Genetically engineered NK cell and application thereof in treatment of fatty liver disease related to metabolic dysfunction

Abstract

The invention discloses a genetically engineered NK cell and application thereof in treatment of metabolic dysfunction related fatty liver diseases, and relates to the field of cellular immunotherapy and genetic engineering. The invention provides a genetically engineered NK cell. The NK cell is genetically engineered to overexpress or heterologous express a CD6 molecule. The invention provides application of the genetically engineered NK cell in preparation of drugs for preventing or treating metabolic dysfunction related fatty liver diseases and progressive diseases thereof. The modified NK cell disclosed by the invention can highly express CD6, can specifically recognize and target pathological hepatocytes which highly express ALCAM in MASH / hepatic fibrosis through a CD6-ALCAM axis, and is clear in treatment target; the therapy is based on brand new discovery of an NK cell dysfunction mechanism in an MASLD disease process, the defect is accurately corrected through genetic engineering, and the immune surveillance function of a body is recovered instead of non-specific anti-inflammation or anti-fibrosis.

Description

Technical Field

[0001] This invention belongs to the fields of cell immunotherapy and genetic engineering, specifically a genetically engineered NK cell and its application in the treatment of fatty liver disease related to metabolic dysfunction. Background Technology

[0002] Nonalcoholic fatty liver disease (NAFLD), also known as metabolic dysfunction-associated fatty liver disease (MASLD), is the most common chronic liver disease worldwide. Its disease spectrum progresses from simple fatty liver (NAFL) to metabolic dysfunction-associated steatohepatitis (MASH), liver fibrosis, cirrhosis, and even hepatocellular carcinoma (HCC). Currently, there are no particularly effective approved drugs for MASH and its associated liver fibrosis, highlighting an urgent clinical need.

[0003] NK cells are key effector cells of the innate immune system, playing a crucial role in tumor immune surveillance and antiviral defense. Recent studies suggest that NK cells also play a complex role in hepatic immune homeostasis and disease progression. Existing research indicates that the function and phenotype of hepatic NK cells undergo dynamic changes during the course of MASLD / MASH. However, the specific mechanisms of action of particular NK cell subsets in disease progression and their potential as therapeutic targets remain unclear.

[0004] The inventors' previous research revealed that liver NK cells exhibit a trend of immaturity during the progression of MASLD to MASH and liver fibrosis. One of the key differences between immature NK cells, such as TD-iNK, and mature NK cells is the absence or reduction of CD6 expression. CD6 can interact with activated leukocyte adhesion molecule (ALCAM) expressed on the surface of hepatocytes. Mature CD6+ NK cells can target and inhibit pathological hepatocytes with high ALCAM expression through this pathway, thereby limiting disease progression. However, in the disease state, immature NK cells have insufficient CD6 expression, leading to the failure of ALCAM inhibition on hepatocytes, resulting in persistently high ALCAM expression, which in turn drives pathological processes such as liver fibrosis.

[0005] Therefore, modifying NK cells through genetic engineering to stably and highly express CD6, thereby restoring or enhancing their ability to target pathological hepatocytes via the CD6-ALCAM axis, provides a novel approach for developing new cell therapies to treat MASH and related liver fibrosis.

[0006] The information disclosed above in this background section is only for enhancing the understanding of the background section of this invention, and therefore may include prior art that is not known to those skilled in the art. Summary of the Invention

[0007] This invention aims to address the lack of effective treatments for MASH and related liver fibrosis in the existing technology, as well as the problem of NK cell dysfunction in the disease state. This invention provides a genetically engineered NK cell method that, by restoring CD6-ALCAM interaction, enhances the NK cell's ability to recognize and clear diseased hepatocytes, thereby enabling its use in the treatment of MASLD and its progressive disease.

[0008] To achieve the above objectives, the present invention provides the following technical solution:

[0009] In a first aspect, the present invention provides a genetically engineered NK cell, wherein the NK cell is genetically modified to overexpress or heterologously express CD6 molecules.

[0010] Furthermore, the genetic modification is achieved by introducing the gene encoding CD6 into NK cells via a viral vector or a non-viral vector.

