Monoclonal antibody targeting nlrp3-ser295 phosphorylation site and application thereof

Abstract

The present application relates to a kind of monoclonal antibody and its application of targeting NLRP3-Ser295 phosphorylation site, the monoclonal antibody includes heavy chain variable region and light chain variable region, the VL sequence of the light chain variable region is as shown in SEQ ID No:1, the VH sequence of the heavy chain variable region is as shown in SEQ ID No:2.The light chain and heavy chain expression ratio of the monoclonal antibody can be accurately controlled, combined with humanization technology, it can be used for specified monoclonal antibody bioreactor amplification, satisfy the basic demand of detection reagent or drug batch production;Light chain variable region and heavy chain variable region can specifically recognize the Ser295 phosphorylation site (p-NLRP3 S295 ) of NLRP3 protein, realize the accurate blocking of NLRP3 inflammasome activation path, significantly reduce the side effect caused by non-specific crosstalk;And the intracellular delivery platform of eTAT can be universal, provide modularization reference for the development and application of intracellular antibody-drug conjugate (ADC).

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a monoclonal antibody targeting the NLRP3-Ser295 phosphorylation site and its applications. Background Technology

[0002] Existing monoclonal antibody (mAb) drugs mainly act on extracellular or membrane surface targets and cannot penetrate the cell membrane to neutralize intracellular pathogenic target proteins. Therefore, in biomedical research, this leads to: ① the inability to reach key intracellular signaling molecules / targets; ② the need for long-term, high-dose administration, which can easily cause off-target toxicity and immunogenic side effects; ③ the reliance on constructing stable mammalian cell lines that express the antibody stably and efficiently, but gene drift, copy number loss, and the expansion of non-target antibody-producing cells can easily cause a sharp drop in expression levels and batch-to-batch quality differences.

[0003] Steroidal anti-inflammatory drugs (SAIDs) and nonsteroidal anti-inflammatory drugs (NSAIDs) are the main treatments for chronic inflammation in clinical practice. SAIDs have systemic toxic side effects such as hypothalamic-pituitary-adrenal axis suppression, osteoporosis, and elevated blood sugar. NSAIDs relieve fever and pain by inhibiting prostaglandin synthesis, reducing leukocyte aggregation, but their anti-inflammatory effects require 1-2 weeks to establish a stable anti-inflammatory effect, and they have an increased incidence of gastrointestinal ulcers and cardiovascular events. In recent years, mAbs have been used in the treatment of chronic inflammatory diseases. Marketed mAbs, such as rituximab, trastuzumab, and dupilumab, have the characteristic of clear targeting; however, they also have shortcomings such as being limited to extracellular antigens, ineffective against intracellular inflammasomes, and unable to block the source of disease.

[0004] NOD-like protein family 3 (NLRP3) is an important innate immune recognition receptor. NLRP3 mutations / overactivation are closely associated with atherosclerosis, neurodegenerative diseases, and non-alcoholic fatty liver disease (NAFLD). Existing small molecule inhibitors, such as MCC950, CY-09, and OLT1177, have hindered their clinical drug development due to hepatotoxicity, off-target kinase inhibition, or pharmacokinetic defects. The NLRP3 inflammasome is considered an intracellular "undruggable" target, and there is currently a lack of intracellular mAbs that can penetrate the cell membrane, resulting in a gap in targeted therapy for environmentally induced liver injury.

[0005] Therefore, there is an urgent need for a method that can efficiently penetrate cell membranes and specifically neutralize p-NLRP3. S295 We will develop intracellular mAbs and establish a high-expression, low-mutation, and scalable recombinant expression system to fill the technological gap in early intervention for MASLD associated with environmental exposure. Summary of the Invention

[0006] This invention aims to at least partially address one of the technical problems in related technologies, namely, the inability of existing monoclonal antibodies to penetrate cell membranes and bind to intracellular targets, and the difficulty in blocking NLRP3 inflammasome activation at its source. Therefore, the purpose of this invention is to provide a monoclonal antibody that can efficiently penetrate cell membranes and target the NLRP3-Ser295 phosphorylation site, along with its intracellular recombinant expression system.

[0007] Therefore, in a first aspect of the present invention, the present invention proposes a monoclonal antibody targeting the NLRP3-Ser295 phosphorylation site, comprising a heavy chain variable region and a light chain variable region, wherein the VL sequence of the light chain variable region is shown in SEQ ID No:1, and the VH sequence of the heavy chain variable region is shown in SEQ ID No:2.

