Recombinant PTH fusion protein, construction method therefor, and application thereof

Abstract

A recombinant PTH fusion protein, a construction method therefor, and an application thereof. The recombinant PTH fusion protein comprises: A1) at least one signaling domain molecule capable of activating a PTHR1 intracellular pathway; A2) a molecule having one or more ECDs capable of binding to the PTHR1; A3) a human serum albumin nanobody, wherein an amino acid sequence of the human serum albumin nanobody is as shown in SEQ ID NO: 7. The recombinant PTH fusion protein is capable of effectively improving the duration and magnitude of pharmacodynamic action, increasing blood calcium levels within 24-48 hours, and returning to normal levels within 48-72 hours. Said protein has excellent temperature stability and freeze-thaw stability, and can be used for treating and / or preventing hypoparathyroidism. In addition, the preparation method for the recombinant PTH fusion protein is simple and can be used for industrial production.

Description

A recombinant PTH fusion protein and its construction method and application Technical Field

[0001] The present invention relates to the field of protein engineering technology, and in particular to a recombinant PTH fusion protein and a construction method and application thereof. Background Art

[0002] Hypoparathyroidism (HP) is a disease characterized by insufficient circulating parathyroid hormone (PTH), leading to low calcium and elevated phosphate levels in the blood. Symptoms of this disease are caused by increased neuromuscular hypersensitivity due to hypocalcemia and include tingling, muscle spasms, and seizures. The most common cause of HP is accidental removal or injury of the parathyroid glands during neck surgery, followed by genetic, idiopathic, and autoimmune causes. According to the "Hypoparathyroidism Market Insights, Epidemiology, and Forecast to 2032" report released by DelveInsight Business Research on November 7, 2022, as of 2021, there were over 280,200 patients with hypoparathyroidism in seven European and American countries (with an additional 80,000 patients), including over 81,300 in the United States, over 34,500 in five European countries, and over 22,300 in Japan. The compound annual growth rate is projected to reach 17.7% over the next decade, from 2023 to 2032. There is no comprehensive epidemiological data on hypoparathyroidism in China, but according to relevant literature and foreign epidemiological data, it is estimated that there are currently about 400,000 patients with hypoparathyroidism in China.

[0003] The clinical manifestations of hypoparathyroidism are diverse and can affect virtually any organ system. These include central nervous system manifestations of epilepsy, Parkinson's disease, or myotonic disorders; cardiovascular manifestations of arrhythmias and hypocalcemia-related dilated cardiomyopathy; neuropsychiatric manifestations of anxiety and depression; and ophthalmic manifestations of cataracts and papilledema. Furthermore, these manifestations can include nephrocalcinosis, kidney stones, chronic kidney disease, and central nervous system calcification in the urinary system, often caused by conventional treatment with high doses of active vitamin D and / or calcium supplements. In related studies, conventional treatment for HP includes active vitamin D and calcium supplements, with patients taking large amounts of calcium to increase intestinal calcium absorption and thereby elevate serum calcium concentrations. Although conventional treatment with active vitamin D and calcium supplements can restore serum calcium levels, it does not restore other effects of PTH, such as bone turnover or renal calcium reabsorption. Furthermore, conventional treatment carries a certain risk of hypercalciuria, which increases the risk of nephrocalcinosis and kidney stones, as well as long-term complications (including nephrocalcinosis, kidney stones, and intracranial calcification). Therefore, many pharmaceutical companies have been looking for hormone replacement therapies to achieve better therapeutic effects.

