Manipulated natural killer cells, and methods for using them in immunotherapy and autophagy inhibition techniques.

Abstract

We provide polynucleotide constructs and multifunctional engineered natural killer (NK) cells that express such constructs for the treatment of cancer, particularly glioblastoma. [Solution] A nucleotide construct is provided comprising a first sequence encoding at least a first binding domain or fragment thereof, which includes a natural killer (NK) activating receptor or a first protein specific to a first cancer-associated antigen, and a second sequence encoding at least a second binding domain or fragment thereof and a cleavable linker, wherein the second binding domain is specific to an adenosine-producing cell surface protein or an adenosine intermediate-producing cell surface protein of a target cell, and the cleavable linker is operatively linked to the first binding domain.

Description

Technical Field

[0001] Priority Claim This application claims the benefit of priority from U.S. Provisional Patent Application No. 62 / 923,644 filed on October 21, 2019 in the name of Matosevic et al. and claims the benefit of priority to this application. The contents of the aforementioned application are hereby incorporated by reference in their entirety into the present disclosure herein.

[0002] The present disclosure generally relates to the field of cancer therapy and treatment, and more particularly to multi-functional immunotherapy utilizing engineered natural killer (NK) cells that have multiple anti-tumor functions and are designed to target the main driving factors of cancer resistance to therapy, particularly glioblastoma resistance.

Background Art

[0003] Glioblastoma (GBM) is by far the most common and aggressive malignant type of primary brain tumor in adults and children, and is one of the most difficult to treat. GBM patients tend to respond poorly to traditional treatments, and GBM has the worst prognosis among all central nervous system malignant tumors. With advanced diagnostic methods as well as aggressive single and multi-faceted treatment options such as surgery, chemotherapy, radiation therapy, small molecule inhibitors, and the use of multiple types of anti-neoplastic agents, survival rates have improved only modestly over the past decades , and the median survival is approximately one year. In fact, all conventional therapies tested to date have failed to improve the overall survival of GBM patients in phase III clinical trials, and there is no effective treatment for GBM. The reason for this failure is multifactorial.

[0004] Firstly, GBM is inherently highly invasive and aggressive, and the treatment for GBM is, This is difficult due to its biological location in the brain, the blood-brain barrier, and the nerve parenchyma. This, along with the complex interactions between various cells within the tumor and the cells surrounding the tumor, Heterogeneity of GBM is designated as one of the main causes of treatment resistance and malignant tumor recurrence. Each of the known GBM subtypes (classical, mesenchymal, nervous, and preneurial) is , diverse genetic and epigenetic cells associated with individual and variable cellular plasticity It exhibits a peculiar texture.

[0005] Glioma stem-like cells (GSCs) are a subpopulation of GMB cells. This also contributes to treatment resistance. GSCs exhibit self-regenerative capacity, pluripotency, and the induction of tumorigenesis. This can be a driving force supporting glioma development, resistance to therapy, and invasive recurrence. It is becoming increasingly recognized that, at least in part, conventional therapies are not effective in targeting specific GSC subgroups. This is due to the inability to eliminate the group. However, these cells are conventional GBM Poor reproducibility with Dell cell lines (including U87MG) hinders the GMB study.

[0006] GBM progression is a highly controlled catabolic regulator of the cellular energy balance. It is also promoted by autophagy. Under normal conditions, cells undergo basal-level autophagy. Using this technology, we can maintain biological functions, homeostasis, quality control of cellular contents, and old cells. It helps to eliminate protein and damaged organelles. However, in cancer cells, Artophagy can promote tumorigenesis by stimulating cancer cell proliferation and tumor growth. In fact, autophagy is involved in GBM metabolism, survival in hypoxia, progression, and This may support resistance to therapy. Beclin, encoded by the BECN1 gene. -1 plays a central role in promoting autophagy.

[0007] Additionally, GBM may participate in antigen evasion or promote immunosuppression, thus affecting multiple immune responses. It triggers checkpoints. One of these is ATP to anti-inflammatory adenosine. Multiple cell types that mediate the gradual hydrolysis of ADP and are upregulated in GBM. Ecto-5 is a surface enzyme expressed on (both infiltrating immune cells and tumor cells). '-Nucleotidase (CD73). CD73 works together with CD39 to adenosynovitis It induces the production of extracellular adenosine from 5'-triphosphate (ATP). Adenosine is Next, it binds to adenosine receptors on natural killer (NK) cells, and NK cell activity This leads to serious immunometabolic dysregulation. Thus, it is extremely important to maintain homeostasis of the immune system. Importantly, immunosuppression mediated by the adenosinergic pathway is The GBM is hijacked.

[0008] In addition to the above, avoiding treatment with GBM has a significant effect on the body's innate immune system. Extremely immunosuppressive, causing both metabolic and functional cascades that suppress the receptor. It is also promoted by the hypoxic tumor microenvironment (TME). Insufficient angiogenesis (vascularzati Pathophysiological conditions of hypoxia and ischemia, such as those found in tumor TMEs caused by (on) This is a critical factor for adenine nucleotides such as ATP and adenosine diphosphate (ADP). It also promotes metabolic changes. Under normal physiological conditions, ATP is localized in intracellular compartments; however, However, the level of extracellular ATP (and therefore adenosine) is associated with hypoxia, ischemia, and malignant tumors. It increases significantly in response to the situation and defines the characteristics of the tumor environment. For example, intratumor extracellular AT P concentrations can be up to 1,000 times higher than those in normal tissue from cells of the same origin. These conditions lead to dysregulation of NK cells, and therefore suppression of NK cell antitumor surveillance and immunity. This contributes to the invasiveness of the tumor, which in turn promotes its growth.

[0009] As a key effector of innate immunity, NK cells are unique and play a crucial role in cancer immune surveillance. It performs a crucial function. The major histocompatibility complex (MHC) presented on the surface of infected cells is not simply Unlike T cells, which are detected, NK cell function depends on the balance between activating and inhibitory receptors. This is promoted, and through this, they interact with pathogens and MHC classes on cancer cells. It recognizes molecule I. NK cells, without prior sensitization, are subjected to various abnormal cells or stress. It can eliminate unwanted cells and even preferentially kill stem-like cells or cancer stem cells. When NK cells form immune synapses, they release cytokines that induce cell lysis. However, GBM employs various tactics to delay and alter the immunosuppressive pathway, and This stops the process and prevents malignant cells from being recognized as dangerous or foreign. These mechanisms prevent cancer from being eliminated by the immune system, leading to a loss of control over tumor growth. This can lead to failure and allow the disease to progress to a fatal state from a very early stage.

[0010] In parallel with GBM-induced inhibition of NK cell response function, downregulation or mutation of target antigens occurs. It is commonly observed in BM and is a contributing factor to immune evasion and resistance to treatment. Including chimeric antigen receptors (CARs) that are targeted to the primary source or programmable for tumor sensing, The previous adoptive T-cell therapy strategy has been preclinically evaluated as a countermeasure against such evasion. Although valued, GBM employs a mechanism that goes beyond antigen escape to avoid targeting. Primal avoidance leads to a decrease in the efficacy of cell-based antigen-specific monotherapy and differential splicing. This can be caused by mechanisms including singular, missense mutations, or lineage switching. It is induced by using most GBM-specific and GBM-related antigens. In all previous clinical studies, proliferation of antigen-avoidance variants has been observed. [Overview of the Initiative] [Problems that the invention aims to solve]

[0011] There is an urgent need for new and effective treatment options for GBM. Multiple complex mechanisms are involved. Recreating NK cell loss of function in this context presents a significant challenge to conventional therapies. However, it also requires a larger presence of NK cells specifically within the tumor tissue, and More meaningful clinical responses may be initiated. Conventional methods have been successful in these areas. Therefore, the ability to enhance GBM tumor targeting and increase NK cell recruitment to TME is Yes, and through it, NK cell dysfunction may be relieved, making it a commercially viable treatment option for GBM. There is a need to develop a method that is both practical and safe. [Means for solving the problem]

[0012] This disclosure describes the development of a novel immunotherapy targeting multiple immune evasion mechanisms in GBM. The invention is described below. The composition, system, and method involve simultaneously targeting two or more GBM antigens. Genetic engineering designed to target and simultaneously release antibodies that block CD73 activity. For the first time, natural killer (NK) cells are combined. At least one embodiment In this disclosure, the present invention relates to this cell-based immunotherapy and small molecule autophagy inhibitors, for example ...and further combine with chloroquine without limitation. Autophagy in this background The use of G-inhibitors further inhibits GBM, as well as further penetrates and affects the overall GBM. This leads to the secretion of chemokines that attract NK cells, enhancing the effectiveness of the therapy. The compositions, systems, and methods of this disclosure may restrict GBM evasion from immune surveillance and tumor This can promote the recruitment of NK cells, thus leading to a sustained anti-GBM response.

[0013] Polynucleotide constructs are provided herein. In at least one embodiment In such a structure, at least a second binding domain or fragment thereof At least a first binding domain or so which is operably concatenated to a second sequence that encodes Includes a first sequence encoding a fragment of the first binding domain or its fragment The nucleotide contains an NK-activated receptor, or a first protein specific to the first cancer-associated antigen. Furthermore, the second binding domain or fragment thereof is located on the adenosine-producing cell surface of the target cell. It is specific to proteins or adenosine intermediate-producing cell surface proteins of target cells. The first domain is a hinge domain (for example, linker or sperm, without limitation). (P2A) one or more self-cleaving peptides (e.g., P2A) , E2A, F2A, T2A), or both may be further coded as an option. Second combination The domain / fragment is cleavable linker operationally linked to the first binding domain. - Further includes. In a particular embodiment, the cleavable linker is a target cell and / or cleavage by one or more proteases present in the tumor microenvironment (TME). It can be configured (without limiting) lysosomal cysteine ​​proteases serine proteases (trypsin, etc.), aspartate protease, threonine pro Such proteases include theases and matrix metalloproteinases. (Widely known and understood in the technical field). At least one exemplary example. In the application form, the second binding domain is specific to CD73, CD39, or CD38. It contains antibody fragments and may optionally contain single-chain antibody fragments (scFv).

[0014] Exemplary embodiments of the structure include at least a third binding domain or fragment thereof. It further includes a third sequence encoding the following: This includes an NK-activated receptor or a second protein specific to a second cancer-associated antigen. For example, the first binding domain or its fragment is specific to the first cancer-associated antigen. It may contain protein, and the third binding domain or fragment thereof is an NK-activating receptor ( For example, without limitation, natural killer group 2 member D receptor (NKG2 D) may contain NKp30, NKp46, NKp40, or DNAM-1). In one exemplary embodiment, the first sequence is at least 90% identical to sequence number 7. The first amino acid is one, and the third sequence is at least 90% identical to sequence number 6. It codes for the second amino acid, which is [the second amino acid].

[0015] The cancer-related antigens of this disclosure (with respect to one of the binding domains or fragments or (Multiple may be specific) Without limitation, disialoganglioside (isia loganglioside (GD2), ganglioside G3 (GD3), Her2 p185), CD19, CD20, CD56, CD123, CD22, CD30, CD3 3, CD171, CS-1, C-type lectin-like molecule-1; EpCAM, G250, proteo Glycan, GD3, GD2, MHC II, TAG-72, milk mucin core protein Lewis A antigen, tyrosine protein kinase transmembrane receptor (ROR1), c-met, Epidermal growth factor receptor (EGFR), EGFR variant III, and / or carcinoembryonic antigen ( It may contain one or more of the CEAs. Furthermore, the first cancer-associated antigen is one It may contain the antigen of the first type, while the second cancer-related antigen may be the same or a different type, as desired. It may contain the antigen of Ip.

[0016] In at least one exemplary embodiment, a first and / or third binding domain Or the fragment contains an extracellular ligand-binding domain including NKG2D, The respective sequences are superior to NKG2D expression in wild-type NK cells. Further encodes regulated expression. Additionally or alternatively, a second binding domain or The fragment is linked to scFv and contains anti-CD73, anti-CD39, or anti-CD3 8 may include, and the first and / or third binding domain or fragment thereof is scF It may contain anti-GD2 linked to v.

[0017] Furthermore, the first sequence may be expressed in the first chimeric antigen receptor (CAR). The second sequence is expressed in the second CAR. Furthermore, the target cells are cancer cells, TM Malignant cells in E, and in at least one exemplary embodiment, the target cells are These are glioblastoma cells or glioblastoma TMEs.

[0018] In at least one exemplary embodiment, one or both of the first and third arrays. When activated, one or more signatures promote cytotoxicity or cytolytic activity. It may additionally encode a signaling domain. Therefore, such a signaling domain is The binding domain or fragment thereof, which is operationally linked to it, binds to target cells. And it is activated. One or more signaling domains are activated in the immunoglobulin γ-Fc region. Regional receptor III-A (FcγRIIIA), cluster of differentiation antigens 28 (CD28), tumor necrosis factor receptor superfamily member 9 (TNFRSF9) (or 4-1BB), tumor necrosis factor receptor superfamily member 4 (TNFRSF) 4 or OX40), Fas ligand (FasL), TNF-related apoptosis-inducing ligand TRAIL, DNAX-activated protein 10 (DAP10), DNAX-activated protein Protein 12 (DAP12), native cytotoxic receptor NKp46, native cytotoxic receptor N Kp44, innate cytotoxic receptor NKp30, lymphocyte function-associated antigen 1 (LFA-1), Differentiation antigens 244 (CD244), CD137, CD3 zeta (CD3ζ), and NK cells. The group may be selected from those consisting of G2D-DAP10 receptor complexes. In one embodiment, one of the signal transduction domains encoded by the first sequence The multiple DAP10s are one of the signaling domains encoded by the second sequence. One or more of these are CD3ζ.

[0019] Manipulated cells or manipulated cell lines are also provided herein. In one embodiment, any one of the polynucleotide constructs disclosed herein is generated Manipulated cells or manipulated cell lines are provided. Furthermore, manipulated cells Alternatively, the manipulated cell line has a first binding domain or its f encoded thereby Hinds that are operationally linked to and located between the lagment and signaling domains It may include a first sequence that further codes for the zidomain. At least one exemplary implementation. In this state, the manipulated cells or cell line are at least 80%, 85%, of SEQ ID NO: 8. Alternatively, they express amino acid sequences that are 90% identical.

[0020] Other embodiments provide a pharmaceutical composition comprising a population of manipulated cells of the present disclosure. In such embodiments, one or more signaling domains are operatively linked thereto The bound domain or fragment thereof may, for example, without limitation, be a cancerous Alternatively, it can be activated when it binds to target cells such as glioblastoma tumor cells. Here, the pharmaceutical composition , pharmaceutically acceptable carriers, and / or preservatives, fillers, disintegrants, wetting agents, emulsifiers , suspending agents, sweeteners, flavoring agents, fragrances, antibacterial agents, antifungal agents, smoothing agents, and dispensing agents (di Spensing agent, etc., any pharmaceutically acceptable diluent, adjuvant, excipient, or This may include a vehicle (depending on the mode of administration and the nature of the dosage form).

[0021] Methods for treating individuals with cancer using immunotherapy are also provided. In at least one embodiment, such a method involves at least: NK-activated receptor , or a first binding domain containing a first protein specific to a first cancer-associated antigen or This includes its fragment, as well as the second binding domain or its fragment, and cleavage. A polynucleotide construct encoding a possible linker, wherein the second binding domain is It is specific to adenosine-producing or adenosine intermediate-producing cell surface proteins of target cells. The cleavable linker is operationally linked to the first binding domain, polynucleate The therapeutic dose of a pharmaceutical composition containing a population of engineered NK cells expressing an otide construct The process includes the step of administering the substance to an elephant or the step of leaving the substance administered to the elephant. In a particular embodiment, The polynucleotide constructs are specific to NK-activated receptors or a second cancer-associated antigen. It may further encode a third binding domain or fragment thereof containing a second protein. Therefore, the first binding domain or its fragment is specific to the cancer-associated antigen. It may contain protein 1, and the third binding domain or fragment thereof is NK activation receptor The substance and the third binding domain or fragment thereof (and vice versa) The polynucleotide construct, upon activation, can cause cytotoxicity or necrosis of manipulated NK cells. To promote cytolytic activity, the first and / or third binding domains / fragments It may additionally encode one or more operationally linked signaling domains.

[0022] The step of administering (or keeping in administration) a therapeutically effective dose of the pharmaceutical composition is performed intravenously. It can be administered intratumorally, parenterally, or by injection. Additionally or alternatively, as a treatment. The step of administering or keeping an effective amount of the pharmaceutical composition to the target is adoptive cell therapy. This may include steps to be implemented or have been implemented.

[0023] Such a method is at least 80%, 85%, or 90% identical to sequence number 8. A population of manipulated NK cells that express specific amino acid sequences can be employed.

[0024] In at least one embodiment, the cancer may be glioblastoma. Additionally or alternatively The first binding domain or its fragment is a first protein specific to GD2. It may include a third binding domain or fragment thereof, and the extracellular ligament containing NKG2D It may include a binding domain, and the third binding domain or fragment thereof may contain CD73. It may be specific to CD39 or CD38.

[0025] Other methods of combination therapy are also provided. In at least one embodiment, the method is O The step of administering or administering additional therapeutic measures including tophagy inhibitors The process may further include steps. Such autophagy inhibitors are, for example, limited. Without therapeutically effective doses of small molecule inhibitors and / or genes in the autophagy pathway This can be due to genetic downregulation (e.g., BECN1, p62, β-actin, ATG5, ATG7, LC3B, ATG12, ATG16L1, PI3K-III, ULK1, UL (e.g., K2, FIP200, and / or LAMP2). Not limited to such small molecule inhibitors. Typical examples include chloroquine, hydroxychloroquine, spautin-1, and SAR4. 05, vertoprofin, and any currently known or subsequently discovered This may include other pharmaceutical autophagy inhibitors or downregulators. At least one example. In an exemplary embodiment, the autophagy inhibitor comprises chloroquine, which is optionally 0 It can be administered at concentrations ranging from 0.01 μM to 200 μM.

[0026] If the method described herein includes combination therapy, a therapeutically effective amount of the pharmaceutical composition is administered to the target. The steps (or administer) are performed intravenously, intratumorally, parenterally, or by infusion. The steps of administering or keeping in place additional therapeutic measures that may be performed are systemic. It can be administered by injection or infusion.

[0027] A kit for treating subjects experiencing glioblastoma is also provided. For example, and limited Without being used, such a kit contains a therapeutically effective amount of the pharmaceutical composition of this disclosure; and treatment An effective dose of an autophagy inhibitor (a small molecule inhibitor or other type of pharmaceutical inhibitor, or something similar) (whether or not), or a composition that achieves genetic downregulation of the autophagy pathway It is possible. If the autophagy inhibitor in the kit includes a small molecule inhibitor, at least 1 In one embodiment, the inhibitor is chloroquine, hydroxychloroquine, and spautin-1 The group may be selected from SAR405 and verteporfin.

[0028] The embodiments disclosed herein, as well as other features, advantages, and aspects contained herein, Furthermore, to achieve these, see the various exemplary embodiments of this disclosure. This will become clear from the detailed explanation below. Such a detailed explanation is in the attached drawings. It will be better understood when considered in conjunction with this. [Brief explanation of the drawing]

