A fusion protein ngf2 with improved half-life in vivo and its use

Abstract

The application discloses a fusion protein NGF2 with improved in-vivo half-life and application thereof, and belongs to the technical field of medical biological engineering, and comprises ABD and FGF2, and the gene and protein sequence thereof are respectively composed of the sequences shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:6 and SEQ ID NO:7, and the complete gene and protein sequence thereof is shown in SEQ ID NO:4 and SEQ ID NO:8. The fusion protein NGF2 can keep activity for more than 7 days in an environment of 37 DEG C, and the half-life of the fusion protein in solution is prolonged. In addition, the ABD is combined with HAS to form a complex, and the total molecular weight reaches 90kDa, which is higher than the cut-off molecular weight of glomerular filtration, so that the in-vivo half-life of the fusion protein is effectively prolonged.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical bioengineering technology and relates to a fusion protein NGF2 that increases the in vivo half-life and its applications. Background Technology

[0002] Human fibroblast growth factor 2 (FGF2) is an important signaling protein that regulates cell metabolism, proliferation, differentiation, and survival. FGF2 is considered an important component for constructing functional tissue engineering. Since the successful synthesis of human recombinant FGF2 in 1988 (GMFox, SGSchiffer, MFRohde, LBTsai, ARBanks, T.Arakawa, Production, biological activity, and structure of recombinant basic fibroblast growth factor and an analog with cysteine ​​replaced by serine, J Biol Chem 263(34)(1988)18452-8.), this protein has played a key role in cell proliferation and angiogenesis in various tissues, including muscle, blood vessels, teeth, skin, nerves, cartilage, bone, and tendons (A.Beenken, M.Mohammadi, The FGF family: biology, pathophysiology and therapy, Nat Rev Drug Discov 8(3)(2009)235-53.). Therefore, FGF2 is considered a highly valuable therapeutic protein.

[0003] However, the half-life of natural FGF2 in solution is less than 1 day (I. Ding, AM Peterson, Half-life modeling of basic fibroblast growth factor released from growth factor-eluting polyelectrolyte multilayers, Sci Rep 11(1)(2021)9808. T. Shiba, D. Nishimura, Y. Kawazoe, Y. Onodera, K. Tsutsumi, R. Nakamura, M. Ohshiro, Modulation ofmitogenic activity of fibroblast growth factors by inorganic polyphosphate, JBiol Chem 278(29)(2003)26788-92.), which limits its medical applications in vitro and in vivo.Furthermore, since the molecular weight of FGF2 is 18 kDa (S. Liao, J. Bodmer, D. Pietras, M. Azhar, T. Doetschman, J. Schultz Jel, Biological functions of the low and high molecular weight protein isoforms of fibroblast growth factor-2 in cardiovascular development and disease, Dev Dyn 238(2)(2009)249-64.), which is much lower than the molecular weight cutoff of glomerular filtration (30-50 kDa) (A. Ruggiero, CH Villa, E. Bander, DA Rey, M. Bergkvist, CA Batt, K. Manova-Todorova, WMDeen, DAScheinberg, MRMcDevitt, Paradoxical glomerular filtration of carbon nanotubes, Proc Natl Acad Sci US A107(27)(2010)12369-74.), its half-life in the human body is only 50 minutes (B.Liebman,C.Schwaegler,ATFoote,KSRao,T.Marquis,A.Aronshtam,SPBell,P.Gogo,RRLaChapelle,JLSpees,Human growthfactor / immunoglobulin complexes for treatment of myocardial ischemia-reperfusion injury,Front Bioeng Biotechnol 10(2022)749787.).These factors significantly limit the therapeutic efficacy of FGF2 in treating liver fibrosis (M. Sato-Matsubara, T. Matsubara, A. Daikoku, Y. Okina, L. Longato, K. Rombouts, L. T. Huy, J. Adachi, T. Tomonaga, K. Ikeda, K. Yoshizato, M. Pinzani, N. Kawada, Fibroblast growth factor 2 (FGF2) regulates cytoglobin expression and activation of human hepatic stellate cells via JNK signaling, JBiol Chem 292(46)(2017)18961-18972.) and renal fibrosis (X. Tan, Q. Tao, S. Yin, G. Fu, C. Wang, F. Xiang, H. Hu, S. Zhang, Z. Wang, D. Li, Single administration of FGF2 after renal ischemia-reperfusion injury alleviates Potential applications in diseases such as post-injury interstitial fibrosis and Nephrol Dial Transplant 38(11)(2023)2537-2549.

