A pharmaceutical composition and its use in the treatment of central obesity

Abstract

The present application relates to the technical field of central obesity related research and treatment, by constructing a central obesity mouse model, it is found that the up-regulation of brain-derived neurotrophic factor (BDNF) expression in adipose tissue is closely related to central obesity resistance. A BDNF fusion polypeptide composed of TAT cell-penetrating peptide, flexible linker and BDNF mature peptide is designed and prepared, the fusion polypeptide has strong cell viability promoting effect at the cellular level, can effectively activate the BDNF signaling pathway and has better stability than natural BDNF protein; an anti-TrkB monoclonal antibody with high affinity and high titer is also prepared. In vivo experiments show that the BDNF fusion polypeptide and its combination with the anti-TrkB monoclonal antibody can effectively inhibit the weight gain of central obesity mice, improve glucose metabolism disorder, increase serum BDNF level and activate the TrkB signaling pathway, and the combination has better synergistic therapeutic effect, which provides a new direction and experimental basis for the treatment of central obesity.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and particularly relates to a pharmaceutical composition and application thereof in the treatment of central obesity. BACKGROUND

[0002] In recent years, the number of obese people in the world has increased explosively. Central obesity, due to its complex pathological mechanism and high treatment difficulty, has become a difficult problem to be solved in the medical field. Central obesity is a metabolic disease characterized by abnormal accumulation of fat in the internal organs and abdomen. Its core mechanism is the dysfunction of hypothalamic energy regulation nuclei (ventromedial nucleus VMH, arcuate nucleus ARC, etc.), leading to increased appetite, reduced energy consumption and lipid metabolism imbalance. Studies have shown that the imbalance of the activity of the hypothalamic melanocortin system (POMC neurons) and neuropeptide Y (NPY) neurons is the key to the occurrence of obesity, and brain-derived neurotrophic factor (BDNF) can activate POMC neurons and inhibit NPY expression by binding to its receptor TrkB, thereby regulating energy homeostasis. Clinical data show that the serum and hypothalamic BDNF levels of obese patients are significantly lower than those of healthy people, and defects in the BDNF / TrkB signaling pathway caused by gene mutations are highly related to obesity.

[0003] At present, there are obvious limitations in the treatment of central obesity. For example, GLP-1 receptor agonists (such as liraglutide) can inhibit appetite through peripheral pathways, but cannot directly target the central BDNF signaling pathway, and long-term use can cause nausea, vomiting and other gastrointestinal side effects. The natural BDNF protein has a short half-life (about 1-2 hours) and is difficult to penetrate the blood-brain barrier (BBB), so its effect is limited when administered centrally. In recent years, researchers have tried to deliver BDNF through gene therapy or nanocarriers, but still face problems such as high immunogenicity and complex preparation process. In addition, the development of monoclonal antibodies against TrkB is mainly focused on the field of cancer, and there is no agonistic antibody for obesity treatment.

[0004] Therefore, it is urgent to develop a treatment plan that is efficient, low-toxic and can target the key pathological link of central obesity. SUMMARY

[0005] The present application reveals the correlation between BDNF and central obesity through a series of experiments, and develops related therapeutic substances and compositions.

[0006] Therefore, the present application discloses a BDNF fusion polypeptide, the amino acid sequence of the fusion polypeptide consists of a TAT cell-penetrating peptide, a flexible linker peptide and a BDNF mature peptide in order, and the specific amino acid sequence is shown in SEQ ID NO. 1.

[0007] Preferably, the amino acid sequence of the TAT penetrating peptide of the present application is YGRKKRRQRRR, the amino acid sequence of the flexible linker peptide is GPGGGGS, and the BDNF mature peptide is a human BDNF mature peptide, and the amino acid sequence thereof corresponds to 119 amino acids of His129-Arg247 in UniProt No. P23560.

[0008] In one aspect, the present application also discloses a composition for treating central obesity, which comprises an effective amount of the BDNF fusion polypeptide and an effective amount of the anti-TrkB monoclonal antibody.

[0009] Preferably, the amino acid sequence of the heavy chain variable region of the anti-TrkB monoclonal antibody of the present application is shown in SEQ ID NO. 2, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO. 3.

[0010] Preferably, the dose of the BDNF fusion polypeptide and the anti-TrkB monoclonal antibody of the present application is 5 mg / kg.

[0011] In one aspect, the present application also discloses the use of the BDNF fusion polypeptide in the preparation of a medicament for treating central obesity.