[0008] According to the monoclonal antibody of the present invention, the expression ratio of the light and heavy chains of the monoclonal antibody can be precisely controlled. Combined with humanization technology, it can be used for bioreactor scale-up of specified monoclonal antibodies, meeting the basic requirements for mass production of detection reagents or drugs; the variable regions of the light and heavy chains can specifically recognize the Ser295 phosphorylation site (p-NLRP3) of the NLRP3 protein. S295 This technology overcomes the technical bottleneck of traditional antibodies being unable to penetrate cell membranes to neutralize intracellular targets, achieving precise blocking of the NLRP3 inflammasome activation pathway and significantly reducing side effects caused by nonspecific crosstalk. Furthermore, eTAT's intracellular delivery platform is universal, providing a modular reference for the development and application of intracellular antibody-drug conjugates (ADCs).

[0009] Optionally, the monoclonal antibody subtype is IgG2a / κ.

[0010] Optionally, the monoclonal antibody has: (a) The light chain variable region shown in SEQ ID No:3, or a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence homology to the light chain variable region sequence shown in SEQ ID No:3; and (b) The heavy chain variable region shown in SEQ ID No:4, or a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence homology with the heavy chain variable region sequence shown in SEQ ID No:4.

[0011] In a second aspect, the present invention provides an isolated polynucleotide encoding the aforementioned monoclonal antibody.

[0012] The polynucleotide includes the sequence encoding the light chain variable region shown in SEQ ID No:5 and the sequence encoding the heavy chain variable region shown in SEQ ID No:6.

[0013] In a third aspect, the present invention provides a construct containing the isolated polynucleotides described above.

[0014] The construct for the light chain variable region consists of a VL sequence (as shown in SEQ ID No:7) linked to a mouse-derived Fc light chain constant region (as shown in SEQ ID No:8), with a signal peptide sequence (as shown in SEQ ID No:9) added to the front end. EcoR I and BamH I enzyme digestion and insertion into the pTT5-CMV-Amp vector form; The construct for the heavy chain variable region is a linking of the VH sequence (SEQ ID No:12) to the mouse Fc heavy chain constant region (SEQ ID No:13), with a signal peptide sequence (SEQ ID No:14) added to the front end. The heavy chain constant region CH is then linked to a covalently bound tag (SEQ ID No:16) SpyTag sequence (SEQ ID No:16) via a flexible linker (G4S)2 (SEQ ID No:15). EcoR I and Xba The I enzyme digests and inserts the vector into the pTT5-CMV-Amp vector to form the vector.

[0015] In a fourth aspect, the present invention provides a host cell comprising the above-described construct.

[0016] In a fifth aspect, the present invention provides a medicament for treating fatty liver disease associated with metabolic dysfunction, comprising the above-described monoclonal antibody and a pharmaceutically acceptable carrier.

[0017] In a sixth aspect of the invention, the invention provides an intracellularly delivered monoclonal antibody complex obtained by fusing the aforementioned monoclonal antibody with the membrane-penetrating peptide eTAT.

[0018] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Attached Figure Description

