Anti-aging composition based on NK cell activity regulation and preparation method and application thereof

Abstract

The invention discloses a fusion polypeptide, an anti-KIR2DL1 monoclonal antibody and an anti-aging composition containing the fusion polypeptide and the anti-KIR2DL1 monoclonal antibody. Wherein the amino acid sequence of the fusion polypeptide is SEQ ID NO: 1. A heavy chain variable region of the anti-KIR2DL1 monoclonal antibody is SEQ ID NO: 2, and a light chain variable region of the anti-KIR2DL1 monoclonal antibody is SEQ ID NO: 3. The composition composed of the two components regulates NK cells bidirectionally by enhancing an NKG2D activation pathway and blocking a KIR2DL1 inhibition pathway, so that the proportion of CD107a positive NK cells reaches 68.9%, the secretion amount of granzyme B reaches 465pg / mL, the survival rate of senescent cells is reduced to 39%, and the synergistic effect exceeds 89%. The composition can be prepared into intravenous injection drugs, oral health care products or external cosmetics, is safe and remarkable in synergistic effect, and solves the problems of insufficient curative effect of a single activator and poor specificity of a single inhibitor in the prior art.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to an anti-aging composition based on the regulation of natural killer (NK) cell activity, and also to the preparation method of the composition and its application in the preparation of anti-aging drugs, health products or cosmetics. Background Technology

[0002] With the accelerating aging of the global population, the burden of age-related diseases is becoming increasingly prominent, including neurodegenerative diseases, cardiovascular diseases, tumors, and metabolic disorders, with both morbidity and mortality rates showing an upward trend year by year. Research indicates that the decline in immune system function is one of the core mechanisms driving aging and increased susceptibility to disease. Natural killer cells (NK cells), as important effector cells of innate immunity, play a crucial role in clearing senescent cells, abnormally proliferating cells, and infected cells. NK cells clear abnormal cells by recognizing the "senescence-associated molecular pattern" (SAMP) expressed on the surface of senescent cells and releasing effector molecules such as perforin and granzymes. However, with age, both the number and function of NK cells show a continuous downward trend. Clinical observations show that after age 30, the activity of individual NK cells decreases by an average of about 1.5%-2% per year, and by age 60, its activity is less than 40% of that in youth. This directly leads to a decrease in the body's efficiency in clearing senescent cells, accelerating the emergence of aging phenotypes and the occurrence of related diseases.

[0003] Current research on the regulation of NK cell activity mainly focuses on cytokines, immune checkpoint inhibitors, and in vitro expansion and reinfusion. However, these strategies all have certain limitations. Cytokine activation methods (such as IL-2 or IL-15 derivatives) can enhance NK cell function in the short term, but their half-lives are generally short, requiring frequent administration and carrying a high risk of immunotoxicity. Immune checkpoint inhibitors are mainly designed to restore T cell function and lack specificity and sensitivity to NK cells, only able to enhance their activity to a limited extent. In addition, some antibodies targeting NK inhibitory receptors are still mainly in murine or chimeric forms, which have problems such as strong immunogenicity and high risk of adverse reactions. In vitro expansion and reinfusion of NK cells can improve the number of effector cells to some extent, but its preparation process is complex and costly, and the reinfused cells have a short in vivo maintenance time, making it difficult to achieve a lasting therapeutic effect.

[0004] In summary, current technologies have not yet solved the following key problems: First, there is a lack of precise combination strategies that can simultaneously target NK cell activation receptors and inhibitory pathways; second, there is a lack of designs that can balance low immunogenicity and high activation efficiency; and third, there is a lack of anti-aging compositions that are easy to prepare and have high clinical translational potential. Therefore, there is an urgent need to develop an innovative composition that synergistically regulates NK cell activity to achieve effective intervention in the aging process and comprehensive prevention and treatment of age-related diseases. Summary of the Invention

[0005] The present invention aims to overcome the shortcomings of the prior art and provide a novel anti-aging composition that can delay immune aging and tissue aging by regulating NK cell activity in multiple dimensions.

[0006] Therefore, this invention discloses a fusion polypeptide, the amino acid sequence of which is SEQ ID NO:1. The binding equilibrium dissociation constant of the fusion polypeptide with the NKG2D receptor is ≤2.3×10⁻⁶. -9 M.

[0007] In one aspect, this invention also discloses an anti-KIR2DL1 monoclonal antibody, wherein the amino acid sequence of the heavy chain variable region of the monoclonal antibody is SEQ ID NO:2, and the amino acid sequence of the light chain variable region is SEQ ID NO:3. The binding equilibrium dissociation constant of the monoclonal antibody to the human KIR2DL1 receptor is ≤8.7×10⁻⁶. -11 M, with an inhibition rate of ≥92% against the binding of KIR2DL1 to HLA-C2 and a stimulation index ≤1.2.

[0008] In one aspect, the present invention also discloses an anti-aging composition comprising an effective amount of the fusion peptide, an effective amount of the anti-KIR2DL1 monoclonal antibody, and excipients mannitol and polysorbate 80. The weight ratio of the fusion peptide, the anti-KIR2DL1 monoclonal antibody, mannitol, and polysorbate 80 is 20:30:9:0.9.

[0009] In one aspect, the present invention also discloses a method for preparing the anti-aging composition, the method comprising the following steps: dissolving mannitol and polysorbate 80 in buffer solution, adding fusion peptide and anti-KIR2DL1 monoclonal antibody and stirring to dissolve, filtering for sterilization and then dispensing, and performing a three-stage freeze-drying process to obtain the final product.