[0004] Given the aforementioned issues, the development of technologies that can extend the half-life of FGF2 is of particular importance. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention discloses a fusion protein NGF2 that improves the in vivo half-life, effectively prolonging the half-life of human fibroblast growth factor 2 (FGF2) in solution and in vivo. At the same time, the fusion protein NGF2 can further improve the stability and activity of FGF2.

[0006] This invention discloses a fusion protein NGF2 with enhanced in vivo half-life, comprising an albumin-binding domain (ABD) and human fibroblast growth factor 2 (FGF2). The FGF2 is either wild-type FGF2 or modified human fibroblast growth factor 2 (FGF2-STAB) (Z. Kadlecova, V. Sevriugina, K. Lysakova, M. Rychetsky, I. Chamradova, L. Vojtova, Liposomes affect protein release and stability of ITA-modified PLGA-PEG-PLGA hydrogel carriers for controlled drug delivery, Biomacromolecules 25(1)(2024)67-76.).

[0007] Furthermore, the fusion protein NGF2 disclosed in this invention further includes a peptide linker connecting the C-terminus of the ABD peptide and the N-terminus of the FGF2 peptide. The peptide linker is a soft-chain polypeptide having 25 amino acids.

[0008] Furthermore, the fusion protein NGF2 disclosed in this invention comprises the gene and protein sequences shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7, respectively. The complete gene and protein sequences of the fusion protein NGF2 are shown in SEQ ID NO:4 and SEQ ID NO:8.

[0009] Compared to natural FGF2, the fusion protein NGF2 of this invention maintains its activity for over 7 days at 37°C. The molecular weight of the fusion protein NGF2 is 23 kDa. Through the binding of ABD with human serum albumin (HSA) to form a complex, the total molecular weight reaches 90 kDa, far exceeding the molecular weight cutoff for glomerular filtration, thus effectively prolonging its half-life in vivo. Based on this, the fusion protein NGF2 of this invention can be applied to cell proliferation and angiogenesis in various tissues to prepare drugs that promote tissue repair and regeneration, such as those in muscles, blood vessels, teeth, skin, nerves, cartilage, bones, and tendons.

[0010] The fusion protein NGF2 of this invention can also be used to prepare drugs for treating liver fibrosis and kidney fibrosis.

[0011] The beneficial effects of this invention are:

[0012] The fusion protein NGF2 of this invention solves two major technical problems of FGF2. Compared with natural FGF2, NGF2 can maintain its activity for more than 7 days at 37°C, thus significantly extending its half-life in solution. On the other hand, NGF2 has a molecular weight of 23 kDa, and through the binding of ABD and HAS (67 kDa) to form a complex, the total molecular weight reaches 90 kDa, which is much larger than the molecular weight cutoff for glomerular filtration, thereby effectively extending its half-life in vivo.

[0013] The fusion protein NGF2 of this invention is expected to significantly enhance the application potential of FGF2 in the treatment of diseases such as liver fibrosis and kidney fibrosis, providing stronger support for clinical treatment.