[0019] Figure 1This is a schematic diagram of the NLRP3 functional domain partitioning and Ser295 phosphorylated peptide design according to an embodiment of the present invention. Figure 2 This is a comparison diagram of the amino acid sequences near the Ser295 site of phosphorylated amino acids in hNLRP3 and mNlrp3 according to embodiments of the present invention; Figure 3 To secrete mAb@p-NLRP3 according to an embodiment of the present invention S295 ELISA detection of multi-line hybridoma cell culture supernatant containing monoclonal antibodies; Figure 4 To purify ascites fluid according to an embodiment of the present invention, mAb@p-NLRP3 S295 The assay included: (A) detecting antibody titers in the supernatant of positive clone hybridoma cell lines (2E5, 6A4, 7G4, 14C7, and 11C11) by ELISA, with PBS as a negative control; and (B) performing SDS-PAGE staining on monoclonal antibodies from 2E5, 6A4, 7G4, 14C7, and 11C11 using Coomassie brilliant blue staining. PAGE analysis showed bands of heavy chains (H) and light chains (L). * Compared with the control group, P <0.05; Figure 5 For the detection of the properties of the 14C7 antibody according to an embodiment of the present invention, wherein ELISA is used to detect antibody subtype (A), titer (B), and affinity (C); Figure 6 This is a schematic diagram of the light chain expression plasmid (pTT5-14C7L) according to an embodiment of the present invention; Figure 7 This is a schematic diagram of the heavy chain expression plasmid (pTT5-14C7H) according to an embodiment of the present invention; Figure 8 To detect the properties of the purified recombinant monoclonal antibody (R-mAb) according to embodiments of the present invention, wherein (A) CHO cells were co-transfected with pTT5-14C7L and pTT5-14C7H plasmids (1 μg / mL) for 24 h, the supernatant was collected and R-mAb was purified, and the expression of heavy chain (H) and light chain (L) of the purified R-mAb was detected by SDS-PAGE; (B) HepaRG cells were co-transfected with pTT5-14C7L and pTT5-14C7H plasmids (1 μg / mL) for 24 h, and the heavy chain (H) and light chain (L) of the intracellular R-mAb were detected by Western blotting; the titer (C), antibody subtype (D), and affinity (E) of the purified R-mAb were detected by ELISA. Figure 9 To detect the neutralization effect of R-mAb according to an embodiment of the present invention, wherein (A) WB detection of p-NLRP3 S295Levels of activation and pyroptosis-related proteins; (B, C) Relative release levels of LDH (B) and IL-1β (C); (D) Representative image of DiO-labeled cell membrane (green) colocalization with GSDMD (red) as shown by IF analysis, scale bar, 10 μm; (E, F) Relative levels of ALT (E) and AST (F) in cells;*, compared with control group. P <0.05; Figure 10 For the pharmacokinetic evaluation of R-mAb according to embodiments of the present invention, wherein: (A) IF image showing representative distribution of R-mAb-eTAT in major organs such as heart, liver, spleen, lung and kidney, scale bar, 100 μm; (B) H&E staining (top image) and Oil Red-O (OR-O) staining (bottom image) of liver tissue, scale bar, 50 μm; (C) IHC detection of p-NLRP3 in liver. S295 Expression and distribution, scale bar, 50 μm; (D) Western blot detection of p-NLRP3 S295 (E) Levels of activation and pyroptosis-related proteins; (F) Relative serum IL-1β levels detected by ELISA; (G) Relative levels of TC (left) and TG (right) in the liver; * Relative serum ALT (left) and AST (right); * Compared with the control group, P <0.05. Detailed Implementation

[0020] The technical solution of the present invention is illustrated below through specific examples. It should be understood that the one or more method steps mentioned in the present invention do not preclude the existence of other method steps before or after the combined steps, or the insertion of other method steps between these explicitly mentioned steps; it should also be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the present invention. Furthermore, unless otherwise stated, the numbering of each method step is merely a convenient tool for identifying each method step, and not for limiting the order of the method steps or defining the scope of the present invention. Changes or adjustments to their relative relationships, without substantially altering the technical content, should also be considered within the scope of the present invention.

[0021] To better understand the above technical solutions, exemplary embodiments of the present invention are described in more detail below. While exemplary embodiments of the present invention are shown, it should be understood that the present invention can be implemented in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided to enable a more thorough understanding of the present invention and to fully convey the scope of the invention to those skilled in the art.

[0022] In this document, the term "antibody" is used in the broadest sense to refer to a protein or polypeptide containing an antigen-binding site, antigen-binding fragment, or antigen-binding portion, encompassing natural and artificial antibodies of various structures, including but not limited to complete antibody forms or antigen-binding fragments of antibodies. According to the specific implementation, the provided antibody is a monoclonal antibody.

[0023] The monoclonal antibodies mentioned in this article are generally either separable or recombinant. "Separable" means that they can be identified, isolated, and / or recovered from cells or cell cultures expressing peptides or proteins. Typically, isolated peptides (e.g., isolated antibodies) can be prepared through at least one purification step. "Isolated antibodies" refers to antibodies or antigen-binding fragments that are substantially free of other antibodies or antigens with different antigen specificities. "Recombinant" means that antibodies can be generated in foreign host cells using gene recombination technology.

[0024] "Specific binding" or "specifically binding to" a specific antigen or epitope, or "specific to" a specific antigen or epitope, means distinguishing it from non-specific interactions. This specific binding can be measured using methods commonly used in the art. The ability of an antibody or antigen to bind can be measured using enzyme-linked immunosorbent assay (ELISA) or other techniques familiar to those skilled in the art.