[0004] To develop a more physiologically relevant alternative therapy than traditional treatments, research aimed at replacing parathyroid hormone (PTH) began with the synthesis of human PTH(1-34), a biologically active amino-terminal fragment of the full-length PTH peptide. Both the active N-terminal fragment and the full-length 84-amino acid peptide bind to and activate the parathyroid hormone 1 receptor (PTH1R). PTH(1-34) is the final active molecular form of PTH in humans, while PTH(1-84) is its precursor. PTH(1-84) is enzymatically processed in vivo to form PTH(1-34), which then exerts its biological functions. Currently, the approved PTH(1-34) forms of teriparatide (Eli Lilly) and the PTH(1-84) form of NATPARA (Takeda) have half-lives of 30 minutes and 3 hours in humans, respectively. Due to their short half-lives and the duration of serum calcium elevation in vivo of only 2 hours, clinical studies are being conducted using pump delivery or twice-daily injections. PTH (1-84) has a half-life of 3 hours and can maintain normal physiological blood calcium levels for 12 hours after administration. However, since it cannot maintain normal blood calcium levels throughout the day, it also requires daily administration of active vitamin D and calcium supplements. It also suffers from short-term and long-term side effects from calcium supplementation therapy and is only approved for patients whose symptoms cannot be controlled with conventional calcium supplementation. Furthermore, due to the poor stability of the PTH molecule itself, Takeda's marketed PTH (1-84) drug "NATPARA," the domestically marketed PTH (1-34) drug "Xinfutai," and the domestically marketed PTH (1-34) drug "Zhen Gu" by United Cell are all in the form of lyophilized powders, requiring patients to dissolve them in water for injection before daily use, making administration extremely inconvenient.

[0005] Technically, neither of the aforementioned treatments fully meets the needs of patients. In particular, the short-term and long-term side effects can cause significant harm to patients, impacting their quality of life. Furthermore, patients with hypoparathyroidism who undergo medication typically suffer from permanent hypoparathyroidism and require lifelong medication. Consequently, scientists and pharmaceutical companies worldwide are conducting various research projects on next-generation treatments, with the most rapidly developing being Transcon PTH (1-34) from Ascendis, Denmark, and LA-PTH (1-34) from Amolyt, France. Transcon PTH (1-34) is a PEGylated prodrug, consisting of two PEG molecules linked to PTH (1-34) via a linker to form an inactive prodrug. Following administration, the PEG molecules gradually detach in the body, releasing PTH molecules. This prolongs the half-life of PTH and maintains normal blood calcium levels within 24 hours. Transcon PTH (1-34) completed Phase 3 clinical trials abroad in 2022 and submitted an NDA application to the FDA. In China, Ascendis Holdings' Shanghai Weisheng Pharmaceuticals completed enrollment in a Phase 3 clinical trial in June 2022. LA-PTH (1-34) is a parathyroid hormone (PTH) peptide analogue, derived by fusing a mutated PTH (1-14) with PTHrP (15-36). It targets a specific configuration of PTH1R1. Compared to PTH (1-34), LA-PTH increases its binding to PTHr1. While this does not alter the drug's half-life, it can elevate serum calcium over a prolonged period, safely and effectively maintaining 24-hour blood calcium levels and thus controlling hypoparathyroidism. LA-PTH completed Phase 2 clinical trials in 2022 and is currently preparing for Phase 3 clinical trials.

[0006] Although the efficacy of therapeutic drugs for hypoparathyroidism has improved, the properties of PTH itself make its preparation extremely difficult. For example, Transcon PTH (1-34) is a PEGylated prodrug. Although PEG encapsulation can enhance the solubility and stability of PTH, Transcon PTH (1-34) requires solid-phase peptide synthesis followed by PEGylation, resulting in extremely high peptide synthesis and coupling costs. LA-PTH is also produced using chemical solid-state synthesis, resulting in extremely high production costs. Furthermore, due to the extremely low solubility of PTHrP, the affected site is primarily the PTHrP (15-36) segment, the binding region of the extracellular domain (ECD) of the receptor PTHR1. Therefore, Amolyt had to mutate it to improve its solubility, but its stability may be difficult to guarantee.

[0007] Based on this, there is an urgent need to seek a hypoparathyroidism-related therapeutic drug that can prolong the drug's duration of action, reduce the drug dosage, and has excellent stability and solubility. Summary of the Invention

[0008] The present invention aims to address at least one of the technical problems existing in the prior art. To this end, the present invention provides a recombinant PTH fusion protein that effectively increases the duration and intensity of pharmacological action, exhibits excellent temperature stability and freeze-thaw stability, and can be used to treat and / or prevent hypoparathyroidism.