[0014] The fusion protein NGF2 of this invention not only solves the problem of the short half-life of FGF2, but also has certain advantages in reducing immunogenicity, improving stability, enhancing targeting, and reducing production costs. These advantages make it promising for applications in the biomedical field, and can provide more effective and safer solutions for the treatment of related diseases. Attached Figure Description

[0015] Figure 1 This invention provides an analysis of NGF2 expression and purification using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Lane 1: Label; Lane 2: Total lysate; Lane 3: Soluble fraction; Lane 4: Passage through Q-column; Lane 5: 100% elution from Q-column; Lane 6: Passage through SP-column; Lane 7: 30% elution from SP-column; Lane 8: NGF2 from 60% elution from SP-column.

[0016] Figure 2 This invention employs non-denaturing polyacrylamide gel electrophoresis (NAGE) to describe the mixing of a fixed molar amount of NGF2 with different molar ratios of human serum albumin (HAS). Specifically, lane 1 has a NGF2 to HSA molar ratio of 1:0.25; lane 2 has a NGF2 to HSA molar ratio of 1:0.5; lane 3 has a NGF2 to HSA molar ratio of 1:0.75; lane 4 has a NGF2 to HSA molar ratio of 1:1; lane 5 has a NGF2 to HSA molar ratio of 1:1.5; lane 6 has a NGF2 to HSA molar ratio of 1:2; lane 7 has a NGF2 to HSA molar ratio of 0:0.25; lane 8 has a NGF2 to HSA molar ratio of 0:2; and lane 9 has a NGF2 to HSA molar ratio of 1:0.

[0017] Figure 3The diagram shows the predicted NGF2-HSA complex structure using ColabFold v1.5.5:AlphaFold2 in this invention.

[0018] Figure 4 The effects of different concentrations of NGF2 on the proliferation of NIH-3T3 cells in this invention are shown.

[0019] Figure 5 This is an SDS-PAGE analysis of NGF2 in this invention after incubation in PBS solution at 4°C and 37°C for 7 days.

[0020] Figure 6 This is the experimental result of NGF2 binding to rat serum albumin in this invention. Detailed Implementation

[0021] All reagents and instruments used in this embodiment are available from commercial sources, and the methods used, unless otherwise specified, are consistent with conventional laboratory methods.

[0022] Example 1: Expression and purification of NGF2

[0023] In Example 1, the fusion protein NGF2 consists of an albumin-binding domain (ABD), human fibroblast growth factor 2 (FGF2), and a peptide linker connecting the C-terminus of the ABD polypeptide and the N-terminus of the FGF2 polypeptide. The peptide linker is a soft-chain polypeptide with 25 amino acids. The gene and protein sequences of this fusion protein NGF2 are represented by the sequences shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7, respectively. The complete gene and protein sequences are shown in SEQ ID NO:4 and SEQ ID NO:8. SEQ ID NO:5 is the protein structure sequence of ABD, SEQ ID NO:6 is the soft-chain polypeptide, and SEQ ID NO:7 is the protein structure sequence of FGF2-STAB, obtained by Enantis, Ltd (Czech Republic). In this invention, BioArrow Technology Limited was commissioned to construct a DNA plasmid containing the NGF2 gene.

[0024] First, the NGF2 gene from the pET29b(+) vector was transformed into BL21(DE3) for expression. A single colony was picked and inoculated into sterile LB medium containing 50 μg / ml kanamycin, and cultured overnight at 30°C with a shaking speed of 250 rpm. The overnight culture was then inoculated 1:100 into 200 mL of sterile TB medium, and 50 μg / ml kanamycin was added. The culture was then grown at 30°C and 250 rpm. The optical density (OD) of the culture was measured... 600 When the protein expression reaches 0.6-0.8, 0.5 mM of isopropyl-β-D-thiogalactoside (IPTG) is added to induce protein expression.