[0025] The names of amino acids used in this article are represented by standard single-letter or three-letter codes commonly used in the art. The term "homology" of sequences as used herein refers to the degree of sequence similarity when comparing protein, polypeptide, or nucleic acid sequences. Homology can be determined using various methods known in the art. For example, publicly available software such as BLAST, ALIGN, and BLAST-2 can be used. The amino acid substitutions mentioned herein are conserved amino acid substitutions. A "conserved amino acid substitution" in this article refers to the substitution of one amino acid residue with a different amino acid residue in a side chain having similar physiological and chemical properties. For example, conserved amino acid substitutions can occur between amino acid residues with hydrophobic side chains (e.g., Met, Ala, Val, Leu, and Ile), between residues with neutral hydrophilic side chains (e.g., Cys, Ser, Thr, Asn, and Gln), between residues with acidic side chains (e.g., Asp and Glu), between amino acids with basic side chains (e.g., His, Lys, and Arg), or between residues with aromatic side chains (e.g., Trp, Tyr, and Phe). As is known in the art, conserved amino acid substitutions generally do not cause significant changes in protein conformation and structure, thus preserving the protein's biological activity.

[0026] The test materials used in this invention are all commercially available products and can be purchased on the market; unless otherwise specified, the experiments involved are all conventional experimental methods.

[0027] Immortalized human liver progenitor cells (HepaRG) were preserved and passaged by our research group (derived from ATCC, Bio-129940). SP2 / 0 and CHO cells were donated by Professor Shengxiang Ge's research group and used to prepare hybridoma cells. HepaRG cells were cultured in William's E medium supplemented with 10% fetal bovine serum (FBS) and differentiated for one week by adding 1% dimethyl sulfoxide (DMSO). Other cells were cultured in RPMI 1640 medium supplemented with 10% FBS. All cells were cultured in a 37°C, 5% CO2 incubator.

[0028] The pCR3.1-NLRP3S295>A (site-directed mutation of serine at position 295 to alanine, S295>A) and pCR3.1-NLRP3S295>D (site-directed mutation of serine at position 295 to aspartic acid, S295>D) plasmids were constructed by inverse PCR and are stored in our laboratory. siTRIM40 and siNC were purchased from GenePharma (Shanghai, China). Cell transfection was performed using Lipofectamine 2000 (ThermoFisher Scientific, Waltham, Massachusetts, USA) according to the manufacturer's instructions.

[0029] Six-week-old female BALB / c mice were purchased from Shanghai Slack Laboratory Animal Co., Ltd. All animals were housed in individually ventilated cages with a 12-hour light cycle, a temperature of 22-25 ℃, and a relative humidity of 45-55%, and were allowed free access to food and water.

[0030] The present invention will now be described with reference to specific embodiments. It should be noted that these embodiments are merely descriptive and do not limit the present invention in any way.

[0031] Example 1: Design of Phosphorylated Antigens and Prediction of B Cell Epitopes (1) Preliminary bioinformatics screening identified potential serine (S) / threonine (T) phosphorylation sites in NLRP3 (such as S5, S198, S295, etc.). An online prediction tool (B-cell epitope prediction website: http: / / sysbio.unl.edu / SVMTriP / result.php?jobid=5bbd9a25d17d98.59500216) was used to score the S295 region as a B-cell epitope, achieving a score of 0.73–0.91, ranking first among candidate sites. Figure 1 As shown, Figure 1A schematic diagram of the NLRP3 functional domain partitioning and Ser295 phosphorylated peptide design shows the amino acid sequence with a specific Ser295 phosphorylation site and KLH.

[0032] (2) Cross-species conservation verification: human hNLRP3 and mouse mNlrp3 are conserved in the 14 aa sequence around S295, such as... Figure 2 As shown.

[0033] (3) Three peptides were synthesized, including: the NLRP3 S295 phosphorylated polypeptide PSRILFLMDGFDELQC-Cys (C-terminus with added cysteine ​​for KLH conjugation), as an immunopeptide; the same sequence as the screening peptide, directly coating ELISA plates; and the negative control peptide, which was the S295A (non-phosphorylated) replacement sequence. All peptides had an HPLC purity ≥95%.

[0034] Example 2: Working solution of polypeptides for immunization Dissolve the immunopeptide in ddH2O to prepare a 5 mg / mL stock solution, and sterilize it using a 0.22 μm filter membrane. Prepare the working solution according to the ratio of stock solution: ddH2O: adjuvant containing zinc aluminum risedronate (refer to CN114504640B) = 1:4:5 (v / v / v), with a final concentration of 500 μg / mL. Prepare and use immediately.

[0035] Example 3: Monitoring of mouse immunity and antiserum (1) Select 6-8 week old female Balb / C mice (n = 8), and subcutaneously inject the working solution of Example 2 at 8 points on the abdominal skin, 25 μL at each point, once a week for 4 consecutive weeks. 72 h after the 3rd and 4th immunizations, collect 200 μL of blood from the orbital venous plexus, let it stand at 37 ℃ for 30 min, and then centrifuge at 5,000 ×g for 5 min to obtain serum.