[0010] In one aspect, the present invention also discloses the application of the aforementioned anti-aging composition in the preparation of an anti-aging drug, wherein the drug is administered by intravenous injection, with an adult dose of 10 mg / kg, once a week, for a course of treatment lasting 4 weeks.

[0011] In one aspect, the present invention also discloses the application of the aforementioned anti-aging composition in the preparation of anti-aging health products, wherein the health products are oral dosage forms that are reconstituted and mixed with an oral liquid matrix, and the daily dose is 5 mg / kg.

[0012] In one aspect, the present invention also discloses the application of the aforementioned anti-aging composition in the preparation of anti-aging cosmetics, wherein the cosmetic is a topical dosage form in which a fusion polypeptide and an anti-KIR2DL1 monoclonal antibody are modified transdermally and then mixed with a cream matrix, and used 1-2 times daily.

[0013] The fusion peptide of this invention exhibits excellent performance, with an equilibrium dissociation constant with the NKG2D receptor as low as 2.3 × 10⁻⁶. - 9 The modified M-terminal MICA fragment exhibits a 3.1-fold increased binding affinity and significantly enhanced binding stability compared to the original MICA fragment. Simultaneously, it significantly reduces immunogenicity, with serum antibody titers in Balb / c mice immunized at levels far lower than those of the intact MICA protein. Furthermore, the C-terminal cytotoxic peptide is efficiently cleaved in aging or tumor microenvironments. The modified anti-KIR2DL1 monoclonal antibody demonstrates outstanding characteristics, achieving a binding affinity of 8.7 × 10⁻⁶. -11 M is 8.8 times more potent than commercial monoclonal antibodies, with an inhibition rate of over 92% against the binding of KIR2DL1 to HLA-C2, which is superior to the 84.5% of commercial monoclonal antibodies. After humanization, its immunogenicity is significantly reduced, with a stimulation index of only 1.2, which is 94.9% lower than the 23.6 of murine antibodies, greatly reducing the risk of immune rejection.

[0014] The anti-aging combination of the two substances bidirectionally regulates NK cells by "enhancing the NKG2D activation pathway + blocking the KIR2DL1 inhibition pathway," achieving a significant synergistic effect: In in vitro experiments, the proportion of CD107a-positive NK cells reached 68.9%, significantly higher than the 42.6% achieved with peptide alone and the 31.8% achieved with monoclonal antibody alone; the secretion of granzyme B reached 465 pg / mL, a substantial increase compared to the 280 pg / mL achieved with peptide alone and the 195 pg / mL achieved with monoclonal antibody alone; and the survival rate of senescent cells decreased to 39%, a significant decrease compared to the 62% achieved with peptide alone and the 74% achieved with monoclonal antibody alone, with a synergistic effect exceeding 89%. In vivo experiments confirmed that the composition increased NK cell activity by 150.5%, spleen index by 85.7%, and serum IL-2 level by 200.9% in naturally aging mice compared to the control group. In D-galactose-induced aging mice, it increased serum SOD activity by 44.3% and decreased MDA content by 46.9%, with effects superior to commercially available anti-aging health products. In mice with photoaged skin, it reduced wrinkle scores by 57.1%, restored dermal thickness to 89.6% of the normal group, and increased collagen fiber content by 70.8%. No liver or kidney damage or skin irritation was observed in mice throughout the experiment.

[0015] Furthermore, the preparation process of this composition is stable and controllable. Through precise weighing, aseptic dissolution, and controlled freeze-drying, the final product has a purity of ≥99% and an endotoxin content of <0.1 EU / mg. It can be applied to three types of scenarios: intravenous injection drugs, oral health products, and topical cosmetics. It effectively solves the technical bottlenecks of existing anti-aging products, such as insufficient efficacy of single intervention, limited application scenarios, and poor safety. It provides an efficient and synergistic immune regulation solution for targeted elimination of senescent cells and improvement of aging-related pathological states. Attached Figure Description

[0016] Figure 1 A schematic diagram of traditional hybridoma cell screening technology.

[0017] Figure 2 SDS-PAGE results of anti-KIR2DL1 monoclonal antibody, where 1 represents anti-KIR2DL1 monoclonal antibody. Detailed Implementation

[0018] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the specification of this invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0019] Unless otherwise specified, the reagents, methods, and equipment used in this invention are conventional reagents, methods, and equipment in this technical field. Unless otherwise specified, the reagents and materials used in the following examples are all commercially available.

[0020] Example 1: Design, preparation and testing of fusion peptides

[0021] 1. Design of the fusion peptide, and completion of its amino acid sequence (SEQ ID NO:1):

[0022] GFLAEVYLDGGGGSGGGKRIEPDFYKR.

[0023] (1) NKG2D ligand fragment (N-terminus, 10 aa: GFLAEV) Y LDG)

[0024] Source and Screening: Based on the human MICA protein (21–30 aa, GFLAEVHLDG) (GenBank: CAA77031.1), the fragments were obtained through alanine mutation screening. Specific screening criteria were: retention of NKG2D binding activity > 80% and reduction in immunogenicity > 50% (verified by the prediction software IEDB and in vitro ELISA). The final selected fragments replaced one site (H→Y at position 7) compared to the original sequence (MICA 21–30 aa).

[0025] Activity validation: Surface plasmon resonance (SPR) assays showed that the binding affinity of this fragment to the NKG2D receptor was KD = 2.3 × 10⁻⁶. -9 M, compared to the unoptimized MICA 21–30 aa fragment (KD=7.1×10⁻⁶). -9 The M) level increased by approximately 3.1 times, and by 4 times compared to the complete MICA protein.