[0012] In one aspect, the present application also discloses the use of the anti-TrkB monoclonal antibody in the preparation of a medicament for treating central obesity.

[0013] The beneficial effects of the present application are summarized as follows:

[0014] 1. The present application clarifies the close relationship between the up-regulation of BDNF expression in adipose tissue and the resistance to central obesity, which provides an important theoretical basis for in-depth understanding of the pathogenesis of central obesity and exploring therapeutic targets.

[0015] 2. On this basis, the present application develops a BDNF fusion polypeptide, which, by introducing a TAT penetrating peptide and a flexible linker peptide, improves the cell uptake efficiency and stability compared with natural BDNF protein, and exhibits stronger cell viability and stronger ability to activate BDNF signaling pathway at the cellular level, thereby providing a new active substance for the treatment of central obesity.

[0016] 3. The anti-TrkB monoclonal antibody prepared by the present application has high affinity and high titer, and has better performance than commercial antibodies, thereby providing a powerful tool for targeting TrkB for the treatment of central obesity.

[0017] 4. The application shows significant synergistic effect of the composition of BDNF fusion polypeptide and anti-TrkB monoclonal antibody in the in vivo experiment on the weight control, sugar metabolism improvement and TrkB signal pathway activation of central obesity mice, which provides a new effective strategy and drug research direction for the clinical treatment of central obesity, and has broad application prospects. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 SDS-PAGE detection results of BDNF fusion polypeptide.

[0019] Figure 2 Western blot detection results of BDNF fusion polypeptide.

[0020] Figure 3 SDS-PAGE detection results of anti-TrkB monoclonal antibody.

[0021] Figure 4 Western blot detection results of anti-TrkB monoclonal antibody (the molecular weight of TrkB recombinant protein is about 78 kDa). DETAILED DESCRIPTION

[0022] 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 application belongs. The terminology used in the description herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. As used herein, the term "and / or" includes any and all combinations of one or more of the associated listed items.

[0023] Unless specifically noted, the reagents, methods, and equipment employed in the present application are those conventional in the art. Unless specifically noted, the reagents and materials used in the following examples are commercially available.

[0024] Example 1: Construction, expression and functional verification of BDNF fusion polypeptide

[0025] I. Fusion polypeptide construction design: the fusion polypeptide sequence is designed as SEQ ID NO. 1, which is composed of three parts:

[0026] (1) Human BDNF mature peptide (UniProt number: P23560, His129-Arg247, 119 aa in total);

[0027] (2) TAT transmembrane peptide (transmembrane transduction, improve cell uptake): YGRKKRRQRRR

[0028] (3) Flexible linker peptide (stability-enhancing peptide SA): GPGGGGS

[0029] The final fusion sequence is: TAT cell-penetrating peptide-flexible linker peptide-BDNF mature peptide, and the specific amino acid sequence is shown in SEQ ID NO. 1.

[0030] II. Preparation and identification of BDNF fusion polypeptide

[0031] 1. Experimental materials

[0032] (1) Host cell: Escherichia coli BL21 (DE3).

[0033] (2) Expression vector: pET-28a (+).

[0034] (3) Reagents: restriction enzymes (NdeI, XhoI), T4 DNA ligase, DNA polymerase, antibiotics (kanamycin), IPTG (isopropyl-β-D-thiogalactoside), protein purification related reagents (nickel column filler, elution buffer, etc.), SDS-PAGE related reagents, Western blot related reagents (primary antibody is anti-BDNF antibody, secondary antibody is HRP labeled goat anti-rabbit IgG).

[0035] 2. Experimental steps

[0036] 2.1 Gene synthesis and vector construction: according to the amino acid sequence of BDNF fusion polypeptide, the corresponding DNA sequence is designed by codon optimization. The DNA sequence is synthesized by a gene synthesis company, and NdeI and XhoI restriction sites are introduced at both ends. The synthesized DNA fragment and pET-28a (+) vector are double-digested with NdeI and XhoI, respectively. The enzyme digestion system is: DNA or vector 5 μg, NdeI 2 μL, XhoI 2 μL, 10× enzyme digestion buffer 5 μL, add ddH2O to 50 μL, 37°C enzyme digestion for 3h). After agarose gel electrophoresis separation, the target fragment and vector fragment are recovered by gel recovery kit. The recovered target fragment and vector fragment are ligated with T4 DNA ligase, and the ligation system is: target fragment 3 μL, vector fragment 1 μL, T4 DNA ligase 1 μL, 10× ligation buffer 1 μL, add ddH2O to 10 μL, 16°C ligation overnight. The ligation product is transformed into Escherichia coli DH5α competent cells, and plated on LB plates containing kanamycin (50 μg / mL), and incubated at 37°C overnight. Single colonies are picked for PCR identification, and positive clones are sent for sequencing verification.

