Novel long-acting recombinant human growth hormone fusion protein and application thereof

Abstract

The invention belongs to the technical field of biological medicines, and discloses a novel long-acting recombinant human growth hormone fusion protein. The fusion protein is obtained by fusion expression of human growth hormone (hGH) and an anti-human albumin nano antibody (VHH). And the long-acting principle is that the clearance rate is reduced by reversibly and widely combining with albumin with endogenous circulation. The fusion protein has the characteristics that 1, the fusion protein is obtained by fusion expression of two functional domains of hGH and VHH, hGH sequence mutation and complex chemical modification are not involved, the preparation method is simpler, and the sample uniformity is better; 2, in the fusion protein, the N end of the hGH protein has no additional structure, and the VHH functional domain is connected to the C end of the hGH, so that the influence on the activity of the hGH is reduced; and 3, animal efficacy results show that the fusion protein has a long-acting effect.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a novel long-acting recombinant human growth hormone fusion protein. Background Technology

[0002] Growth hormone (GH) is a protein hormone secreted by the anterior pituitary gland. Composed of 191 amino acids with a molecular weight of approximately 22 kDa, it is a key hormone regulating human growth, development, and metabolism. In regulating growth and development, GH precisely targets chondrocytes in the epiphyses of long bones, promoting proliferation, differentiation, and bone matrix deposition, thus driving longitudinal bone growth. Simultaneously, it also promotes the growth of muscles and internal organs (such as the heart and kidneys) by increasing the number and volume of muscle fibers and stimulating the proliferation of visceral cells, ensuring that the body's various systems develop synchronously with bone growth. In regulating metabolism, GH activates amino acid transporters, promoting cellular amino acid uptake and accelerating protein synthesis by enhancing ribosome activity. It also activates lipases, promoting the breakdown of adipocytes and releasing free fatty acids for energy. GH also participates in glucose metabolism regulation by improving the efficiency of glucose utilization in muscles and modulating the fat-glucose metabolism linkage. Therefore, GH is an important factor connecting growth, metabolism, and health.

[0003] Certain congenital causes (such as gene defects, pituitary developmental abnormalities) or intracranial tumors, infections, and trauma can lead to insufficient synthesis and secretion of GH, a condition known as growth hormone deficiency (GHD). Clinical manifestations include delayed growth and development, abnormal metabolic function, fatigue, and poor mental state. The incidence of GHD varies across different studies and regions, with a global incidence of approximately 1 / 4000-1 / 10000 in children. In the general adult population, the incidence is approximately 1 / 10000-1 / 40000, while in patients with pituitary diseases, the incidence can be as high as 15%-20%. The primary treatment for GHD is growth hormone replacement therapy. Recombinant human growth hormone (rhGH) is injected to promote growth and development in children, significantly increasing their final height; and to improve metabolism and quality of life in adults. Recombinant human growth hormone preparations include short-acting and long-acting formulations. Short-acting formulations are prepared using genetic engineering techniques to create recombinant human growth hormone with the same sequence as the natural protein for human application. Short-acting formulations are highly active, well-established, and have a large market share, currently holding a dominant position. However, their short half-life necessitates daily injections, posing a significant challenge, especially for long-term use, particularly in children. Long-acting formulations extend the half-life of short-acting formulations through chemical modification and carrier protein fusion expression, thereby increasing dosing intervals. For example, Jinsai Pharmaceutical's Jinsai Zeng and Tebo Bio's Yipeisheng both use PEG-modified rhGH to reduce glomerular clearance, enabling weekly dosing; Novo Nordisk's Parcy growth hormone uses fatty acid modification to enhance binding to plasma albumin, thus extending its half-life; Ascendis Pharma's Longpei growth hormone uses a unique TransCon temporary linking technology to temporarily link carrier molecules to growth hormone proteins, forming an inactive and non-cleared prodrug, achieving slow release of the active protein in vivo for a long-acting effect. Although long-acting formulations offer significant advantages in terms of administration convenience, they also have lower protein activity, require larger clinical dosages, and have higher production costs, increasing the burden on patients. Meanwhile, all existing long-acting formulations are administered weekly, and there is still room for further reduction in the frequency of administration and clinical demand. Summary of the Invention