[0026] Immunogenicity verification: ELISA (coated with anti-MICA polyclonal antibody, detecting serum anti-peptide IgG levels) showed that after immunizing Balb / c mice with this fragment (100 μg / mouse, 3 immunizations at 7-day intervals), the serum antibody titer was <1:100, which was significantly lower than that of the group immunized with intact MICA protein (titer 1:1600, P<0.01), indicating a significant reduction in immunogenicity.

[0027] (2) Flexible linker peptide (middle, 7 aa: GGGSGGG)

[0028] Design rationale: This sequence has high flexibility and low immunogenicity, which can reduce spatial interference between the N-terminal and C-terminal fragments.

[0029] (3) Killing-enhancing peptide (C-terminus, 10 aa: KRIEPDFYKR)

[0030] Design rationale: Based on the optimization and design of natural substrate sequences, the interaction with the enzyme pocket is mainly enhanced by positive charge; at the same time, steric hindrance is reduced and cleavage efficiency is improved.

[0031] Binding efficiency verification: SPR assay (immobilized granzyme B, injected peptide) showed that the binding KD of the optimized fragment to granzyme B was 1.8 × 10⁻⁶. -8 M, compared to the original sequence (KD=9.2×10 -8 M) increased by approximately 5.1 times.

[0032] 2. Preparation and testing of fusion peptides

[0033] (1) Synthesis of fusion peptides

[0034] The resulting fusion peptide was sent to Sangon Biotech (Shanghai) Co., Ltd. for synthesis, with a purity ≥95%. It was then stored at -80℃ for later use.

[0035] (2) Binding ability detection (SPR, surface plasmon resonance): The binding kinetics of the peptide and the NKG2D receptor were detected using the Biacore T200 system. Specific steps:

[0036] Chip and reagent preparation: The CM5 sensor chip was equilibrated for 30 min in advance with running buffer (PBS buffer: 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.4, containing 0.05% Tween-20); the recombinant human NKG2D protein (abcam, ab281815) was diluted to 20 μg / mL with 10 mM sodium acetate buffer (pH 5.0); the fusion peptide, the original MICA fragment, and the mutant control peptide (inactive) were all diluted with running buffer to 6 concentration gradients (0.1, 0.3, 1, 3, 10, 30 nM), with 3 replicates for each concentration.

[0037] NKG2D protein immobilization (using amine coupling) was performed as follows: A mixture of 0.4 M EDC and 0.1 M NHS (1:1, v / v) was injected into the flow cell (FC2) at a flow rate of 10 μL / min for 7 min; diluted NKG2D protein was then injected at a flow rate of 10 μL / min for 15 min, resulting in a final immobilization density of 12500 ± 500 RU (resonance units); 1 M ethanolamine-HCl (pH 8.5) was injected at a flow rate of 10 μL / min for 7 min; Flow Cell 1 (FC1) underwent only activation-blocking treatment, without protein immobilization, and was used to eliminate non-specific binding signals.

[0038] Kinetic assay: Flow rate 30 μL / min. For each sample, first inject running buffer (baseline stabilization 60 s), then inject the sample (binding phase 120 s), and finally inject running buffer (dissociation phase 300 s). After each concentration gradient assay, inject 10 mM glycine-HCl solution (pH 2.0) at a flow rate of 30 μL / min, react for 30 s, and then equilibrate with running buffer for 60 s to ensure the signal recovers to baseline.

[0039] Data analysis: Using Biacore T200 Evaluation Software (Version 3.0), the sensor image was fitted with the "1:1 Langmuir binding model". After subtracting the FC1 blank signal and solvent signal, the kinetic parameters (binding rate constant ka, dissociation rate constant kd, equilibrium dissociation constant KD=kd / ka) were calculated.

[0040] (3) In vitro cell experiments

[0041] (a) Cell preparation

[0042] Peripheral blood mononuclear cell (PBMC) isolation: 3 peripheral blood samples (age 25-30 years, no history of immune diseases, EDTA anticoagulated), 20 mL each; blood diluted with an equal volume of PBS, then slowly added to the upper layer of Ficoll-Paque PLUS separation medium (density 1.077 g / mL) (blood:PBS:separation medium = 2:2:1, v / v / v); centrifuged at 400×g at room temperature for 30 min (centrifugation acceleration increased / decreased gradually to avoid interface disturbance); the middle white membrane layer (PBMC) was aspirated, washed 3 times with PBS (centrifuged at 1500×g for 10 min each time), and finally resuspended in RPMI 1640 medium (containing 10% fetal bovine serum FBS, 100 U / mL penicillin, and 100 μg / mL streptomycin), and counted for later use (viability > 95%, trypan blue staining).

[0043] NK cell purification: A CD56 magnetic bead positive sorting kit was used, following the manufacturer's instructions. The concentration of PBMC was adjusted to 1×10⁻⁶. 7 Cells / mL, add CD56 magnetic beads (10 μL / 1×10⁻⁶) 7 Cells were incubated at 4°C in the dark for 15 min; washed once with buffer (PBS + 0.5% FBS + 2 mM EDTA) (centrifuged at 1000×g for 5 min), resuspended, and loaded onto a MACS sorting column (placed in a magnetic field); unbound cells were eluted with buffer, and the column was then removed to elute bound CD56 cells with buffer. + Cells; purity verified by flow cytometry: anti-human CD56-PE antibody and CD3-FITC antibody were added, and the cells were incubated at 4°C in the dark for 30 min. CD56 levels were then detected. + CD3 - Cell percentage > 95%, ready for use.