[0037] 2.2 Protein expression and induction: The recombinant plasmid was transformed into E. coli BL21(DE3) competent cells, and then plated on LB agar plates containing kanamycin (50 μg / mL) and incubated at 37 °C overnight. A single colony was inoculated into 5 mL LB liquid medium containing kanamycin (50 μg / mL) and incubated at 37 °C, 220 rpm overnight. 1 mL of the overnight culture was transferred into 100 mL LB liquid medium containing kanamycin (50 μg / mL) and incubated at 37 °C, 220 rpm until the OD 600 of the culture was about 0.6-0.8. IPTG was added to a final concentration of 0.5 mM, and the expression was induced at 16 °C, 180 rpm for 16 h.

[0038] 2.3 Protein purification: The bacterial solution after induction was centrifuged at 4 °C, 8000 rpm for 10 min, and the bacterial cells were collected. The bacterial cells were resuspended with an appropriate amount of binding buffer (20 mM Tris-HCl, 500 mM NaCl, 20 mM imidazole, pH 7.4) and ultrasonically broken (power 300 W, work 3 s, interval 5 s, total 30 min). The broken bacterial solution was centrifuged at 4 °C, 12000 rpm for 30 min, and the supernatant was collected. The supernatant was passed through a nickel column, and the column was washed with binding buffer until the OD 280 of the effluent was basically unchanged. The target protein was eluted with elution buffer (20 mM Tris-HCl, 500 mM NaCl, 500 mM imidazole, pH 7.4), and the elution peak was collected. The eluted protein was analyzed by SDS-PAGE to detect the purity and molecular weight of the protein.

[0039] 2.4 Protein identification: The protein separated by SDS-PAGE was transferred to a PVDF membrane and blocked with 5% skim milk for 1 h. Anti-BDNF antibody (1:1000 dilution) was added and incubated at 4 °C overnight. TBST was washed 3 times for 10 min each time. HRP-labeled goat anti-rabbit IgG (1:5000 dilution) was added and incubated at room temperature for 1 h. TBST was washed 3 times for 10 min each time. Color development was performed with ECL luminescent solution, and exposure and development were performed to confirm that the protein was a BDNF fusion polypeptide.

[0040] 3. Experimental results

[0041] 3.1 Protein expression and purification results: SDS-PAGE analysis showed that the bacterial solution after induction appeared a clear band at about 15 kDa, which was consistent with the theoretical molecular weight of the BDNF fusion polypeptide. After nickel column purification, the purity of the target protein reached more than 90% ( Figure 1 ).

[0042] 3.2 Protein identification results: Western blot results showed that specific bands appeared at about 15 kDa, further confirming that the purified protein was a BDNF fusion polypeptide (shown in the figure). Figure 2

[0043] III. Verification of the activity of BDNF fusion polypeptide at the cellular level

[0044] 1. Experimental materials

[0045] (1) Cell line: SH-SY5Y human neuroblastoma cells.

[0046] (2) Culture medium: DMEM / F12 medium, supplemented with 10% fetal bovine serum, 1% penicillin-streptomycin.

[0047] (3) Reagents: BDNF fusion polypeptide, natural BDNF protein (purchased from Huawmei Biological, its item number is CSB-EP002655HU(A4)), MTT reagent, CCK-8 reagent, BDNF receptor antagonist K252a, POMC detection kit.

[0048] 2. Experimental steps

[0049] 2.1 Cell culture: SH-SY5Y cells were inoculated in culture bottles containing DMEM / F12 medium and cultured in a 37°C, 5% CO2 incubator. When the cell confluence reached 80%-90%, the cells were passaged or used for experiments.

[0050] 2.2 Cell viability detection: SH-SY5Y cells were inoculated in a 96-well plate at a density of 1×10 4 cells per well, and cultured for 24 h. Different concentrations (0, 10, 20, 50, 100 ng / mL) of BDNF fusion polypeptide and natural BDNF protein were added, respectively, and each group had 6 replicate wells, and the culture was continued for 24 h. 10 μL of MTT reagent (5 mg / mL) was added to each well, and the culture was continued for 4 h. The supernatant was removed, 150 μL of DMSO was added to each well, and the mixture was shaken for 10 min to fully dissolve the crystals. The absorbance value was measured at 570 nm wavelength using a microplate reader, and the cell viability was calculated.