[0036] (2) Serum was serially diluted (1:100~1:12800) and antibody titers were detected by indirect ELISA (50 ng / well of screening peptide was used for coating).

[0037] (3) Select OD 450 Mice with a P / N ratio ≥1.0 and P / N ≥5.0 were used for final enhancement. They were injected into the spleen with 50 μg of soluble antigen (immunopeptide) (PBS 100 μL). The spleen was harvested 72 h later for hybridoma preparation.

[0038] Example 4: Hybridoma Preparation and Initial Screening (1) Spleen cells: SP2 / 0 cells = 5:1, add 1 mL of 50% polyethylene glycol (PEG, molecular weight 1450), and complete the fusion within 90 s. Terminate and resuspend in pre-warmed RPMI-1640 medium (HAT-1640) containing hypoxantin, aminopterin, and thymidin.

[0039] (2) 96-hole plate laying (2 10 5 Cells / well), cultured at 37 ℃ and 5% CO2 for 3 days, then replaced with HT medium.

[0040] (3) On day 4, 50 μL of the supernatant was collected for ELISA detection (50 ng / well of screening peptide). Positive wells were transferred to 24-well plates for expansion culture. Limiting dilution was used to perform subcloning 3-5 times consecutively until the monoclonal positivity rate reached 100%. mAb@p-NLRP3 was screened in positive hybridoma cell lines by ELISA. S295 The titer. Based on the initial detection threshold OD 450 With nm > 2.1, a series of monoclonal antibodies were progressively screened, and finally five hybridoma cell lines (2E5, 6A4, 7G4, 14C7, 11C11) were retained. Figure 3 As shown.

[0041] Example 5: Ascites preparation and antibody purification Balb / C mice, pre-sensitized by intraperitoneal injection of medical liquid paraffin, were selected and intraperitoneally injected with hybridoma cells of various types, including 2E5, 6A4, 7G4, 14C7, and 11C11. 5 10 6 / animal. One week later, when the abdomen was noticeably distended, 8-10 mL of ascites fluid was aspirated per animal; centrifuged at 12,000 rpm and 4 ℃ for 10 min, and the supernatant was collected for Protein A / G affinity chromatography to purify and recover the monoclonal antibody. The gel was stained with Coomassie Brilliant Blue (Beyotime, Shanghai, China) for 1 h, and then destained overnight at room temperature. Images were captured using a chemiluminescence imaging system (model c-300) (Azure Biosystems, Dublin, California, USA). Results are as follows. Figure 4 As shown, the SDS-PAGE reduction bands are clear (HC ~50 kDa, LC ~26 kDa), with a purity ≥95%.

[0042] Example 6: Identification of the properties of the 14C7 antibody Hybridoma cell supernatants were collected and antibody subtypes were detected by ELISA according to the manufacturer's instructions (Thermo Fisher Scientific, Waltham, Massachusetts, USA): mouse antibody (GAM-HRP) was used as a positive control, and the optical density (OD) of each hybridoma cell supernatant was measured at 450 nm. 450 nm); mAb@p-NLRP3 was detected by ELISA. S295 Set up a negative control for the titer; if OD 450 A nm ≥ 2.1 nm indicates the highest antibody dilution is considered positive. This is for the detection of mAb@p-NLRP3. S295 The affinity was used to coat the plate with 0.5 μg / mL and 1 μg / mL S295 antigen phosphopeptides, and mAb@p-NLRP3 was applied. S295 Dilute to different concentrations (0.5-1.5 μg / mL) for ELISA detection. OD 450 Plotting nm on the y-axis, when the curve reaches the plateau phase, it indicates that the antigen has completely bound. At this point, OD... 450 nm is set to 100%, and the concentration corresponding to 50% (half maximum) is determined to calculate the affinity constant.

[0043] like Figure 5 As shown, mouse antibody subtype detection was performed, and 14C7 was identified as the IgG2a / κ subtype; the antibody was coated with pS295-BSA (0.5 µg / mL) and serially diluted [Log]. 10 (2)–Log 10 (6) The LogEC50 of 14C7 was measured to be 8.531; with different concentrations (0.5 and 1 µg / mL) of pS295-BSA coating, the Kd (0.5) of 14C7 was measured to be 0.01018 and Kd (1.0) was 0.006914; the value was the smallest among the 5 candidate strains, and it will be used as the representative example in the following.