[0029] [Figure 1-1] This figure illustrates the results of correlation analyses of gene expression in glioblastoma (GBM) patient data, surface expression of CD73, GD2, and NKG2DL in patient-derived GBM, and the design of multifunctional NK-based GBM immunotherapy; sub-section A shows correlations between normalized gene expression (FPKM) of selected genes using data from 156 GBM patients (Pearson correlation coefficients are shown using continuous gradient colors); sub-section B shows correlations between normalized gene expression (FPKM) of the entire NK gene set and individual genes (correlations are expressed as normalized enrichment scores (NES)); sub-section C shows GBM patients with high expression of at least one of the four identified genes: NT5E, B4GALNT1, MICA, and MICB. A Venn diagram showing the number of cells is shown; subsection D shows a bar graph showing the patient distribution of gene expression in GBM tumors based on the four genes identified in subsection C; subsection E shows data on the surface expression of CD73, GD2, and NKG2DL on various types of patient-derived GBM cells, including SJ-GBM2 (juvenile), GBM43 (primary), and GBM10 (recurrent), as determined by flow cytometry (results are reported as change factor (FC) compared to control; data are shown as mean ± SEM); and subsection F shows a schematic diagram representing at least one embodiment of the multifunctional tumor-responsive engineered NK cells of this disclosure and their operating mechanisms (TAP represents tumor-associated proteases; αCD73 scFv represents anti-CD73 scFv; αGD2 scFv represents anti-GD2 scFv). [Figure 1-2] (As stated above.) [Figure 1-3] (As stated above.) [Figure 1-4] (As stated above.) [Figure 2] These are schematic data on the effects of adenosine on pNK cell viability and activation marker expression; sub-section A shows schematic data on the cell viability (%) of pNK cells after 24 hours of treatment with 1000 μM adenosine; and sub-section B shows schematic data on NKG2D expression on pNK cells after 24 hours of treatment with 1000 μM adenosine (data are shown as mean ± SEM). [Figure 3] This figure shows in silico modeling of the structure of a particular construct of the present disclosure; subsection A shows a molecular model of serial anti-GD2 and anti-CD73 scFv extracellular domains linked together via a GS spacer and a cleavable peptide linker (identified by arrows), the model obtained based on vector sequences using RaptorX; and subsection B shows the docking of the extracellular scFv domain with GD2 (indicated by dashed circles), the docking performed using PatchDock and refined in FireDock using the model of the entire extracellular region shown in subsection A and the cleavable peptide indicated by arrows (image produced in Chimera). [Figure 4] This is a schematic representation of at least one embodiment of a multifunctional structure according to at least one embodiment of the present disclosure. [Figure 5-1]This disclosure provides schematic data on the creation of multifunctional genetically engineered NK cells; subsection A shows a schematic representation of the transgenes to the NKG2D.DAP10.CD3ζ-CAR construct (construct 1B) according to this disclosure, targeting the NKG2D ligand (NKG2DL); subsection B shows a schematic representation of the structure of construct 1B; subsection C shows schematic data on the cell viability (%) of NK cells engineered to express construct 1B according to this disclosure (NKG2D.CAR-NK92) or unengineered control (NK92) 48 hours after transfection; subsection D shows NKG2D expression as determined by flow cytometry on the transfected cells in subsection C; subsection E shows GBM43 expression at an E / T ratio of 5 over 4 hours. Subsection C shows data on in vitro cytotoxicity of cells (NKG2D.CAR-NK92) and untransfected NK-92 cells (NK92) against cells; subsection F shows a schematic representation of the transgene structure representing the anti-GD2.CD28.CD3ζ-CAR construct (construct 1A) according to this disclosure, which targets GD2; and subsection G shows a schematic representation of the structure of construct 1A (anti-GD2.CD28.CD3ζ-CAR); subsection H shows the cell viability (%) of NK-92 cells engineered to express construct 1A (GD2.CD28.CD3ζ-CAR) or unengineered control (NK92) 48 hours after transfection; subsection I shows anti-GD2 as determined by flow cytometry on transfected cells of subsection H. scFv expression is observed; subgroup J cells show a 5 E / T ratio over 4 hours, compared to subgroup H cells (anti-GD2.CD28).The data shows in vitro cytotoxicity of CD3ζ-CAR-NK92) and unmodified NK-92 control; subsection K shows a schematic representation of the transgene representing the complete multifunctional construct 1 according to this disclosure, construct 1 comprises two CARs isolated by P2A, the first CAR having CD28 and CD3ζ signaling domains targeting GD2 and linked to anti-CD73 scFv by a tumor-associated protease-cleavable linker which, upon local release, inhibits or instead blocks adenosine production from GBM, the second CAR construct comprises NKG2D-DAP10-CD3ζ, targeting the NKG2D ligand and being GBM tumor-responsive; subsection L represents tumor-responsive anti-CD73 This shows a schematic representation of the scFv secretion bispecific CAR (construct 1); subsection M shows data on cell viability (%) of NK-92 cells engineered to express the entire multifunctional construct 1 48 hours after transfection; subsection N shows NKG2D expression determined by flow cytometry 48 hours after transfection; subsection O shows anti-CD73 scFv and anti-GD2 on NK cells determined by flow cytometry after proteolytic cleavage at uPA releasing anti-CD73 scFv fragments Subsection P shows scFv expression; subsection Q includes flow cytometry data indicating the purity of isolated peripheral blood-derived NK (pNK) cells (CD56+CD3-); subsection Q shows data on the cell viability (%) of pNK cells engineered to express the entire construct after two rounds of lentiviral transduction; subsection R shows NKG2D expression on engineered pNK cells as determined by flow cytometry after two rounds of lentiviral transduction; subsection S shows schematic data on the expression of anti-CD73 scFv and anti-GD2 scFv on pNK cells as determined by flow cytometry after two rounds of lentiviral transduction; and subsection T shows untransduced pNK and CD73 in NK MACS® medium.This depicts representative data for the growth rate of mCAR-NK cells; (The data shown through subdivisions P-T pertain to pNK cells isolated in triplets from representative donors; the data in Figure 5 are shown as mean ± SEM. *p<0.05, **p<0.0). [Figure 5-2] (As stated above.) [Figure 5-3] (As stated above.) [Figure 5-4] (As stated above.) [Figure 5-5] (As stated above.) [Figure 5-6] (As stated above.) [Figure 6] This figure shows schematic data related to the investigation of primary NK cells; subcategories A-D and E-H each show data for pNK cells isolated from two separate donors, and characterization and expression measurements were examined as described in relation to Example 1 (data are shown as mean ± SEM; *p<0.05, **p<0.0). [Figure 7-1]These are schematic data regarding the in vitro effector activity of multifunctional genetically engineered NK cells against patient-derived GBM according to at least one embodiment of this disclosure; subsection A shows in vitro cytotoxicity of NK-92 (control) and CD73.mCAR-NK92 cells (expressing construct 1) against GBM43 cells, expressed E / T ratio over 4 hours; subsection B shows a bar graph representing degranulation [% CD107 and (MFI)CD107] of NK-92 and CD73.mCAR-NK92 cells after 4 hours of co-culture with GBM43 cells (E / T ratio, 5:1) (NK cells were analyzed by flow cytometry for surface CD107a expression as a marker of degranulation); subsection C shows IFN-γ production (% %) by NK-92 and CD73.mCAR-NK92 cells after 4 hours of co-culture with GBM43 cells (E / T ratio, 5:1) Sub-section D shows in vitro cytotoxicity of CD73.mCAR-NK92 and CD73.mCAR-NK92 (after aCD73 scFv cleavage) cells against GBM43 cells at the displayed E / T ratio over 4 hours; Sub-section E shows CD73 activity of GBM43 cells after incubation with cleaved aCD73 scFv after release from uPA-treated CD73.mCAR-NK92 cells; Sub-sections F-H show in vitro cytotoxicity of pNK and CD73.mCAR-pNK cells against various GBM cells, including SJ-GBM2, GBM43, and GBM10 cells, at the displayed E / T ratio over 4 hours; Sub-section I shows degranulation (%) of pNK and CD73.mCAR-pNK cells after 4 hours of co-culture with GBM43 cells (E / T ratio, 5:1) Sub-section J shows the IFN-γ production (% IFN-γ) of pNK and CD73 mCAR-pNK cells after 4 hours of co-culture with GBM43 cells (E / T ratio, 5:1); sub-section K shows the E / T ratio of pNK and CD73 compared to GBM43 cells over 4 hours.This shows in vitro cytotoxicity of mCAR-pNK (aCD73 scFv cleavage) cells; subsection L shows cleaved aCD73 cells after cleavage from uPA-treated CD73.mCAR-NK cells. The data shows CD73 activity in GBM43 cells after incubation with scFv; subcategory M shows a relative decrease in CD16 expression (%) of MFI on pNK and CD73.mCAR-pNK cells after 12 hours of co-culture with GBM43 cells (E / T ratio, 5:1); subcategory N shows a relative increase in NKG2A expression (%) of MFI on pNK and CD73.mCAR-pNK cells after 12 hours of co-culture with GBM43 cells (E / T ratio, 5:1); and subcategories O and P show in vitro cytotoxicity of pNK and CD73.mCAR-pNK cells against non-malignant neuronal cell lines hCMEC / D3 and HCN-2 at the indicated E / T ratio over 4 hours (data shown through subcategories F-P relate to at least three triplets of isolated pNK cells from representative donors; data are shown as mean ± SEM; *p<0.05, **p<0.01). . [Figure 7-2] (As stated above.) [Figure 7-3] (As stated above.) [Figure 8] This shows a schematic diagram of the reaction used to detect phosphate formation by CD73 (sub-section A), and a standard phosphate curve in a 96-well plate (sub-section B) obtained by reading the OD value at 620 nm after 30 minutes of incubation. [Figure 9] Live imaging microscope images (taken using IncuCyte S3) of co-cultures of GBM43 cells and pNK or CD73.mCAR-pNK cells (pNK cells expressing construct 1 of the present disclosure), where small, irregularly shaped circular dots in the image represent NK cells and large, spindle-shaped cells represent GBM43 cells (scale bar = 200 μm). [Figure 10]This figure shows data on the surface expression of CD73, GD2, and NKG2DL on hCMEC / D3 and HCN-2 cells. The expression levels of the three markers on normal brain cell lines were determined by flow cytometry and reported as the change ratio (FC) compared to the control; data are shown as mean ± SEM. [Figure 11] This is a schematic representation of the inhibition of autophagy in GBM cells by either BECN1 gene knockdown or pharmacological treatment with CQ; subsection A shows data from GBM43 cells transfected with BECN1 shRNA(h) lentiviral particles or treated with 50 μM CQ for 24 hours, after which the cells were lysed and the expression levels of BECN1, LC3B, and p62 were analyzed by flow cytometry (using β-actin as a loading control); and subsection B shows the quantification of the relative expression levels of BECN1 (BECN1 / β-actin), LC3B (LC3B / β-actin), and p62 (p62 / β-actin) (it should be noted that beclin 1 (BECN1), LC3-II (LC3B), and p62 are the three main markers of autophagy). [Figure 12-1]This figure shows data on the effects of targeting autophagy in GBM on NK cell function and homing; sub-section A represents the viability of GBM43 cells after 24 hours of treatment with various concentrations of CQ in vitro; sub-section B shows cell viability (%) plotted against log [CQ concentration (μM)] with respect to IC50 calculated using Prism 5 (GraphPad Software Inc., California); sub-sections C and D show CCL5 and CXCL10 in GBM43 cells transfected with BECN1 shRNA lentivirus (BECN1-GBM43) or in cells treated with CQ. Subsections E and F show mRNA expression (results are reported as change factor (FC) compared to an untransfected control); subsections E and F show ELISA quantification of CCL5 and CXCL10 in the supernatant of GBM43 cells after 24 hours of treatment with CQ, in or out of the presence of various inhibitors including LY294002, BAY11-7082, and SP600125; subsection G outlines an in vitro experimental bilayer BBB model representing the experimental setup used to evaluate the effect of CCL5 or CXCL10 on pNK cell migration across the BBB; subsection H shows the migration of pNK cells through the experimental BBB; subsection I shows data on the in vitro viability of GBM43 and BECN1- GBM43 cells as determined by the CCK-8 assay; subsection J outlines an experimental design to evaluate the effect of targeting autophagy by BECN1 knockdown on tumor growth and NK cell invasion (i.e., GBM43 cells with Rag1- / -Injected into the right flank of the mouse, and BECN1-GBM43 cells injected into the left flank; Subsection K shows data from tumor growth monitored and recorded on the day of display;Subsection L shows images of immunohistochemical (IHC) staining of NK cells (top row), CCL5 (middle row), or CXCL10 (bottom row) performed on the shown tumor sections using anti-NKp46, anti-CCL5, and anti-CXCL10 antibodies, respectively (bars = 50 μm; 200 × magnification); subsection M shows quantification of NK cells infiltrating control and BECN1-tumors, with cell counts recorded at 200 × magnification in four consecutive high-magnification fields (HPFs); subsections N and O graph the IHC scores of CCL5 and CXCL10 in control and BECN1-tumors (Note: Data shown in subsection H pertain to at least three triplets of isolated pNK cells from one representative donor); data are shown as mean ± SEM; *p<0.05, **p<0.01. [Figure 12-2] (As stated above.) [Figure 12-3] (As stated above.) [Figure 13] This is a schematic representation of chemokine expression in response to autophagy inhibition in GBM cells. CCL2 and CXCL12 mRNA expression in BECN1-GBM43 or CQ-treated GBM43 cells was determined by RT-PCR, with GAPDH used as the reference gene (control). Results are reported as FC compared to the control; data are shown as mean ± SEM; *p<0.05, **p<0.01. [Figure 14] This figure illustrates the use of an ELISA array to determine the concentrations of various types of chemokines in GBM43 culture medium; subsection A shows a schematic diagram of the entire experimental process; and subsection B shows the determined concentrations of each chemokine examined. [Figure 15]Subsection A shows immunohistochemical (IHC) staining of mouse (Rag1- / -)NK cells performed on GBM43 (control) subcutaneous xenograft tumor sections; and subsection B shows IHC staining of mouse (Rag1- / -)NK cells performed on BECN1-GBM43 subcutaneous xenograft tumor sections, where IHC staining indicates enhanced NK cell infiltration into both the peripheral and internal regions of the tumor tissue of BECN1-GBM43 subcutaneous xenograft tumors. [Figure 16] This is schematic data on the sensitization of GBM cells to NK cell-mediated killing after targeting of autophagy; sub-section A shows NKG2DL expression (MFI and %) on GBM43 cells after 24 hours of treatment with various concentrations of CQ; sub-section B shows CD73 expression (MFI and %) on GBM43 cells after 24 hours of treatment with various concentrations of CQ; sub-section C shows CD73 activity on GBM43 cells after 24 hours of treatment with various concentrations of CQ; sub-section D shows GD2 expression (MFI and %) on GBM43 cells after 24 hours of treatment with various concentrations of CQ; sub-section E shows the pNK cell-killing activity against GBM43 cells after 24 hours of treatment with various concentrations of CQ; sub-section F shows BECN1- after 4 hours of co-incubation at an E / T ratio of 5. This shows the toxic activity of NK92 and pNK cells against GBM43 cells; all data in Figure 16 are shown in at least three sets from one representative donor; data are shown as mean ± SEM; *p<0.05, **p<0.01. [Figure 17-1]This figure shows the anti-GBM activity of the multifunctional engineered NK cells of this disclosure in a GMB43 xenograft model; subsection A shows a schematic diagram illustrating the in vivo treatment program; subsection B shows schematic data on tumor growth in individual treatment groups, including PBS, CQ, pNK cells, and CD73.mCAR-pNK cells expressing construct 1 of this disclosure (tumor size was determined by caliper measurement); subsection C shows schematic data on the mean tumor weight of mice in each treatment group after necropsy 28 days after the start of treatment; subsection D graphs the change in mouse body weight in each group during the treatment period; subsection E shows NK cells (bright dots) and cleaved anti-CD73 cells performed on the shown tumor sections in each treatment group using anti-NKp46 antibody and protein L. The image shows immunofluorescence (IF) staining of scFv (indicated by the arrow) (bar = 50 μm; 200 × magnification); subsection F shows IHC staining of CD73 performed on the shown tumor sections using anti-CD73 antibody in various treatment groups (bar = 100 μm; 200 × magnification); subsection G graphs the IHC scores of CD73 expression performed on the shown tumor sections in various treatment groups; subsections H and I show IHC staining of CD73 expression in various treatment groups using anti-CCL5 and anti-CXCL10 antibodies, respectively. The displayed tumor sections show IHC staining for CCL5 (sub-section H) and CXCL10 (sub-section I) (bar = 50 μm; 200 × magnification); sub-section J shows the IHC scores for CCL5 or CXCL10 performed on the displayed tumor sections in various treatment groups (Note: The data exemplified in this study pertain to at least three sets of isolated pNK cells from one representative donor; data are shown as mean ± SEM; *p < 0.05, **p < 0.01). [Figure 17-2] (As stated above.) [Figure 18]This figure shows data related to the creation of luciferase-expressing GBM43 (GBM43-Luc) cells. GBM43 cells were transfected with commercial luciferase (Luc) lentivirus particles expressing the firefly luciferase 3 gene under an inducible suCMV promoter. After transduction, puromycin-resistant cells were harvested, and luciferase expression was detected by bioluminescence measurements using IVIS spectroscopy. Subsection A shows a representative bioluminescence image demonstrating the ability of GBM43-Luc to express luciferase (signal intensity is shown as a function of seeded cells); subsection B shows representative schematic data of fluorescence intensity as a function of GBM43-Luc cell number (total flux (photons / second) was quantified using AURA software); subsection C shows growth curves of GBM43-Luc and parental control GBM43 cells (data are shown as mean ± SEM). [Figure 19-1]This figure shows data on the in vivo activity of CD73.mCAR-pNK cells in combination with autophagy inhibition in an orthotopic GBM43 xenograft model; subsection A shows a schematic diagram illustrating the in vivo treatment program; subsections B and C show tumor volume in individual mice in each group, monitored over time using bioluminescence imaging; subsection D shows a line graph depicting the average tumor size of mice in each treatment group after necropsy 28 days after the start of treatment; subsection E shows a line graph of the change in mouse body weight in each group during the treatment period; subsection F shows images of IHC staining for CCL5 (left two panels) and CXCL10 (right two panels) performed on the displayed tumor sections in various treatment groups using anti-CCL5 and anti-CXCL10 antibodies, respectively. (Bar = 50 μm; 200 × magnification); Subsection G shows images of IHC staining of NK cells performed on the displayed tumor sections using anti-NKp46 antibody (Bar = 50 μm; 200 × magnification); Subsection H shows a bar graph depicting the quantification of NK cell infiltration into intracranial tumors treated with CD73.mCAR-pNK or CQ+CD73.mCAR-pNK (here, cell counts were recorded at 200 × magnification in four consecutive high-magnification fields (HPFs)); Subsection I shows images of IHC staining for CD73 performed on the displayed tumor sections using anti-CD73 antibody in various treatment groups (Bar = 50 μm; 200 × magnification); Subsection J shows schematic data depicting the IHC score of CD73 expression performed on the displayed tumor sections in various treatment groups; Subsection K shows GBM A schematic diagram illustrating extracellular adenosine production in TME is shown; subsection L shows schematic data depicting adenosine concentrations in localized brain tissue of mice in each treatment group (Note: The data shown in Figure 19 pertain to at least three isolated pNK cells; data are shown as mean ± SEM; *p<0.05, **p<0.01). [Figure 19-2] (As stated above.) [Figure 19-3] (As stated above.) [Figure 19-4] (As stated above.) [Figure 19-5] (As stated above.) [Modes for carrying out the invention]

[0030] This disclosure is susceptible to various modifications and alternative forms, but its exemplary implementation is... Examples of implementations are shown in the drawings and are described in detail herein.

[0031] A brief explanation of sequence listings Sequence ID 1 is the amino acid sequence of the protease-sensitive linker, as follows:G GGGSGGGGSGGGGS; Sequence ID 2 is the amino acid sequence of a cleavable peptide fragment, as follows: :LSGRSDNH; Sequence ID 3 is the amino acid sequence of the "self-cleaving" P2A peptide, as follows:G SGATNFSLLKQAGDVEENPGP; Sequence ID 4 is the following nucleic acid sequence of the primer: ACATCGCTCAG ACACCATG; Sequence ID 5 is the following nucleic acid sequence of the primer: TGTAGTTGAGG TCAATGAAGGG; and, Sequence ID 6 contains the natural killer group 2 member D receptor (NKG2D), Artificial A of at least one embodiment of the antigen-binding domain or fragment thereof of the present disclosure This is a mino acid sequence (hCD3 zeta-hNKG2D-P2A-hDAP10 / Flag). :

[0032] [ka] Sequence ID 7 specifically binds to the cancer-associated antigen disialoganglioside (GD2), s at least one of the antigen-binding domains or fragments of the present disclosure, including cFv This is an artificial amino acid sequence in the form of (anti-GD2 scFv-CD8H-hCD28-hCD 3 Zetas):

[0033] [ka] Sequence ID 8 is a cleavable linker that connects to a second binding domain specific to CD73. The first is a fragment that specifically binds to the cancer-associated antigen GD2, which is operatively linked to it. The antigen-binding domain or its fragment (SEQ ID NO: 7), and the part containing NKG2D The CAR structure of the present disclosure includes the antigen-binding domain or a fragment thereof (SEQ ID NO: 6) This is an artificial amino acid sequence of at least one embodiment of the structure (anti-CD73 scFv- Linker-Cleavable Peptide-Linker-GD2.CAR-NKG2D.CAR):

[0034] [ka]

[0035] In addition to the foregoing, the above sequence is included in the file submitted with this specification. Provided in a computer-readable format and incorporated herein by reference. Information recorded in computer-readable form is subject to U.S. Patent Rule 1.821(f), This is identical to the sequence list in the document provided above.

[0036] Detailed explanation For the purpose of promoting understanding of the principles of this disclosure, the following are illustrated in the drawings. A description of the embodiment will be given, and specific words will be used to describe it. Nevertheless, the description of these embodiments does not intend to limit the scope. It will be understood that this is not the case. On the contrary, this disclosure is based on the attached claims. Substitutions, modifications, and equivalents that may be included within the spirit and scope of this application as defined herein It is intended to cover all. As previously described, this technology covers one or more preferred The compositions, systems, and The method may include many different configurations, forms, materials, and accessories.

[0037] In the following explanation, in order to provide a thorough understanding of this disclosure, numerous specific examples are provided. Specific details are provided. Certain examples may not use some or all of these specific details. This disclosure may be implemented in any biological system, and is not limited to any particular biological system. It should be understood that this can fluctuate.

[0038] Unless otherwise defined, all technical and scientific terms used herein The term has the same meaning as commonly understood by those skilled in the art in the relevant technical field. Any method and materials similar to or equivalent to those described herein may be used in this application. Preferred methods and materials may be used in the practice or examination of the subject, as specified herein. It is described in this specification and the appended claims. The singular forms "a," "an," and "the" are used in this text unless otherwise explicitly stated. To the extent that it is specified, it includes plural referents. Furthermore, unless otherwise specifically stated, "approximately" The terminology refers to a value of plus or minus 10% in relation to a percentage, and also to a unit value. This allows for a certain degree of variability in the value or range, including a range of plus or minus 1.0 units. For example, approximately 1.0 means a range of values ​​from 0.9 to 1.1, or the limit range for which the range is indicated. The term "substantially" refers to a certain degree of variability in a value or range, for example, Allows the value or range to be within 90%, 95%, or 99% of the specified limit. It is possible.

[0039] "Subject" or "patient" means, as used herein, a mammal, a patient Humans are defined as individuals, including males, females, adults, and children.

[0040] "Nucleic acids" are deoxyribonucleotides that exist in either single-stranded or double-stranded form. This term refers to ribonucleotides and their polymers, as well as their complements. Naturally occurring and naturally occurring nucleic acids, having similar binding properties to the reference nucleic acid. Furthermore, known nucleotide analogs or modified nucleotides that are metabolized in a similar manner to the reference nucleotide. It contains nucleic acids that have altered skeletal residues or ligations.

[0041] The terms "polypeptide," "peptide," and "protein" refer to amino acid residues. polymers, polypeptides, or polypeptide fragments, peptides, or melts In this specification, interchangeable (unless otherwise explicitly stated) refers to synthetic polypeptides. It can be used. The term refers to a situation in which one or more amino acid residues correspond to naturally occurring ones. Amino acid polymers are artificial chemical mimics of amino acids, as well as naturally occurring amino acids. This applies to amino acid polymers and amino acid polymers that do not exist in nature.

[0042] As used herein, "adenosinergic" means acting on adenosine. It means that.

[0043] As used herein, "antibody fragment" refers to a portion of an intact antibody, preferably a portion of an intact antibody. This refers to the antigen-binding region or variable region of an intact antibody. Examples of antibody fragments include, for example, single-chain antibody molecule (scFv), nanobody, F(ab')2, F(ab)2, Fab', Includes Fab, Fv, or dAb. Regardless of the structure, used herein. The antibody fragment binds to the same antigen recognized by the full-length antibody. For example, antibody Fragments include variable regions such as the "Fv" fragment, which consists of variable regions of heavy and light chains. An isolated fragment consisting of regions, or light chain and heavy chain variable regions, acts as a peptide linker. Recombinant single-chain polypeptide molecules ("scFv proteins") linked by It occurs. Single-chain antibodies, often abbreviated as "scFv," interact with antigens to target antigen-binding sites. V to form H and V L It consists of a polypeptide chain containing both domains. H and V L The domain is typically linked by a peptide consisting of 1 to 25 amino acid residues. The components also include diabodies, triabodies, and single-domain antibodies (dAbs). Although this disclosure refers to antibodies and various properties of antibodies, it does not refer to them. Unless otherwise explicitly stated, this disclosure also applies to functional antibody fragments. It can be done.