[0051] 2.3 BDNF signaling pathway activation detection: SH-SY5Y cells were inoculated in a 96-well plate at a density of 1×10 5 ​The cells were seeded in 6-well plates at a density of 1 x 105 cells per well and cultured for 24 h. 100 ng / mL of BDNF fusion polypeptide, native BDNF protein and an equal volume of medium (control group) were added, respectively, and a BDNF receptor antagonist K252a treatment group (K252a was added for pre-treatment for 30 min, and then the BDNF fusion polypeptide was added) was set up. Each group had 3 replicate wells, and the cells were cultured for another 24 h. The cells were collected, total protein was extracted, and the protein concentration was determined by the BCA method. The expression level of POMC in the cells was detected by a POMC detection kit, and the operation was performed according to the kit instructions.

[0052] 3. Experimental results

[0053] 3.1 The cell viability detection results are shown in Table 1. The BDNF fusion polypeptide and the native BDNF protein can both promote the viability of SH-SY5Y cells, and the BDNF fusion polypeptide has a stronger cell viability promoting effect.

[0054] Table 1. Cell viability detection results

[0055]

[0056] 3.2 The BDNF signal pathway activation detection results are shown in Table 2. The BDNF fusion polypeptide can significantly activate the BDNF signal pathway and promote the expression of POMC, and this effect can be inhibited by the BDNF receptor antagonist K252a.

[0057] Table 2. BDNF signal pathway activation detection results

[0058]

[0059] IV. Stability evaluation

[0060] The fusion protein was placed in PBS at 37°C for 24, 48 and 72 hours, and the protein residual amount was detected. The results are as follows:

[0061] (1) The residual proportion of the fusion polypeptide was 92.1% at 24 h, 79.6% at 48 h and 65.3% at 72 h.

[0062] (2) The residual proportion of the native BDNF protein was 64.6% at 24 h, 45.2% at 48 h and 38.4% at 72 h.

[0063] The above results show that the fusion polypeptide is significantly better than the native BDNF protein.

[0064] Example 2: Preparation and testing of anti-TrkB monoclonal antibody

[0065] I. Experimental materials

[0066] 1. Experimental animals: 6-8 weeks old female Balb / c mice.

[0067] 2. Cell lines: SP2 / 0 myeloma cells.

[0068] 3. Reagents: Complete Freund's adjuvant, incomplete Freund's adjuvant. TrkB antigen protein (ab132927) (purity >95%). Polyethylene glycol (PEG, MW 1500). HAT medium (containing hypoxanthine, aminopterin and thymidine), HT medium (containing hypoxanthine and thymidine), RPMI-1640 medium. Fetal bovine serum (FBS). Goat anti-mouse IgG-HRP secondary antibody. TMB developing solution, stop solution (2M H2SO4). Protein A affinity chromatography column filler. SDS-PAGE related reagents, Western blot related reagents. Commercial anti-TrkB monoclonal antibody (ab134155).

[0069] II. Experimental steps

[0070] 1. Antibody preparation

[0071] (1) Animal immunization: Mix TrkB antigen protein with complete Freund's adjuvant in equal volume, emulsify thoroughly, then subcutaneously inject Balb / c mice with multiple points, each mouse with a dose of 100 μg of antigen protein. Three weeks later, mix TrkB antigen protein with incomplete Freund's adjuvant in equal volume, emulsify, and then subcutaneously inject with multiple points, each mouse with a dose of 50 μg of antigen protein. Then every 2 weeks for one booster, a total of 3 times, each dose of 50 μg of antigen protein, all using incomplete Freund's adjuvant emulsification. Seven days after the last booster, take the tail vein blood of the mice, separate the serum, and detect the serum antibody titer by indirect ELISA. When the antibody titer reaches the requirement (>1:10 5 ), perform cell fusion.