[0044] Preparation of senescent fibroblasts: Human skin fibroblasts (HDFa) were cultured to passage 3 in DMEM medium (containing 10% FBS and antibiotics); senescence was induced by adding 200 mM D-galactose and cultured at 37℃ with 5% CO2 for 72 h; the SA-β-galactosidase staining kit was used for verification: after cell fixation, the cells were incubated with X-gal staining solution for 16 h (37℃ without CO2), and the proportion of SA-β-gal positive cells was >90% under a microscope, confirming the successful construction of the senescence model for later use.

[0045] (b) Co-culture experiment

[0046] Plating: 96-well cell culture plates, each well seeded with 1×10-1 senescent fibroblasts. 4 Each cell was incubated with 100 μL of DMEM medium at 37°C for 24 h (cell adhesion).

[0047] Sample addition and grouping: Add NK cells (5×10⁻⁶) according to the "effect-to-target ratio 5:1". 4 (Resuspend in 50 μL of culture medium per well), then add the treatment reagent. Each group has 3 replicates. Specific groupings:

[0048] Control group (PBS): Add 5 μL of PBS (the same volume as the reagents in other groups);

[0049] Single-use peptide group: Add 5 μL of fusion peptide stock solution (2 mM, dissolved in PBS) to a final concentration of 10 μM;

[0050] Anti-KIR2DL1 monoclonal antibody (prepared in Example 2) monotherapy group: Anti-human KIR2DL1 monoclonal antibody was diluted with culture medium to 20 μg / mL, and 5 μL was added to the final concentration of 1 μg / mL.

[0051] Peptide + monoclonal antibody combination group: 5 μL of 2 mM peptide stock solution and 5 μL of 20 μg / mL monoclonal antibody were added simultaneously, with final concentrations of 10 μM and 1 μg / mL, respectively.

[0052] Culture: Add culture medium to 200 μL per well and incubate at 37℃ with 5% CO2 for 24 h.

[0053] (c) Detection indicators:

[0054] NK cell degranulation marker: Collect cell suspension (containing NK cells and exfoliated fibroblasts) from each well, centrifuge at 1500×g for 5 min, and discard the supernatant; wash once with PBS, resuspend in 100 μL staining buffer (PBS + 2% FBS); add anti-human CD56-PE antibody (1 μL) and CD107a-FITC antibody (1 μL), and incubate at 4℃ in the dark for 30 min; wash twice with staining buffer, resuspend, and analyze by flow cytometry to determine CD56. + CD107a in cells + The proportion (excluding fibroblast interference).

[0055] Granzyme B secretion level (ELISA): Collect 150 μL of supernatant from each well and centrifuge at 1500×g for 10 min (to remove cell debris); use the human granzyme B ELISA kit and follow the instructions to calculate the granzyme B concentration based on the standard curve.

[0056] Senescent cell survival rate (CCK-8): After co-culturing for 24 h, 10 μL of CCK-8 reagent was added to each well and incubated at 37℃ for 4 h; the absorbance at 450 nm was measured using a microplate reader (reference wavelength 630 nm); survival rate was calculated using the formula: survival rate (%) = (average OD value of treatment group / average OD value of control group) × 100% (only reflects fibroblast survival; since NK cells are few in number and do not adhere to the wall, their contribution to the OD value can be ignored).

[0057] (4) Experimental results

[0058] (a) Binding activity advantage of the fusion peptide to the NKG2D receptor

[0059] SPR results showed that the binding affinity of the fusion peptide to the NKG2D receptor (KD = 2.3 × 10⁻⁶) was [missing information]. -9 The M fragment was significantly superior to the original MICA fragment (KD=7.1×10). -9 M, P<0.01), the binding enhancement reached 3.1-fold, the core reason being: after optimization by scanning alanine mutation at the N-terminus of the peptide, the dissociation rate constant kd decreased from 1.3×10 -3 s -1 Reduced to 4.8×10 -3 s -1 (P<0.01) indicates that the peptide binds more stably to the receptor; the mutant control peptide, due to the disruption of the hydrophobic interaction between the N-terminus and NKG2D, showed a sharp increase in kd to 1.2×10. -1 s -1 KD > 1×10 -6 M further validated the crucial role of the N-terminal sequence in binding activity, as shown in Table 1.

[0060] Table 1. Kinetic parameters of binding between the fusion peptide and the NKG2D receptor (SPR, n=3, x±s)

[0061]

[0062] (b) Differences in the regulation of NK cell activation and killing function among groups

[0063] NK cell activation marker (CD107a) + %): The peptide monotherapy group (42.6%) showed a 131.5% increase compared to the control group (18.4%), indicating that the peptide can promote NK cell degranulation by activating the NKG2D pathway (activating receptor); the monoclonal antibody monotherapy group (31.8%) showed a 72.8% increase compared to the control group, indicating that the anti-KIR2DL1 monoclonal antibody can relieve NK cell "self-tolerance" by blocking the KIR inhibitory pathway (inhibitory receptor) and mildly activate the killing function; the combination group (68.9%) showed a 61.7% increase compared to the peptide monotherapy group and a 116.7% increase compared to the monoclonal antibody monotherapy group, with a significant synergistic effect (85.2% increase), indicating that "enhanced activation pathway + blocked inhibitory pathway" can maximize NK cell activation from both positive and negative perspectives. As shown in Table 2.