[0044] A "chimeric antigen receptor" (CAR, also known as a chimeric T cell receptor) is a type of receptor that is responsible for the development of the body. To be expressed in principal T cells or NK cells, as well as specific target antigens (e.g., To induce an immune response against cells expressing CD39 / CD79 and its antigens. It is a designed synthetic construct. CARs are typically CD3-ζ, CD28, OX40, CD3-ζ including the end domains of 4-1BB, Lck, and / or ICOS This also includes additional components such as the CD28 transmembrane domain and the selective T cell activation moiety. The fusion protein incorporates a single-chain antibody (scFv) or Fab fragment, etc. It contains antibody fragments. Various combinations of such elements can also be used. Therefore CAR utilizes an immune receptor activation motif (ITAM) located in the cytoplasmic tail. It can also be designed to convert activation signals via co-stimulatory domains. Gene constructs that utilize the primordial binding site are human leukocyte antigen (HLA) independent. The formula binds to the natural antigen on the surface of target cells, and therefore matches the patient or specific HLA. It has the added advantage of being "universal" in that it does not need to be retrieved from the source. ru.

[0045] A CAR according to the embodiments of this disclosure can be produced by any means known in the art. It can be produced, however, it is preferable that it be produced using recombinant DNA techniques. Nucleic acid sequences encoding several regions of CAR were obtained using standard molecular cloning techniques (G Nomu library screening, PCR, primer-assisted ligation, yeast and It can be prepared by bacterial scFv libraries, site-directed mutagenesis, etc., and is a complete It can be collected into a code sequence. The resulting coding region can be inserted into the expression vector. It can be used to transform host allogeneic or autologous NK cells with appropriate expression. In other words, Then, transduction or transfection is performed to introduce the CAR expression construct into NK cells. It can be introduced and then used to induce an immune response in the target. Additionally or alternatively Alternatively, CRISPR / Cas9 genome editing technology can be used to knock out specific genes. It is also possible. Techniques for genetically modifying NK cells for cancer immunotherapy are relevant to this field. It is generally known in [location / area].

[0046] Furthermore, as described above, CAR-NK cells also respond to disease-associated antigens (TAAs) It includes a targeting molecule such as scFv or Fab that binds to a hapten on the targetable construct. It is possible. Unlike T cells, NK cells target cells that are to be killed. To avoid the problem of lacking antigen specificity, cell-targeted scFv or Fab can be transmembrane-transmissible. It ligates to one or more intracellular signaling domains via its domain, and activates lymphocyte activity. It can lead to sexualization. The signaling domain used with CAR-NK cells includes: For example, CD3-ζ, CD28, etc. may be included. The CAR structures of this disclosure are in the art. Any such publicly known structures may be included. A wide variety of CAR structures have been reported. It is available commercially.

[0047] The target of interest "binds" to the "antigen-binding domain or its fragment" of this disclosure. "Binding domain" or "binding domain or its fragment" refers to a protein, binding domain or manipulated cells target cells or tissues that express proteins or antigens. In doing so, sufficient affiliate It binds to the antigen / target via a cellular mechanism. The protein, binding domain, and target molecule... and / or with respect to the binding of manipulated cells, a specific polypeptide or a specific polyp "Specific binding to" or "specific binding to" an epitope on a ptide target or "specific binding to" The term "specific" means a binding that is measurably different from a nonspecific interaction. Specific binding is determined, for example, by competition with a control molecule similar to the target. It can be measured. In at least one embodiment, “specifically binding” means that its finger Antigen binding to designated adenosine-producing enzyme target receptors (e.g., CD73 or CD39) This refers to binding to the binding domain, and not to binding to other designated non-target receptors.

[0048] Nucleic acids are "operationally linked" when they are placed in a functional relationship with another nucleic acid sequence. For example, DNA for a presequence or secretion leader is a polypeptide. When expressed as a preprotein that participates in secretion, it acts on the DNA of polypeptides. They are linked together; promoters or enhancers are positioned to facilitate translation. If so, it is operationally linked to the code array. Generally, "operationally linked" This means that the linked DNA sequences are adjacent, and in the case of a leader, they are adjacent. This means that it is in the leading phase. However, enhancers are not necessarily adjacent. It is not necessary. Ligation can be achieved by ligation at a convenient restricted area. If such a site does not exist, a synthetic oligonucleotide adapter or linker —However, it can be used in accordance with conventional practices.

[0049] The "amino acid sequence identity percentage (%)" in reference to polypeptide sequences refers to the sequence Align the sequences to achieve the maximum percentage of identity, and introduce gaps where necessary. Furthermore, after not considering any conservative substitutions as part of sequence identity, the reference polypeptide is... The percentage of amino acid residues in the candidate sequence that are identical to the amino acid residues in the column. It is defined as an alignment for the purpose of determining amino acid sequence identity percentage. For example, using publicly available computer software, in this technical field It can be achieved in various ways within the scope of one's skills. Those skilled in the art will be able to compare them. Any algorithm required to achieve maximum alignment across the entire length of the array Including this, appropriate parameters for aligning the array can be determined.

[0050] "Downshifted" or "downshifted" can be used interchangeably, at an established level. Genes, nucleic acids, and metabolism compared to (for example, healthy cohorts or subjects of interest). Decreased levels of markers such as products, transcripts, enzymes, proteins, or polypeptides Refers to. "Upregulated," "upregulated," or "overexpressed" can also be used interchangeably. It can be used and compared to established levels (e.g., those of healthy controls or subjects of interest). In addition, genes, nucleic acids, metabolites, transcripts, proteins, enzymes, or polypeptides, etc. This refers to an increase in the level of the erogen. For example, in the context of this application, CD73 is a healthy control and In contrast, it may be overexpressed in patients experiencing solid tumors or other cancers. Either directly or alternatively, NK T cells upregulate the expression of NKG2D in manipulated T cells. It can be manipulated to do so.

[0051] "Marker" or "biomarker" means as used herein. Furthermore, the level of expression in subjects experiencing an active disease state is the same as that of the subject or healthy subjects. It is described as being expressed differently when it is significantly different from that of a sample taken from a normal subject. Markers may be listed. Differentiated markers may be compared to normal or control samples or subjects. Compared to the baseline expression level (i.e., downregulated), it may be overexpressed. It may be expressed at a low level or at a high level. An increase or decrease in the marker in a biological sample, Quantification measures the presence and / or relative abundance of gene products or transcripts. This can be determined by one of several methods known in the art. - The level is expressed as an absolute value, or compared to the baseline level, and the cutoff level. It can be determined as the level of the target marker compared to the target. Alternatively, one or more types. The relative abundance of a marker can be determined by comparing it to a control that may be clinically normal.

[0052] The terms “treatment” or “therapy” as used herein (and “treatment "To treat," "to treat," and "therapeutic," etc. (these grammatical variations) refer to the individual being treated. This includes therapeutic and / or preventive interventions in attempts to alter the body's natural course. More specifically, therapeutic treatment refers to the reduction, improvement, and / or elimination of symptoms of a particular disorder. Downward and / or stabilization (e.g., inability to progress to a more advanced stage), as well as symptoms This refers to any of the delays in the progression of the condition. Preventive treatments include: for specific disorders, To prevent the onset of the disease, to reduce the risk of developing the disease, to reduce the incidence rate, to prevent the onset of the disease To delay the onset of symptoms, reduce the incidence of the disease, and increase the time until symptoms appear. This refers to one of the following. The desired effect of treatment is to prevent the onset or recurrence of the disease. This includes alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, and preventing metastasis. To reduce the rate of disease progression, improve or alleviate the disease state, and achieve remission. This includes, but is not limited to, improvements in prognosis. In some embodiments, Using the compositions of this disclosure, one can delay the onset of disease and / or tumor, or To slow (or even halt) the progression of disease and / or tumor growth.

[0053] As used herein, the term "antitumor effective dose" refers to cancer cells or tumors. To reduce tumor growth, or to decrease tumor volume or the number of tumor cells in the subject This refers to the effective amount of construct-expressing NK cells needed to increase life expectancy. "Antitumor effective amount" refers to the effective amount of NK cells needed to increase life expectancy. To induce or reduce the physiological effects associated with tumors or cancer, This may also refer to an effective amount of synthesized NK cells or engineered NK cell lines.

[0054] In this specification, when used, "therapeutic effective dose," "therapeutic effective amount," and "effective amount" are used. The phrase "(unless otherwise specifically stated) refers to a single treatment or treatment cycle." If administered in this manner, this disclosure may affect the health, well-being, or mortality of the subject. This means the quantity of polypeptides and / or manipulated cells (for example, not limited to...) (and any reduction or prevention of effects associated with cancerous conditions). Diseases and illnesses described herein To treat a condition or disorder (including, but not limited to, cancerous conditions such as solid tumors). Appropriate polypeptides, manipulated cells, or other compounds to be administered to the target. The appropriate dosage or amount depends on the type and severity of the condition being treated, and the pathology of the disease. Whether it is progressing, formulation of the composition, patient response, judgment of the prescribing physician or healthcare provider, one or This includes whether multiple constructs are being administered, the route of administration, and the patients being treated. or characteristics of the subject (such as overall health status, age, sex, weight, and drug tolerance) It will vary according to several factors. Therefore, the manipulated content contained in a given unit dosage form The absolute number of cells can vary widely depending on the age, weight, and physical condition of the subject, as well as the administration. It depends on factors such as the method used.

[0055] The therapeutically effective dose is the amount at which the therapeutically beneficial effects of a therapeutic agent outweigh any toxic or harmful effects. It is also the case that, in at least one embodiment, the antitumor effective dose is the therapeutic effective dose. It is possible.

[0056] For the treatment of cancer, cancerous tumors, or other diseases or disorders, as described herein The dosage administered to the manipulated cells is the dosage practiced by those skilled in the art. Follow the schedule regimen. Typically, 10 9 Doses exceeding individual cells / patients, adoptive cells It is administered to patients undergoing cytoplasmic transfer therapy. Determining the effective dose or amount is difficult. Based on the detailed disclosure provided herein, within the full scope of the skills of a person skilled in the art ru.

[0057] The term "pharmaceutical composition" refers to the manipulated cells or manipulated cells described herein. One or more NK cell lines, as well as preservatives, fillers, disintegrants, wetting agents, emulsifiers, and suspension agents. Turbidity agents, sweeteners, flavoring agents, fragrances, antibacterial agents, antifungal agents, lubricants, and dispensing agents, etc., pharmaceuticals A carrier, diluent, adjuvant, excipient, or vehicle that is generally acceptable, comprising at least This refers to a composition containing one type of component (depending on the mode of administration and the properties of the dosage form).

[0058] The term "pharmaceutically acceptable" and its grammatical variations refer to the fact that they are compositions, carriers, When referring to diluents, reagents, etc., they must be interchangeable and have a reasonable benefit / risk ratio. Therefore, the materials may cause excessive toxicity, irritation, allergic reactions, and / or nausea, dizziness, Without undesirable physical effects such as acute gastric upset, in mammals This indicates that it can be administered upwards. In other words, it means that it can be administered biologically or otherwise desired. It is not a material that causes any undesirable biological effects. Without rubbing, or in any significant manner with any of the other components of the pharmaceutical composition in which it is contained. NK modified to express the constructs of this disclosure without interacting in a manner harmful to them It can be administered to an individual along with cells (and / or stem cells or iPSCs).

[0059] The term "isolated" means that a material is in its original environment, for example, when it is naturally occurring. In that case, it means that it is extracted from the natural environment. For example, if it exists within a living organism. Although naturally occurring NK cells have not been isolated, they coexist in the natural system. The same NK cells isolated from some or all of the material have been isolated.

[0060] Treatment for glioblastoma (GBM), without limitation, including surgery, radiation, and chemotherapy. Conventional treatments have proven ineffective, largely due to the severe immunosuppression characteristic of GBM. GBM possesses a variety of molecular, genetic, and epigenetic characteristics. The cells' ability to evade conventional treatments is partly due to their intratumoral heterogeneity. It is a particularly difficult tumor to treat. Furthermore, GBM is a monotherapy that targets a single antigen. Characterized by antigen-evading variants that resist the target.

[0061] The limitations of single-antigen-based therapy have been recognized in clinical research through tumor proliferative variants. Yes, there are. Up until now, single GBM antigens such as EGRF, EGRFvIII, or ErbB2 CAR-NK cells targeting HER2 are limited to the use of NK cell lines, and are fully responsive. The response rate was disappointingly low and inconsistent. These results are for single antigens against GBM. This seems to reflect the clinical hurdles of targeted CAR-T therapy.

[0062] Stringent requirements for target selection impose constraints on targeting strategies. Multi-targeting Combination antigen targeting using CAR-manipulated cells is effective against antigen escape in GBM. Although it shows some ability to cope, it is mainly drawn out by hypoxic GBM TME. The resulting poor immune cell infiltration and immunometabolic reprogramming interfere with the persistent response. Furthermore, and perhaps not surprisingly, the proliferation of antigen-evading variants has been linked to most GBM-related issues. Regarding linked antigens, there is a clinical record of them leading to immune evasion and resistance to treatment. Unsustainable responses were observed in GBM treated with CAR-T therapy in a Phase III trial. This has resulted in a lack of improvement in patients' overall survival (OS).

[0063] Dual antigen-targeting or programmable tumor-sensing CARs are in the background of adoptive T-cell therapy. Although GBM has been preclinically evaluated for treatment in only one area, GBM is... It employs a mechanism that goes beyond antigen escape, which avoids immune surveillance that the conventional methods cannot address. In fact, dual antigen programming alone is not sufficient for clinically effective treatment. Furthermore, avoiding treatment with GBM creates an unfavorable niche for NK cell effector function. It is further enhanced by the extremely immunosuppressive hypoxic TME that provides. Furthermore, many The extremely hypoxic nature of tumors (such as GBM) is due to ectonucleoside triphosphates. Acid diphosphohydrase-1 (CD39), ecto-5'-nucleotidase (CD73) , and also promotes the activity of other adenosinergic signaling variants (e.g., CD38). It produces the immunosuppressive metabolite adenosine, which is a crucial purine for NK cell activity. This can lead to a loss of dynamic signaling-mediated dysphagia.

[0064] In addition, GBM cells utilize autophagy for their growth, progression, and therapeutic effects. It promotes resistance to [the disease]. Finally, glioma stem-like cells, a subset of GBM cells. (GSC) is also a contributing factor to treatment resistance, and conventional GBM model cell lines, including U87MG, are affected. Therefore, reproducibility is poor. This complexity is due to the coordinated or synergistic effects of these factors in meaningful clinical responses. This necessitates various therapeutic strategies that can initiate the process of finding the answer.

[0065] The inventive concepts of this disclosure generally do not target only one or more cancer-specific antigens. Furthermore, in at least one embodiment, autophagy is also inhibited, thereby curing GBM Cancer, particularly GBM, is treated with sensitized, multifunctional, manipulated NK cells. This invention relates to methods, compositions, and manipulated peptides for the treatment of [unspecified condition]. Furthermore, this disclosure relates to [unspecified condition]. In certain embodiments, adenosinergic signaling is directly inhibited. Therefore, this invention The compositions, systems, and methods shown herein relate to novel engineered NK cells. For example, antigen evasion, immunometabolism suppression, and / or poor presence of NK cells in tumors. A treatment for GBM that simultaneously targets multiple clinically relevant pathways of GBM progression, including This is the first multifunctional, engineered NK cell-based therapy. The data in this disclosure are particularly G Although relating to BM, the inventive concept described herein relates to cancer-related antigens and adenosine or adenosine. Those skilled in the art will understand that this relates to any cancer that expresses the nosine intermediate. .

[0066] In at least one exemplary embodiment, for example, a structure with at least two functions ( For example, CAR), that is, cancer-related antigens (for example, without limitation, disialoga At least one structure for encoding a protein specific to ngrioside (GD2) Structures, and NK activating receptors (e.g., potent activating NK cell receptors, without limitation) Cognitive ligands target the natural killer group 2D (NKG2D) of the drug. A single NK cell construct is provided, comprising at least a second construct for NK cells. When expressed, these two binding domains become the transmembrane domain and / or intracellular domain. One or more stimulative or co-stimulative domains of NK cells, which may include the main (i.e.) It can be coupled with signal transduction domains. One or more such constructs may be As desired, it can be expressed in single NK cells. In this background, at least two different The use of CAR avoids the antigen escape by cancer cells that is seen with conventional treatments.

[0067] Furthermore, at least one of the structures is operationally connected to the structure via a severable linker. Linked, for example, one or more CD73, CD39, or By including CD38 blocking antibody fragments, immunosuppressive purinergic / adeno It can be further manipulated to impair syngnathic signaling. For example, at least one In one embodiment, the first CAR is connected to the anti-CD73scFv via a severable linker. They can be conjugated. Thus, in operation, antibodies and / or fragments are CAR It is released completely independently of signaling and / or activation based on it. Then it is released The released antibody / fragment is the adenosine-producing cell surface protein or adenosine-producing cell protein of cancer cells. It functions to inhibit the activity of denosine intermediate-producing cell surface proteins, thereby inhibiting the activity of the cell surface protein. The local concentration of extracellular adenosine decreases, which in turn affects the immunosuppressive purine signaling pathway. It impairs tumor sensitivity. In at least one exemplary embodiment, the release of the linker impairs tumor sensitivity. Therefore, it is possible to cleave it by the activity of the protease that is upregulated in TME. Yes, this results in the local release of adenosine-blocking antibody fragments in the TME. Therefore, systemic toxicity must be avoided.

[0068] Furthermore, insufficient homogenesis of NK cells into the tumor bed, particularly caused by GBM To address the issue and to sensitize cancer cells for immunotherapy, this disclosure The embodiment also impairs autophagy by administering at least one additional therapeutic treatment. It can be made to and / or to be disabled. In at least one embodiment, additional therapeutic treatment The treatment involves administering one or more pharmacological autophagy inhibitors, such as chloroquine (CQ), to the patient. This includes administering. Additionally or alternatively, at least one additional therapeutic measure is: By generating GBM cells derived from gene knockdown patients, the autophagy pathway This includes targeting one or more genes of patients associated with the present disclosure. When used in conjunction with manipulated NK cells, the additional therapeutic treatment enhances the GBM environment. Such reorganization can be achieved, and therefore, through the involvement of the CCL5 and CXCL10 chemokine axes This can substantially enhance NK cell infiltration.

[0069] A brief description of the relevant cellular pathways and mechanisms will aid in understanding the inventive concepts described herein. Provided for the purpose of, and subsequently provided herein, the constructs, compositions, and A detailed explanation of the new method will follow.

[0070] Adenosine One of the contributing factors to the pathogenesis of solid tumors is the result of anaerobic glycolysis in the hypoxic solid tumor core. This is an increase in adenosine concentration. In solid tumors, ATP is abundant in the extracellular space. It is released into the atmosphere, where its concentration is more than 1000 times higher than in healthy tissue. It can reach several hundred micromoles per liter. This phenomenon is mainly due to the tumor core. This leads to cell death and stimulates metabolic or hypoxic stress, as well as active ATP export. is caused by stress and pro-inflammatory signals. In the TME, extracellular ATP acts as a danger signal involved in the priming of innate immune cell recruitment and anti-tumor activity; how ever, at the same time, extracellular ATP is degraded into immunosuppressive adenosine via the coordinated enzymatic activity of at least CD39 and CD73 as well as CD38. As a result , in various solid tumors, the accumulation of extracellular adenosine and subsequent engagement of adenosine receptors on tumor-reactive NK cells is a highly immunosuppressive mechanism that promotes tumor growth .

[0071] CD39 and CD73 are anchored cell surface proteins that are ectonucleoside triphosphate diphosphohydrolases presenting catalytic domains facing the extracellular space. CD38 and CD157 are also alternative pathway surface molecules with extracellular catalytic domains, except that they consist of ADP-ribosyl cyclases. Expression of these ectoenzymes by solid tumors such as GBM and in the T ME results in the production of extracellular adenosine.

[0072] CD38 and CD157 are part of the same family of NADase / ADPR cyclase enzymes . CD38 is characterized by a relatively large extracellular domain bearing a catalytic site and is a surface glycoprotein. In contrast, CD157 is attached to the membrane via a glycosylphosphatidyl inositol anchor. The extracellular domains of both molecules contain conserved critical residues . Both molecules also metabolize nicotinamide adenine dinucleotide (NAD +), which also affects purinergic receptors, and drive immune effector cells toward tolerance + ) For example, the present invention focuses on adenosine production, which has a tremendous effect in generating NK cells. In practice, extracellular NAD + can be degraded by an integrated network of ectonucleotidases, including CD38 and CD157 , which generates intermediates that modulate signal transduction and activate immune regulatory circuits. Extracellular adenosine can be produced from NAD through the cooperative action of + CD38, which generates ADP-ribose (ADPR) and PC-1 (ectonucleotide pyrophosphatase / phosphodiesterase family member 1 ), which generates AMP. Like CD38, CD157 generates cADPR and subsequently ADPR when incubated + together with NAD.

[0073] In human peripheral blood, both CD39 and CD73 are typically expressed on approximately 2 to 5% of NK cells within non-malignant blood cells. Therefore, expression of both CD39 and CD73 is virtually absent in circulating human NK cells in healthy individuals. However, significant expression of CD39 by human tumors and tumor-infiltrating immune cells has been widely described, which is associated with adenosine production that has an inhibitory role against effector anti-tumor immunity and exposure to pro-inflammatory cytokines, oxidative stress, and hypoxia. Similarly, CD73 expression remains constitutively high on many types of cancer cells. High CD73 expression has been shown to correlate with unfavorable clinical outcomes, which is consistent with the immunosuppressive role of adenosine. Expression of CD38, CD73, and / or CD157 can also be upregulated in TME, particularly in hypoxic TME.