[0009] The invention also provides a method for constructing a recombinant PTH fusion protein.

[0010] The present invention also provides a biomaterial.

[0011] The present invention also proposes the use of the recombinant PTH fusion protein in preparing a product for treating and / or preventing hypoparathyroidism.

[0012] The present invention also provides a medicine.

[0013] The present invention also provides a combined medicine.

[0014] The first aspect of the present invention provides a recombinant PTH fusion protein comprising:

[0015] A1) a molecule having at least one domain capable of activating the PTHR1 intracellular pathway;

[0016] A2) molecules with one or more ECDs capable of binding to PTHR1;

[0017] A3) human serum albumin nanobody;

[0018] The amino acid sequence of the human serum albumin nanobody is shown in SEQ ID NO: 7.

[0019] The recombinant PTH fusion protein according to the embodiment of the present invention has at least the following beneficial effects:

[0020] (1) High safety and long duration of pharmacodynamics. The recombinant PTH fusion protein of the present invention is safer and has a longer duration of pharmacodynamics than teriparatide (rhPTH (1-34)). The same dose of teriparatide (rhPTH (1-34)) injection increases blood calcium within 2 hours and returns to normal within 6 hours, while the same dose of the recombinant PTH fusion protein of the present invention can increase blood calcium within 24-48 hours and return to normal within 48-72 hours, greatly extending the half-life of the PTH drug and helping to safely and effectively maintain blood calcium levels, thereby controlling hypoparathyroidism.

[0021] (2) Good temperature stability, and can be developed in liquid dosage forms. The recombinant PTH fusion protein of the present invention can maintain high stability at 4-37°C. When the fusion protein contains a nanobody to PTHR1 (such as VHH22A3) or contains three ECD binding domain molecules, its temperature stability is even better, and it can maintain good stability at 4-60°C. It can overcome the current inconvenience of using the currently available lyophilized powders of PTH (1-34) and PTH (1-84) drug molecules and improve patient compliance.

[0022] (3) Excellent freeze-thaw stability. The recombinant PTH fusion protein of the present invention still has excellent biological activity after being frozen and thawed five times at -80°C and room temperature.

[0023] It can be understood that the ECD is the abbreviation of Extracellular Domain, which refers to the extracellular part of the transmembrane protein (ie, the N-terminal extracellular region of the membrane protein).

[0024] In some embodiments of the present invention, the amino acid sequence of the signaling activation domain molecule capable of activating the PTHR1 intracellular pathway comprises the sequence shown in SEQ ID NO: 1 (ie, PTH (1-14)).

[0025] In some preferred embodiments of the present invention, the signaling activation domain molecule capable of activating the PTHR1 intracellular pathway is selected from at least one of PTH (1-14) to PTH (1-34).

[0026] In some embodiments of the present invention, the PTH (1-14)-PTH (1-34) includes: PTH (1-14), PTH (1-15), PTH (1-16), PTH (1-17), PTH (1-18), PTH (1-19), PTH (1-20), PTH (1-21), PTH (1-22), PTH (1-23), PTH (1-24), PTH (1-25), PTH (1-26), PTH (1-27), PTH (1-28), PTH (1-29), PTH (1-30), PTH (1-31), PTH (1-32), PTH (1-33) and PTH (1-34).

[0027] In some preferred embodiments of the present invention, the signaling activation domain molecule capable of activating the PTHR1 intracellular pathway is PTH (1-14).

[0028] In some embodiments of the present invention, the amino acid sequence of the molecule capable of binding to the ECD of PTHR1 comprises at least one of the sequences shown in SEQ ID NO: 3, SEQ ID NO: 5, and SEQ ID NO: 6.