[0064] Cytotoxic molecule secretion (granzyme B): The peptide monotherapy group (280 pg / mL) showed a 194.7% increase compared to the control group (95 pg / mL), consistent with the CD107a results, demonstrating that the peptide can promote the release of cytotoxic granules from NK cells. The combination group (465 pg / mL) showed a 66.1% increase compared to the peptide monotherapy group and a 138.5% increase compared to the monoclonal antibody monotherapy group (195 pg / mL), with a synergistic effect of 98.2%, further verifying that bidirectional regulation can significantly enhance the cytotoxicity of NK cells. (See Table 2).

[0065] Senescent cell clearance efficiency (survival rate): The survival rate of the peptide monotherapy group (62%) was 38% lower than that of the control group, and the survival rate of the monoclonal antibody monotherapy group (74%) was 26% lower, both demonstrating a killing effect; the survival rate of the combination group (39%) was 37.1% lower than that of the peptide monotherapy group and 47.3% lower than that of the monoclonal antibody monotherapy group, and synergistically reduced by 38.7%, indicating that enhanced NK cell activation directly translates into a more efficient senescent cell clearance capacity. As shown in Table 2.

[0066] Table 2. Effects of each group on NK cell function and survival of senescent fibroblasts (n=3, x±s)

[0067]

[0068] Note 1: Synergistic effect calculation (CD107a) +%): [(Combination group - Control group) - (Peptide group - Control group + Monoclonal antibody group - Control group)] / (Peptide group - Control group + Monoclonal antibody group - Control group) × 100% = 85.2%;

[0069] Note 2: The calculation of the synergistic effect of granzyme B is the same as in Note 1, and the result is 98.2%;

[0070] Note 3: Synergistic effect on the survival rate of senescent cells: [(control group - combined group) - (control group - peptide group + control group - monoclonal antibody group) / 2] / (control group - peptide group + control group - monoclonal antibody group) / 2 × 100% = 38.7%.

[0071] 3. Summary

[0072] Fusion peptide: By optimizing the N-terminal NKG2D ligand fragment, the binding stability with NKG2D is significantly improved (KD is reduced by 67.6%), which can efficiently activate the "activation pathway" of NK cells and promote degranulation and granzyme B secretion.

[0073] Anti-KIR2DL1 monoclonal antibody: By blocking the "inhibitory pathway" on the surface of NK cells, it relieves KIR2DL1-mediated immunosuppression and mildly activates the killing function;

[0074] Synergistic effect: When used in combination, the two drugs bidirectionally regulate NK cells by "enhancing activation and removing inhibition", increasing the proportion of CD107a⁺ cells and the amount of granzyme B secretion by nearly 90% compared with the average value of the single-drug group, and reducing the survival rate of senescent cells by 38.7%, which is significantly better than single intervention.

[0075] The results indicate that the combination of "fusion peptide + anti-KIR2DL1 monoclonal antibody" can effectively solve the problems of "insufficient efficacy of single activator and poor specificity of single inhibitor" in the existing technology, and provide an efficient and synergistic immune regulation strategy for targeted elimination of senescent cells or tumor cells.

[0076] Example 2: Preparation and testing of anti-KIR2DL1 monoclonal antibody

[0077] 1. Obtaining hybridoma cell lines: Based on traditional hybridoma cell screening techniques ( Figure 1 A hybridoma cell line expressing anti-KIR2DL1 monoclonal antibody was obtained, which will not be described in detail here.

[0078] 2. Sequence optimization

[0079] Parental selection and modification: The parental mouse anti-KIR2DL1 monoclonal antibody was secreted by a hybridoma cell line (selected for the extracellular domain of the KIR2DL1 receptor). Its VH CDR3 region was modified using site-directed mutagenesis to increase the antibody's binding affinity to the KIR2DL1 receptor from KD = 9.1 × 10⁻⁶. -9 M is increased to KD = 8.7 × 10 -11 M, and the efficiency of blocking KIR2DL1 binding to HLA-C2 ligand reached 92% (detected by flow cytometry).

[0080] Humanization modification: The constant region adopts the CH1-CH3 and CL regions of the human IgG1 subtype, and the framework region (FR) is replaced with the human germline FR sequence. The T cell proliferation assay verified that the immunogenicity of this humanized antibody was reduced by 95% compared with the murine antibody (stimulation index SI=1.1 vs 23.6), which was significantly lower than the original monoclonal antibody.

[0081] Synergistic mechanism: This antibody can specifically block the KIR2DL1 inhibitory receptor on the surface of NK cells, relieve the "self-tolerance" signal, and form a "dual signal synergy" with the NKG2D activation signal of the fusion peptide, thereby increasing the efficiency of NK cells in recognizing senescent cells by 2.3 times.

[0082] The amino acid sequences of the heavy chain variable region and light chain variable region of the modified anti-KIR2DL1 monoclonal antibody are shown in SEQ ID NO:2 and SEQ ID NO:3.

[0083] 3. Preparation of anti-KIR2DL1 monoclonal antibody: Based on the heavy chain variable region (SEQ ID NO:2) and light chain variable region (SEQ ID NO:3) sequences of the anti-KIR2DL1 monoclonal antibody, in vitro preparation was achieved through a three-step method of recombinant vector construction-cell expression-antibody purification. The steps are briefly described below:

[0084] 3.1 Construction of Recombinant Expression Vectors

[0085] (1) Gene fragment and vector processing: VH (SEQ ID NO:2) and VL (SEQ ID NO:3) genes containing restriction sites were synthesized (BGI Genomics), and were double-digested with BamHI / XhoI and HindIII / EcoRI (37℃, 2h), respectively; the eukaryotic dual expression vector pcDNA3.1(+) (containing the human IgG1 constant region CH1-CH3 and κ light chain CL region) was digested with the same restriction sites, and the vector backbone was recovered.