[0004] To address the technical problems of the prior art, the present invention aims to provide a novel long-acting recombinant human growth hormone fusion protein, which is expressed by fusing human growth hormone (hGH) with an anti-human albumin nanobody (VHH). The principle of long-acting efficacy is to reduce clearance by reversibly and extensively binding to endogenous circulating albumin. The characteristics of this fusion protein are: 1. It fuses the expression of both hGH and VHH functional domains, without involving mutations in the hGH sequence or complex chemical modifications, resulting in a simpler preparation method and better sample uniformity; 2. In the fusion protein, the N-terminus of the hGH protein has no additional structure, and the VHH functional domain is linked to the C-terminus of hGH, reducing the impact on hGH activity; 3. Animal efficacy results show that this fusion protein has a long-acting effect and has the potential for bi-weekly administration in humans.

[0005] To achieve the above objectives, the present invention adopts the following technical solution: A novel long-acting recombinant human growth hormone fusion protein, wherein the fusion protein is expressed by fusing human growth hormone with an anti-human albumin nanobody.

[0006] Preferably, the fusion protein consists of, from N-terminus to C-terminus, a growth hormone protein sequence, a linker peptide, and an anti-human albumin nanobody sequence; its amino acid sequence comprises 315 amino acids, of which positions 1-191 are the growth hormone protein sequence, positions 192-200 are the linker peptide sequence, and positions 201-315 are the anti-human albumin nanobody sequence.

[0007] Preferably, the amino acid sequence of the fusion protein is shown in SEQ ID NO.1.

[0008] Preferably, the fusion protein has a signal peptide attached to its N-terminus, the nucleotide sequence of which is shown in SEQ ID NO.5; The amino acid and nucleotide sequences of the signal peptide are shown in SEQ ID NO.3-4.

[0009] The aforementioned fusion protein may or may not contain a linker peptide between its human growth hormone domain and its anti-human albumin nanobody domain.

[0010] A nucleic acid molecule encoding a novel long-acting recombinant human growth hormone fusion protein, the nucleotide sequence of which is shown in SEQ ID NO.2.

[0011] A novel long-acting recombinant human growth hormone fusion protein expression vector, wherein the expression vector comprises the aforementioned nucleic acid molecule.

[0012] Application of a novel long-acting recombinant human growth hormone fusion protein in the preparation of drugs for the treatment and / or prevention of growth hormone-related diseases.

[0013] Preferably, the purity of the novel long-acting recombinant human growth hormone fusion protein is greater than 95%.

[0014] A pharmaceutical composition comprising the fusion protein or the nucleic acid molecule described herein.

[0015] Preferably, the pharmaceutical composition further includes a pharmaceutically acceptable carrier.

[0016] Compared with the prior art, the present invention has at least the following technical effects: This invention provides a novel long-acting recombinant human growth hormone fusion protein, which is expressed by fusing human growth hormone (hGH) with an anti-human albumin nanobody (VHH).

[0017] This fusion protein has a long-lasting effect, which is achieved by reversibly and extensively binding to endogenous circulating albumin to reduce clearance.