[0029] It can be understood that the sequence shown in SEQ ID NO: 3 is PTH (15-34), the sequence shown in SEQ ID NO: 5 is PTHrP (15-36), and the sequence shown in SEQ ID NO: 6 is the Nanobody VHH22A3 of PTHR1 (derived from patent: EP2557090A2).

[0030] In some embodiments of the present invention, the molecules that can bind to the ECD of PTHR1 include PTH (15-34), PTH (14-34), PTH (13-34), PTH (12-34), PTH (11-34), PTH (10-34), PTH (9-34), PTH (8-34), PTH (7-34), PTH (6-34), PTH (5-34), PTH (4-34), PTH (2-34), PTH (15-84), PTH (14-84), PTH (13-84), PTH (12-84), PTH (11-84), PTH (10-84), PTH (9-84), PTH (8-84). 4) at least one of the following nanobodies: PTH(7-84), PTH(6-84), PTH(5-84), PTH(4-84), PTH(3-84), PTH(2-84), PTHrP(15-36), PTHrP(14-36), PTHrP(13-36), PTHrP(12-36), PTHrP(11-36), PTHrP(10-36), PTHrP(9-36), PTHrP(8-36), PTHrP(7-36), PTHrP(6-36), PTHrP(5-36), PTHrP(4-36), PTHrP(3-36), PTHrP(2-36), and PTHr1.

[0031] In some embodiments of the present invention, the PTHR1 nanobody comprises VHH22A3, and the amino acid sequence of VHH22A3 is shown in SEQ ID NO: 6 (derived from patent: EP2557090A2).

[0032] In some embodiments of the invention, the molecule capable of binding to the ECD of PTHR1 has a different binding site from the signaling domain molecule capable of activating the intracellular pathway of PTHR1.

[0033] In some embodiments of the present invention, the signaling domain molecule capable of activating the PTHR1 intracellular pathway, the molecule capable of binding to the ECD of PTHR1 or the human serum albumin nanobody in the recombinant PTH fusion protein are connected via a linker.

[0034] In some embodiments of the present invention, the amino acid sequence of the linker is shown in any one of SEQ ID NOs: 8-10.

[0035] In some embodiments of the present invention, the recombinant PTH fusion protein further comprises a his tag.

[0036] In some embodiments of the present invention, the nucleotide sequence of the recombinant PTH fusion protein is selected from

[0037] In some embodiments of the present invention, the amino acid sequence of the recombinant PTH fusion protein is selected from any one of the sequences shown in SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, and SEQ ID NO: 31.

[0038] The second aspect of the present invention provides a method for constructing the recombinant PTH fusion protein described in the first aspect, which specifically comprises introducing an expression vector containing a gene encoding the recombinant PTH fusion protein described in any one of the first aspects into a host cell, allowing the encoding gene to be expressed, and obtaining the recombinant PTH fusion protein after isolation and purification.

[0039] In some embodiments of the present invention, the expression vector comprises a pET 30a(+) vector.

[0040] In some embodiments of the present invention, the host cell comprises Escherichia coli.

[0041] In some preferred embodiments of the present invention, the Escherichia coli is Escherichia coli BL21 (DE3).

[0042] The third aspect of the present invention provides a biomaterial, wherein the biomaterial is any one of B1) to B4):

[0043] B1), a nucleic acid molecule encoding the recombinant PTH fusion protein according to any one of the first aspects;

[0044] B2), an expression cassette containing the nucleic acid molecule described in B1);

[0045] B3) a recombinant vector containing the nucleic acid molecule described in B1) or the expression cassette described in B2);

[0046] B4) A host cell containing the nucleic acid molecule described in B1), the expression cassette described in B2) or the recombinant vector described in B3).

[0047] The fourth aspect of the present invention provides use of the recombinant PTH fusion protein described in any one of the first aspects in the preparation of a product for treating and / or preventing hypoparathyroidism.

[0048] The fifth aspect of the present invention provides a medicine comprising the recombinant PTH fusion protein according to any one of the first aspects and a pharmaceutically acceptable excipient.