[0074] Therefore, CD39 and CD73 are excess on many solid tumor cells, especially in GBM. It is expressed and, through the upregulation of adenosine signaling after dephosphorylation of extracellular AMP, It is involved in promoting cancer progression. As described herein, adenosinergic signaling This is due to heterogeneous desensitization of chemokine receptors and a decrease in pro-inflammatory cytokines. It interferes with the transport and activity of NK cells and inhibits the exocytosis of cytotoxic NK granules. This creates a pro-angiogenic niche that supports tumor development.

[0075] Adenosine-induced immunosuppression is mitigated by antibody-mediated blockade of CD73 or its variants. This is possible; however, this alone depends on the recruitment of NK cells into the hypoxic tumor niche. It exists. Conventional efforts have focused on adenosinergic signaling in conjunction with NK-based immunotherapy. They are not our target.

[0076] NK activity NK cells, specialized effectors of the innate immune system, are pathogen-derived or stress-induced. The generation of germline-encoded activating receptors that can directly bind to inducible autoantigens. As a result, it can respond rapidly to cancer cells. NK cell activity is activated by receptors and inhibitors. It is controlled by the balance of signals from the repertoire of harmful receptors. Activated receptors This includes, without limitation, innate cytotoxic receptors (NCRs) and natural killer glues. P2 Member D (NKG2D), CD16 (FcγRIIIA), FasL, TRAIL , as well as co-insertions such as LFA-1, CD244 (2B4), and CD137 (41BB). This includes highly reactive receptors. These activated cell surface receptors are involved in the cell lysis program, as well as via intracytoplasmic ITAMs such as 2B4, 4-1BB and / or via other transmembrane signaling transduction adapters, has the ability to induce secretion of cytokines and chemokines. As used herein, the term "signal transduction domain", unless otherwise specified, means and includes both stimulatory and co-stimulatory domains.

[0077] Conversely, inhibitory NK cell receptors mainly recognize cognate MHC class I proteins and provide self-tolerance to healthy cells. Cells that lack or have reduced expression of MHC class I proteins, which is often observed after transformation or viral infection, are unable to trigger sufficient inhibitory signals and become susceptible to NK cell attack.

[0078] Upregulated expression of ligands for activating NK cell receptors can sensitize cells to NK cell attack. Such activating receptors include the C-type lectin-like receptor NKG2D. The NKG2D receptor is expressed on NK cells as well as on many T cells including NKT cells, CD8+ T cells, and γδ T cells. However, in T cells, NKG2D usually acts only as a co-stimulatory receptor and does not directly mediate cytotoxicity, which differs from its function in NK cells.

[0079] Expression of NKG2D ligands, which is often found on tumor cells, generally marks cells for immune attack and is considered a "danger signal" that activates NK cells by binding to the NKG2D receptor. In fact, ex vivo studies using human cells and in vivo tumor models in mice show that expression of NKG2D ligands on tumor cells We demonstrated that its expression leads to increased susceptibility to NK cell attack. If the system is functioning properly, ligation of NKG2D on NK cells activates NK cells. It promotes sexualization and influences the adaptive immune response; however, the NKG2D receptor Various mechanisms that inhibit the action of NKG2D ligand and enable immune evasion ( (Regarding GBM)

[0080] As discussed herein, adenosine signaling is received on NK cells. This results in downregulation of cytotoxicity (for example, without limitation, adenosine, cytotoxicity). It has been confirmed that it downregulates NKG2D on human NK cells primed by [the drug]. In addition to extracellular adenosine concentration, the expression of the NKG2D receptor on NK cells is also important. A variety of other factors, including changes in the active ingredients and the physicochemical properties of TME (e.g., hypoxia). TME can be regulated by factors such as tumor-associated fibroblasts and tumor-associated macrophages. Treg, immunomodulatory enzymes (e.g., arginase and cyclooxygenase-2), and immunosuppressive factors (e.g., interleukin-10 (IL-10), transform TGF-β (transluminal growth factor-β), vascular endothelial growth factor (VEGF), prostaglandins Diverse cells and molecules, including E2 (PGE2) and programmed death ligand 1). It consists of tumor cells (GMB cells, etc.) and immunosuppressive cells, in TME, Docalyxin-like protein 1 (PCLP1), activin-a, India Calcium amine-pyrrole 2,3-dioxygenase (IDO), PGE2, TGF-β, And by expressing or secreting macrophage migration inhibitors (MIFs) under NKG2D It mediates directional regulation.

[0081] Furthermore, hypoxia can directly or indirectly induce the secretion of immunosuppressive molecules in the TME. This is a key characteristic, and as a result, NK cells can utilize IL-2 and other cytokines to deliver N They lose the ability to upregulate KG2D expression. Under hypoxic conditions, tumor cells undergo various chemotherapy. It can secrete NK cells, which in turn can mobilize immunosuppressive cells that secrete cytokines, thereby NK It downregulates G2D expression.

[0082] Structures and related methods The present disclosure includes inventive constructs, manipulated NK cells and NK cell lines, compositions, and methods. This involves at least one cancer antigen and / or NK-activating receptor, or a combination thereof. It uniquely targets adenosine-producing cell surface proteins or adenosine in cancer / tumor cells. By blocking the activity of intermediate-producing cell surface proteins, immunosuppressive purine-mediated Further providing releaseable soluble antibody fragments that disrupt signal transduction. Multiple types If an antigen and / or activating receptor is employed, this is the tumor's response to antigen escape. Limits capabilities. Furthermore, soluble antibody fragments that block adenosine are spatially controlled. In at least one embodiment, it is emitted only when it is inside the TME. This allows for its sustained and non-toxic release. Therefore, this is different from other conventional therapies. To avoid the systemic toxicity problems that can occur in this context.

[0083] The novel methods described herein involve the specificity of manipulated NK cells and the blockade of adenosine-producing enzymes (e.g.) For example, it uniquely combines immune involvement induced by anti-CD73. Furthermore, As further details below, such treatments involve one or more autophagy When combined with the administration of G-inhibitors, it may further sensitize tumors and further enhance therapeutic efficacy. Therefore, the constructs, manipulated NK cells, pharmaceutical compositions, and results of this disclosure are as follows. The resulting therapies address multiple clinically recognized mechanisms of GBM progression while avoiding toxicity. This results in a combination immunotherapy approach that can simultaneously target multiple factors.

[0084] Referring here to Figure 4, at least one exemplary embodiment of the synthetic gene construct 100 A state is provided. Gene construct 100 expresses it (bioengineered and other known states). NK cells and NK cell lines (achieved by the state) are normally expressed on the surface of native NK cells. It is engineered to express at least one domain and / or receptor that is not otherwise expressed. These modified NK cells and N The binding of K cell lines is through a novel domain not present in native NK cells. In at least one embodiment, the structure 100 includes one or more CAR structures. Obtained. In at least one exemplary embodiment, the structure 100 is a first CAR structure. Includes objects and a second CAR structure.

[0085] Perhaps in its simplest form, the gene construct 100 has at least a second binding A second distribution that intends to include the domain or its fragments and the detachable linker 104 At least one binding domain or fragment 102 operationally linked to the column A first construct comprising a first sequence encoding a first binding domain or its Ragment 102 is an NK activating receptor (e.g., NKG2D; SEQ ID NO: 6) or cancer It may contain proteins specific to the related antigen (e.g., GD2; SEQ ID NO: 7, etc.). Figure 5, bottom. Positional divisions F and G are the first binding domain or its f encoded by the first sequence At least one embodiment of the Ragment 102 is schematically illustrated.

[0086] In at least one exemplary embodiment, the first construct is the adenosine of the target cell. Alternatively, a second binding domain specific to adenosine intermediate-producing cell surface proteins. It is further manipulated to also code its fragment 104. For example, such as Denosine or adenosine intermediate-producing cell surface proteins are adenosine or its intermediates. CD39, CD73, CD38, or any other cell surface tannins on target cells that produce body tissue. It may contain protein. In at least one exemplary embodiment, adenosine or adenosine. The nosine intermediate-producing cell surface protein contains CD73, and the second binding domain or Fragment 104 contains anti-CD73 fragments such as anti-CD73 scFv (Figure 5). (See subcategory K.)

[0087] Notably, the second binding domain or its fragment 104 is a cleavable linker. This further includes any such severable linker known in the art. This may include a linker, and in at least one exemplary embodiment, is located within the TME. It contains a linker that can be cleaved by one or more proteases. (See details above) As such, cancer cells produce adenosine through CD73 and other surface proteins. The second binding domain 104 produces such adenosine or adenosine intermediates. Because it may be specific to living tumor cell surface proteins, its linker in TME If cleaved, the resulting soluble antibody fragments are released from the manipulated NK cells. It is released and binds to adenosine (or adenosine intermediate) surface proteins of tumor cells. This becomes possible.

[0088] Gene construct 100 further comprises the second construct shown in Figure 5, subdivisions K and L. It is possible. Similar to the first construct, the second construct is an NK-activating receptor, or cancer-related anti A binding domain (or its fragment) containing a protein specific to the source It may include a second sequence that codes for (t). Figure 5, subdivisions A and B are by the second sequence at least one implementation of a third binding domain or fragment thereof that is coded as The state is illustrated in a general diagram.

[0089] One or both of the first and third sequences (where applicable) are used in NK cell signaling They can encode domains, and thereby, when activated, they are in manipulated cells or It may promote the cytotoxic and / or cytolytic activity of cell lines (for example, not limited to...) Figure 5, CDζ in subdivisions A and F, and Figure 5, DA in subdivision A (See P10). The first and third binding domains 102 and 106 are as desired. complementarity-determining regions, variable regions, and / or their antigen-binding flags. It may include a variety of linkers, etc., as may be required to achieve the desired result. Pacer, scFv, hinge, and / or protein complexes are the first and second arrangements It will be understood that the columns may be additionally coded by one or both of them. .

[0090] The first and third sequences are specific to activating receptor 102 and cancer-associated antigens, respectively. Although shown in Figure 5 as containing heterologous protein 106, the first and third The sequences could both encode proteins against cancer-associated antigens, or both could be NK cells. It can encode cell activation receptors (i.e., it does not necessarily have to be one of each type). Furthermore, a second sequence encoding the second binding domain or its fragment 104 It can be operably linked to the NK cell activating receptor of the first domain 102. If both the first and third sequences encode antigens or receptors 102 and 106, then an example is given. In embodiments, they are two different cancer-specific antigens or two different NK cells. It can encode an activating receptor; however, although often desirable, this is required. That's not the case.

[0091] Figure 5, Subdivisions K and L are the first, second, and third binding domains (or their f At least one of the gene constructs of this disclosure, including 102, 104, and 106 A schematic diagram of an exemplary embodiment is shown. There, as a non-limiting example, the first coupling Main 102 contains the CD8α hinge and two signaling domains (CD28 and C). Includes an anti-GD2 scFv fragment operably linked to D3ζ (collectively, G D2-CAR; Sequence ID 7), the second binding domain 104 is a cleavable linker (sequence). The anti-CD73 scFv fragment is linked to the first binding domain 102 via (number 1). It contains a third binding domain 106, NKG2D, and two signal transduction domains ( (DAP10 and CD3ζ), and at least one P2A self-cleaving peptide ( NKG2D-CAR (SEQ ID NO: 6), GD2-CAR is one of the self-cleaving P2A peptides. It is linked to NKG2D-CAR via (Sequence ID 4). At least one exemplary In one embodiment, the structure 100 is at least 80%, 85%, or It codes for 90% sequence identity.

[0092] Target cells include cancer-associated antigens, NKG2D, or NK-activated receptors and cognitive receptors, among others. Ligands of and / or adenosine or its intermediates via cell surface proteins Any cell that produces, for example, without limitation, cancer cells or, instead, within TME It may contain malignant cells. Ligands for GD2 and NKG2D are particularly human G It is widely expressed on BM and is therefore referred to in this disclosure as a non-limiting example. However While being limited, ganglioside G3 (GD3), Her2 (p185) , CD19, CD20, CD56, CD123, CD22, CD30, CD33, CD1 71, CS-1, C-type lectin-like molecule-1; EpCAM, G250, proteoglycan, GD3, GD2, MHC II, TAG-72, milk mucin core protein, Lewis A Antigen, tyrosine protein kinase transmembrane receptor (ROR1), c-met, epidermal growth factor Other receptors include EGFR, EGFR variant III, and carcinoembryonic antigen (CEA). A related antigen may be encoded in a construct of the present disclosure, and a particular antigen may be a particular type It may be particularly associated with pu-type cancer.

[0093] Similarly, NKG2D is shown in Figure 5 as a representative NK cell activation receptor. This includes, without limitation, NKp30, NKp46, NKp40, DNAM-1, etc. Any NK-activating receptor having a cognitive ligand presented on a desired target cell , may be coded in structure 100. In a particular embodiment, the structure and NK cells, which are produced as a result, are, for example, antigens that are mainly expressed in the cancer of interest or The first sequence 102 and / or encoding a protein specific to the cognitive ligand By including a third sequence 106, it can be specifically manipulated for certain cancer types. .

[0094] Additionally, certain embodiments of the construct 100 encoding the NK-activated receptor are related to the technology. It may be further manipulated for upregulated expression using techniques known in the field. For example, in at least one exemplary embodiment, the binding domain or its flange One or more sequences encoding the NKG2D gene trigger the development of NKG2D in wild-type NK cells. (i.e., the expression of such receptors in the typical form of naturally occurring NK cells) Compared to that, it is engineered to encode upregulated expression of NKG2D. Such manipulated, upregulated expression increases the available local NKG2D. Therefore, manipulated NK cells are employed to treat other tumors with GBM and hypoxic TME. It is particularly useful when treating ulcers.

[0095] Many cancer-associated antigens and cognitive ligands of NK cell activators are found in cancer cells (specifically) Since it is adjusted upward in GBM and TME, the coupling in structure 100 Such antigen in one or more of the main or its fragments 102, 106 Including proteins specific to cancer cells results in manipulated NK cells that can treat cancer. This enables the direct targeting and recognition of other such cells. Therefore, this construct 100 enhances specificity and directly targets tumors, cancers, and other malignant cells. To enable targeting and engagement safely.

[0096] The binding domains 102, 104, and 106 are shorter phosphorus molecules of about 10 to 25 amino acids. The weight of immunoglobulins linked using carpeptides (V H ) and light (V L ) chain A fusion protein between variable regions, consisting of one or more single-chain variable fragments (scFv). It may further contain other antibody fragments such as sequences or nanobodies, scFv or other The specific composition of the antibody fragment determines the desired properties of the resulting peptide (e.g., softness). (Rich in glycine for flexibility, and serine or threonine for solubility) They can be selected. As is well known in the art, scFv or another antibody flavus The gument is V H The N-terminus and V L It can be connected to the C-terminus, and vice versa. The protein is used for the removal of the constant region and the introduction of scFv or other antibody fragments. Nevertheless, it retains the specificity of the original immunoglobulin.

[0097] Therefore, in at least one embodiment, the second binding region or domain 10 4, and the first binding region or domain 102 are, respectively, specific to a mouse or a human. Derived from, or from, humanized monoclonal antibodies, or from other known sources and public It contains scFv fragments following the methodology. The fragments are optional antigen-specific antibodies. It may also be an antigen-binding domain with a different number of elements. In a more specific embodiment, the fragment The expression is optimized for human codon usage for expression in human NK cells. Antigen-specific scFv encoded by a sequence (e.g., αCD73 scFv or αCD73 scFv) GD2 scFv) is. In at least one exemplary embodiment, the first array is , has at least 80%, 85%, or 90% sequence identity with sequence number 7, and it is C The binding domain or fragment 102 encoding D73 scFv is sequence number It has at least 80%, 85%, or 90% sequence identity with 2.

[0098] Returning to one or more signaling domains that can be encoded by construct 100 To mention, the first and / or third binding regions or domains 102, 106 are It can be operationally connected to (directly or via the hinge region described below), in fact The first binding domain itself is a signaling domain (e.g., CD28, 4-1B May include B, DAP10). One or more signaling domains may include, for example, binding. When the attached binding domain or fragment binds to target cells, NK cells lyse Includes NK activator receptors or receptor complexes that can induce decomposition and cytotoxicity programs. It is possible. For example, the signaling domains are CD28, CD3ζ, or DAP10. It may include a signaling molecule or any other signaling domain known in the art. In a particular embodiment, the signal transduction domain is FcγRIIIA, Fas L, TRAIL, 4-1BB, OX40, LFA-1, CD244, CD137, or It may contain the NKG2D-DAP10 receptor complex. Furthermore, the signaling domain is limited Without being specified, DAP12, NKp46, NKp44, NKp30, and DAP1 It may also include additional co-stimulatory domains, including one or more of the following:

[0099] In application, at least a first and optionally a third of the construct 100 with respect to the target cells. The involvement of binding domains 102 and 106 is through the signaling domain of the manipulated NK cell. This promotes signal transduction and activates the ITAM motif on the CD3ζ adapter chain, NK cells target solid tumors and other adenosine-producing or adenosine intermediate-producing targets. This results in cell-mediated cytotoxicity. Therefore, manipulated NK cells produce GD2 and / or Alternatively, directly target NKG2D ligand-producing surface cell proteins (e.g., on solid tumors). And if binding occurs, the signal is transmitted to the manipulated NK cells via the signaling domain. It is sent to the target (cancer) cells to which it is bound, and the cell-lysing and / or cytotoxic mechanisms It induces a 'nism'.

[0100] Optionally, the structure 100 may, for example, include a third binding domain 106. At a position between αGD2 scFv and the signaling domains CD28 and CD3ζ, Hinge domains located within combined domains 102 and 106 (labeled as "CD8α hinge") The hinge domain may additionally include the linker or (see Figure 5). A construct that includes one or more arrays encoding spacers, for example, the first / A sufficient distance between the third binding domains 102, 106 and the membrane and / or cell surface It may provide; additionally or alternatively, it may facilitate and / or modify the desired tertiary structure. Possible steric damage that could adversely affect the antigen binding or effector function of NK cells The hinge domain may be included (and / or configured) to mitigate the problem. Didomains may be used and / or manipulated for optimal expression in human cells. ru.

[0101] Additional or alternative intracellular signaling domains may be added to construct 100. This can enhance the killing stimulus (i.e., the resulting NK media of the manipulated NK cells). (Further enhances intercellular cell injury). For example, as shown in subdivision L of Figure 5, human C The D3ζ intracellular domain is operative with both the first and third binding domains 102 and 106. It can be linked to other cytoplasmic domains, if desired, for additive or synergistic effects. Along with one or more of the fused cytoplasmic domains, as desired, It can be used.

[0102] During operation, binding domains 102, 104, and 106 target cells of interest. When binding domains 102 and 106 bind to targeted cells, the manipulated NK cells , via signaling domains, they send signals to lyse and / or lyse target cells. It induces cell damage. Furthermore, at least one of the binding domain or its fragments If it includes an upregulated activating receptor (e.g., NKG2D), this is the case for NK cells. It enhances the antitumor response and suppresses not only the local concentration of extracellular adenosine but also NK cell activation. It also reduces the control. This is further enhanced by the release of the second binding domain 104. As previously mentioned, if the second binding domain 104 is located in the TME, then the prote Aase is a linker that connects the second domain / fragment 104 to the first domain 102. This causes the molecule to be cleaved, thereby releasing the fragment into the TME and targeting the cell. Initiates local blockade of adenosine or adenosine intermediate activity, particularly in NKG2. The combination of increased D activity and local CD73 target blockade is effective in GBM and other cancers. It is highly effective in achieving controlled and localized responsiveness.

[0103] Therefore, the structure 100 is a complex network of pathways that promote GBM and other cancer progression. Enables multi-directional attacks against twerks. Two or more types in a single structure (exemplary implementation) In terms of morphology, if three types of targets are incorporated, one of them can be released, and the operation of this disclosure The NK cells produced not only activate and enhance the anti-cancer immune response, but also perform antigen evasion and other functions. This will make it possible to suppress GBM's evasive tactics.

[0104] These inventive techniques offer unique advantages over conventional methods. Firstly, allogeneic stem cells NK cells do not cause graft-versus-host disease, and their wide range of readily available uses This makes it possible. Mature NK cells have a relatively limited lifespan and effective antitumor activity. This makes it possible, while also considering the possibility of long-term adverse events such as on-target / off-tumor effects. It reduces. Furthermore, the expression of this construct is localized, so CD73 and It prevents the systemic toxicity typically seen with similar antibodies and inhibits TME mechanisms such as hypoxia. It also rescues the downregulation of activating receptors induced by the sympathetic mechanism.

[0105] Constructions according to the embodiments can be prepared using conventional techniques. The majority are natural arrangements. Because they can be adopted, natural genes can be isolated, enabling proper conjugation of various components. It can be manipulated as needed, for example, to result in the deletion of an undesirable part of the gene. By employing polymerase chain reaction (PCR) using primers, nucleic acids The sequence can be isolated. Alternatively, restriction digestion of the cloned gene can be used to isolate the sequence. It is possible to create structures. In either case, they have blunt ends or complementary overlaps. Sequences may be selected to provide a restriction site.

[0106] Various operations for preparing the constructs described herein may be performed in vitro, and in specific implementations In this state, the construct is suitable for use with standard transformation or transfection methods. It is introduced into a vector for cloning and expression in the host. Therefore, each operation After the process, the resulting construct, created by DNA sequence fusion, was cloned and used as a vector. The sequences were isolated and screened to identify the desired transgene and expression regulatory sequences. Ensure that the code is written correctly. The sequence is obtained as desired by restriction analysis, sequencing, etc. This can be used for screening.

[0107] The vectors of the embodiments presented herein are true vectors known in the art. Further nuclear promoters may be employed. Also, the vector is, unless there is another reason, in To facilitate these operations in vitro, they may contain selectable markers. In the application method, the introduced gene is derived from mRNA transcribed in vitro from a DNA template. It can be revealed.

[0108] In exemplary nucleic acid constructs (polynucleotides) employed according to the embodiments, The motor is operationally linked to the nucleic acid sequence encoding the introduced gene of the embodiment, i.e. These are messenger RNAs from the DNA encoding the single-agent construct. It is positioned to promote the transcription of. The promoter may also be of genomic origin. It can be produced synthetically. Alternatively, several well-known viral promoters are also suitable. .