[0011] Secondly, the present invention provides a method for preparing genetically engineered NK cells as described above, comprising the following steps:

[0012] Step 1: Obtain NK cells;

[0013] Step 2: Construct a gene delivery vector to express CD6, and clone the gene sequence encoding the CD6 molecule into a suitable expression vector;

[0014] Step 3: Introduce the vector constructed in Step 2 into the NK cells obtained in Step 1;

[0015] Step 4: Screen NK cells that successfully express CD6 and expand them in vitro to obtain a population of genetically engineered NK cells that highly express CD6.

[0016] Thirdly, the present invention provides the use of the genetically engineered NK cells described above in the preparation of drugs for the prevention or treatment of metabolic dysfunction-associated fatty liver disease (MASLD) and its progressive disease.

[0017] Furthermore, the progressive diseases include metabolic dysfunction-associated steatohepatitis (MASH), MASH-associated liver fibrosis, and cirrhosis.

[0018] Fourthly, the present invention provides a pharmaceutical composition comprising the genetically engineered NK cells as described above and a pharmaceutically acceptable carrier or excipient.

[0019] Furthermore, the pharmaceutical composition is a formulation for intravenous injection.

[0020] Fifthly, this invention provides the application of CD6 molecules as a target for enhancing the efficacy of NK cells in treating metabolic dysfunction-associated fatty liver disease (MASLD) in genetically engineered NK cells.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] 1. The modified NK cells of this invention highly express CD6 and can specifically recognize and target pathological hepatocytes that highly express ALCAM in MASH / liver fibrosis through the CD6-ALCAM axis, with a clear therapeutic target. This therapy is based on a novel discovery of the NK cell dysfunction mechanism in the progression of MASLD and precisely corrects this defect through genetic engineering to restore the body's own immune surveillance function, rather than non-specific anti-inflammatory or anti-fibrotic effects.

[0023] 2. As described in the background section, in vivo experiments have demonstrated that reinfusion of CD6-expressing NK cells can reverse the progression of MASH-related liver fibrosis, showing strong therapeutic potential. This invention provides a novel cell immunotherapy approach for MASH and related liver fibrosis, which currently lacks effective treatment methods, and has significant clinical translational value. Attached Figure Description

[0024] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in this invention. For those skilled in the art, other drawings can be obtained based on these drawings.

[0025] Figure 1 Verification of NK cell immaturation and its pro-fibrotic effect in the progression of MASLD:

[0026] (ae) Single-cell data from two mouse liver cirrhosis samples and two mouse HCC liver samples;

[0027] (fk) The separation of immune cell changes with the progression of MASLD, with only the changes in NK cell subsets showing the most significant separation with the progression of MASLD;

[0028] (lp) Results of single-cell analysis;

[0029] (q) Schematic diagram of how NK cell immaturity promotes the progression of MASH-related liver fibrosis;

[0030] (rs) Body weight and liver weight of mice in different groups;

[0031] (t) Biochemical indicators of mice;

[0032] (u) Pathology of mice;

[0033] (v) NK cell subtypes in the livers of mice from different groups;

[0034] (w) NK cell subtypes in the spleen of mice from different groups;

[0035] Figure 2 The mutual repulsion between CD6+ NK cells and ALCAM+ hepatocytes and the discovery of the CD6-ALCAM axis:

[0036] (a) Cell communication between different NK cell and hepatocyte subsets;

[0037] (b) TD-iNK appears in the MASH-associated liver fibrosis stage;

[0038] (cd) Multicolor fluorescence staining confirmed that CD6+NK cells and ALCAM+ hepatocytes exhibited mutual repulsion in liver fibrosis and hepatocellular carcinoma sections.