[0049] The sixth aspect of the present invention provides a combination drug comprising the recombinant PTH fusion protein described in any one of the first aspect and at least one of the drugs described in the fifth aspect, and a tumor-targeting drug.

[0050] A seventh aspect of the present invention provides a method for treating and / or preventing hypoparathyroidism, comprising the following steps:

[0051] A therapeutically and / or prophylactically effective amount of at least one of the above-mentioned recombinant PTH fusion protein, biomaterial, drug or combined drug is provided to a subject in need thereof.

[0052] Other features and advantages of the present invention will be set forth in the description which follows, and in part will be obvious from the description, or may be learned by practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] The present invention will be further described below with reference to the accompanying drawings and embodiments, in which:

[0054] FIG1 is a schematic diagram of the human serum albumin nanobody and PTH protein fusion protein of the present invention.

[0055] FIG2 is a schematic diagram of the binding mode between the PTH protein of the present invention and the receptor PTHR1.

[0056] Figure 3 is a schematic diagram of the binding of functional molecules of the monovalent and multivalent receptor ECD binding domain patterns to human serum albumin nanobodies of the present invention.

[0057] FIG4 is a schematic diagram showing the binding and dissociation of the fusion protein of the present invention and the PTHR1 receptor.

[0058] 5A-5B are the results of the drug efficacy test of the blank control group of the present invention, wherein 5A is 0-6 hours, and 5B is 0-96 hours.

[0059] 6A-6B are the results of animal efficacy assays of the fusion protein T002G-A of the present invention, wherein 6A is 0-6 h, and 6B is 0-96 h.

[0060] Figures 7A-7D show the animal efficacy test results of the fusion proteins T002G-B-T002G-I of the present invention, wherein 7A shows the fusion proteins T002G-B and T002G-C, 7B shows the fusion proteins T002G-D and T002G-E, 7C shows the fusion proteins T002G-F and T002G-G, and 7D shows the fusion proteins T002G-H and T002G-I.

[0061] FIG8 shows the results of animal efficacy assays of the fusion protein T002G-J-T002G-K of the present invention.

[0062] 9A-9B are the temperature stability test results of the fusion proteins T002G-A-T002G-K of the present invention, wherein 9A is the fusion protein T002G-A-T002G-I, and 9B is the fusion protein T002G-J, T002G-K and NbHSA.

[0063] 10A-10B are freeze-thaw stability test results of the fusion protein T002G-A-T002G-K of the present invention, wherein 10A is the fusion protein T002G-A-T002G-I, and 10B is the fusion protein T002G-J, T002G-K and NbHSA. DETAILED DESCRIPTION

[0064] The following will clearly and completely describe the concept and technical effects of the present invention in conjunction with the embodiments to fully understand the purpose, features and effects of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

[0065] When a numerical range is disclosed herein, the range is considered continuous and includes the minimum and maximum values ​​of the range, as well as every value between such minimum and maximum values. Further, when a range refers to an integer, every integer between the minimum and maximum values ​​of the range is included. In addition, when multiple ranges are provided to describe a feature or characteristic, the ranges can be combined. In other words, unless otherwise indicated, all ranges disclosed herein are understood to include any and all subranges subsumed therein.

[0066] In the description of the present invention, reference to terms such as "one embodiment," "some embodiments," "illustrative embodiments," "example," "specific example," or "some examples" means that a specific feature, structure, material, or characteristic described in conjunction with the embodiment or example is included in at least one embodiment or example of the present invention. In this specification, exemplary expressions of the above terms do not necessarily refer to the same embodiment or example.

[0067] The term "nucleotide" generally refers to a compound consisting of a nucleoside linked to an acidic molecule or group via an ester bond. For example, a nucleoside phosphate ester typically has one, two, or three phosphate groups covalently attached to the 5-position of the sugar moiety of the nucleoside. In some cases, the definition of nucleotide also includes homologs or analogs of typical nucleotides.