[0018] The characteristics of this fusion protein are as follows: 1. It integrates the expression of two functional domains, hGH and VHH, without involving mutations in the hGH sequence or complex chemical modifications, making the preparation method simpler and the sample homogeneity better; 2. In the fusion protein, the N-terminus of the hGH protein has no additional structure, and the VHH functional domain is linked to the C-terminus of hGH, reducing the impact on hGH activity; 3. Animal efficacy results show that this fusion protein has a long-lasting effect and has the potential to be administered to humans every two weeks. Attached Figure Description

[0019] Figure 1 Chromatogram of hGH-VHH purification and SDS-PAGE electrophoresis detection; Figure 2 The binding curves of hGH-VHH and hGHR-Fc are shown. Figure 3 The binding curve of hGH-VHH to albumin; Figure 4 hGH-VHH promotes the proliferation of Nb2-11 cells; Figure 5 This represents the blood drug concentration in mice after subcutaneous administration. Figure 6 Changes in body weight after administration in a pituitary deprivation rat model. Detailed Implementation

[0020] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the present invention will be briefly introduced below in conjunction with the accompanying drawings and descriptions of the embodiments or the prior art. Obviously, the following description of the structure of the accompanying drawings is only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort. It should be noted that the description of these embodiments is for the purpose of helping to understand the present invention, but does not constitute a limitation of the present invention.

[0021] Example 1. Construction of Sequence and Expression Strains A novel long-acting recombinant human growth hormone fusion protein, namely hGH-VHH protein, has the amino acid sequence shown in SEQ ID NO.1, where 1-191 is the hGH protein sequence, 192-200 is the linker, and 201-315 is the VHH antibody sequence against human albumin.

[0022] The encoding DNA sequence of SEQ ID NO.1 is shown in SEQ ID NO.2.

[0023] SEQ ID NO.3-4 are the amino acid and DNA sequences of the pelB signal peptide secreted and expressed by Escherichia coli.

[0024] The signal peptide sequence was designed into the N-terminus of the hGH-VHH protein sequence, and the DNA sequence is shown in SEQ ID NO.5.

[0025] SEQ ID NO.5 was synthesized by Cysofa Biotechnology Co., Ltd. and seamlessly cloned into the multiple cloning site of the pET30a vector. The recombinant plasmid was transformed into Escherichia coli BL21(DE3).

[0026] Example 2. Expression and purification of hGH-VHH protein The expression strain was inoculated into a 5L fermenter containing 3L LB medium for fermentation. When the OD600 of the bacterial culture reached 20, IPTG was added to a final concentration of 0.1mM and induced for 6-8 hours. After induction, the fermentation supernatant was collected by centrifugation.

[0027] The supernatant was purified using 10 ml of Protein A (BorgLone Biotechnology Co., Ltd.) packing material. The column was washed with 20 mM MPB, 150 mM NaCl, pH 7.4 equilibration buffer before loading the sample. After loading, the column was washed with equilibration buffer for 5 column volumes, followed by elution with 0.1 M glycine at pH 3.5. The eluent was purified using an SP Bestarose FF column (BorgLone Biotechnology Co., Ltd.) with equilibration buffer of 10 mM PB, pH 4.0 and elution buffer of 10 mM PB, 1 M NaCl, pH 4.0. After loading, the column was washed with equilibration buffer for 2-3 column volumes, followed by 0%-100% linear elution, and the elution peak was collected. 2 M NaCl was added to the eluent from the SP Bestarose FF chromatography, and the column was further purified using a Butyl Bestarose FF column (BorgLone Biotechnology Co., Ltd.). The equilibration buffer was 10 mM PB, 2 M NaCl, pH 7.0, and the elution buffer was 10 mM PB, pH 7.0. After loading the sample, wash with equilibration buffer for 2-3 column volumes, then wash with 80% elution buffer for 3 column volumes, and finally elute with 100% elution buffer and collect the elution peak.

[0028] like Figure 1 The image shows the chromatogram of hGH-VHH purification and the SDS-PAGE electrophoresis detection. Lane M represents the protein molecular weight marker; lane 1 is the elution of Protein A (loaded into SP Bestarose FF chromatography); lane 2 is the flow-through of SP Bestarose FF chromatography; lanes 3-11 are the collection tubes of SP Bestarose FF elution; lane 12 is the loading of Butyl Bestarose FF chromatography; lane 13 is the flow-through of Butyl Bestarose FF chromatography; and lane 14 is the elution of Butyl Bestarose FF chromatography.