[0025] Cell pellets were collected by centrifugation, and *E. coli* cells were lysed using an ultrasonic homogenizer (Lichen ultrasonic cell disruptor) in suspension buffer (20 mM Tris buffer, 100 mM NaCl, pH 7.0). Cell debris and soluble protein NGF2 were then separated using a high-speed centrifuge (Beckman Coulter Avanti JXN-26). The NGF2 supernatant was then preliminarily purified using a HiTrapQ HP strong anion exchange chromatography column (GE Healthcare), and the eluent was collected. Finally, the eluent was further purified using a HiTrap SP HP strong cation exchange chromatography column (GE Healthcare). NGF2 was eluted in 60% elution buffer (20 mM Tris buffer, 1 M NaCl, pH 7.0), and the protein purity and expression were analyzed by SDS-PAGE.

[0026] Combined with SDS-PAGE plot ( Figure 1 Following the chromatographic purification steps, it was confirmed that the NGF2 protein had been successfully expressed in bacteria and effectively purified using ion exchange chromatography. The final product (lane 8) showed a single, clear protein band with a molecular weight of 23 kDa for NGF2, indicating high purity and suitability for further biomedical applications.

[0027] Example 2: Determination of NGF2 binding to HSA

[0028] Example 2 analyzed the binding between NGF2 and human serum albumin (HSA). A fixed amount of NGF2 (2 μg) was mixed with HSA at different molar ratios and incubated at 37°C for 10 min. Subsequently, the mixture was analyzed by non-denaturing polyacrylamide gel electrophoresis to evaluate the binding of NGF2 to HSA.

[0029] like Figure 2The results showed that NGF2 can bind to human serum albumin (HSA), forming a complex with a higher molecular weight. The experimental results indicated that 1 mole of NGF2 can completely bind to a maximum of 0.5 moles of HSA. This demonstrates that the fusion of the albumin-binding domain (ABD) with human fibroblast growth factor 2 (FGF2) does not affect the binding ability between ABD and HSA.

[0030] Figure 3 The study demonstrated that ColabFold v1.5.5:AlphaFold2 predicted the formation of a large complex structure with a molecular weight of 90 kDa by the binding of NGF2 (23 kDa) and HAS (67 kDa), which is much larger than the molecular weight cutoff of glomerular filtration (30-50 kDa). Figure 3 In the NGF2 protein sequence, the red segment represents ABD (sequence as shown in SEQ ID NO:5), the blue segment represents the soft chain polypeptide (sequence as shown in SEQ ID NO:6), the yellow segment represents FGF2-STAB (sequence as shown in SEQ ID NO:7), and the green segment represents HAS (protein sequence as shown in SEQ ID NO:9).

[0031] Example 3: Assay of NGF2-induced NIH-3T3 cell proliferation

[0032] NIH-3T3 cells were seeded into 96-well plates at a density of 2000 cells per well and cultured in complete DMEM medium (DMEM 31966). The cells were cultured overnight at 37°C in newborn calf serum. After 24 hours, the cell culture medium was replaced with DMEM medium containing a low concentration of serum (DMEM 31966). Newborn calf serum was used. Next, NIH-3T3 cells were starved for 24 hours, and then different concentrations of NGF2 (0, 0.0625, 0.125, 0.25, 0.5, 1, 2, 4, 8, 16 ng / μl) were added to each well, and the cells were cultured for another 72 hours. Then, 10 μL of 5 mg / ml 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide reagent (MTT reagent) was added to each well, and the cells were cultured at 37°C for 4 hours. After culture, the solution in each well was completely removed, and 100 μL of dimethyl sulfoxide (DMSO) was added. After the formazan crystals were completely dissolved, the absorbance was measured at 540 nm using a Thermo Multiskan FC microdispersive analyzer. Finally, cell proliferation was calculated using Prism software. This experiment was repeated three times to ensure the reliability of the results.

[0033] Figure 4This study demonstrated the significant promoting effect of NGF2 on the proliferation of NIH-3T3 cells at different concentrations. In the presence of the albumin-binding domain (ABD), all NGF2 treatment groups showed a high percentage change in cell proliferation compared to the control group (0 ng / μl), particularly starting from a concentration of 0.25 ng / μl, which significantly induced the proliferation of NIH-3T3 cells. This confirms the proliferative effect of NGF2. The fact that NGF2 could still significantly induce the proliferation of NIH-3T3 cells in the presence of the albumin-binding domain (ABD) indicates its significant cell repair capacity. Compared to the control group, NGF2 could increase cell viability by a maximum of 180%. These results suggest that NGF2 has potential applications in promoting cell proliferation and repair, particularly in the fields of tissue engineering and regenerative medicine.