[0109] For the expression of the constructs of this disclosure in NK cells or NK cell lines, the transgene is used Using the naturally occurring or endogenous transcription start region of the nucleic acid sequence, in the target host This can produce the desired expression. Alternatively, exogenous factors that enable constitutive or inducible expression can be used. The transcription start region may be used, expression may depend on the target host, the desired level of expression, and the sex of the target host. It can be controlled depending on the quality, etc.

[0110] Similarly, in some cases, the target construct encoded by the transgene is refined. A leader that is directed toward the cell surface, specific to human protein expression, and attached to the N-terminus. A slash / or signal sequence may be used.

[0111] Isolated nucleic acid segments and expression cases incorporating the DNA sequences of the constructs of this disclosure. A set is also provided. Those skilled in the art can use such constructs to introduce viral mutations, mRNA Alternatively, DNA electroporation, as well as other viral and nonviral transduction. , and also employed in conjunction with known gene modification techniques, including transfection techniques, Engineered NK cells and / or engineered N cells expressing the constructs described herein. This will demonstrate that it is possible to create a K cell line.

[0112] Methods for producing and / or increasing manipulated NK cells are also provided in this disclosure. In at least one embodiment, the construct provided herein is coded Renucleotides can be used either with a suitable vector or without a vector, in conventional trans Infection and / or transduction methods are used to introduce (or different) cells into the target's own cells. It can be introduced into cells derived from a donor subject by electroporation or otherwise. Methods for stably transfecting or inducing NK cells are available in the present art. It is publicly known. In a further aspect, this structure uses a transposon-based system. It is used to introduce the cell's genomic DNA, a non-viral vector, or a viral vector into the cell. Vectors (for example, retroviral vectors, adenovirus vectors, adeno-associated viruses) It can mediate the incorporation of the construct into a vector (or lentiviral vector). In at least one embodiment, CAR is taken up by NK cells, and therefore NK It can be modified to promote the expression of construct-derived fusion proteins in cells.

[0113] Sources of natural NK cells can include both homogeneous and autologous sources. In some cases, NK cells can differentiate from stem cells or induced pluripotent stem cells (iPSCs). For example, The constructs described in the document may be expressed in stem cells or iPSCs, which then, They can be differentiated into NK cells using methods known to those skilled in the art in the relevant technical field. Therefore The cells to be manipulated according to the embodiments of this specification include umbilical cord blood, peripheral blood, and human embryonic stem cells. It can be isolated from cells or iPSCs.

[0114] In other embodiments, NK cells are derived from human peripheral blood mononuclear cells or umbilical cord blood. These are primary human NK (pNK) cells, etc. In at least one exemplary embodiment, The manipulated NK cells were then subjected to recurrent and primary NK cell regeneration according to methods known in the art. It can be produced from patient-derived cells. Alternatively, engineered NK cells expressing the constructs of this disclosure. Cells and / or manipulated NK cell lines are produced from standardized cell populations for clinical use. It can provide a homogeneous population of NK cells that can grow to a certain scale.

[0115] NK cells, stem cells, and pNK cells modified to express the constructs described herein. Cells, or iPSCs, are at least partially affected by cells that may be targets of NK cell damage. Delivery to subjects with a characteristic disease condition (e.g., a disease state that overexpresses adenosine). It can be formulated into a pharmaceutical composition together with a "carrier" for delivery. When using this method, the "carrier" can be any solvent, dispersion medium, diluent, antibacterial agent, coating, or bioc L, and / or antifungal agents, isotonic agents, absorption retarders, buffers, carrier solutions, suspensions, colloids This includes such media and / or active substances. The use of such media and / or active substances in the art It is well known that, except insofar as any conventional medium or active substance is incompatible with the active ingredient, Its use in the pharmaceutical compositions described herein is intended. Supplementary active ingredients are also included in the composition. It can be incorporated.

[0116] Furthermore, the pharmaceutical compositions of this disclosure (e.g., engineered cells expressing the constructs herein) (including) are useful alone or for treating cancer and / or solid tumor cancers. It can be used in combination with a well-established active agent. At least one exemplary In embodiments, one or more pharmaceutical compositions of the present disclosure are one or more auto It can be administered to a single patient in combination with a fuzzy inhibitor.

[0117] As will be discussed in detail in the embodiments below, the novel multifunctional manipulated N of the Disclosure K cell homing was significantly enhanced when administered in combination with an autophagy inhibitor. It has been newly discovered that autophagy inhibition affects CCL5 and CXCL1 It induces significant NK cell chemotaxis in parallel with upward regulation of the 0 gradient, while simultaneously promoting tumor formation. It reduces the amount of progressive CCL2 and CXCL12. In other words, these chemokines The release attracts manipulated NK cells to cancer TMEs, which are needed to fight the disease. Efficiently transport them to the designated location.

[0118] Furthermore, disabling autophagy is beneficial for enhancing the NK effector's functionality. The sophisticated and complex reorganization of the anti-GBM immunological response contributing to GB was also revealed. The clinical efficacy of adding autophagy inhibitors to M therapy is generally FDA approved. A prospective randomization ratio using chronic administration of the toxicophagy inhibitor chloroquine (CQ) This was demonstrated in a prospective controlled randomized trial. The results showed Furthermore, it demonstrated a significantly enhanced response of GBM to antineoplastic therapy and against exogenous activators. This sensitizes cancer cells and demonstrates that such treatment is clinically safe and well-tolerated. .

[0119] Therefore, in at least one exemplary embodiment, the pharmacology of the autophagy process A scientific defect is a composition (or active ingredient) comprising engineered cells expressing the constructs of the present disclosure. In conjunction with the administration of [another drug], immediately following it, or before or after it, an autophagy inhibitor is administered. This can be achieved through the administration of therapeutic measures, including autophagy inhibitors, for example. This may involve administering small molecule inhibitors, which may inhibit or reduce autophagy in the subject. This may include any possible therapies. In at least one embodiment, the small molecule inhibitor is C Q, hydroxychloroquine, spoutin-1, SAR405, verteporfin, or Currently known information appropriate for administration to patients to inhibit or block autophagy Alternatively, it may include any other small molecule inhibitors to be developed later. Additionally or alternatively, Autophagy is a process that involves the downregulation of one or more genes associated with autophagy. For example, it can be genetically inhibited through gene knockdown techniques. At least one In an exemplary embodiment, the gene is the following gene BECN1, p62, β-act Chin, ATG5, ATG7, LC3B, ATG12, ATG16L1, PI3K-III ULK1, ULK2, FIP200, and LAMP2, or autophagy pathways Of any other related genes (and which can be inhibited, either through downregulation or otherwise) It may include one or more types.

[0120] The composition itself contains a combination of active ingredients, or it can be used alone or in combination with other ingredients. Whether delivered in combination with a substance or therapy, the pharmaceuticals described herein The composition is transmitted through various pathways in the mammalian, preferably human, body and various It can be delivered to a site and achieve a specific effect. Those skilled in the art will know that more than one route of administration is possible. Although they can be used, certain pathways produce a more immediate and / or effective response than other pathways. They will recognize that it can be provided. For example, intratumoral delivery is used in the treatment of solid tumor cancer. This is possible (and may be advantageous in terms of minimizing off-target effects). Local or systemic delivery involves administering the pharmaceutical composition into a body cavity, infusion, or parenteral delivery. This can be carried out by the compositions of this disclosure, which can be used for additional therapeutic treatments such as autophagy inhibition. When administered in addition to, autophagy inhibitors are administered via systemic injection or infusion, and This may be performed as otherwise desired by the healthcare provider.

[0121] Pharmaceutical compositions can be formulated in a variety of forms adapted to the preferred route of administration. However, The composition is not limited to parenteral (e.g., intradermal, subcutaneous, intravenous, transdermal) Through known routes, including intramuscular, intraperitoneal, or local (e.g., intratracheal, intrapulmonary, etc.) It may be administered by means of [method]. The composition may also be administered by sustained release or delayed release.

[0122] The formulation can be conveniently presented in unit dosage form, and by methods well known in the field of pharmaceutical technology. A method for preparing a composition using a pharmaceutically acceptable carrier is described herein. NK cells (and / or stem cells or iPSCs) that have been modified to express a substance, The process includes the step of binding one or more auxiliary components to a carrier. Generally, The agent helps to uniformly and / or closely bind the manipulated cells to, for example, a liquid carrier. Therefore, it can be prepared.

[0123] NK cells (and / or stem cells or) modified to express the constructs herein Pharmaceutical compositions containing iPSCs can be in the form of solutions, suspensions, emulsions, sprays, aerosols, and It may be provided in any suitable form, including but not limited to any form of mixture. The composition is a formulation having any pharmaceutically acceptable excipient, carrier, or vehicle. It can be delivered in this state. N modified to express the construct of this specification to be administered to a subject. The effective amount of K cells (and / or stem cells or iPSCs) is discussed herein. It can vary depending on the various medication factors involved.

[0124] In some embodiments, the method is, for example, about 10 9 In or above individual cells / subjects Rotate, or about 10 5 Individual cells / kg~approx. 10 10 This disclosure provides doses in individual cells / kg. A therapeutically effective dose of modified cells that express the construct is administered to the target. Steps may be included, however, in some embodiments, the method falls outside these domains. This can be carried out by administering a dose of manipulated cells.

[0125] In some embodiments, modified and engineered particles have been modified to express the constructs described herein. Pharmaceutical compositions containing cells can be administered, for example, in single or multiple doses per week, but No, in some embodiments, the method administers the pharmaceutical composition at a frequency that falls outside this range. It can be implemented by means of.

[0126] In any case, the amount of manipulated cells administered should take the route of administration into consideration. Yes, and a sufficient number of manipulated cells are introduced to achieve the desired therapeutic objective. It should be like that. Generally, pharmaceutical compositions are based on the symptoms or clinical signs of a disease. It is administered to the subject in an effective dose and dosage regimen to treat the symptom, and it is (limited) To reduce it (to any degree), to limit its progression, This may include improving the situation or eliminating it.

[0127] The constructs, manipulated cells, and NK cell lines described herein may be used for therapeutic purposes, without limitation. To reduce the size of tumors or other targeted cells in the target, or to reduce tumor size. By inhibiting the growth or regrowth of sores or other cancerous or malignant cells, Many applications include treating subjects with nosine overexpression cancer or other disease conditions. It can be used in the following cases. Therefore, adenosine overexpression cancer or related disease conditions Embodiments of methods for treating subjects are also provided.

[0128] Methods of treating individuals with cancer using immunotherapy are also offered. Such a method involves administering a therapeutically effective dose of the pharmaceutical composition described herein (or The procedure may include the step of administering the drug to target cells. For example, the pharmaceutical composition may be present on at least the target cells. A first binding domain or flag that targets one type of cognitive ligand. A ment (e.g., NKG2D), at least a second binding domain that is specific to GD2 or its fragments (e.g., anti-GD2, and optionally scFv), and target cells At least one cell surface protein that is specific to adenosine production or adenosine intermediate production. The third binding domain or its fragment (e.g., anti-CD73, and optionally sc Engineered NK cells expressing polynucleotide constructs encoding Fv (as specified herein) May include a group of (as described herein). A bound domain Activation occurs when it binds to target cells, which can lead to cytotoxicity or cell damage in the manipulated cells. Signaling domains containing one or more domains involved in promoting lysis activity It can also be coded. Target cells are, for example, cancer cells (e.g., GBM) or TME. It may contain malignant cells.

[0129] The administration steps are not limited to intravenous, intratumoral (locally), or parenteral. Using any of the previously described administration techniques, including by injection (systemically). It may be implemented using [a specific method]. Furthermore, it may be implemented to express one or more of the constructs of the present disclosure. The pharmaceutical compositions herein, which include modified and manipulated cells, are used for adenosine overload such as glioblastoma. The novel drugs described herein may be collected into kits for treating subjects experiencing cancer. In conjunction with the scientific composition, such a kit simplifies the administration of such composition to the subject. And / or one or more tools and / or devices that are desirable to facilitate this It is understood that this may further include (for example, vials, syringes, tubes, etc.) cormorant.

[0130] A method for treating a subject suffering from cancer, in at least one exemplary embodiment. This includes one or more autophagy inhibitors described herein, which are added to the target. The procedure may further include administering (or keeping in place) a specific therapeutic treatment. For example, Such autophagy inhibitors include therapeutically effective doses of small molecule inhibitors and / or autophagy inhibitors. This may include genetic downregulation of genes in the azithromycin pathway. At least one exemplary implementation. In terms of form, the method involves administering chloroquine to the subject at a concentration of 0.01 μM to 200 μM. The procedure includes (or administers) additional therapeutic measures. It may be performed as needed, including by systemic injection or infusion.

[0131] The method involves at least a dual approach (i.e., the pharmaceutical compositions of this disclosure and autophagy) If it includes the administration of additional therapeutic measures, including inhibitors, the means of administering both therapeutic modes are the same. It does not need to be, nor does it need to occur simultaneously or by the same medication pattern. For example, the pharmaceutical composition may be administered intravenously, intratumorally, parenterally, or by infusion. The administration of additional therapeutic measures is carried out by systemic injection or infusion. Therefore, treatment The timing and dosage of medication can be tailored to the specific patient and their medical condition. ru.

[0132] Furthermore, kits to facilitate the multiple treatment methods described herein may also be provided. At least one In an exemplary embodiment, the kit comprises a therapeutically effective amount of a pharmaceutical composition in accordance with the present disclosure, It may also contain a therapeutically effective amount of an autophagy inhibitor. In at least one embodiment... Such therapeutic components are housed separately within the kit. Therefore, the kit simplifies and / or facilitates the administration of such compositions to the subject. It may further include one or more desirable tools and / or devices (for example) (This refers to vials, syringes, tubes, etc.)

[0133] Optionally, the method may also include a step of preparing a pharmaceutical composition for the subject. For example, Any step may include taking out or setting aside a sample, such The sample includes stem cells, blood cells, or iPSCs. Such isolated cells are Subsequently, it is isolated from the sample (i.e., in the case of a sample including peripheral blood collection, one type Alternatively, multiple NK cells may be isolated, and if necessary or desired, they may be augmented. The sample is obtained from the subject (e.g., autologous cancer immunotherapy), and the adoptive cell therapy is performed using the same method. This is used for the procedure. Alternatively, the sample may be provided by a donor separate from the subject. (For example, allotherapy). In at least one embodiment, isolation, genetic modification, and / or any augmentation step is performed in vitro.

[0134] The method involves transducing isolated cells with an expression vector containing the construct of the present disclosure. This may also include any step involving transfecting. Then the desired construct A population of manipulated cells that express is achieved. Then, a collection of such manipulated cells The drug may be administered to the subject as previously described. In at least one embodiment, Such therapies include adoptive cell therapy.

[0135] Furthermore, the method is for the treatment of cancer, solid tumors, and / or symptoms or related conditions. Regarding improving or eliminating side effects associated with eel therapy, currently known or It may be (or may include) the administration of additional therapies that will be developed later. As illustrated. In at least one exemplary embodiment, the method is the auto described above. Additional therapeutic measures, including fuzzy inhibitors, are administered (or kept in place) to target the patient. This may include certain steps.

[0136] In at least one embodiment of such a method, the construct of the present disclosure isolates the subject. The transformed NK cells are introduced, and then the transformed NK cells are reintroduced into the target cells. This induces an antitumor and / or anticancer response, thereby reducing the disease state in the subject. Or eliminate them. The appropriate NK cells that may be used are listed above and are not limited to those listed above. Includes blood-derived NK cells. Even non-NK cells as described herein may be used. To those skilled in the art As is well known, various methods such as leukocyte apheresis isolate these cells from the subject. Therefore, it is readily available.

[0137] The constructs, manipulated cells and cell lines, pharmaceutical compositions, and methods described herein Although various embodiments are described in considerable detail, the embodiments are not limited to... Examples are given. Many variations and modifications of the embodiments described herein are included in this disclosure. This should be obvious to those skilled in the art. Therefore, without departing from the scope of this disclosure, Various changes and modifications may be made, and equivalents may be substituted for those elements, as is the case for those skilled in the art. This will be understood as such. In fact, this disclosure is either too comprehensive or too restrictive. It is not intended to be. The scope of this disclosure is as provided by the attached claims and those It should be determined by equivalents.

[0138] Furthermore, when describing a typical embodiment, this disclosure may refer to the method as a specific sequence of steps. It may present and / or a process. However, if the method or process is not this Unless the method or process depends on the specific order of the steps described in the specification, the method or process is described. It should not be limited to a specific sequence of steps. As a person skilled in the art would understand, Other arrangements of the steps may be possible. Therefore, the specific steps disclosed herein are not limited to those disclosed herein. The order of these should not be interpreted as a limitation on the claims. In addition, the method Claims relating to and / or processes are not limited to the execution of those steps in the order they are written. It should not be fixed, and those skilled in the art will know that the arrangement can vary, and the spirit and scope of this disclosure remain unchanged. It is easy to understand that one can remain within the enclosure.

[0139] Therefore, the claims in this description and the attached claims are obvious to those skilled in the art based on this disclosure. It is intended to encompass all modifications and changes.

[0140] Tumor-promoting antigens and construct design considerations The antigen target selection described herein involves combinations of pro-tumor antigens in GBM patient samples. Based on its presence, GD2 is a marker that plays a specific role in contributing to GBM pathology. The gene-level transcriptional expression of NKG2DL and CD73 is observed in The Cancer Ge GBM RNA-seq patient days from the nome Atlas (TCGA) patient cohort It was found to be present individually in over 96% of Taseth (156 patients) (151 out of 156 patients). Observed, and while double combinations were detected in 68% of patient samples, both in children and adults. Primary and adult-relapsed patient-derived GBM cells further supported this expression pattern. .

[0141] Modulating the intensity of antigen recognition for target antigens present in low concentrations is important for reducing the risk of poor antibody It may enhance activation signals under normal density conditions, but this is because it is present in normal cells. This also enhances off-target toxicity to such antigens. Direct antigen recognition (for example, CAR) (by activation based on) and sustained spatially controlled release of soluble antibody fragments Matching, targeting specific antigen combinations, can suppress potential off-target effects. These antigens show changes in expression over time or in individual tumors, targeting in patients. A combination is provided. Therefore, according to this disclosure, at least one exemplary embodiment In this state, heterogeneous combinations of antigens can be targeted locally in response to their expression levels. On the other hand, NK cells were manipulated to express a trifunctional construct that does not harm healthy cells. These multifunctional CAR-NK cells are human brain microvascular endothelial cells (hCMEC / D 3) And clearly compared to normal cells in the brain, including human cortical neurons (HCN-2). It did not exhibit toxicity (see the examples below). This is because, at least in part, This is supported by the low expression of at least one targeted marker on these cells. The induced release of CD73 antibody fragments by GBM TAP is dependent on CAR expression. A persistent response that exists but is independent of its activation, low concentrations in local TME CD73 is involved, providing two related but independent mechanisms for tumor recognition.

[0142] Several studies have shown that blood-derived NK cells from both healthy donors and GBM patients are different. Significant phenotypic similarity has been demonstrated. However, the phenotype of GBM tumor-resident NK cells And the functions have been significantly altered, and the activating receptors CD16, NKG2D, NKp30, NK Characterized by significantly lower levels of p46, DNAM-1, CD2, and 2B4. Mechanistically, GBM patients utilize their own immunosuppressive TMEs to... Infiltrative NK cell function and impaired NK-mediated cytotoxicity supported GBM escape from epidemic surveillance. Now. These immune evasion mechanisms are rooted in the heterogeneity of GBM and promote the progression of GBM. This represents a complex network of paths.

[0143] The NKG2D-NKG2DL interaction is crucial in activating the anti-cancer immune response. It plays a role. However, as discussed earlier in this specification, the surface of NK cells NKG2D expression in the surface is produced by the exocene CD73 in GBM TME. It decreases significantly in response to high extracellular concentrations of denosine. Against this backdrop, the inventors of this invention... CD73 is a significant prognostic biomarker for GBM, and is important for the survival time of GBM patients. We have previously confirmed that it can be used as a correlation factor. In addition, this disclosure states that NK NKG2D through the use of inventively engineered NK cells expressing G2D-based CAR Compositions, cells, and methods have been achieved that can induce upregulation of CD73 blockade. Previous studies have shown that the combination enhances the antitumor response. However, adenos Immunosuppression of NK cells via the agonistic axis goes beyond activating receptor inhibition and affects NK cell metabolism and It encompasses a variety of effector functions. Due to the broad expression of CD73, this enzyme is targeted. Localizing the blocked signal may be preferable for systemic administration of anti-CD73 therapy. ru.

[0144] To achieve a controlled and localized response, induce release in GBM TME. In the presence of tumor-responsive locally released anti-CD73, local CD73 activity can be blocked. NK cells were manipulated to possess scFv. The data presented herein Furthermore, the manipulated NK cells described herein not only reduce the local concentration of extracellular adenosine, but also This supports the idea that it also reduces immunosuppression of NK cell activation in a tumor-specific manner. Sustained low-concentration release of CD73 enzyme activity (i.e., TME) Therefore, it sustains the metabolic function of NK cells that would otherwise be lost. In that respect, local CD73 blocking CAR-mediated signaling in combination with disconnection is used to cause cytotoxicity in cancer cells. Furthermore, these impaired metabolic activities also contribute to an antitumor response that can be measured by their impaired metabolic activity. And, in turn, this helps to sustain the longer-term retention of NK cells in tumors.

[0145] Transient modulation of antigens can also occur in response to treatment. Two MHC class I Chain-related proteins (MICA and MICB) and six UL16-binding proteins (U Expression of NKG2DL, including LBP1-6), is associated with chemotherapy (TMZ) or irradiation (IR). After the standard treatment used, it is upregulated in GBM. Provided in the example below. As supported by the data, NKG2DL expression is reduced by autophagy inhibitors. It can also be regulated, and treatment using it can significantly reduce NKG2DL on patient-derived GBM cells. It can induce an increase (both MFI and % levels). NKG2DL expression is associated with GBM cells. Previously, it was found that stem-like GBM cells are inversely correlated with cellular maturity, and these ligands Although the expression was heterogeneous, a more substantial pattern was expressed by autophagy inhibitors. The induced change is that the GBM is sensitized to the effector function via the NKG2DL-R axis. Furthermore, through a phenomenon that can be reduced by CAR-mediated induction of NKG2D expression, the effect It also promotes the indirect loss of NKG2D on ter cells. Furthermore, autophagy inhibitors The reorganization of GBM TMEs induced by this involves a decrease in GD2 expression, which is related to GBM Reduce the apoptotic load imposed on tumor-infiltrating cells induced by gangliosides. This is associated with therapy resistance caused by glioma stem-like cells in patient GBM tumors. To reproduce some of the stem cell-like properties, cells were sourced from patients with primary GBM. Patient-derived xenografts produced using [a specific method / technology] were utilized.