[0039] Figure 3 In vitro and in vivo verification of mature NK cells inhibiting hepatocyte ALCAM via CD6 and CD6-NK cells reversing liver fibrosis:

[0040] (a) Flowchart of in vivo and in vitro validation of NK inhibitors targeting and inhibiting ALCAM in hepatocytes via CD6;

[0041] (b) Flow cytometry analysis of the purity of NK cells sorted by magnetic beads;

[0042] (c) Using CFDA-SE markers to label NK cells;

[0043] (de)CD6 recombinant protein can inhibit ALCAM expression in G2 cells;

[0044] (f) Construct CD6-NK;

[0045] (gh)CD6-ALCAM promotes NK cell and hepatocyte adhesion;

[0046] (i) CD6-NK cells are more capable of killing G2 cells;

[0047] (jl) Quantitative analysis of the above experiments;

[0048] (m) In vivo experiments have demonstrated that CD6-ALCAM promotes the adhesion of NK cells and hepatocytes;

[0049] (n) Molecular docking diagram of CD6-ALCAM binding;

[0050] (oq)CD6-NK reverses MASH-related liver fibrosis in mice. Detailed Implementation

[0051] To enable those skilled in the art to better understand the technical solutions of the present invention, the present invention will be further described in detail below with reference to the accompanying drawings. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0052] Example 1: Verification of NK cell immaturation and its pro-fibrotic effect in the progression of MASLD

[0053] To review the progression of MASLD, we analyzed single-cell data from 3 normal mouse liver samples, 5 MASLD mouse liver samples (distinguished as ordinary fatty liver and MASH), 2 mouse cirrhosis samples, and 2 mouse HCC liver samples. Figure 1 Among numerous immune cells, we found that only NK cell subsets showed the most significant separation as MASLD progressed. Figure 1 We validated the results of single-cell analysis in paraffin-embedded liver sections from patients with MASLD at different stages using multiple immunohistochemistry. Figure 1 Finally, mice were injected with neutralizing antibodies against NK1.1 and IL-15 to obtain NK cell immature and mature mice, while mice injected with IgG (immunoglobulin G) served as controls.

[0054] The results showed that NK cell immaturity promotes the progression of MASH-related liver fibrosis.

[0055] Example 2: The mutual repulsion between CD6+ NK cells and ALCAM+ hepatocytes and the discovery of the CD6-ALCAM axis

[0056] To investigate the key factors disrupting NK cell maturation and naivety, we used the GeneTrajectory (v1.0.0) algorithm to analyze the spatiotemporal dynamics of gene expression in single-cell transcriptome data. We observed that NK cells, upon developing to an abnormal subtype, remain at that stage and cease further maturation. We obtained the characteristic marker of this subtype and explored the stage at which this subtype of cells appears in MASLD using multicolor immunofluorescence. We found that this subtype of cells appeared in paraffin-embedded sections of liver fibrosis. Figure 2b) We named this cluster of cells Terminally Differentiated-Immature NK (TD-iNK). We then used intercellular communication to further investigate how the dynamic remodeling of the immune microenvironment affects hepatocytes as liver disease progresses. Different NK cell subsets and hepatocyte subsets all pointed to the CD6-ALCAM axis as a key signaling axis for communication between NK cells and hepatocytes. Multicolor fluorescence staining confirmed the mutual repulsion between CD6+ NK cells and ALCAM+ hepatocytes in liver fibrosis and hepatocellular carcinoma sections. Figure 2 :cd).

[0057] Example 3: Preparation of human NK cells with high CD6 expression

[0058] Step 1: Obtain NK cells, which can be derived from peripheral blood, umbilical cord blood, induced pluripotent stem cell (iPSC) differentiation, or NK cell lines.

[0059] We chose to isolate PBMCs from the peripheral blood of healthy volunteers and obtain high-purity primary NK cells by magnetic bead sorting or flow cytometry sorting.

[0060] Step 2: Construct the lentiviral expression vector pLVX-EF1α-CD6-IRES-GFP, in which the CD6 gene contains the full-length human CD6 coding sequence (NCBI reference sequence). Lentiviral production is achieved using 293T cells.

[0061] Step 3: Infect primary NK cells with the above-mentioned lentivirus while activating them, with a multiplicity of infection (MOI) of 10-50.

[0062] Step 4: 72 hours after infection, the infection efficiency was assessed by detecting the GFP positivity rate by flow cytometry, and the high expression of CD6 protein was confirmed by staining with anti-human CD6 antibody.

[0063] Step 5: Sorting GFP+ (i.e., CD6-high expression) NK cells and expanding them in a medium containing IL-2 and IL-15 for subsequent functional experiments.