[0044] Example 7: Variable Region Cloning and Expression Vector Construction (1) Total RNA was extracted from 14C7 hybridoma and reverse transcribed into cDNA. Antibody κ chain and heavy chain PCR amplification and sequencing were performed (mouse monoclonal antibody subtype identification kit, E607067, Sangon Biotech (Shanghai) Co., Ltd.). The obtained sequences were evaluated by the online IMGT and SOMPA platforms and confirmed to be free of frameshift, and the light chain and heavy chain variable region (VL and VH) sequences were obtained.

[0045] (2) Synthesize the corresponding sequences of VL and VH and construct the recombinant expression system; obtain the light chain expression plasmid pTT5-14C7L and the heavy chain expression plasmid pTT5-14C7H; all constructions were confirmed to be free of mutations by sequencing.

[0046] A schematic diagram of the light chain expression plasmid pTT5-14C7L is shown below. Figure 6 As shown, pTT5-14C7L comprises a VL sequence (nucleotide sequence as shown in SEQ ID No:7) and a murine Fc light chain constant region (mCK) (nucleotide sequence as shown in SEQ ID No:8) to form a complete recombinant antibody light chain, with a signal peptide sequence (nucleotide sequence as shown in SEQ ID No:9) added to the front end. EcoR I (nucleotide sequence: gaattc) and BamH I (nucleotide sequence: ggatcc) was inserted into the pTT5-CMV-Amp multiple cloning site region by enzyme digestion.

[0047] The pattern of the heavy chain expression plasmid pTT5-14C7H is as follows: Figure 7 As shown, pTT5-14C7H comprises a VH sequence (nucleotide sequence as shown in SEQ ID No:10) and a mouse Fc heavy chain constant region (IgG2a) (nucleotide sequence as shown in SEQ ID No:11) to form a complete recombinant antibody heavy chain. A signal peptide sequence (nucleotide sequence as shown in SEQ ID No:14) is added to the front end. The heavy chain constant region (CH) is linked to a covalently bound tag SpyTag sequence (nucleotide sequence as shown in SEQ ID No:13) via a flexible linker (G4S)2 (nucleotide sequence as shown in SEQ ID No:12). EcoR I (nucleotide sequence: gaattc) and Xba I (nucleotide sequence: tctaga) was inserted into the pTT5-CMV-Amp multiple cloning site region by enzyme digestion.

[0048] Example 8: CHO cell expression and purification of recombinant antibody In Example 7, pTT5-14C7L and pTT5-14C7H were transfected into CHO cells (2 cells) at a 1:1 ratio (total DNA 1 μg / mL). 10 6 Cells / mL), after 24 h, were replaced with fresh DMEM complete medium and cultured for 3 days. The expression of recombinant monoclonal antibody in the culture supernatant was detected. The supernatant was collected and purified into R-mAb by dual-column tandem purification. SDS-PAGE was used to detect the expression of heavy chain (H) and light chain (L) of the purified R-mAb.

[0049] HepaRG cells were co-transfected with pTT5-14C7L and pTT5-14C7H plasmids at a 1:1 ratio (1 μg / mL) for 24 h to construct intracellular R-mAb recombinant expression. Western blotting was used to detect the heavy chain (H) and light chain (L) of intracellular R-mAb to confirm endogenous expression. 14C7 mAb not treated with eTAT was used as an exogenous mAb control. Western blotting was performed by electrophoresis. After transfer to a PVDF membrane, the membrane was blocked with 5% skim milk, washed, and then mixed with anti-p-NLRP3. S295 Primary antibodies against NLRP3, ASC, GSDMD, Caspase-1, or IL-1β were incubated overnight at 4°C. The membranes were then incubated with the corresponding secondary antibodies, and images were acquired using a chemiluminescence imaging system (model c-300). ELISA was used to detect the titer, antibody subtype, and affinity of the purified R-mAbs; S295 phosphorylated peptide antigens (0.5 and 1.0 μg / mL) were used to pre-coat microplates for antibody affinity assays.

[0050] The results are as follows Figure 8 As shown, the product was identified by reducing SDS-PAGE, ELISA titer, and affinity, and the results were consistent with those of the 14C7 hybridoma cell-derived antibody mAb@p-NLRP3. S295 Consistent.

[0051] Example 9 Neutralization function in in vitro experiments (HepaRG cell pyroptosis model) HepaRG cells were treated with pTT5-14C7L and pTT5-14C7H (1 µg / mL each). –1 The plasmid was transfected for 24 hours; after changing the medium, the cells were exposed to palmitic acid (PA, 400 μmol / L, 24 h) to construct p-NLRP3 hepatocytes. S295 In vitro experimental model of pyroptosis dependence.