[0146] Antigen targeting may not be sufficient to induce a sustained anti-GBM response. Clinical data The study showed that the presence of NK cells within tumors is positively associated with patient outcomes and survival. However, NK cells are present in GBM at low levels. Diverse pathophysiology in GBM. Among the mechanisms of regulating chemokine function, chemokine receptor / ligand interactions are involved in their signature In parallel with the signaling gradient, NK cell transport is promoted to GBM by CXCR4 / CXCL12 This may result in improved homing, a demonstrated response to directed NK cell homing. However, little evidence of chemokine-dependent transport has been shown in intracranial GBM. This disclosure describes the process of targeting CCL5 in GBM after pharmacological targeting of autophagy. The study revealed a significant correlation between the CXCL10 chemokine pathway and the chemokine pathway. Mechanistically, Inhibition of autophagy in GBM cells affects the PI3K / NF-κB signaling pathway. Through activation, it induces the upregulation of chemokines, particularly CCL5 and CXCL10. It was determined that this subsequently conferred significant chemotactic ability to NK cells and was observed in the brains of treated mice. This resulted in the remarkable detection of adopted CD73.mCAR-pNK cells. Although significant upregulation of these chemokines was detected, other factors related to NK cell intratumoral transport were observed. The contribution of tumor-associated macrophages (TA) cannot be definitively ruled out at this time. Conversely, tumor-associated macrophages (TA) Both are recognized for their contributing roles in the recruitment of immunosuppressive cells such as M. The expression of CL2 and CXCL12 was significantly reduced in response to autophagy blockade. The preclinical in vivo data presented in the specification are related to autophagy. Inhibitor-mediated targeting activates antigen-level expression and pro-apoptotic pathways in the GBM. Through a mechanism that modulates sexualization, which may involve sensitization to their elimination Furthermore, it demonstrates that it enhances the anti-GBM activity of NK cells. These data are from autof Novel, practical responses induced by azithromycin inhibition and CAR-based antigen targeting The data presented herein indicate the effects of administering autophagy inhibitors. It also highlights the complex and sophisticated reorganization of GBM TMEs that is induced. Conversely, two distinct confounding factors affecting the effects of autophagy inhibitors have been clinically recognized. As an adjuvant, CQ (a representative example of an autophagy inhibitor) was used in a double-blind II study. In a Phase I study, it was shown to enhance the clinical response to anti-GBM therapy. Data This supports the idea that CQ could cross the blood-brain barrier in vivo, while preclinical studies have shown that 50 The concentration of mg / kg is appropriate for the administration regimen for orthotopic glioblastoma xenografts. It has been proven.

[0147] However, along with its limited ability to control only tumor burden, GBM in vivo Multiple autophagy inhibitors targeting phenotypic signatures and chemokine profiles The effect of this combination may nevertheless independently enhance CAR-based adoptive transfer immunotherapy. This refers to the subtle role in combination regimens. Many autophagy inhibitors such as CQ Ideally, potential toxicity should be incorporated into the therapeutic administration regimen. The use of the low-level metabolite hydroxychloroquine is one way to mitigate toxicity, especially associated with CQ. This is the strategy. Here, when mice are treated with a combination of CQ and NK cells... NK cells were injected intracranially, while CQ was administered systemically. Importantly, the treatment group No observable CQ-dependent toxicity was observed in this case.

[0148] This disclosure and related data are from multifunctional genetically engineered human NK(CD73.mCA) The development of R-pNK cells addresses tumor heterogeneity and multiple immunosuppressive characteristics of GBM TME. This demonstrates that effective anti-GBM activity can be achieved through a collaborative approach to overcome the problem. This disclosure describes how targeting autophagy can function as an immune modulator. This promotes the homing of effector CAR-NK cells to GBM tumor sites, while, Reprogramming GBM TME to counter sensitization to CAR-based targeting is also a possibility. It stopped. In fact, all treated mice showed delayed or complete cessation of tumor growth. I have experienced both, and the most effective results were obtained when CAR-NK cells were co-administered along with autophagy inhibitors. A significant response was obtained. Regarding the use of human CAR-NK cells in immunotherapy for GBM. As evidence, the data shows that human CAR-NK therapy is a viable option, while In certain embodiments, the optimal response is determined by the adoptive transfer administration regimen and dosage. This indicates that it may depend on frequency. [Examples]

[0149] The following examples illustrate certain specific embodiments of the present disclosure and do not necessarily represent all other embodiments. This invention is not intended to limit its scope.

[0150] mouse Female Rag1, 6-8 weeks old - / - Mouse and NOD.Cg-Prkdc scid I L2 rgtm1Wjl / SzJ(NSG) mice were used at the Purdue Cancer Research Center. Maintained at the Center for Cancer Research. All animal experiments described herein are based on Approved by the Purdue University Animal Experimentation Committee.

[0151] Isolation and cell culture of peripheral blood NK cells Blood samples were obtained from healthy adult donors, and pNK cells were extracted using EasySep(trademark) Dir. ect Human NK Cell Isolation Kit (StemCell Technologies, Can Cells were isolated from whole blood by negative selection using ADA. Then, the cells were processed according to the manufacturer's instructions. Use the NK MACS® culture medium system (130-114-429) according to the instructions. The cells were cultured and augmented using Miltenyi Biotec (Germany). NK-92 cells (purchased directly from the American Cell Culture and Preservation Center (ATCC)) 0% FBS, 100 U / mL penicillin, 100 μg / mL streptomycin, 2 ml M L-Glutamine, 400 U / mL IL-2, and 0.1 mM 2-Mercaptol Tanol was maintained in RPMI1640 cells that had been captured. HCN-2 cells (directly from ATCC) (Purchased) 10% FBS, 100 U / mL penicillin, and 100 μg / mL Streptomycin was maintained in supplemented DMEM. hCMEC / D3 cells (Padue) - University, Indiana) 5% FBS, 100 U / mL penicillin, 100 μg / mL streptomycin, 1.4 μM hydrocortisone, 5 μg / mL ascorbic acid, 1% chemically defined lipid concentrate, 10 ml M HEPES and 1 ng / mL bFGF were grown in EBM-2 supplemented. SJ-GBM2, GBM43, and GBM10 cells (Indiana Univer City School of Medicine, Indiana) 10% FB S and 1% HEPES were grown in supplemented DMEM. All cell lines were added The cells were incubated at 37°C in a humid 5% CO2 environment.

[0152] Chemical reagents Chloroquine diphosphate (CQ) (98%) and adenosine 5'-monophosphate sodium Alkaline Hydroxide (AMP) (99%), ACROS Organics (Trademark) (Fair Purchased from Lawn, New Jersey. Hydrocortisone (98%), Al I bought it from fa Aesar (Haverhill, Massachusetts). Ascorbic acid, sodium chloride (NaCl), and bovine serum albumin (BSA) , Thermo Fisher Scientific (Waltham, Massac Purchased from Husetts. Potassium chloride (KCl), magnesium chloride (MgCl 2) Sodium bicarbonate (NaHCO3), adenosine (99% or more), DEAE-extract Stran hydrochloride and glucose, Sigma (St. Louis, Missour i) Purchased from: Brefelzin A solution 1000× and Monensin solution 1000× I purchased it from BioLegend (San Diego, California). - Luciferin potassium salt (over 99%), Syd Labs (Natick, Mass Purchased from achusetts. LY294002, BAY11-782, and S P600125, Cayman Chemical (Ann Arbor, Michi Purchased from gan. Fetal bovine serum (FBS) and dimethyl sulfoxide (DMSO) I purchased it from Corning (Corning, New York). Tointerleukin-12 (IL-2) is produced by Akron Biotech (Boca R Transferred from aton, Florida. Recombinant human interleukin-15(I L-15) and fibroblast growth factor-basic (bFGF), GoldBio (St. Purchased from Louis (Missouri). Recombinant human RANTES (CCL5) I purchased it from PeproTech (Rocky Hill, New Jersey). Recombinant human CXCL10, recombinant human u-plasminogen activator / uroquina -ze, CF(uPA), and recombinant human NKG2D / CD314 Fc chimera, R esearch and Diagnostic Systems, Inc. (Minn I purchased Biotin-Protein L from eapolis (Minnesota). From nScript Biotech (Piscataway, New Jersey) I bought it. BsiWI from New England Biolabs (Ipswich, Purchased from Massachusetts. RPMI1640, DMEM, IMDM, Penicillin / streptomycin solution 100×(PS), 2-mercaptoethanol (5 0 mM), HEPES (1 M), chemically defined lipid concentrate, trypan blue solution, and trypsin-EDTA, Gibco®, Thermo Fisher S Purchased from scientific (Waltham, Massachusetts). pti-MEM Reduced Serum Media is used with Invitrogen(C I bought it from arlsbad (California). EBM-2, Lonza G I purchased human AB serum from roup AG (Basel, Switzerland). , Valley Biomedical (Winchester, Virginia) I purchased it. Collagen I, rat tail, Enzo Life Sciences, I Purchased from nc. (Farmingdale, New York). RIPA dissolution bag Far System (sc-24948) is manufactured by Santa Cruz Biotechnol I purchased it from ogy, Inc. (Dallas, Texas).

[0153] Plasmid construction and lentivirus production The (1), (2), (3), and (4) below were custom cloned and vectorbu Produced by ilder.com. (5) Santa Cruz Biotec Purchased from hnology, Inc. (Dallas, Texas). (6) Gen I purchased it from Target Inc. (San Diego, California). (1) pT7[mRNA]-NKG2D-CAR: In this plasmid, human NKG Under 2D-specific CAR (NKG2D-DAP10-CD3ζ), it is identified as construct 1B. The gene was expressed under the control of the T7 promoter. The NKG2D sequence is derived from previous work. did. (2) pT7[mRNA]-GD2-CAR: In this plasmid, human GD2 specific Target CAR (anti-GD2 scFv-CD8 hinge-hCD28-CD3ζ; under construction 1A (identified as) was expressed under the control of the T7 promoter. Anti-GD2 scFv The line originated from a previous job. (3) pT7[mRNA]-CD73-CAR: In this plasmid, human CD73 Specific cleavable anti-CD73 scFv, human GD2 specific CAR(GD2 scFv) -CD8 hinge-hCD28-CD3ζ; construct 1A) and human NKG2D specific CA The entire structure, including R(NKG2D-DAP10-CD3ζ; Structure 1B), is included in the T7 promo. It was expressed under the control of the chemist. Here, the anti-CD73 scFv fragment was expressed as SEQ ID NO: A linker containing 1, a cleavable peptide fragment (SEQ ID NO: 2), and a short space It was coupled with GD2-CAR via SURR (GSSGT). GD2-CAR was "self The NKG2D-CAR was bound to the anti-C peptide via the "cleavage" P2A peptide (SEQ ID NO: 4). The D73 scFv sequence originated from previous work. (4) pLV[Exp]-Puro-EF1A>{CD73-CAR}: As described in (3) The lentiviral vector encoding the entire structure (structure 1) is EF1 alpha- It was expressed under the control of a romodor. (5) Beclin 1 (BECN1) shRNA (h) lentivirus particle (sc-2979 7-V): BECN1 gene knockdown BECN1 - GBM43 cells, manufacturer's It was produced by lentiviral transduction according to the rotocall. (6) CMV-luciferase (firefly), (Puro) lentivirus particles (LVP3 25): Firefly luciferase-labeled GBM43: GBM43 (Luc) cells, manufacturer Following the protocol, a lentiviral vector containing luciferase was used. It was created by viral transduction.

[0154] material Table 1 lists the antibodies and stains used in the experiments described herein. The assay kits and primers described in this disclosure are listed below.

[0155] [Table 1]

[0156] The following assay kits were used in the examples described herein: (1) TransIT (registered trademark) - mRNA transfection kit (Mirus Bio LLC, Madison, Wisconsin); (2) EasySep(trademark) Direct Human NK Cell Isolation Kit (StemCell Technologies, Vancouver, Canada); (3) Human CCL5 (RANTES) Biolegend-ELISA MAX (trademark) )Deluxe Set(BioLegend, Sand Diego, California nia); (4) Human CXCL10 (IP10) Biolegend-ELISA MAX (trademark) )Deluxe Set(BioLegend, Sand Diego, California nia); (5) Malachite Green Phosphate Assay Kit (BioAssay Sy stems, Hayward, California); (6) Adenosine assay kit (Cell Biolabs Inc., San Di ego, California); (7) mirVana(trademark) miRNA isolation kit (Life Technologi es, Carlsbad, California); (8) HiScribe (trademark) T7 ARCA mRNA kit (with tailing) New England Biolabs, Ipswich, Massachusetts s); (9) EZ-10 Spin Column RNA Cleanup & Concentration Bio Basic Inc. (Ontario, Canada); (10)qScript (trademark) One-Step SYBR (registered trademark) Green qRT-PCR kit, Low ROX (Trademark) (Quanta BioSciences I nc., Gaithersburg, Maryland); (11) Fixation / Permeation Solution Kit (BDBiosciences, San Jose, California); (12) 7-AAD / CFSE cell-mediated cytotoxicity assay kit (Cayman Ch chemical, Ann Arbor, Michigan)

[0157] The following RT-PCR primers were used in the examples described herein: CCL5 qPCR primer pair (HP100784), human CXCL10 qPCR R pair (HP100690), human CCL2 qPCR primer pair (HP10485 4) Human CXCL9 qPCR primer pair (HP100773), and human CX CL12 qPCR primer pair (HP100192) From cal US Inc. (Chesterbrook, Pennsylvania) Obtained. GAPDH was used as an endogenous control. Its primer was primer 1 (distributed Column number 4) and primer 2 (SEQ ID NO: 5) are used in Integrated DNA Te Obtained from Technologies, Inc. (Coralville, Iowa). RT- All primers used in the PCR assay were selected according to the manufacturer's instructions. I used it.

[0158] statistical analysis Data are presented in this specification as mean ± SEM. Regarding all studies... Statistical analysis using Excel 2007 software (Microsoft Office) The comparison between two normally distributed test groups was performed using the two-sided Stud method (2007). The analysis was performed using a t-test. For analyses of three or more groups, a one-way ANOVA solution was used. Comparisons were performed using analysis. A p<0.05 value was considered statistically significant.

[0159] [Example 1] NK cells engineered using a multifunctional, responsive CAR-based construct were found to be involved in human GBM. It can target multiple antigens that are widely present. Genes corresponding to NK cells, as well as various pro-tumor or anti-tumor genes in GBM. The gap between the expression profiles of a set of functional genes encoding ulcer-causing ligands and targets. To identify correlations, we use the gene set scoring method to analyze Genomic Data. The Cancer Genome Atlas (TCGA) We examined a GBM RNA-seq patient dataset (156 patients) from ) and found that certain characteristics We identified the relationship between the expression of certain functional genes and the presence of NK cells. The genes are not limited to the NK cell signature gene set (e.g., NCR1 (including NCR3, KLRB1, CD160, and PRF1), NT5E (CD73) (encodes), B4GALNT1 (encodes the enzyme that produces GD2), MICA / B (which encodes a common ligand for NKG2D), as well as CCL5 and CXCL It included 10 (which codes for two chemokines named after different species).

[0160] Linear correlations between normalized gene expression (FPKM) of selected genes are shown in corrplo The determination was made using the t R package. GBM patients (N=156) were divided into high and low expression groups. Using the upper and lower quartiles as cutoffs, respectively, Based on the expression of individual genes (B4GALNT1, MICA, MICB, and NT5E) They were then classified into high / low expression groups. The high expression group was of interest, and of the four genes, at least A Venn diagram was created using the number of patients with high expression of one type (see Figure 1, subcategory C). I want to be.

[0161] NT5E and B4GALNT1 are NK signature sets in GBM, respectively. There is a negative correlation with the individual genes that represent the antigens encoded by these genes. This suggests that it acts to suppress NK cell function and proliferation in GBM. Alternatively, NKG2D Rigan codes MICA / B, CCL5, and CXCL10. Genes known to promote NK effector responses, including those related to individual NK cells, are known to drive NK cell responses. A positive correlation was observed with signature genes (r<0) (see Figure 1, subcategory A). .

[0162] Classification of GBM patients into high / low groups based on the upper and lower 50% quartiles (TCGA R (Using NA-seq data) further supports these findings, and 1 in 156 patients 51 people tested positive for four targeted genes: NT5E, B4GALNT1, MICA, or M We identified that at least one of the ICBs is overexpressed. Enrichment of the NK signature gene set using data from GBM patients Synthesis analysis showed a negative correlation (NE) in the expression of the NT5E and B4GALNT1 genes. S<1), and for the MICA / MICB, CXCL10, and CCL5 genes A positive correlation (NES>1) was observed (see Figure 1, subcategory B, and Table 2). .

[0163] [Table 2]

[0164] The Venn diagram showing the relationship between gene expression among patients represents the expression of multiple gene combinations. This shows a significant number of patients and a number lower than expected (see Figures 1C-1D).

[0165] Two tumorigenic markers, GD2 and CD73, are used to identify patient-derived primary adult-onset (GB) tumors. Includes M43), pediatric (SJ-GBM2), and recurrent adult (GBM10) brain tumor cells. Furthermore, it is widely and heterogeneously expressed on various types of GBM (see Figure 1, subcategory E). (I would like to be treated). Among these, the exocene CD73 has been previously demonstrated to be a negative prognostic factor. It was done.

[0166] Conversely, cognitive ligands for the potent activated NK cell receptor NKG2D also affect GBM. Although highly present, NKG2D is often downregulated in TME. To verify this, pNK cells were divided into 5 × 10 units in 500 μL of culture medium. 5 Individual cell / we Seeds were seeded in 24-well plates at a density of 1000 μM. Then, adenosine was added at a final concentration of 1000 μM. The solution was added at [temperature]. After 24 hours, cells were collected and their viability (%) was measured using the CCK-8 assay. This was determined by (sub-category A in Figure 2). NKG2D expression (%) and MFI were measured using flow saturation. The determination was made by tometry (sub-category B in Figure 2). As shown in Figure 2, adenosine The presence of [this substance] has a clear negative impact on cell viability and similarly reduces NKG2D expression. These findings suggest that it is necessary to increase NKG2D expression on NK cells through manipulation. I support the existence of sexuality.

[0167] To achieve responsive and combined targeting of these GBM antigens, two cistrobes are used. First, the chromogenic vector is manipulated to simultaneously express two individual CARs, and then GD2.C D28.CD3ζ CAR (Structure 1A) and NKG2D.DAP10.CD3ζ C We produced NK cells expressing both AR (construct 1B). Then, we used construct 1A against anti-C Along with D73scFv, further manipulation is performed in tandem after the resectable tumor-sensitive linker. Create CD73-GD2.CD28.CD3ζ-CAR (structure 1) (see Figures 3-4). (I would like to be), CD73 blocking antibody fracturing in GBM TME, independent of CAR activation. This resulted in structures designed for the localized release of spores.

[0168] Next, structure 1B (Figure 5, subdivision A) incorporates a severable anti-CD73 scFv. Structure 1A (CD73-GD2.CD28.CD3ζ-CAR) (Figure 5, Subdivision F) ), and a multifunctional CAR with the entire structure (Structure 1; Figure 5, Sub-section K) By creating distinct types of each element, the individual genetic components of the multifunctional construct 1 are created. We evaluated its functionality.

[0169] Each CAR was expressed separately in NK-92 cells, and the expression of the corresponding CAR structure was verified. Figure 5, subcategories B, G, and L). More specifically, plasmids (1), (2), and (3) The mRNA derived from each individual (as mentioned above) is processed by HiScribe(trademark)T 7 ARCA mRNA kit (with tailing) (New England Biol Synthesized by abs, Ipswich, and Massachusetts. All mR NA products are processed using the EZ-10 spin column RNA Cleanup & Concentrate. Concentrated by the on-kit (Bio Basic Inc., Markham, Canada) They were reduced and purified. Then, their concentrations were measured using a Qubit 4 fluorometer (Thermo Fisher Scientific, Waltham, Massachusetts ) was used for measurement.

[0170] To create manipulated NK-92 cells expressing various CAR constructs, Following Carr's recommended protocol, TransIT(registered trademark)-m was administered to NK-92 cells. RNA transfection kit (Mirus Bio LLC, Madison, W mRNA was transfected with isconsin. In short, NK-92 cells The seeds were seeded into a 12-well plate (5 × 10 in 1 mL of complete growth medium). 5 (Individual cell / well). m Add RNA (1 μg) to Opti-MEM (100 μL) in a sterile polystyrene tube. Add and mix the wells. Then, add mRNA Boost Reagent (3 μL) Trans Reagent (3 μL) was added and mixed sequentially. The tube was then opened. The complex was incubated at warm temperature for 5 minutes and then added to the cells as a droplet. Transfection Next, the cells were incubated at 37°C and 5% CO2 for 48 hours, followed by CAR structure development. It was collected for the current assessment.

[0171] Trans To measure infection efficiency, cells were stained with APC-conjugated NKG2D antibody. BD Accuri(trademark) C6 Plus (Becton, Dickinson (and Company, Franklin Lakes, New Jersey) The data was analyzed using flow cytometry.

[0172] Regarding the detection of GD2.CAR expression (expression of construct 1A and / or construct 1), The cells were sequentially stained with biotin-protein L and APC-streptavidin. The expression level of GD2.CAR was analyzed by flow cytometry. The overall CAR structure Regarding CD73.mCAR-NK-92 cells expressing the construct (construct 1), the cells are as follows: Incubate with various concentrations of uPA to extract from the cell surface and the remainder of the CAR construct. By inducing the removal of anti-CD73 scFv, the expression of anti-CD73 scFv is suppressed. The determination was made. After this, the cells were treated with biotin-protein L and APC-streptavidin. The cells were stained. Next, the expression of the remaining constructs was analyzed by flow cytometry. Cell viability after infection was analyzed using the CCK-8 assay (Dojindo Mol ecular Technologies, Inc., Rockville, Maryl Measured by (and).