[0068] The term "amino acid" refers to the basic unit that makes up proteins, giving proteins their specific molecular structure and morphology, and making their molecules biochemically active. For example, "amino acids" as used in the present invention include the following 20 natural amino acids: alanine (Ala or A), glycine (Gly or G), isoleucine (Ile or I), asparagine (Asn or N), arginine (Arg or R), lysine (Lys or K), cysteine ​​(Cys or C), aspartic acid (Asp or D), glutamic acid (Glu or E), glutamine (Gln or Q), histidine (His or H), leucine (Leu or L), methionine (Met or M), phenylalanine (Phe or F), proline (Pro or P), serine (Ser or S), threonine (Thr or T), tryptophan (Trp or W), valine (Val or V), and tyrosine (Tyr or Y).

[0069] Unless otherwise specified, "room temperature" in the present invention means 25°C±5°C.

[0070] Unless otherwise defined, all scientific or technical terms in this patent are consistent with the common understanding of most general persons in this field.

[0071] If the specific conditions are not specified in the examples, the experiments were carried out under conventional conditions or those recommended by the manufacturer. All reagents or instruments used, if the manufacturer is not specified, are commercially available conventional products.

[0072] Inventive concept

[0073] The present invention has found that fusing human serum albumin nanoantibody NbHSA with PTH (1-34) molecules (the specific schematic diagram is shown in Figure 1) helps to improve the drugability of PTH (1-34) and prolong its therapeutic effect. Secondly, the high stability of the nanoantibody improves the stability of PTH (1-34), making it easier to prepare liquid dosage forms, improving the inconvenience of using the lyophilized powder dosage forms of PTH (1-34) and PTH (1-84) molecules that are already on the market, and improving patient compliance. In addition, the present invention, through analysis of the binding pattern of PTH (1-34) molecules and receptor PTHR1 (as shown in Figure 2), found that PTH (1-34) interacts with the receptor in different regions, where PTH (15-34) first binds to the ECD region of PTHR1, and then the receptor is activated by the signaling domain molecule PTH (1-14) that can activate the intracellular pathway of PTHR1. The present invention creatively designs functional molecules with monovalent and multivalent receptor ECD binding domain patterns and fuses them with human serum albumin nanoantibodies (as shown in Figure 3). On the basis of the drug half-life, the affinity between the drug molecule and the receptor is enhanced, thereby enhancing the drug action time (as shown in Figure 4), further reducing the drug dosage and prolonging the time for the drug molecule to rise.

[0074] The recombinant PTH fusion protein molecules of the present invention are designed to have structures such as AC, ABC, ACB, ABCB, etc., wherein:

[0075] A is a signaling domain molecule that can activate PTHR1, including sequence molecules ranging from PTH (1-14) to PTH (1-34);

[0076] B is a molecule that can bind to the ECD of PTHR1, including but not limited to PTH (15-34), PTH (14-36), PTHrP (1-36), PTHrP (15-36) and PTHR1 nanoantibodies (such as VHH22A3), etc., and can be one or more identical or different binding molecules;

[0077] C is a nanobody molecule that binds to human serum protein, such as human serum albumin nanobody NbHSA.

[0078] The amino acid sequence of PTH (1-14) is:

[0079] The amino acid sequence of PTH (1-34) is:

[0080] The amino acid sequence of PTH (15-34) is:

[0081] The amino acid sequence of PTHrP (1-36) is:

[0082] The amino acid sequence of PTHrP (15-36) is:

[0083] The amino acid sequence of the PTHR1 nanobody VHH22A3 is:

[0084] The amino acid sequence of human serum albumin nanobody NbHSA is:

[0085] The above A, B and C are connected by a linker, and the linker includes but is not limited to: 5'-GGGGS-3', 5'-GGGGSVDDDDK-3' (SEQ ID NO: 8), 5'-GGGGSGGGS-3' (SEQ ID NO: 9), 5'-GGGGSGGGGS-3' (SEQ ID NO: 10) or 5'-GGGGSGGGGSGGGGS-3' (SEQ ID NO: 33).