[0064] Example 4: In vitro killing experiment of CD6-NK cells on ALCAM-overexpressing hepatocyte lines

[0065] Human hepatocellular carcinoma cell lines with high ALCAM expression were selected as target cells. Target cells were labeled with CFSE. CD6-highly expressing NK cells prepared in Example 3 were used as the experimental group, and unmodified control NK cells were used as the control group. The experimental and control groups were co-cultured with target cells at different effector-to-target ratios. After co-culturing for 4-6 hours, cells were collected, and the apoptosis rate of CFSE-positive target cells was detected by flow cytometry.

[0066] As a result, the killing rate of ALCAM-overexpressing target cells in the experimental group (CD6-NK) was significantly higher than that in the control group, demonstrating that CD6 overexpression enhances the targeted killing ability of NK cells against specific hepatocytes.

[0067] Example 5: Therapeutic effect of CD6-NK cells in a mouse MASH-liver fibrosis model

[0068] A mouse model of MASH-related liver fibrosis was established by a high-fat diet (HFD) combined with intraperitoneal injection of Ccl4. The model mice were randomly divided into three groups: a PBS control group, a control NK cell therapy group, and a CD6-NK cell therapy group. PBS, unmodified NK cells, or mouse CD6-modified NK cells prepared according to the method in Example 3 were injected via tail vein once a week for 3-4 weeks. After treatment, the mice were sacrificed, and serum and liver tissue were collected.

[0069] Detection: a) Serum biochemical indicators (ALT, AST); b) Liver tissue H&E staining and Sirius red staining to assess the area of ​​steatosis and fibrosis; c) Immunohistochemistry or immunofluorescence to detect the expression level of ALCAM and NK cell infiltration in the liver.

[0070] As a result, compared with the PBS group and the control NK cell group, the CD6-NK cell treatment group showed significantly lower serum transaminase levels, and significantly reduced hepatic steatosis, inflammatory infiltration, and collagen deposition. ALCAM expression in the liver may have been suppressed. This demonstrates that CD6-NK cells can effectively reverse the MASH-liver fibrosis process in mice.

[0071] The foregoing has only described certain exemplary embodiments of the present invention by way of illustration. Undoubtedly, those skilled in the art can modify the described embodiments in various ways without departing from the spirit and scope of the present invention. Therefore, the foregoing drawings and descriptions are illustrative in nature and should not be construed as limiting the scope of protection of the claims of the present invention.

Claims

1. A genetically engineered NK cell, characterized in that: The NK cells were genetically modified to overexpress or heterologously express CD6 molecules.

2. The genetically engineered NK cell according to claim 1, characterized in that: The genetic modification is achieved by introducing the gene encoding CD6 into NK cells using a viral or non-viral vector.

3. A method for preparing genetically engineered NK cells as described in any one of claims 1-2, characterized in that, Includes the following steps: Step 1: Obtain NK cells; Step 2: Construct a gene delivery vector to express CD6, and clone the gene sequence encoding the CD6 molecule into a suitable expression vector; Step 3: Introduce the vector constructed in Step 2 into the NK cells obtained in Step 1; Step 4: Screen NK cells that successfully express CD6 and expand them in vitro to obtain a population of genetically engineered NK cells that highly express CD6.

4. The use of the genetically engineered NK cells as described in any one of claims 1-2 in the preparation of medicaments for the prevention or treatment of metabolic dysfunction-related fatty liver disease and its progressive disease.

5. The application according to claim 4, characterized in that, The progressive diseases include metabolic dysfunction-associated steatohepatitis (MASH), MASH-associated liver fibrosis, and cirrhosis.

6. A pharmaceutical composition, characterized in that, It includes the genetically engineered NK cells as described in any one of claims 1-2, and a pharmaceutically acceptable carrier or excipient.

7. The pharmaceutical composition according to claim 6, characterized in that, The pharmaceutical composition is a preparation for intravenous injection.

8. Application of a CD6 molecule as a target for enhancing the efficacy of NK cells in treating metabolic dysfunction-related fatty liver disease in genetically engineered NK cells.

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