[0173] As shown in Figure 5, subcategories C, H, and M, in all cases, CAR expression NK The cells maintained a high survival rate (over 95%). At the same time, robust structures were formed on the NK cells. 1B expression was achieved (approximately 44%) (see Figure 5, sub-category D), and construct 1B was expressed. The cells that were transfected showed higher GBM43 cells compared to untransfected cells. It showed cytotoxic activity (Figure 5, subcategory E). Similar expression was achieved with construct 1A ( Approximately 42% (see Figure 5, subcategory I), corresponding to patient-derived GBM target cells. This resulted in high cytotoxicity (see Figure 5, subcategory J). This data was constructed This supports the idea that substance 1A can effectively recognize and eliminate GD2-expressing GBM cells.

[0174] When manipulated to express the complete multifunctional sequence (construction 1), NK-92 cells, NKG2D expression (Figure 5, subgroup N), anti-GD2 scFv expression, and anti-CD73 s This showed a significant increase in cFv expression (Figure 5, subcategory O).

[0175] Compared to NK-92 cells, primary NK (pNK) cells derived from the peripheral blood of healthy donors are Generally, it has higher lytic activity against patient-derived GBM target cells. M43 cells were harvested from multiple donors and subsequently engineered to express construct 1. To verify that it can be killed by pNK cells, pNK cells were collected from healthy adult donors. Isolated from (Figure 5, subdivision P, and Figure 6), the entire multifunctional structure 1 (CD73.m Lentivirus expresses CAR-pNK in the presence of DEAE-dextran The organisms were manipulated through transduction.

[0176] Cytokine-activated pNK cells (culturing in NK-MACS® medium for one week) The activated lentiviral was transfused in two rounds with the help of dextran. Over the course of the process, the CAR was manipulated to express a functional CAR (pLVp[Exp]-Puro- EF1A>{CD73-CAR}). In short, pNK cells are seeded in a 24-well plate. (10% FBS and 500 U / mL IL-2 supplemented with 0.5 mL RPMI) 5 × 10 in 1640 culture medium5 Individual cells / well). Lentiviral supernatant 10 infection multiplicity (M The solution was added using OI, and then a dextran aqueous solution was added to a final concentration of 8 μg / mL. The plate was centrifuged at 1000g for 60 minutes and incubated at 37°C with 5% CO2 injected. It was incubated overnight in beta. After transfection, CD73.mCAR -pNK cells are harvested and grown in NK-MACS® medium until further use. Next, the expression levels of all constructs were flow-scaled according to the protocol described above. Cell viability after transfection was measured and analyzed by tometry. Measurement was performed using Pan Blue staining.

[0177] After two rounds of transduction, the expected donor-specific decline in donor NK cell survival rate ( Figures 5, sub-category O, and 6) show NKG2D, anti-GD2 scFv, and anti-CD7 3. Significantly increased expression of scFv (Figure 5, subgroups R and S, and Figure 6) Furthermore, CD73.mCAR-pNK cells expressing construct 1 use feeder Without any additional ingredients, it can substantially grow over two weeks in commercial NK-MACS® medium. On the other hand, stable gene expression was maintained (see Figure 5, subcategory T).

[0178] Collectively, these results suggest that CD73, GD2, and as combined GBM targets are important. To validate the use of NKG2DL and their use in NK cell-based GBM immunotherapy Provide a logical basis for consideration.

[0179] [Example 2] Multifunctional, manipulated NK cells target patient-derived GBM cells while simultaneously harming normal cells. Do not cause harm To investigate the killing efficacy, a killing assay was performed using NK-92, NKG2D, and CAR-NK9. 2 (Structure 1B), GD2.CAR-NK92 (Structure 1A), CD73.mCAR-N Along with either K92 (construct 1), pNK, or CD73.mCAR-pNK cells These cells are used to target various cells (SJ-GBM2, GBM43, GBM10) They were co-cultured for 4 hours with hCMEC / D3 and HCN-2 at various E:T ratios. To evaluate the effect of the anti-CD73 scFv moiety on the killing activity, the expression of the entire construct was performed. Either CD73.mCAR-NK92 or CD73.mCAR-pNK cells possessing Incubate the solution with uPA to remove all anti-CD73 scFv from the surface. Subsequently, the ability of these cells to kill GBM43 target cells was measured.

[0180] To examine degranulation, use CD73.mCAR-NK92 or CD73.mCAR-p One of the NK cells was subjected to APC-CD107a antibody and monensi according to a known technique. The cells were co-cultured with GBM43 cells for 4 hours in the presence of CD107 (E:T=5:1). a(% and MFI) was detected by flow cytometry.

[0181] To investigate IFN-γ production, we followed a known protocol and used CD73.mCAR-N Either K92 or CD73.mCAR-pNK cells in the presence of brefeldin A They were then co-cultured with GBM43 cells for 4 hours (E:T=5:1). Next, the cells were harvested. Wash, fix / permeabilize, then stain with APC-IFN-γ antibody, and then the flow cells It was detected by metric analysis.

[0182] To investigate CD73 activity and its ability to produce adenosine, GBM43 cells were used. This was then processed in complete DMEM into a 96-well plate, with 2 x 10⁶ samples per well. 4 Seeded as individual cells After overnight incubation, GBM43 cells were incubated for 6 hours. From either CD73.mCAR-NK92 or CD73.mCAR-pNK cells Incubated with anti-CD73 scFv after uPA (100 nM) mediated cleavage. Next, the cell medium is removed and the cells are diluted in phosphate-free buffer (ddH2O). 117mM NaCl, 5.3mM KCl, 1.8mM MgCl2, 26mM N The samples were rinsed three times using aHCO3 (10 mM glucose, pH 7.4). Phosphate-free. Diluted AMP (250 μM final) is added to the buffer, and the ink is incubated at 37°C for 10 minutes. Finally, the concentration of phosphoric acid (Pi) produced by AMP hydrolysis was determined by the manufacturer. Follow the instructions for use and use the Malachite Green phosphate assay kit. It was measured.

[0183] Changes in CD16 and NKG2A expression on NK cells in response to GBM cells (i.e.) To investigate the NK cell phenotypic changes, CFSE-labeled GBM43 cells were first completely... In DMEM, 4 x 10 per well are placed in a 24-well plate. 4 Individual cells were seeded. Overnight After incubation, either pNK or CD73.mCAR-pNK cells are added. Then, it was left for 4 hours (E:T=5:1). Next, the cells were collected, washed, and APC-CD16 Alternatively, stain with either APC-NKG2A antibody and perform flow cytometry to determine expression. It was measured.

[0184] Compared to control NK-92 cells, CD73.mCAR-NK92 cells (expressing construct 1) The treatment showed significantly improved killing of GBM43 cells after 4 hours at various E / T ratios. (Figure 7, Subcategory A). Furthermore, co-culture with GBM43 cells is CD73.mCAR-N Stimulating K92 cells induces degranulation, which is measured by cell surface CD107a expression. Figure 7, subcategory B), IFN-γ secretion was upregulated (Figure 7, subcategory C).

[0185] CD73.mCAR-NK92 cells were treated with uPA (100 nM) to produce anti-CD73 After releasing scFv, the number of GBM43s killed decreased (Figure 7, sub-category D). Additionally, cleaved anti-CD73 scFv is converted to extracellular adenosine by GBM43 cells. This resulted in a significantly reduced production (Figure 7, subcategory E, and Figure 8), and a decrease in CD compared to GBM. This showed that the enzymatic activity of 73 was impaired. Additionally, this data suggests that anti-CD73 s cFv is functional and specific to CD73 and can discard adenosine accumulation. This supports the claim.

[0186] Additionally, the gene expresses multifunctional construct 1 (CD73.mCAR-pNK). Manipulated human peripheral blood-derived NK cells have more properties than unmanipulated control NK cells. To a high level, SJ-GBM2 (childhood), GBM43 (adult), and GBM 10. This also resulted in the efficient elimination of (recurrent) cells (Figures 6 and 7, sub-category F~). (See H). On killing GBM targets by natural or engineered NK cells. Live imaging reveals the dynamic nature of this process, and the involvement of native human NK cells. In comparison, CD73.mCAR-pNK cells exhibited higher specificity in killing GBM cells. To prove (see Figure 9; GBM cells, 4 × 10 4 24 cells / well density Seeds were sown in a gel plate. After overnight incubation, pNK or CD73.m.CAR-pNK cells were cultured. One of the cells was added to an E / T ratio of 5. The co-culture was then prepared using IncuCyte S Image acquisition was performed using 3, and scans were conducted every 10 minutes for 4 hours.

[0187] Stimulation by GBM cells significantly increased NK cell degranulation and CD73.mCAR - It contributed to intracellular production of IFN-γ by pNK cells (Figure 7, sub-sections I and J). CD73.mCAR-NK cells lacking anti-CD73 scFv after uPA treatment were 2.5 After co-culturing for 4 hours at an effector / target (E / T) ratio of 5, target GBM43 cells It exhibited significantly reduced cytotoxicity (see Figure 7, subcategory K). In addition, C After treatment with anti-CD73 scFv cleaved from D73 mCAR-pNK cells, GBM43 cells show a significant decrease in adenosine production due to the loss of active CD73. This demonstrated the ability (Figure 7, lower category L).

[0188] Therefore, the antitumor specificity bestowed upon NK cells by gene expression of the multifunctional construct is evident. This is compared to the changes observed in unmanipulated human NK cells in response to GBM. This resulted in enhanced resistance to CD16 loss after contact with GBM cells (Figure 7, sub-category M). This also resulted in a decrease in the upward regulation of NKG2A (Figure 7, subcategory N).

[0189] Finally, due to the multispecific targeting of any potential expression of these antigens on non-tumor tissues, To address any potential off-target effects, target healthy cells with CD73. The capabilities of mCAR-pNK cells were also evaluated using the protocol described herein. CD73.mCAR-pNK cells are normal cells, specifically hCMEC / D3 and H It did not preferentially kill cells belonging to the nervous system, including CN-2 cells. Instead, CD73.mCAR-pNK cells are a control of unmodified pNK cells compared to healthy brain cells. It exhibited effector activity comparable to that of cells (see Figure 7, subcategories O and P). sea ​​bream).

[0190] For example, multiple mechanisms, including individual patterns of inhibitory ligand expression, Although it may be involved in cell targeting, it is induced by CD73.mCAR-pNK cells. The low rate of cytotoxicity is related to targeted ligands on these cells compared to GBM cells. This may be due to a relatively lower expression profile (see Figure 10). Summary These observations demonstrate the functional superiority of CD73.mCAR-pNK cells, and This represents a promising pathway for highly specific anti-GBM immunotherapy with no observable toxicity to conventional cells. vinegar.

[0191] [Example 3] Functional targeting of autophagy in GBM involves NK activity and homing (Honi Enhance ng Autophagy is a crucial cell survival mechanism that leads to and promotes the development and progression of cancer. This is a ideology. It promotes GBM resistance to therapy, and GB against immunotherapy. To modify its ability to establish M sensitization, autophagy in GBM is blocked. The effects were examined using two methods. In one study, GBM derived from knockdown patients was investigated. Cell (BECN1 - Through the targeting of the BECN1 gene by creating GBM43, Tophagy was genetically blocked. More specifically, GBM43 cells were subjected to the previously described process. They were grown in this manner. Lentivirus BECN1 shRNA particles, according to the manufacturer's instructions for use. Use according to the instructions, BECN1 - GBM43 cells were created. BECN1 - GBM The in vitro growth behavior of 43 plants was verified by CCK8 assay analysis.

[0192] Alternatively, chloroquine (CQ), a common FDA-approved autophagy inhibitor, can be used. This treatment pharmacologically inhibited autophagy (see Figure 11). We investigated their targeting method for both NK cell infiltration and effector function. Regarding the method, GBM43 cells were treated with various concentrations of CQ. In response to CQ, in vitro stimuli were observed. To determine the viability of trophoblast cells, GBM43 cells were divided into 1 × 10⁶ cells. 4 Cell density per well The cells were then seeded into 96-well plates. After overnight incubation, the cells were treated with CQ at various concentrations. The cells were incubated for another 24 hours. Next, cell viability was analyzed using the CCK-8 assay. They made the determination.

[0193] Use the RIPA lysis buffer system to process the cell lysates according to the manufacturer's instructions. Prepared using [the specified method]. Subsequently, the sample was run and provided by RayBiotech, Inc. The analysis was performed using the auto-western service. Target autophagy markers It includes BECN1, LC3B, p62, and β-actin (Beclin 1 (B ECNI), LC3-II (LC3B), and p62 are the three main types of autophagy. (It is a marker.)

[0194] Importantly, in all cases, CQ administration was observed 24 hours later in GBM43 cells The survival rate and proliferation of (IC) were impaired in a dose-dependent manner. 50 =67.10 μM; Figure 12. (See subcategories A and B).

[0195] The gradient between chemokines and their cognitive receptors is directed towards sites with higher concentrations. It initiates directional migration of cells, ultimately regulating the transport of immune cells to tumors. Spurred by the lack of insight into chemokine-mediated transport by NK cells to tumors, Regarding the expression levels of several chemokines known to be associated with NK cell transport, We investigated the effects of inhibiting autophagy in GBM.

[0196] RNA extraction and RT-PCR Total RNA was extracted using the mirVana® miRNA isolation kit, and the concentration was adjusted. The determination was made using a Qubit 4 fluorescence spectrometer. RNA (80 ng) from each sample was... , qScript (trademark) One-Ste in the ViiA-7 RT-PCR system p SYBR(registered trademark) Green qRT-PCR kit (Thermo Fish (Using er Scientific, Waltham, Massachusetts) The genes were then reverse-transcribed. The GAPDH gene was used as an endogenous control. Comparison of the genes of interest. The Ct values ​​were normalized relative to the Ct values ​​of GAPDH. 2 -Δct Using the method, the genes Determine the relative expression, and on the other hand, 2 -ΔΔctChanges in gene expression compared to the control using the method. The magnification was calculated.

[0197] BECN1 - Both GBM43 and CQ-treated GBM43 cells, their mRNA The actual increase in CCL5 and CXCL10 at transcriptional levels is measured by a significant increase in these levels. This resulted in qualitative upregulation (Figure 12, subdivisions C and D). At the same time, chemo-ky CCL2 and CXCL12 are found to be present in GBM43 cells when autophagy is inhibited. This showed a decrease in the transcription level (Figure 13).

[0198] ELISA measurement To evaluate the changes in the protein levels of these chemokines, and the underlying molecules To gain more insight into the mechanism, we will examine the chemokine concentrations of these chemokines. In the absence and in the presence of various pharmacological inhibitors that promote the signaling pathway to , Quantification was performed using ELISA in conditioned medium of CQ-treated GBM43 cells. , PI3K inhibitor LY294002, NF-κB inhibitor BAY11-7082, and It also contained the JNK inhibitor SP600125.

[0199] More specifically, to measure secreted CCL5 and CXCL10, GBM4 The three cells included LY294002, BAY11-7082, and SP600125. Treatment with a final concentration of 50 μM CQ for 24 hours, with or without various small molecule inhibitors. The supernatant was collected, and the levels of CCL5 and CXCL10 were adjusted according to the manufacturer's instructions. Human CCL5 and CXCL10 Biolegend-ELISA MAX® D Quantification was performed using the eluxe Set. Multiple chemokine secretions from GBM43 cells. To observe the levels, the cell culture supernatant was also collected, and all selected targets were targeted by Eve T Chemokai provided by echnologies (Calgary, Canada) The determination was made using an array.

[0200] The conditioned medium for control GBM43 cells was CCL2 (1324.95 pg / mL) and CX Compared to the expression of CL12 (69.2 pg / mL), CCL5 (12.55 pg Both CXCL10 (21.03 pg / mL) and CXCL10 (21.03 pg / mL) showed relatively low levels. (See Figure 14, especially its sub-section B). However, CQ processing GBM4 The three cells showed significantly higher levels of CCL5 overall compared to untreated control cells. Both CXCL10 and CXCL10 were secreted (see Figure 12, sub-categories 4E and 4F). The increased production of these chemokines is due to LY294002 or BAY11-7082. It was significantly inhibited, but not with SP600125, which is because of GBM In contrast, the expression of CCL5 and CXCL10 was enhanced after inhibition of autophagy, and PI3K This suggests a mechanistic link between the activation of the / NF-κB pathway and this process.

[0201] Transwell BBB Migration Assay To determine the involvement of CCL5 and CXCL10 in NK cell migration, the following steps were taken: Therefore, the migration of activated pNK cells to the CCL5 and CXCL10 gradients was investigated using primary human astronomers. Established by direct contact co-culture of trocytes and hMEC / C3 human brain microvascular endothelial cells. Tracking was performed using the in vitro BBB model (Figure 12, subgroup G).

[0202] An in vitro direct contact co-culture blood-brain barrier (BBB) ​​model, as previously described, 5 3 It has been established. In short, human astrocytes are given 2 μg / cm³ 2 With poly-L-lysine Recorded 24-well Transwell® insert (pore size 5μ) m) 4 × 10 4 individual cells / cm 2 Seeds were sown at a certain density and grown / differentiated in culture medium for 48 hours. Next, the culture medium is removed, and the hCMEC / D3 cells suspended in EBM-2 medium are divided into 1 × 10⁻¹⁶ cells. 5 individual cells / cm 2 The seeds were sown at this density.

[0203] The co-cultures were grown in EBM-2 with medium changes every other day for an additional 7 days, A follow-up investigation was conducted. It contained recombinant CCL5 or CXCL10 at 100 ng / mL. 600 μL of RPMI1640 medium supplemented with 1% FBS, Transwell (Registered Trademark) 100 μL of 1% FBS was placed in the lower chamber of the plate. Activated pNK cells (5 × 10) in RPMI1640 medium 5 Place the (pieces) into the upper chamber. (Pore size of 5 μm). Incubated at 37°C and 5% CO2 for 6 hours. Afterward, the number of pNK cells that migrated to the lower chamber was determined by flow cytometry. The data is presented as a percentage of migration based on total cell input.

[0204] As shown in Figure 12, sub-section H, activated pNK cells contain CCL5 (100 ng / They migrate along the ligand gradient of (mL) and CXCL10 (100 ng / mL). It significantly increased the ability of BECN1. Interestingly, - GBM43 cell survival rate Growth was not affected by BECN1 knockdown (Figure 12, subcategory I) ).

[0205] In vivo research The effects of BECN1 knockdown on GBM tumor growth and NK cell infiltration, mature B And Rag1, which lacks T lymphocytes but instead has a mature NK cell compartment. - / - Further evaluation was performed in vivo in mice (Figure 12, subcategory J).

[0206] GBM cells (3×10 6 (pieces) to Rag1 - / - Subcutaneous (SC) in both flanks of mice Inoculated (GBM43 control cells in the right flank, and BECN1 in the left flank) - GB (M43 cells). Monitor tumor growth and measure tumor length (L), width (W), and height (H). The tumor volume (mm³) was measured using a digital caliper. 3 ) is given by the equation: V=0.52 The calculation was performed using ×L×W×H. Tumor growth was measured until the mouse met the specified evaluation criteria. It was monitored. At that point, tumor tissue was collected and processed for histological (IHC) analysis. did.

[0207] NSG mice were placed in the right flank with 3 × 10⁶ units. 6 By inoculating individual GBM43 cells A subcutaneous GBM xenograft was established. Ten days later, the tumor was approximately 80 mm. 3 When it reaches that volume The mice were randomly assigned to five groups (n=4 / group), and one of the following protocols was used. The following were processed according to the instructions: (1) PBS only; (2) 5 × 10 6Individual pNK cells in a single vein (IV) injection; (3)5×10 6 Individual CD73.mCAR-pNK cells (construction 1) (4) Intravenous (IV) injection; (5) CQ (50 mg / kg) alone intraperitoneal (IP) injection. NK The cells were administered once a week for three weeks. Mice that received adoptive NK cell therapy (groups 2 and 3) The mice also received IL-15 (0.5 μg / mouse) via IP injection every 3-4 days. Tumor volume is tracked by caliper measurement using the formula described above: V = 0.52 × L × W Calculated using ×H. Mouse body weight was recorded throughout the treatment. At the end of therapy... Next, the mice were sacrificed and the tumors were collected for histological (IHC / IF) analysis.

[0208] Additionally, orthotopic GBM xenografts were created using the previously described NSG mice. In short, on day 0, GBM43(Luc) cells (1 × 10⁶) 5 (individual) to be digitized The device was stereotactically implanted in the right forebrain using a localized delivery system. Nine days after implantation, the mice were divided into four groups. The subjects are randomly assigned to groups (n=4 / group) and processed according to one of the following protocols. (1) Untreated (as a control); (2) Intraperitoneal administration of CQ (50 mg / kg) alone (IP) Injection; (3)2×10 6 A single CD73.mCAR-pNK cell (expressing construct 1) German intracranial (IC) injection; (4) CQ(IP) and CD73.mCAR-pNK cells (IC) ) in combination with: CQ three times a week (1 time / (Daily) Continuous injections were administered. CD73.mCAR-pNK cells were administered once a week for 3 weeks. Tumor The volume was measured using the Spectral Ami Optical imaging system. Monitors were observed and recorded. The weight of the mice was also recorded during the treatment period. At the end of the treatment, the mice The mice were slaughtered, and whole brain tissue from each mouse was used for adenosine measurement and IHC / IF analysis. It was recovered.

[0209] Immunohistochemistry (IHC) and immunofluorescence (IF) staining are performed by the Department of Histology at Purdue University School of Veterinary Medicine. Histology Research Laboratory at the Purdue University College of Veteri This was conducted at (West Lafayette, Indiana) Medical Center. For this procedure, the tumor is fixed in 10% neutral buffered formalin, embedded in paraffin, and cut into 3-5 μm sections. It was cut into pieces.

[0210] BECN1 - Regarding GBM43 xenografts, mouse NK cells in tumors are used. NK cells in tumors were detected by staining using Uss NKp46 / NCR1 antibody. For quantification, stained cells were compared to five randomly selected tumor cells on each slide. The field of view was counted at 200x magnification.

[0211] CCL5 and CXCL10 expression in tumors are controlled by human CCL5 / RANTE, respectively. Detection was performed by staining using S antibody and CXCL10 antibody. Staining was then performed using chemokine immunoassay. Staining intensity (weak=1, moderate=2, and high=3), and density of positive tumor cells (0%= The evaluation was based on the following criteria: 0, 1-40% = 1, 41-75% = 2, over 76% = 3. Each sample The final score of the unit was multiplied by its intensity and density.