[0072] (2) Cell fusion: The spleen of the immunized mouse was taken, washed with sterile PBS, ground into a single cell suspension, filtered through a 200-mesh cell sieve, and the spleen cells were collected. At the same time, the SP2 / 0 myeloma cells in the logarithmic growth phase were taken, washed with PBS for 2 times. The spleen cells and SP2 / 0 myeloma cells were mixed at a ratio of 10:1, centrifuged at 1200 rpm for 5 min, and the supernatant was discarded. Under the condition of 37°C water bath, 1 mL of preheated 50% PEG 1500 solution was slowly added, and stirred gently while adding, for 1 min. Then 1 mL of RPMI-1640 medium was slowly added within 1 min, followed by 9 mL of RPMI-1640 medium within 2 min, to terminate the effect of PEG. Centrifuged at 1200 rpm for 5 min, discarded the supernatant, resuspended the cells with HAT medium, inoculated in a 96-well cell culture plate with 100 μL per well, and cultured in a 37°C, 5% CO2 incubator.

[0073] (3) Hybridoma cell screening: On the 3rd day after fusion, the cell growth was observed, and the HAT medium was replaced in time. On the 7th-10th day after fusion, when the hybridoma cells grew to 1 / 3-1 / 2 of the hole bottom area, the culture supernatant was taken, and the secretion of anti-TrkB antibody was detected by indirect ELISA method. The specific operation is as follows:

[0074] The TrkB antigen protein was diluted with coating buffer (0.05M carbonate buffer, pH 9.6) to 1 μg / mL, 100 μL per well was added to a 96-well enzyme-labeled plate, and coated at 4°C overnight. The next day, the coating solution was discarded, washed with PBST (PBS containing 0.05% Tween-20) for 3 times, 5 min each time. 200 μL of 5% skim milk was added to each well, and blocked at 37°C for 1 h. Discard the blocking solution, wash with PBST for 3 times, 5 min each time. Add 100 μL of hybridoma cell culture supernatant, incubate at 37°C for 1 h. Wash with PBST for 3 times, 5 min each time. Add 100 μL of diluted goat anti-mouse IgG-HRP secondary antibody, incubate at 37°C for 1 h. Wash with PBST for 5 times, 5 min each time. Add 100 μL of TMB color developing solution to each well, develop at room temperature for 15 min. Add 50 μL of stop solution to terminate the reaction, and measure the absorbance value at 450 nm wavelength with a microplate reader. The hybridoma cell wells with absorbance value significantly higher than the negative control wells were selected for the next step of subcloning.

[0075] (4) Subcloning: The positive hybridoma cells were subcloned by limiting dilution. The positive hybridoma cells were diluted to 1 cell / well, 3 cells / well and 5 cells / well with HT medium, and inoculated into 96-well cell culture plates at 100 μL / well, and incubated in a 37°C, 5% CO2 incubator. After 7-10 days of culture, the culture supernatant was taken and the secretion of the anti-TrkB antibody was detected by indirect ELISA, and the cell wells with monoclonal growth and antibody positivity were selected for expansion culture.

[0076] (5) Antibody production and purification: the hybridoma cells stably secreting the anti-TrkB monoclonal antibody screened were inoculated into the abdominal cavity of Balb / c mice, 1 x 10 6 -5 x 10 6 cells per mouse. After 7-10 days, the mouse ascites was collected. The ascites was diluted 1-2 times with PBS, centrifuged at 12000 rpm for 30 min, and the supernatant was taken. The supernatant was passed through a Protein A affinity chromatography column, the column was equilibrated with binding buffer (20 mM sodium phosphate buffer, pH 7.0), and then the antibody was eluted with elution buffer (0.1 M glycine-HCl buffer, pH 3.0). The elution peak was collected and neutralized to pH 7.0 with neutralization buffer (1 M Tris-HCl, pH 9.0). The purity of the antibody was detected by SDS-PAGE, and the concentration of the antibody was determined by BCA method.

[0077] 2. Antibody test

[0078] (1) Antibody specificity identification (Western blot): the TrkB antigen protein and other irrelevant proteins were separated by SDS-PAGE electrophoresis, and then transferred to a PVDF membrane. The PVDF membrane was blocked with 5% skim milk for 1 h, and then the anti-TrkB monoclonal antibody of the application and the commercial anti-TrkB monoclonal antibody were added respectively after dilution, and incubated at 4°C overnight. PBST was washed 3 times, each for 10 min. Diluted goat anti-mouse IgG-HRP secondary antibody was added and incubated at room temperature for 1 h. PBST was washed 5 times, each for 10 min. Color development was performed with ECL luminescent solution, and exposure and development were performed, and whether a band appeared only at the position corresponding to the TrkB antigen protein was observed, and the clarity and intensity of the band were compared.

