, Jagged2, Delta-1, Pref-1 , DNER, Jedi, SOM-11, wingless, CCN3, MAGP2, MA GP1, TSP2, YB-1, EGFL7, CCR7, DAP12, and DAP10, N och ligand, NKp46 agonist, NKp44 agonist, NKp30 agonist The following is a selection from other NCR agonists and CD16 agonists as described in claim 27. How to treat it.

29. The aforementioned at least one additional NK cell effector agent is IL-21, 4-1BBL, IL-15, IL-21 and 4-1BBL, IL-21 and IL-15, or IL-15 A method for treating a solid tumor according to claim 28, comprising 4-1BBL.

30. The NK cells are present in the presence of the manipulated feeder cells, plasma membrane particles, or exosomes. Under these conditions, at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 1 ８、１９、２０、２１、２２、２３、２４、２５、２６、２７、２８、２９、３０、４５ or the one described in any one of claims 22 to 29, which is incubated for 60 days. How to treat it.

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