[0212] For subcutaneous GBM43 xenografts, NK cell infiltration can be detected by staining with the following: Protein L( Alexa 647 (far red), NKp46 (Alexa 488, green), and DA The findings were examined by IF staining performed using PI nuclear counterstaining. CCL5 and CXCL CD73 expression was evaluated by IHC staining as described above. Expression was detected by staining using a human CD73 antibody.

[0213] Regarding intracranial GBM xenografts, NK cells in the tumor were identified as human NKp46 / NC CCL5 and CXCL10 expression in tumors was detected by staining using R1 antibody. These were then stained using human CCL5 / RANTES antibody and CXCL10 antibody, respectively. Further detection was performed. CD73 expression in tumors was detected by staining with human CD73 antibody. I took it out.

[0214] BECN1 - Tumors from the left flank of mice transplanted with GBM43 cells were compared to those from control GBM43 cells. They exhibited significantly slower growth and smaller size than those derived from 3 cells (Figure 12, lower). (Category K). In parallel with staining for CCL5 and CXCL10, the excised tumor was stained. Immunohistochemical staining was then performed to measure the presence of mouse NK cells by examining the expression of NKp46. In tumors lacking BECN1, significantly higher expression of NKp46 was detected. Deeper NK cell infusion into GBM, which lacks the ability to perform autophagy compared to control tumors. Infiltration was demonstrated (Figure 12, sub-sections L and M). In addition, BECN1 - N in tumors K cells showed a higher distribution in both the peritumoral and intratumoral areas (see Figure 15). Furthermore, the expression of CCL5 and CXCL10 was relatively low in the control tumor. From the baseline level, BECN1- Significantly higher levels of improvement in tumors Sections were added (Figure 12, subsections L, N, and O).

[0215] In addition to increased NK cell infiltration into tumors, 24 hours after treatment with various concentrations of CQ, NK2G2DL expression (MFI and percentage) was evaluated on GBM43 cells.

[0216] GBM43 cells were first treated with CQ (various concentrations), then harvested, and re-implanted in 96 wells. Seeds were seeded. After overnight culture, NK cells (BECN1) were cultured. - GBM43 vs. NK92 comparison Cells (and pNK cells) were added at an E / T ratio of 5 and co-incubated for 4 hours.

[0217] The resulting data showed that CQ-mediated inhibition of autophagy on GBM43 cells. It has a measurable and significant effect on the expression of NKG2DL, CD73, and GD2. This supports the following: Specifically, NKG2DL expression (% and M) on GBM43 cells. Both FI levels increased significantly after 24 hours of treatment with various concentrations of CQ (Figure 16). (Subcategory A). On the other hand, CD73 expression (MFI) was observed even at a low concentration of 6.25 μM. The levels decreased after CQ treatment (approximately 70% of the control group) (Figure 16, Subcategory B).

[0218] In addition to the above, the intratumoral adenosine concentration was also assessed. Brain tissue was collected after processing and cooled. The suspension was rinsed with PBS and homogenized in PBS. Then, the suspension was left to stand at 4°C for 10,000°C. The sample was centrifuged at 0g for 10 minutes, and the supernatant was collected. The adenosine concentration was adjusted according to the manufacturer's instructions. This was determined using an adenosine assay kit. IHC staining was performed as described above. It was implemented on [date / time].

[0219] Adenosine production in human GBM cells mediated by CD73 was also investigated using CQ. The expression of GD2 (MFI) decreased significantly after treatment (Figure 16, subcategory C). It decreased after treatment with CQ at or above the concentration of M. Conversely, GD2 expression decreased. The percentage (%) decreased in response to CQ up to 100 μM, and then it became the control. Compared to cellular organisms (approximately 58%), the percentage remained constant (approximately 46%) (Figure 16, subcategory D). ).

[0220] To further investigate the effect of autophagy inhibition on the cytotoxic capacity of NK cells, CQ-treated GBM43 cells or BECN1 - GBM43 cells, along with pNK cells, 5 The cells were incubated for 4 hours at an E / T ratio. The results showed that treatment with CQ was effective against human NK cells. This supports the finding that GBM cells were indeed sensitized to superior killing (Figure 16, subdivision). (See E and F)

[0221] In summary, the data from these studies suggests that targeting autophagy is effective. Improves NK cell infiltration into GBM tumor beds and increases the level of chemokine CCL5 This demonstrates that it is supported by CXCL10. In addition, autophagy is disabled. This supports the enhancement of NK cell functionality, such as increased NKG2DL expression. By reprogramming the signature, NK cell-mediated response to GBM cells It can also intensify cell damage. GBM is induced by targeting autophagy. TME's complex and unique reprogramming of these reprogrammed paths This could further enhance the sensitization of GBM tumors using NK cells that have been manipulated for optimal utilization.

[0222] [Example 4] Multifunctional, engineered NK cells efficiently target patient-derived GBM tumors in vivo. In vivo antitumor activity of multifunctional manipulated NK cells, as well as against GBM, To evaluate the effects induced by targeting tophagy, patient-derived GBM A subcutaneous xenograft model was established by transplanting 43 cells into NSG mice.

[0223] The processing schedule is summarized in Figure 17, sub-section A. In short, 3 × 10 6 pieces GBM43 cells were subcutaneously (SC) implanted in the right flank of mice (Day 0). 10 days later... (Day 10) Mice in the CQ group were given 50 mg / kg of CQ intraperitoneally once a week for 3 weeks. IP) injection was administered. One day later (day 11), the pNK and CD73.mCAR-pNK groups... A mouse that can be moved, 5x10 6 Individual adopted pNK or CD73.mCAR-pNK Cells were treated intravenously (IV) once a week for 3 weeks. Starting from the day of the first NK cell injection, All mice received 0.5 μg of IL-15 via intravenous therapy (IP) once every 3-4 days.

[0224] Treatment with pNK cells significantly slowed tumor growth compared to mice in the PBS control group. Extended (average tumor volume: 815.75 mm) 3 1475mm 3 , ** p<0.01; flat Equal tumor weight: 1.13g vs. 2.31g ** p<0.01; Figure 17, Subdivisions B and C ). Between pNK cell-treated mice and CD73.mCAR-pNK cell-treated mice, CD 73. mCAR-pNK cells mediate a stronger antitumor response and significantly increase tumor growth rate. A decrease was observed (average tumor volume: 401.5 mm). 3 Ratio 815.75mm 3 , ** p<0.0 1; Average tumor weight: 0.59g vs. 1.13g ** p<0.01). CQ processing is also PBS. Compared to the group, it showed a significant inhibition of tumor growth (mean tumor volume: 953.25 mm²). 3 vs 1 475mm 3 , * p<0.05; mean tumor weight: 1.16g vs. 2.31g. ** p<0. 01; Figure 17, subgroups B and C). Throughout the entire treatment period, in all groups, There was no significant decrease in Us's body weight (Figure 17, subcategory D).

[0225] Next, NK cell infiltration into GBM xenograft tumors was examined by IF staining. Figure 17 As shown in subcategory E, in tumor tissue from pNK cell-treated mice, adoptive transfer Injected NK cells (bright spots) were observed. In comparison, CD73.mCAR-pNK cells were observed. Higher NK cell counts were detected in tumors from cyst-treated mice.

[0226] Additionally, CD73 in vivo + Locally cleaved anti-CD73 that can bind to tumor cells scFv (indicated by the arrow and darker dot) is CD73.mCAR-pNK It was detected near tumor cells in cell-treated mice. In vitro Data and previous in vivo studies have shown that intratumor protease activity induces ligand cleavage. The necessity was demonstrated. For that reason, its activation in the blood was not expected, and as a result, circulation There should be no detectable presence of scFv in the ring. Therefore, using IF, Tumor-specific presence of CD73 scFv was detected.

[0227] The presence of CD73 scFv is co-localized only with CD73.mCAR-pNK staining, and CD This demonstrated that the 73 scFv was successfully disconnected. Furthermore, the CD73.mCAR-pNK process was completed. A significant decrease in CD73 expression on tumors was observed in the control group compared to the other treatment groups. (Figure 17, subdivisions F and G). Additionally, tumor tissue from CQ-treated mice was chemo We revealed significantly higher levels of cain CCL5 and CXCL10 (Figure 17, lower (Categories H-J). These tumors also exhibited lower levels of CD73 expression (Figure 17, lower). Categories F and G).

[0228] [Example 5] Multifunctional genetically engineered NK cells and C to orthotopic patient-derived GBM xenografts Activity in combination with Q The combination of CQ and CD73.mCAR-pNK cells (expressing construct 1) To further analyze the synergistic effects achieved, GBM43 heterologous transfer in NSG mice was performed. A graft orthotopic model was established. First, GBM43 cells expressing firefly luciferase were introduced. By genetically modifying the organism (GBM43-Luc) to enable bioluminescence imaging in vivo, This enabled the monitoring of tumor growth (see Figure 18). More specifically, cells (1000 cells) were grown for 4 days in regular growth medium without puromycin. The total viability of cells over time was measured using the CCK-8 assay and plotted on a logarithmic scale. The cells were divided into lots. Both groups showed similar growth patterns and doubling times.

[0229] A schematic diagram illustrating the in vivo processing program used in this specification is shown in Figure 19, below. This is shown in category A. In short, GBM43-Luc cells (approximately 8×) are introduced into NSG mice. 10 4 (1) is implanted sympathetically, and 10 days after implantation, CQ is administered once a week for 3 weeks into the abdominal cavity. IP) injection (50 mg / kg per day for 3 consecutive days) and / or CD73.mC Intracranial (IC) injection of AR-pNK cells (2 x 10) 4 The individual samples were processed. The control group also In comparison, mice treated with CQ alone showed a significant effect on tumor growth. It was not found (see Figure 19, subcategories B-D). Conversely, CD73.mCAR-pN Mice treated with either K cells or CQ+CD73.mCAR-pNK cells Bioluminescence imaging revealed a clearly reduced tumor growth (Figure 19, bottom). (See Category B). Regarding CQ+CD73.mCAR-pNK cell-treated mice. The most potent antitumor response was observed, particularly in CQ+CD73.mCAR-pNK cells. Tumors in half of the treated mice showed significant cessation during the treatment period. However, no significant changes in mouse body weight were found in any of the groups (Figure 19). Subcategory E).

[0230] Similar to the subcutaneous xenograft study, the tumor tissue of mice in the CQ-treated group was CCL5 and It exhibited robustly upregulated chemokine expression, including CXCL10 (Figure 19, lower group). (F) This may contribute to increased NK cell infiltration, potentially leading to improved therapeutic efficacy. Figure 1 9. As shown in subdivisions G and H, CD73.mCAR-pNK cells alone Compared to mice treated with CQ+CD73.mCAR-pNK cells, GBM tumors in CQ+CD73.mCAR-pNK cell-treated mice Significantly higher NK cell infiltration was observed compared to the control or CQ-treated group. CD73.mCAR-pNK cells and CQ+CD73.mCAR-pNK cell treatment group In both groups, mouse tumors showed decreased CD73 expression, while in the latter group, CD73 expression This showed the most substantial loss of tumors in treated mice (Figure 19, subgroups I and J). This was associated with significantly reduced levels of extracellular adenosine detected in ulcers. Specifically, CD73-mediated adenosine production in local brain tissue is CQ+CD73.mC The most significant reduction was observed in mice treated with AR-pNK cells (Figure 19, subgroup). K and L).

[0231] In summary, these in vivo xenograft studies have shown that CD7 is a good match for patient-derived adult GBM. 3. Demonstrate the potent and specific activity of mCAR-pNK cells. The response is co-therapy with CQ. This is associated with the induction of significant changes in chemokine secretion and CD73 expression on GBM after the procedure. Disabling autophagy induced reorganization of GBM TMEs. [Explanation of Symbols]

[0232] 100 Synthetic Gene Constructs 102 First binding domain or fragment 104 Second binding domain or fragment thereof 106 Third binding domain or fragment

Claims

1. Natural killer (NK) activating receptor, or a first cancer-associated antigen specific to the first A first binding domain or fragment thereof containing a protein, encoding the first The sequence of 1, and A coupling domain or fragment thereof and a cleavable linker The second sequence A nucleotide construct comprising the second binding domain of a target cell adenosine Specially for cell surface proteins that produce adenosine intermediates or cell surface proteins that produce adenosine intermediates in target cells. The linker is different, and the detachable linker is operationally connected to the first bonding domain. Nucleotide constructs are present.

2. A small amount of NK-activated receptors, or a second protein specific to a second cancer-associated antigen. The claim includes at least a third sequence encoding a third binding domain or a fragment thereof. The polynucleotide construct described in item 1.

3. One of the first and third binding domains or fragments thereof is associated with cancer-associated antigens. A specific first protein comprising the first and third binding domains or their flags. The polynucleotide construct according to claim 2, wherein the other of the ment comprises an NK-activated receptor.

4. The second binding domain is an antibody specific to CD73, CD39, or CD38. A polynucleotide construct according to any one of claims 1 to 3, comprising a component.

5. The cleavable linker is activated by one or more proteases in the target cell. The polynucleotide construct according to claim 1, which is cleavable.

6. The first sequence described above is a natural killer group 2 member D receptor (NKG2D), N Extracellular ligand-binding domains containing Kp30, NKp46, NKp40, or DNAM-1 A polynucleotide construct according to claim 1, encoding an NK-activated receptor containing yin.

7. The first cancer-related antigens are disialoganglioside (GD2) and ganglioside G3. (GD3), Her2 (p185), CD19, CD20, CD56, CD123, CD 22, CD30, CD33, CD171, CS-1, C-type lectin-like molecule-1; EpCA M, G250, proteoglycan, GD3, GD2, MHC II, TAG-72, mill Cumucin core protein, Lewis A antigen, tyrosine protein kinase transmembrane receptor (R OR1), c-met, epidermal growth factor receptor (EGFR), EGFR variant III, and A polynucleotide according to claim 1, selected from the group consisting of carcinoembryonic antigens (CEAs). Structure.

8. The second cancer-related antigens mentioned above are disialoganglioside (GD2) and ganglioside G3. (GD3), Her2 (p185), CD19, CD20, CD56, CD123, CD 22, CD30, CD33, CD171, CS-1, C-type lectin-like molecule-1; EpCA M, G250, proteoglycan, GD3, GD2, MHC II, TAG-72, mill Cumucin core protein, Lewis A antigen, tyrosine protein kinase transmembrane receptor (R OR1), c-met, epidermal growth factor receptor (EGFR), EGFR variant III, and Selected from the group consisting of carcinoembryonic antigens (CEAs), the first protein and the second The proteins are not the same, the polynucleotide construct according to claim 7.

9. The first binding domain or fragment thereof is a natural killer group 2 men. The extracellular ligand-binding domain containing the BUR D receptor (NKG2D) is included, and the first sequence Compared to the expression of NKG2D in wild-type NK cells, the upregulation of NKG2D is A polynucleotide construct according to claim 1, further encoding the expressed expression.

10. The second binding domain or fragment thereof is a single-chain antibody fragment (scFv ) comprising anti-CD73, anti-CD39, or anti-CD38 linked to the above third binding domain The in or a fragment thereof is linked to scFv and is an anti-dicialoganglioside (GD The polynucleotide construct according to claim 1, comprising (2).

11. The first sequence is expressed in the first chimeric antigen receptor (CAR), and the second The polynucleotide construct according to claim 2, wherein the sequence is expressed in the second CAR.

12. If activated, one or both of the first and third sequences cause cytotoxicity or cytolysis. Claim 2, which encodes one or more signaling domains for promoting deactivation The polynucleotide construct described above.

13. The one or more signaling domains are operatively linked thereto The main or fragment is activated when it binds to the target cell, according to claim 12. The polynucleotide construct described.

14. The target cells are cancer cells or malignant cells in the tumor microenvironment, as described in claim 1 to A polynucleotide construct as described in any one of item 13.

15. The aforementioned cancer cells or malignant cells are glioblastoma cells or malignant cells in the glioblastoma tumor microenvironment. A polynucleotide construct according to claim 14, which is a cell.

16. One or more signaling domains are immunoglobulin γ-Fc region receptor III -A (FcγRIIIIA), differentiation antigen group 28 (CD28), tumor necrosis factor receptor super Family member 9 (TNFRSF9 or 4-1BB), tumor necrosis factor receptor super - Family member 4 (TNFRSF4 or OX40), Fas ligand (FasL) ), TNF-related apoptosis-inducing ligand (TRAIL), DNAX-activated protein 1 0 (DAP10), DNAX-activated protein 12 (DAP12), native cytotoxic receptor Somatic NKp46, innate cytotoxic receptor NKp44, innate cytotoxic receptor NKp30, LFA-1, differentiation antigen group 244 (CD244), CD137, From the group consisting of CD3 zeta (CD3ζ) and the NKG2D-DAP10 receptor complex A polynucleotide construct according to any one of claims 8 to 13, selected.

17. The one or more signaling domains encoded by the first sequence are D AP10 is the one or more signal transmission encoded by the second sequence. The polynucleotide construct according to claim 14, wherein the domain is CD3ζ.

18. The polynucleotide structure according to claim 4, further comprising the antibody fragment scFv building.

19. The first sequence is a hinge domain, one or more self-cleaving peptides, or The polynucleotide construct according to claim 1, further encoding both of the above.

20. The self-cleaving peptide is selected from the group consisting of P2A, E2A, F2A, and T2A. The polynucleotide construct according to claim 19.

21. The hinge domain includes a linker or spacer, as described in claim 19. Cleotide structure.

22. The first sequence is a first amino having at least 90% sequence identity with sequence number 7. The third sequence codes for an acid and has at least 90% sequence identity with sequence number 6. A polynucleotide construct according to claim 17, encoding a second amino acid.

23. A device that expresses a polynucleotide construct according to any one of claims 1 to 22. A cell or cell line.

24. A claim expressing an amino acid sequence having at least 90% sequence identity with SEQ ID NO:

8. The manipulated cells or cell lines described in 23.

25. The first sequence is the first binding domain encoded by the first sequence or It is operationally linked to its fragment and signaling domain and positioned between them The manipulated cell or cell according to claim 23, which further encodes a hinge domain. KK.

26. The one or more signaling domains are operatively linked thereto Claim 23, wherein the main or fragment is activated when it binds to the target cell. A pharmaceutical composition comprising a population of the manipulated cells described above.

27. The pharmaceutical composition according to claim 26, further comprising a pharmaceutically acceptable carrier.

28. A method of treating a subject suffering from cancer using immunotherapy, at least 、 Natural killer (NK) activating receptor, or a first cancer-associated antigen specific to the first antigen A first binding domain or fragment thereof containing protein; and A second binding domain or its fragment, and a cleavable linker. A polynucleotide construct encoding the second binding domain of a target cell It is specific to adenosine-producing or adenosine intermediate-producing cell surface proteins, and the cut The disconnectable linker is operationally linked to the first binding domain, polynucleotide Targeting therapeutic doses of a pharmaceutical composition containing a population of engineered NK cells expressing a cytoplasmic construct. A method comprising the steps of administering or keeping administered.

29. The aforementioned polynucleotide construct is specific to an NK-activated receptor or a second cancer-associated antigen. A third binding domain or fragment containing a specific second protein further codes The method according to claim 28.

30. One of the first and third binding domains or fragments thereof is associated with cancer-associated antigens. A specific first protein comprising the first and third binding domains or their flags. The method according to claim 29, wherein the other of the ment comprises an NK activating receptor.

31. The aforementioned population of manipulated NK cells is at least 80%, 85%, or 9% of SEQ ID NO:

8. An amino acid sequence having 0% amino acid sequence identity is expressed, according to any of claims 28 to 30 The method described in item 1.

32. Upon activation, the aforementioned polynucleotide construct causes cytotoxicity of the manipulated NK cells. Alternatively, to promote cell lysis activity, one is operatively linked to the first binding domain. The method according to claim 30, further encoding a species or a number of signaling domains.

33. The cancer is glioblastoma, and the first binding domain or fragment thereof is dicya The first protein is specific to loganglioside (GD2), and the third binding domain The receptor or fragment thereof is a natural killer group 2 member D receptor (NKG2). D) comprises an extracellular ligand-binding domain, and the third binding domain or its flange The method according to claim 30, wherein the compound is specific to CD73.

34. The step of administering an additional therapeutic treatment, including an autophagy inhibitor, to the subject or The method according to claim 31, further comprising the step of administering the substance.

35. The autophagy inhibitor is a small molecule inhibitor or autophagy pathway that is effective in therapeutic doses. The method according to claim 34, comprising genetic downregulation of a gene.

36. The genes involved in the autophagy pathway are BECN1, p62, β-actin, and AT. G5, ATG7, LC3B, ATG12, ATG16L1, PI3K-III, ULK1 A selected from the group consisting of ULK2, FIP200, and LAMP2, according to claim 35. Method of description.

37. The autophagy inhibitors mentioned above are chloroquine, hydroxychloroquine, and spautin-1. A small molecule inhibitor selected from the group consisting of SAR405 and verteporfin is included. The method according to claim 36.

38. The chloroquine is administered at a concentration of 0.01 μM to 200 μM, as described in claim 37. method.

39. The method according to claim 28, wherein the cancer is glioblastoma.

40. The steps of administering or keeping administered may be intravenous, intratumor, parenteral, and The method according to claim 28, wherein the method is carried out by injection.

41. The step of administering or keeping a therapeutically effective amount of the pharmaceutical composition to a target is performed intravenously. The additional therapeutic treatment is administered internally, intratumorally, parenterally, or by injection. The aforementioned step of administering or keeping the elephant is carried out by systemic injection or infusion. The method according to claim 34.

42. The step of administering or keeping a therapeutically effective amount of the pharmaceutical composition to the target is adopted The method according to claim 28, comprising the step of performing or having performed cell therapy.

43. This is a kit for treating patients who have experienced glioblastoma. A therapeutically effective amount of the pharmaceutical composition according to claim 26; and Therapeutic doses of autophagy inhibitors A kit that includes this.

44. The autophagy inhibitors mentioned above are chloroquine, hydroxychloroquine, and spautin-1. A small molecule inhibitor selected from the group consisting of SAR405 and verteporfin is included. The kit according to claim 43.

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