[0079] (2) Antibody affinity determination: the affinity of the antibody was determined by ELISA competition method. Specifically as follows:

[0080] TrkB antigen protein was diluted with coating buffer to 1 μg / mL, 100 μL per well was added to 96-well enzyme-labeled plate, and was coated at 4°C overnight. The next day, the coating solution was discarded, and PBST was washed for 3 times, 5 min each time. 200 μL of 5% skimmed milk was added to each well, and was blocked at 37°C for 1 h. The blocking solution was discarded, and PBST was washed for 3 times, 5 min each time. The anti-TrkB monoclonal antibody of the application and the commercial anti-TrkB monoclonal antibody were respectively diluted with PBS to different concentrations (such as 10 -5 -10 -12 M), and were mixed with equal amounts of different concentrations of TrkB antigen protein (such as 10 -5 -10 -12 M), and were incubated at 37°C for 1 h. The mixture was respectively added to the enzyme-labeled plate coated with TrkB antigen protein, 100 μL per well, and was incubated at 37°C for 1 h. PBST was washed for 3 times, 5 min each time. 100 μL of diluted goat anti-mouse IgG-HRP secondary antibody was added, and was incubated at 37°C for 1 h. PBST was washed for 5 times, 5 min each time. 100 μL of TMB color developing liquid was added to each well, and was developed at room temperature for 15 min. 50 μL of stop solution was added to stop the reaction, and the absorbance value was determined by an enzyme-labeled instrument at 450 nm wavelength. The competitive inhibition curves of the antibody of the application and the commercial antibody were respectively drawn according to the absorbance value, and the affinity constant (Kd) of the antibody was calculated.

[0081] (3) Antibody titer determination: the titer of the antibody was determined by indirect ELISA method. TrkB antigen protein was diluted with coating buffer to 1 μg / mL, 100 μL per well was added to 96-well enzyme-labeled plate, and was coated at 4°C overnight. The next day, the coating solution was discarded, and PBST was washed for 3 times, 5 min each time. 200 μL of 5% skimmed milk was added to each well, and was blocked at 37°C for 1 h. The blocking solution was discarded, and PBST was washed for 3 times, 5 min each time. The anti-TrkB monoclonal antibody of the application and the commercial anti-TrkB monoclonal antibody were respectively diluted with PBS by a ratio (such as 1:100, 1:200, 1:400……), 100 μL per well was added, and was incubated at 37°C for 1 h. PBST was washed for 3 times, 5 min each time. 100 μL of diluted goat anti-mouse IgG-HRP secondary antibody was added, and was incubated at 37°C for 1 h. PBST was washed for 5 times, 5 min each time. 100 μL of TMB color developing liquid was added to each well, and was developed at room temperature for 15 min. 50 μL of stop solution was added to stop the reaction, and the absorbance value was determined by an enzyme-labeled instrument at 450 nm wavelength. The highest dilution multiple with the absorbance value greater than 2.1 times of that of the negative control well was taken as the titer of the antibody.

[0082] III. Experimental results

[0083] 1. Antibody production and purification results

[0084] (1) The anti-TrkB monoclonal antibody of the present application: SDS-PAGE detection shows that the purified antibody presents a single band, with a purity of >90% (as shown in the figure). Figure 3 The antibody concentration is 3.23 mg / mL, as determined by BCA method.

[0085] (2) Antibody specificity identification results (Western blot): The anti-TrkB monoclonal antibody of the present application appears a clear and strong band at the position corresponding to the TrkB antigen protein, with little background interference (as shown in the figure). Figure 4

[0086] (3) Antibody affinity determination results: The affinity constant (Kd) of the anti-TrkB monoclonal antibody of the present application is 5x10-11M, while the affinity constant (Kd) of the commercial anti-TrkB monoclonal antibody is 2x10-10M. The results show that the affinity of the anti-TrkB monoclonal antibody of the present application is significantly higher than that of the commercial antibody.

[0087] (4) Antibody titer determination results: The antibody titer of the anti-TrkB monoclonal antibody of the present application is 1:10 6 , while the titer of the commercial anti-TrkB monoclonal antibody is 1:10 5 . Therefore, the titer of the anti-TrkB monoclonal antibody of the present application is 10 times that of the commercial antibody.

[0088] Four, sequence determination of the anti-TrkB monoclonal antibody of the present application

[0089] Through detection, the amino acid sequences of the heavy chain variable region and the light chain variable region of the anti-TrkB monoclonal antibody of the present application are shown in SEQ ID NO. 2 and SEQ ID NO. 3, respectively.