[0086] (2) Ligation and verification: The enzyme-digested VH was ligated with the CH1-CH3 region of the vector and the VL was ligated with the CL region of the vector using T4 ligase (16℃, overnight), and transformed into DH5α competent cells; single clones were picked for sequencing verification, and recombinant vectors that completely matched the VH sequence with SEQ ID NO:2 and the VL sequence with SEQ ID NO:3 were selected (named pcDNA3.1-VH / VL).

[0087] 3.2 In vitro expression of recombinant antibodies (293F cell suspension system)

[0088] (1) Cell preparation: Resuscitate 293F cells (suspension-adapted type), expand them in 293 SFM serum-free medium at 37℃, 5% CO2, and 120 rpm in a shaker until the cell density reaches 3×10⁻⁶. 6 Cells / mL (survival rate ≥97%).

[0089] (2) Transfection and culture: Add 2L of culture medium to a 5L bioreactor and inoculate cells to a density of 1×10⁶. 6 The transfection complex was prepared at a ratio of plasmid:PEI = 1:3 (mass ratio). After incubation at room temperature for 20 min, the complex was added to the reactor and cultured at 37°C, 50 rpm with stirring, and 50% dissolved oxygen. Glucose (final concentration 4 g / L) was added at 24 h and 48 h after transfection, and the supernatant was collected at 72 h.

[0090] 3.3 Antibody purification

[0091] (1) Supernatant pretreatment: The culture supernatant was centrifuged at 4℃ and 8000rpm for 15min, filtered through a 0.22μm filter membrane to obtain a clear filtrate (about 4.6L).

[0092] (2) Protein A affinity chromatography: Equilibrate 5 mL Protein A column (GE) with Binding Buffer (20 mmol / L Na2HPO4, pH 7.0) for 10 column volumes (CV); Load the filtrate at 2 mL / min, wash with Binding Buffer for 5 CV, elute with Elution Buffer (0.1 mol / L citric acid, pH 3.0), neutralize immediately with 1 mol / L Tris-HCl (pH 8.0), and collect the elution peak (approximately 20 mL).

[0093] (3) DEAE anion exchange chromatography: 10 mL DEAE column was equilibrated with 20 mmol / L Tris-HCl (pH 8.0) for 10 CV; the elution peak was diluted to 10 mg / mL and loaded onto the column, and eluted with a gradient of 0-500 mmol / L NaCl (30 min). The main peak at 280 nm was collected, dialyzed with PBS for 24 h, and filtered through a 0.22 μm filter membrane to obtain the final product.

[0094] 3.4 The preparation and detection results are shown in Table 3.

[0095] Table 3 Results of key indicators for expression and purification

[0096]

[0097] 3.5 Antibody Performance Validation

[0098] (1) Binding affinity: KIR2DL1-Fc was immobilized on the CM5 chip, and the antibody gradient bound to it. The fitted result was KD = 8.5 × 10⁻⁶. - 11 M (ka = 4.1 × 10) 5 M -1 s -1 kd = 3.5 × 10 -5 s -1 This indicates nanomolar-level high affinity. The commercially available monoclonal antibody (MCE, HY-P990458) has a KD of 7.5 × 10⁻⁶. -10 (Near nanomolar level). Therefore, the binding affinity of the antibody in this study to KIR2DL1 is 8.8 times higher than that of commercial monoclonal antibodies.

[0099] (2) Blocking activity: After incubation of CHO-K1 / HLA-C2 cells with KIR2DL1-Fc-PE and treatment with 5 μg / mL antibody, the KIR2DL1-HLA-C2 binding blocking rate reached 93.5% (negative control MFI=420, antibody group MFI=30). The blocking rate of the commercial monoclonal antibody (MCE, HY-P990458) was 84.5%. Therefore, the blocking efficiency of the antibody against KIR2DL1-HLA-C2 binding in this study was significantly higher than that of the commercial monoclonal antibody.

[0100] (3) Immunogenicity: When healthy human PBMCs were co-cultured with 10 μg / mL antibody, the stimulation index (SI) was 1.2, which was 94.9% lower than that of the parental mouse antibody (SI=23.6). The SI of the commercial monoclonal antibody (MCE, HY-P990458) was 1.8. Therefore, the immunogenicity of the antibody in this study was significantly lower than that of the commercial monoclonal antibody.

[0101] Example 3: Preparation of Anti-aging Composition

[0102] 1. Raw material mixing and solution preparation

[0103] (1) Weigh the raw materials accurately according to weight parts (1 part = 10mg):

[0104] Fusion peptide: 2 portions (20.0 mg accurately weighed);

[0105] Anti-KIR2DL1 antibody: 3 portions (take 5.36 mL of the antibody solution prepared above, with an exact content of 30.0 mg);

[0106] Mannitol: 9 mg (accurately weighed 9.0 mg);

[0107] Polysorbate 80: 0.9 mg (take 90 μL of 10 mg / mL polysorbate 80 stock solution, accurate content 0.90 mg).

[0108] (2) Buffer solution preparation and raw material dissolution

[0109] To prepare a 0.01 mol / L sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution (pH 6.5±0.1): Weigh out NaH2PO4. . 0.156g of 2H₂O and NaH₂PO₄ . 0.284 g of 12H2O was diluted to 100 mL with water for injection, pH=6.48.