[0052] The levels of IL-1β in cells or mouse serum were detected using an ELISA kit (ABclonal, Wuhan, Hubei, China) following the manufacturer's instructions.

[0053] Immunofluorescence (IF) assay: HepaRG cells were seeded on coverslips in 24-well culture plates and treated as specified. Lipid droplet staining was performed using a BODIPY probe (Beyotime Biotechnology Co., Ltd., Shanghai). After fixation with paraformaldehyde for 15 minutes at room temperature, the nuclei were counterstained with DAPI. To observe pyroptosis, cells were counterstained with DiO dye, fixed, and permeabilized with 0.5% Triton X-100 at 4°C for 5 minutes. Coverlips were blocked with 1% bovine serum albumin and then incubated with anti-GSDMD antibody. Anti-TRIM40 antibody and mAb@p-NLRP3 were used. S295Samples were incubated to observe colocalization. After washing, coverslips were incubated with the appropriate fluorescent secondary antibody. Images were acquired using a Zeiss LSM 880 with Airyscan microscope (Carl Zeiss AG, Oberkochen, Jena, Germany).

[0054] WB detection of p-NLRP3 S295 The levels of activation and pyroptosis-related proteins, such as Figure 9 As shown, p-NLRP3 in the PA + R-mAb group S295 The levels were significantly lower than those in the PA group (reduced by 50%), indicating a significant inhibition of Caspase-1 cleavage activation. The p10 subunit level was 65% lower than that in the PA group, accompanied by a significant 51% reduction in GSDMD levels. Figure 9 A); Compared with the PA treatment group, the LDH release level in the supernatant of HepaRG cells in the PA + R-mAb group was significantly decreased (by 34.38%), and the IL-1β release level was decreased by 16.67%. Figure 9 B, 9C); Laser confocal microscopy showed that PA treatment significantly enhanced the co-localization of GSDMD and DiO, while co-localization was significantly weakened in the R-mAb intervention group, with the co-localization coefficient decreasing by 62.5% (B, 9C). Figure 9 D); ALT and AST decreased by 17.5% and 15% respectively. Figure 9 E, 9F). This suggests that intracellular R-mAb can specifically bind to p-NLRP3. S295 Block p-NLRP3 S295 It induces hepatocyte pyroptosis, protecting cells from lipotoxic damage.

[0055] Example 10: Mouse NAFLD Model and In Vivo Intervention Effects C57BL / 6J mice (n = 6) were fed a methionine and choline-deficient diet (MCD) for 4 weeks to establish a NAFLD model, while the control group was fed a standard diet (SD). After 2 weeks of feeding, 2.5, 5, and 10 mg / kg (Cy3 prelabeled) of R-mAb-eTAT were injected intraperitoneally every three days for 2 weeks. Samples were collected and analyzed 24 hours after the last administration.

[0056] The results are as follows Figure 10 Fluorescence imaging showed that the fluorescence signal of R-mAb (Cy3) was strongest and dose-dependent in the liver and spleen. Figure 10 A); Liver tissue H&E and Oil Red-O (OR-O) staining showed a decrease in lipid droplet area in the 10 mg / kg group ( Figure 10 B); IHC of liver tissue showed p-NLRP3 S295 The levels of p-NLRP3 were significantly elevated in the liver of mice induced by the MCD diet. Compared with the MCD group, the R-mAb group significantly inhibited p-NLRP3.S295 The levels showed a dose-dependent decrease; in the 2.5, 5, and 10 mg / kg BW groups, p-NLRP3... S295 Positive signals decreased by 25.2%, 48%, and 62.5%, respectively, and the staining intensity in the high-dose group was close to the SD level. Figure 10 C) WB detection of p-NLRP3 S295 The levels of activation and pyroptosis-related proteins were dose-dependently decreased in the R-mAb group compared to the MCD group, with p-NLRP3 being significantly reduced. S295 The expression levels of ASC, Caspase-1 p10, GSDMD, and mature IL-1β were measured; among them, in the 2.5, 5, and 10 mg / kg BW groups, the p-NLRP3 level decreased by 26%, 48.3%, and 63.8%, respectively, while the Caspase-1 p10 and IL-1β levels decreased by up to 72.5%. Figure 10 D); ELISA was used to detect the relative levels of serum IL-1β. Compared with the MCD group, the serum LDH levels in the 2.5, 5, and 10 mg / kg BW R-mAb groups decreased by 36.2%, 44.1%, and 53%, respectively. Figure 10 E); Compared with the MCD group, the TC level in liver tissue decreased by 54% and 73.1% in the 5 mg / kg BW and 10 mg / kg BW R-mAb groups, respectively; the TG level in liver tissue decreased by 25%, 57%, and 67% in the 2.5, 5, and 10 mg / kg BW groups, respectively. Figure 10 F); Compared with the MCD group, serum ALT levels decreased by 11.7%, 28.5%, and 39.1% in the 2.5, 5, and 10 mg / kg BW R-mAb groups, respectively; serum AST levels decreased by 7.3%, 22.6%, and 21.7%, respectively. Figure 10 G). The results suggest that R-mAb reduces p-NLRP3 in mouse liver in a dose-dependent manner. S295 Pyroptosis-dependent effects alleviated lipid accumulation and inflammatory damage in the liver of NAFLD mice.