[0090] The above results show that the anti-TrkB monoclonal antibody of the present application has a lower affinity constant and a higher titer, which means that it can bind to the TrkB antigen protein at a lower concentration and with stronger binding ability. In practical applications, the amount of antibody used can be reduced, and the cost can be reduced. Therefore, the anti-TrkB monoclonal antibody of the present application is superior to the commercial antibody in performance, and has a broader application prospect.

[0091] Example 3: In vivo experiment of the composition

[0092] I. Experimental materials

[0093] 1. Experimental animals: 60 male C57BL / 6 mice, 6 weeks old, weighing 18-20 g. The mice were raised in a specific pathogen-free (SPF) level animal room with a temperature of (22±2) ℃, humidity of 50%-60%, and a 12h light / 12h dark cycle, and free access to food and water.

[0094] ​2. Reagents: BDNF fusion polypeptide was prepared according to the method of Example 2; anti-TrkB monoclonal antibody was prepared according to the method of Example 3; TrkB, p-TrkB antibodies and corresponding secondary antibodies (abcam); RIPA lysis buffer, BCA protein quantification kit and other kits were all commercial products.

[0095] II. Experimental procedures

[0096] 1. Animal grouping: 60 male C57BL / 6 mice were randomly divided into 4 groups, 15 in each group:

[0097] (1) Normal diet group (Group 1): normal diet feed was given, lasting for 8 weeks.

[0098] (2) Model control group (Group 2): high-fat diet feed was given, lasting for 8 weeks.

[0099] (3) Fusion polypeptide group (Group 3): high-fat diet feed was given, and BDNF fusion polypeptide was orally gavaged at 9-10 am every day, at a dose of 10 mg / kg, lasting for 8 weeks. Special mouse gavage needles were used to ensure that the drug was accurately delivered to the stomach.

[0100] (4) Composition group (Group 4): high-fat diet feed was given, and BDNF fusion polypeptide and anti-TrkB monoclonal antibody were injected intraperitoneally at 9-10 am on Monday and Thursday every week, at a dose of 5 mg / kg, lasting for 8 weeks. When injected intraperitoneally, the mice were in a head-low-foot-high posture, and the needle was inserted into the abdomen at an angle of 30° to ensure that the drug was injected into the abdominal cavity.

[0101] 2. Monitoring indicators and methods

[0102] (1) Weekly body weight and food intake monitoring: at a fixed time every week, the body weight of the mice was weighed using an electronic balance, the feed consumption of the mice in each cage in the previous week was recorded, and the average daily food intake of each mouse was calculated.

[0103] (2) Fasting blood glucose detection: at the 8th week of the experiment, the mice were fasted for 12 h (without water restriction), and the fasting blood glucose was detected using a blood glucose meter by tail tip blood sampling method, 2-3 μL of blood was collected from each mouse for detection.

[0104] (3) Sample collection and processing: at the end of the 8th week of the experiment, the mice were again fasted for 12 h, and blood was collected from the eyeball, collected in an anticoagulant tube, centrifuged at 4°C, 3000 rpm for 15 min, the serum was separated and stored in a -80°C refrigerator for testing. After blood collection, the mice were quickly sacrificed, the hypothalamus tissue was separated on ice, weighed and placed in a cryopreservation tube, then transferred to a -80°C refrigerator after quick freezing in liquid nitrogen for storage, for subsequent Western blot detection.

[0105] 3. Index detection method

[0106] (1) Serum BDNF detection: According to the BDNF detection kit instructions, the serum BDNF concentration was calculated according to the standard curve.

[0107] (2) Hypothalamic tissue p-TrkB / TrkB ratio detection:

[0108] Protein extraction: The hypothalamic tissue was taken out, and an appropriate amount of RIPA lysis buffer was added for homogenate lysis on ice. Centrifugation at 12000 rpm for 15 min at 4°C, and the supernatant was taken. The protein concentration was determined using a BCA protein quantification kit.

[0109] Western blot detection: The protein sample was subjected to SDS-PAGE electrophoresis separation, and then transferred to a PVDF membrane. The PVDF membrane was blocked with 5% skim milk for 1 h, and TrkB and p-TrkB primary antibodies (diluted 1:1000) were added, respectively, and incubated at 4°C overnight. The next day, the membrane was washed with TBST for 3 times, 10 min each time, and the corresponding secondary antibody (diluted 1:5000) was added, and incubated at room temperature for 1 h. The membrane was washed with TBST for 3 times, 10 min each time, and finally developed using a chemiluminescence imaging system. The band gray value was analyzed by ImageJ software, and the p-TrkB / TrkB ratio was calculated.