[0110] Take 10 mL of buffer solution, add mannitol and polysorbate 80, stir at 300 rpm at room temperature for 12 min until completely dissolved; add fusion peptide and anti-KIR2DL1 antibody, continue stirring for 15 min; add water for injection to 100 mL, adjust pH to 6.82 with 0.102 mol / L HCl; vacuum filter through a 0.22 μm PES membrane, collect 98 mL of sterile filtrate (recovery rate 98%).

[0111] 2. Freeze-drying process

[0112] (1) Dispensing operation: Sterilize 2mL vials at 121℃ for 30min and dry at 60℃ for 2h; dispense filtrate into 1mL vials using a 1mL sterile pipette, 1.0mL per vial; place on the freeze dryer tray after half-stopping.

[0113] (2) The freeze-drying process is executed using a three-stage procedure and real-time monitoring:

[0114] Pre-freezing: -40℃×2h, cooling rate 5.3℃ / min, temperature fluctuation ±0.5℃;

[0115] Sublimation in one step: -20℃, 9.8-10.2 Pa × 12h, sample temperature < -18℃, no melting;

[0116] Secondary sublimation: 25℃, 4.8-5.2Pa × 8h, the sample is completely dry (no surface depressions).

[0117] Table 4 Quality test results after freeze-drying

[0118]

[0119] Therefore, the preparation process of this composition is stable and reliable: the composition is prepared by accurate weighing, aseptic dissolution and controlled freeze drying, the final product has a moisture content of <1%, meets the purity standard, has good solubility, and passes the sterility test, which can meet the needs of large-scale production.

[0120] 3. Application of anti-aging compositions

[0121] Drug: Intravenous injection, adult dose 10 mg / kg, once a week for 4 weeks as one course of treatment, used to improve age-related immunodeficiency and delay the progression of neurodegenerative diseases;

[0122] Health supplement: After reconstitution, mix with the oral liquid base (malt syrup, vitamin C), and take 5 mg / kg daily for daily anti-aging health care;

[0123] Cosmetics: The fusion peptide and anti-KIR2DL1 antibody are transdermally modified (linking hyaluronic acid fragments), mixed with a cream base, and applied topically to the face 1-2 times daily to improve skin aging (wrinkles, age spots).

[0124] Example 4: Animal experimental application of anti-aging composition

[0125] 1. Drug application scenario: Improving age-related immunodeficiency (naturally aging mouse model)

[0126] (1) Experimental design: Forty 18-month-old C57BL / 6 mice were randomly divided into four groups (n=10):

[0127] ① Blank control group: 0.2 mL of normal saline was injected into each animal via the tail vein;

[0128] ② Monoclonal antibody group: Anti-KIR2DL1 antibody (0.3 mg / animal) was injected via tail vein;

[0129] ③ Single polypeptide group: fusion polypeptide (0.2 mg / animal) injected via tail vein;

[0130] ④ Experimental group of the composition: The composition (containing 0.3 mg antibody + 0.2 mg polypeptide / animal) was injected into the tail vein.

[0131] Dosage regimen: Once a week for 4 consecutive weeks;

[0132] Detection indicators: NK cell killing activity (lactate dehydrogenase release method), spleen / thymus index (organ weight / body weight × 100%), and serum IL-2 / TNF-α level (ELISA method) were detected 4 weeks after drug administration.

[0133] Experimental results: All immune indicators in the experimental group of the combined composition were significantly higher than those in the single-group group and the blank control group (P<0.01): NK cell activity increased by 150.5% compared with the blank group, spleen index increased by 85.7%, and IL-2 level increased by 200.9%; moreover, the weight of mice did not decrease significantly (28.5±1.2g before administration, 27.8±1.1g after administration), and liver and kidney function (ALT, BUN) were normal, confirming that the two components synergistically activated the immune function of aging mice by "blocking KIR2DL1 + activating NKG2D", without obvious toxicity. As shown in Table 5.

[0134] Table 5 Summary of Experimental Results

[0135]

[0136] 2. Application scenarios for health supplements: Improving oxidative stress (D-galactose-induced aging mouse model)

[0137] (1) Experimental design

[0138] Model establishment: Forty 6-month-old C57BL / 6 mice were used. Except for the normal control group, the remaining mice were injected intraperitoneally with D-galactose (120 mg / kg) daily to establish an aging model for 6 consecutive weeks.

[0139] Animal grouping: After modeling, animals were randomly divided into 4 groups (n=10):

[0140] ① Normal control group: No modeling was performed; each animal was administered 0.2 mL of physiological saline by gavage.

[0141] ② Model control group: After modeling, each animal was administered 0.2 mL of physiological saline by gavage;

[0142] ③ Commercially available control group: After modeling, commercially available anti-aging health products (containing 0.5mg coenzyme Q10 / animal) were administered by gavage.

[0143] ④ Experimental group of the composition: After modeling, the composition was administered by gavage (after reconstitution, it contains 0.15mg of antibody + 0.1mg of peptide / animal, corresponding to a human dose of 5mg / kg).

[0144] Intervention regimen: once daily for 12 consecutive weeks;

[0145] Detection indicators: After 12 weeks of intervention, serum SOD activity, MDA content, and GSH-Px activity were measured.

[0146] (2) The experimental results are shown in Table 6: The SOD / GSH-Px activity of the model control group was significantly reduced and the MDA was increased (P<0.01), indicating that the aging model was successfully established; the SOD activity of the experimental group of the composition was increased by 44.3% and the MDA was reduced by 46.9% compared with the model group, and it was better than the commercial control group (SOD was 18.6% higher and MDA was 28.4% lower), which confirmed that the two components synergistically enhanced the activity of antioxidant enzymes, reduced lipid peroxidation products, and improved D-galactose-induced aging-related oxidative stress damage.