[0029] Figure 1 The results showed that the purity of hGH-VHH obtained through purification was greater than 95%.

[0030] Example 3. Binding of hGH-VHH to human growth hormone receptor Add 3 μg / ml human growth hormone receptor-Fc fusion protein (hGHR-Fc, Yiqiao Shenzhou Biotechnology Co., Ltd.) at a rate of 100 μl / well to a 96-well plate and block and coat overnight. The next day, wash the plate three times with PBST. Add serially diluted recombinant human GH (hGH), PEG-modified human GH (hGH-PEG), and purified hGH-VHH to the 96-well plate and incubate overnight at 37°C. Wash the plate three times with PBST. Add HRP-labeled anti-hGH monoclonal antibody (Yiqiao Shenzhou Biotechnology Co., Ltd.) and incubate at 37°C for 1 h. After washing, perform color development according to the instructions of the HRP colorimetric kit (Solepro Biotechnology Co., Ltd.) and read the plate, then plot the curve.

[0031] like Figure 2 The figure shows the binding curves of hGH-VHH and hGHR-Fc.

[0032] Results combined Figure 2 The EC50 values ​​were calculated as follows: hGH = 0.06 μg / ml, hGH-PEG = 1.5 μg / ml, and hGH-VHH = 0.12 μg / ml. The EC50 value of hGH-VHH is twice that of hGH and 1 / 12.5 of that of hGH-PEG.

[0033] Example 4. Binding of hGH-VHH to albumin Add 3 μg / ml human albumin and rat albumin (both purchased from Huamei Biotechnology Co., Ltd.) to 96-well plates at a rate of 100 μl / well, and block and coat overnight. The next day, wash the plates three times with PBST, add serially diluted hGH-VHH to the 96-well plates, incubate at 37°C for 3 h, wash three more times with PBST, add HRP-labeled anti-hGH monoclonal antibody (Yiqiao Shenzhou Biotechnology Co., Ltd.), incubate at 37°C for 1 h, wash the plates, and perform color development according to the instructions of the HRP colorimetric kit (Solepro Biotechnology Co., Ltd.). Plot the curves.

[0034] like Figure 3 The figure shows the binding curve of hGH-VHH to albumin.

[0035] Results combined Figure 3 The results showed that hGH-VHH did not exhibit significant differences in binding to human albumin and rat albumin.

[0036] Example 5: hGH-VHH promotes the proliferation of Nb2-11 cells Nb2-11 cells in logarithmic growth phase were washed twice with PBS and the cell density was adjusted to 1.0 × 10⁶ cells by adding basal culture medium (RPMI-1640 medium containing 1% bovine serum). 5Cells / ml. Add 50 μl of cells to each well of a 96-well cell culture plate. Serially dilute hGH, hGH-PEG, and purified hGH-VHH protein, and add 50 μl to each well of the cells, with three replicates per sample. Incubate the cells at 37°C in a 5% CO2 incubator for 28 h. Add 20 μl of 5 μg / ml MTT reagent to each well and incubate at 37°C for 4 h. Discard the supernatant, add 150 μl of DMSO to each well, shake for 10 min, and then measure the absorbance at 490 nm and calculate the EC50 concentration.

[0037] like Figure 4 As shown, hGH-VHH promotes the proliferation of Nb2-11 cells.

[0038] Results combined Figure 4 It can be seen that the EC50 concentrations of hGH and hGH-VHH are 3.0 μg / ml and 3.9 μg / ml, respectively, both higher than those of hGH-PEG (EC50 is 16.9 μg / ml).

[0039] Example 6. Pharmacokinetics of hGH-Fc in mice Fifty female BALB / c mice aged 6-8 weeks were randomly divided into two groups. The mice were subcutaneously injected with hGH-PEG (20 μg / mouse) and purified hGH-VHH (20 μg / mouse), respectively. Blood samples were collected from five mice alternately via the orbital sinus at 4, 6, 8, 12, 24, 48, 72, 96, 120, and 144 hours after administration, and serum was separated. Serum drug concentrations were determined using a human growth hormone ELISA kit (purchased from R&D Systems).