[0110] III. Experimental results are shown in Table 3.

[0111] 1. Body weight and food intake analysis: The body weight gain of group 2 mice was significantly higher than that of group 1 (P<0.01), indicating that high-fat diet successfully induced obesity (i.e. central obesity) in mice. The body weight gain of groups 3 and 4 was significantly lower than that of group 2 (P<0.05 and P<0.01), and the body weight gain of the combined treatment group was decreased by 68% compared with group 2, indicating that BDNF fusion polypeptide and its combination with anti-TrkB monoclonal antibody can effectively inhibit the increase of body weight caused by central obesity, and the combined treatment has better effect. In terms of food intake, the average daily food intake of groups 3 and 4 was also significantly lower than that of group 2 (P<0.05 and P<0.01), indicating that the two intervention methods may reduce energy intake by regulating appetite, thereby controlling body weight.

[0112] 2. Fasting blood glucose analysis: The fasting blood glucose level of group 2 mice was significantly higher than that of group 1 (P<0.01), reflecting the abnormal glucose metabolism in central obesity mice. The fasting blood glucose levels of groups 3 and 4 were significantly lower than that of group 2 (P<0.05 and P<0.01), and the blood glucose level of the combined treatment group was close to that of group 1, indicating that BDNF fusion polypeptide and its combination with anti-TrkB monoclonal antibody treatment can help improve the glucose metabolism disorder induced by central obesity, and the combined treatment has better effect in regulating blood glucose.

[0113] 3. Serum BDNF level analysis: The serum BDNF level of group 2 was significantly lower than that of group 1 (P < 0.01), which was consistent with the results of previous studies that the BDNF level of obese people or animal models was decreased. The serum BDNF levels of group 3 and group 4 were significantly higher than that of group 2 (P < 0.05 and P < 0.01), and the serum BDNF level of the combined treatment group was close to that of group 1, indicating that the BDNF fusion polypeptide and the combined anti-TrkB monoclonal antibody treatment could effectively improve the serum BDNF level, and the combined treatment had a more significant effect on the recovery of the BDNF level.

[0114] 4. p-TrkB / TrkB ratio analysis: The p-TrkB / TrkB ratio of hypothalamic tissue of group 2 was significantly lower than that of group 1 (P < 0.01), indicating that central obesity inhibited the activation of the TrkB signaling pathway. The p-TrkB / TrkB ratios of group 3 and group 4 were significantly higher than that of group 2 (P < 0.05 and P < 0.01), and the p-TrkB / TrkB ratio of the combined treatment group was significantly higher than that of group 1 (P < 0.05), indicating that the BDNF fusion polypeptide and the combined anti-TrkB monoclonal antibody treatment could activate the TrkB signaling pathway, and the combined treatment had a stronger effect on the activation of the TrkB pathway, which might be an important mechanism for the better effect of the combined treatment on improving body weight, blood glucose and other indicators.

[0115] Table 3 summarizes the experimental results

[0116]

[0117] (*P < 0.05 vs. group 2; **P < 0.01 vs. group 2)

[0118] In summary, the combined use of the BDNF fusion polypeptide and the anti-TrkB monoclonal antibody has a significant synergistic effect on improving the body weight, blood glucose metabolism and activating the TrkB signaling pathway of obese mice induced by central obesity, and provides a strong experimental basis for the treatment of central obesity.

[0119] The above embodiments are the preferred embodiments of the present application, but the embodiments of the present application are not limited to the above embodiments, and any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present application shall be equivalent replacement modes, and all shall be included in the protection scope of the present application.

Claims

1. A composition for the treatment of central obesity, characterized in that, The composition comprises an effective amount of a BDNF fusion polypeptide and an effective amount of an anti-TrkB monoclonal antibody; The BDNF fusion polypeptide consists of a TAT transmembrane peptide, a flexible linker peptide and a BDNF mature peptide in sequence, and the specific amino acid sequence is shown as SEQ ID NO.

1. The amino acid sequence of the heavy chain variable region of the anti-TrkB monoclonal antibody is shown as SEQ ID NO. 2, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO.

3.

2. The composition of claim 1, wherein, The dose of the BDNF fusion polypeptide and the anti-TrkB monoclonal antibody is 5 mg / kg.

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