[0034] Example 4: Stability test of NGF2 after incubation in PBS solution at 4°C and 37°C for 7 days

[0035] A fixed amount of NGF2 (2 μg) was dissolved in PBS solution and incubated at 4 °C and 37 °C. On day 0 of the experiment, NGF2 samples were collected and SDS-loading dye was added as a control. After 7 days of incubation, the same amount of NGF2 was collected at 4 °C and 37 °C, respectively, and analyzed by SDS-PAGE.

[0036] Figure 5 The SDS-PAGE analysis of NGF2 after incubation in PBS solution at 4°C and 37°C for 7 days is shown. Figure 5 It can be seen that NGF2 is extremely stable, and no protein degradation occurred after incubation at 4℃ and 37℃ for 7 days.

[0037] Example 5: Performance of NGF2 and natural FGF2 in rats

[0038] Normal Sprague-Dawley rats were randomly divided into an NGF2 group and an FGF2 group, with two rats in each group. They were intravenously injected with 300 μg / kg of NGF2 and wild-type FGF2, respectively, and blood samples were collected at specified time points (5, 180, and 240 minutes). The protein concentration at 5 minutes was used as the peak value to calculate the remaining percentage at 180 and 240 minutes. A human FGF2 ELISA kit was used. Quantitatively measure the protein level in serum.

[0039] Figure 6The results of the binding experiment of NGF2 to rat serum albumin were presented, and this binding prolonged the half-life of NGF2 in rats. The percentage of protein remaining after 180 and 240 minutes showed that the residual amount of NGF2 was significantly higher than that of native FGF2.

Claims

1. A fusion protein NGF2 with an increased in vivo half-life, characterized in that, It includes an albumin-binding domain and human fibroblast growth factor 2.

2. The fusion protein NGF2 with increased in vivo half-life according to claim 1, characterized in that, The human fibroblast growth factor 2 is wild-type FGF2 or modified human fibroblast growth factor 2, and the protein sequence of the modified human fibroblast growth factor 2 is shown in SEQ ID NO:

7.

3. The fusion protein NGF2 with increased in vivo half-life according to claim 1, characterized in that, The fusion protein NGF2 also includes a peptide linker that connects the C-terminus of the albumin-binding domain peptide to the N-terminus of the human fibroblast growth factor 2 peptide, wherein the peptide linker is a soft chain peptide having 25 amino acids.

4. The fusion protein NGF2 with increased in vivo half-life according to claim 1, characterized in that, The complete gene and protein sequences of the fusion protein NGF2 are composed of the sequences shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:6 and SEQ ID NO:7, respectively.

5. The fusion protein NGF2 with increased in vivo half-life according to claim 4, characterized in that, The complete gene and protein sequences of the fusion protein NGF2 are shown in SEQ ID NO:4 and SEQ ID NO:

8.

6. The fusion protein NGF2 with increased in vivo half-life according to claim 1, characterized in that, The molecular weight of the fusion protein NGF2 is 23 kDa.

7. The fusion protein NGF2 with increased in vivo half-life according to claim 1, characterized in that, The fusion protein NGF2 binds to human serum albumin through its albumin-binding domain to form a complex with a total molecular weight of 90 kDa.

8. The use of the fusion protein NGF2 with an increased in vivo half-life as described in any one of claims 1-7 in the preparation of a drug that promotes tissue repair and regeneration.

9. The use of the fusion protein NGF2 with increased in vivo half-life as described in any one of claims 1-7 in the preparation of drugs for treating liver fibrosis and kidney fibrosis.

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