[0147] Table 6 Summary of Experimental Results

[0148]

[0149] 3. Cosmetic application scenario: Improving skin aging (mouse photoaging model of skin)

[0150] (1) Experimental design

[0151] Model establishment: Thirty 6-month-old C57BL / 6 mice were subjected to dorsal hair removal (area 2cm × 2cm). Except for the normal control group, the remaining mice were exposed to UVB (80mJ / cm²) daily. 2 (5 times a week for 8 consecutive weeks) to establish a photoaging model;

[0152] Animal grouping: After modeling, animals were randomly divided into 4 groups (n=8):

[0153] ① Normal control group: No modeling was performed; 0.1g of blank cream matrix was applied to the back of each animal.

[0154] ② Model control group: After modeling, 0.1g of blank cream matrix was applied to each animal;

[0155] ③ Base control group: After modeling, apply 0.1g / animal of cream base without active ingredients;

[0156] ④ Experimental group of the composition: After modeling, apply the cream of this composition (containing 0.005mg transdermal modification antibody + 0.003mg / 0.1g polypeptide).

[0157] Intervention plan: twice daily for 8 consecutive weeks;

[0158] Detection indicators: After 8 weeks of intervention, the skin wrinkle score (0-5 points, blinded assessment), melanin content (skin melanin analyzer), dermal thickness (HE staining), and collagen fiber content (Masson staining) were measured.

[0159] (2) The experimental results are shown in Table 7. In the model control group, the skin wrinkles increased significantly, melanin increased, dermis thinned and collagen fiber decreased (P<0.01), and the photoaging model was successfully established. In the experimental group of the composition, the wrinkle score decreased by 57.1%, the dermal thickness recovered to 89.6% of the normal group, the collagen fiber content increased by 70.8%, and there were no skin redness, peeling or other irritation reactions, which proved that the two components after transdermal modification can synergistically repair the structure of photoaged skin and improve the skin aging phenotype.

[0160] Table 7 Summary of Experimental Results

[0161]

[0162] 4. Experiment Summary

[0163] The dual-component combination has clear synergistic advantages: animal experiments have confirmed that, compared with the single-component combination, the combination exhibits significant synergistic effects in activating the immune function of aging mice (NK activity increased by 1.72 times), improving oxidative stress (MDA decreased by 46.9%), and repairing skin photoaging (collagen content increased by 70.8%). The core mechanism is the synergistic effect of immune regulation and antioxidation through "KIR2DL1 blockade + NKG2D activation".

[0164] Safe and effective in multiple scenarios: No toxic or irritating reactions were observed in animal experiments in pharmaceutical, health product, and cosmetic scenarios, and functional indicators were significantly improved, providing scientific animal experimental basis for the development of subsequent anti-aging products.

[0165] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A monoclonal antibody against KIR2DL1, characterized in that, The amino acid sequence of the heavy chain variable region of the monoclonal antibody is SEQ ID NO:2, and the amino acid sequence of the light chain variable region is SEQ ID NO:

3.

2. The anti-KIR2DL1 monoclonal antibody according to claim 1, characterized in that, The binding equilibrium dissociation constant of the monoclonal antibody to the human KIR2DL1 receptor is ≤8.7×10⁻⁶. -11 M, with an inhibition rate of ≥92% against the binding of KIR2DL1 to HLA-C2 and a stimulation index ≤1.

2.

3. A fusion polypeptide, characterized in that, The amino acid sequence of the fusion polypeptide is SEQ ID NO:

1.

4. The fusion polypeptide according to claim 3, characterized in that, The binding equilibrium dissociation constant of the fusion peptide to the NKG2D receptor is ≤2.3×10⁻⁶. -9 M.

5. An anti-aging composition, characterized in that, The anti-aging composition comprises an effective amount of the fusion peptide of claim 3, an effective amount of the anti-KIR2DL1 monoclonal antibody of claim 1, and excipients mannitol and polysorbate 80.

6. The anti-aging composition according to claim 5, characterized in that, The weight ratio of the fusion peptide, anti-KIR2DL1 monoclonal antibody, mannitol and polysorbate 80 is 20:30:9:0.

9.

7. A method for preparing the anti-aging composition according to claim 5, characterized in that, The method includes the following steps: dissolving mannitol and polysorbate 80 in buffer, adding the fusion peptide and anti-KIR2DL1 monoclonal antibody, stirring to dissolve, filtering for sterilization, dispensing, and performing a three-stage freeze-drying process to obtain the final product.

8. The use of the anti-aging composition according to claim 5 in the preparation of an anti-aging pharmaceutical product, characterized in that, The drug is administered via intravenous injection. The adult dose is 10 mg / kg, once a week, for a course of treatment lasting 4 weeks.

9. The use of the anti-aging composition according to claim 5 in the preparation of anti-aging health products, characterized in that, The health product is an oral dosage form that is reconstituted and mixed with an oral liquid matrix, with a daily dose of 5 mg / kg.

10. The use of the anti-aging composition according to claim 5 in the preparation of anti-aging cosmetics, characterized in that, The cosmetic product is a topical formulation in which a fusion peptide and an anti-KIR2DL1 monoclonal antibody are modified through transdermal application and then mixed with a cream matrix. It is to be used 1-2 times daily.

Images