[0057] In summary, according to embodiments of the present invention, a method is provided to target the specific phosphorylation site p-NLRP3. S295 The entire process of designing the monoclonal antibody, from antigen design to animal testing, including all parameters and operational details, can be replicated by those skilled in the art. Furthermore, through this application example, based on the same technical concept, the intracellular recombinant expression system of this invention can be extended to other intracellular phosphorylation-specific targets simply by replacing the light / heavy chain variable region sequence. This enables the intracellular expression, high-throughput preparation, and precise delivery of antibodies specific to different functional intracellular targets, thereby significantly improving the availability and functionalization efficiency of intracellular targeted monoclonal antibody drugs.

[0058] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the invention. The illustrative expressions of the above terms in this specification should not be construed as necessarily referring to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. In addition, those skilled in the art can combine and integrate the different embodiments or examples described in this specification.

[0059] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A monoclonal antibody targeting the phosphorylation site of NLRP3-Ser295, characterized in that, It includes a heavy chain variable region and a light chain variable region, wherein the VL sequence of the light chain variable region is shown in SEQ ID No:1, and the VH sequence of the heavy chain variable region is shown in SEQ ID No:

2.

2. The monoclonal antibody according to claim 1, characterized in that, The monoclonal antibody subtype is IgG2a / κ.

3. The monoclonal antibody according to claim 1, characterized in that, have: (a) The light chain variable region shown in SEQ ID No:3, or a sequence having at least 85% sequence homology with the light chain variable region sequence shown in SEQ ID No:3; and (b) The heavy chain variable region shown in SEQ ID No:4, or a sequence having at least 85% sequence homology with the heavy chain variable region sequence shown in SEQ ID No:

4.

4. An isolated polynucleotide, characterized in that, The isolated polynucleotide encodes the monoclonal antibody according to any one of claims 1 to 3.

5. The isolated polynucleotide according to claim 4, characterized in that, The polynucleotide includes the sequence encoding the light chain variable region shown in SEQ ID No:5 and the sequence encoding the heavy chain variable region shown in SEQ ID No:

6.

6. A construct, characterized in that, Contains the isolated polynucleotide as described in claim 4 or 5.

7. The construct according to claim 6, characterized in that, The construct for the variable region of the light chain is a linker of the VL sequence (as shown in SEQ ID No:7) and the constant region of the mouse Fc light chain (as shown in SEQ ID No:8), with a signal peptide sequence (as shown in SEQ ID No:9) added to the front end. EcoR I and BamH I enzyme digestion and insertion into the pTT5-CMV-Amp vector form; The construct for the heavy chain variable region is a linking of the VH sequence (SEQ ID No:12) to the mouse Fc heavy chain constant region (SEQ ID No:13), with a signal peptide sequence (SEQ ID No:14) added to the front end. The heavy chain constant region CH is then linked to a covalently bound tag (SEQ ID No:16) SpyTag sequence (SEQ ID No:16) via a flexible linker (G4S)2 (SEQ ID No:15). EcoR I and Xba The I enzyme digests and inserts the vector into the pTT5-CMV-Amp vector to form the vector.

8. A host cell, characterized in that, It includes the construct described in claim 6 or 7.

9. A medicament for treating fatty liver disease associated with metabolic dysfunction, characterized in that, It includes the monoclonal antibody according to any one of claims 1 to 3; and a pharmaceutically acceptable vector.

10. A monoclonal antibody complex delivered intracellularly, characterized in that, It is obtained by fusing the monoclonal antibody described in any one of 1 to 3 with the membrane-penetrating peptide eTAT.

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