[0040] like Figure 5 The figure shows the blood drug concentration in mice after subcutaneous administration.

[0041] Results combined Figure 5 It can be seen that the scavenging half-life of hGH-VHH is 28.5h, which is higher than that of hGH-PEG (19.2h).

[0042] Example 7. Determination of hGH-VHH protein activity by body weight method in pituitary-deprived rats. Female SD rats aged 26–28 days underwent pituitary removal surgery and were housed in a barrier environment for 2 weeks post-surgery. Rats whose body weight changed by ±10% compared to pre-surgery levels were randomly divided into 6 groups of 6 rats each. Each group received the following administration via subcutaneous injection in the neck: 1. hGH-PEG 0.02 mg / rat weekly; 2. hGH-VHH 0.005 mg / rat weekly; 3. hGH-VHH 0.01 mg / rat weekly; 4. hGH-VHH 0.02 mg / rat weekly; 5. hGH-VHH 0.04 mg / rat every two weeks; 6. The same volume of saline was injected weekly. Six other animals underwent surgery without pituitary removal and served as a sham-operated group, receiving weekly saline injections. All animals were weighed weekly after administration to calculate their body weight change compared to pre-administration levels.

[0043] like Figure 6 The diagram shown illustrates the changes in body weight in a pituitary deprivation rat model after drug administration.

[0044] Results combined Figure 6 It can be seen that the rats in each dose group of hGH-VHH showed significant weight gain and a clear dose-response relationship.

[0045] Two-week administration of hGH-VHH at 0.04 mg / rat was comparable to weekly administration of 0.02 mg / rat. The low-dose hGH-VHH group showed better weight gain than the 0.02 mg / rat dose of hGH-PEG.

[0046] Finally, it should be noted that the above description is merely a preferred embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A novel long-acting recombinant human growth hormone fusion protein, characterized in that, The fusion protein is expressed by fusing human growth hormone and anti-human albumin nanobody.

2. The novel long-acting recombinant human growth hormone fusion protein according to claim 1, characterized in that, The fusion protein comprises, from N terminus to C terminus, growth hormone protein sequence, connecting peptide, anti-human albumin nanobody sequence; the amino acid sequence comprises 315 amino acids, wherein the 1-191th amino acid is growth hormone protein sequence, the 192-200th amino acid is connecting peptide sequence, and the 201-315th amino acid is anti-human albumin nanobody sequence.

3. The novel long-acting recombinant human growth hormone fusion protein according to claim 1, characterized in that, The amino acid sequence of the fusion protein is shown as SEQ ID NO.

1.

4. The novel long-acting recombinant human growth hormone fusion protein according to claim 1, characterized in that, The fusion protein is connected with a signal peptide at the N terminus, and the nucleotide sequence of the signal peptide is shown as SEQ ID NO.

5. The amino acid and nucleotide sequences of the signal peptide are shown as SEQ ID NO. 3-4.

5. A nucleic acid molecule encoding the novel long-acting recombinant human growth hormone fusion protein of claim 1, characterized in that, The nucleotide sequence is shown as SEQ ID NO.

2.

6. An expression vector of a novel long-acting recombinant human growth hormone fusion protein, characterized in that, The expression vector comprises the nucleic acid molecule of claim 5.

7. Use of the novel long-acting recombinant human growth hormone fusion protein of any one of claims 1-4 in the preparation of a medicament for treating and / or preventing diseases related to abnormal growth hormone.

8. Use according to claim 7, characterized in that, The purity of the novel long-acting recombinant human growth hormone fusion protein is greater than 95%.

9. A pharmaceutical composition, characterized by, The pharmaceutical composition comprises the fusion protein of any one of claims 1-4 or the nucleic acid molecule of claim 5.

10. The pharmaceutical composition of claim 9, wherein, The pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

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