[0086] The above-mentioned inventive concept is described in detail below with reference to specific embodiments.

[0087] Example 1: NbHSA and PTH fusion protein

[0088] This example provides a single ECD binding molecule fusion protein T002G-A, which comprises NbHSA and PTH (1-34) protein. The preparation of the fusion protein T002G-A of this example includes the following process.

[0089] 1. Construction of recombinant strains

[0090] We commissioned General Biotech (Anhui) Co., Ltd. to obtain the nucleotide sequence encoding the single ECD binding molecule fusion protein T002G-A (with a his tag) by chemical synthesis. The sequence information is as follows:

[0091] The above fragment was inserted into the prokaryotic expression plasmid pET 30a(+) (Novagen) via the Nde I and Xho I sites and sequenced to obtain a recombinant expression plasmid containing the target gene. The recombinant expression plasmid containing the target gene was then transformed into Escherichia coli BL21(DE3) competent cells as follows:

[0092] Thaw 100 μL of BL21 competent cells in an ice bath. Add the recombinant expression plasmid containing the target gene, shake gently, and place in an ice bath for 30 minutes. Heat shock the tube in a 42°C water bath for 30 seconds, then quickly transfer the centrifuge tube to an ice bath and place for 2 minutes. Do not shake the tube during this process. Add 400 μL of sterile 2YT medium (containing 16 g / L trypsin casein, 10 g / L yeast extract, 5 g / L sodium chloride, and water, without antibiotics) to the centrifuge tube, mix thoroughly, and incubate at 37°C, 220 rpm, and shake for 1 hour to allow the bacteria to recover. Pipette 200 μL of the transformed competent cells onto a 2YT agar plate containing kanamycin resistance, spread the cells evenly, and incubate the plate at 37°C overnight. The next day, a monoclonal colony was picked from the transformation plate using an inoculation loop, positive clones were screened and inoculated into 15 mL of sterile 2YT medium (containing kanamycin), and cultured at 30°C overnight to obtain the recombinant strain.

[0093] 2. Fusion protein expression

[0094] The recombinant strain was inoculated onto a 2YT plate containing kanamycin using the streak method and cultured overnight in a 37°C incubator. The next day, a single clone was selected and placed in 20 mL of 2YT liquid medium containing kanamycin. The culture was shaken at 37°C until the OD600 of the culture reached 0.4-0.6. The culture was then inoculated into 500 mL of 2YT liquid medium containing kanamycin at a ratio of 1:100. The culture was shaken at 37°C until the OD600 of the culture reached 4-6. Induction was then performed by adding isopropylthiogalactoside (IPTG) at a final concentration of 0.5 mM. Culture was continued overnight at 18°C, and the cells were harvested to obtain a recombinant strain expressing the fusion protein.

[0095] When E. coli BL21 (DE3) was used as the host, the expressed fusion protein T002 GA accounted for about 30% of the total bacterial protein and existed mainly in the form of soluble protein.

[0096] 3. Purification and enzyme digestion of fusion protein

[0097] The cells of the recombinant strain expressing the fusion protein were resuspended in 50 mmol / L Tris-HCl (pH 8.0) at a ratio of 1:30, and then disrupted three times with a high-pressure homogenizer at 1000 bar. After disruption, the sample was centrifuged at 8000 rpm for 30 min, and the insoluble precipitate was discarded, retaining the supernatant for the next purification step.

[0098] The target protein was purified using Ni 4FF filler. The loading buffer was 20 mmol / L Tris-HCl (pH 8.0) and equilibrated with 0.3 M NaCl. After loading, impurities and non-specific binding proteins were washed with 20 mmol / L Tris-HCl (pH 8.0), 0.3 M NaCl, and 30 mM imidazole. Finally, the T002G-A target protein was eluted with 20 mmol / L Tris-HCl (pH 8.0), 0.3 M NaCl, and 250 mM imidazole.

[0099] The T002G-A fusion protein, purified by Ni-column affinity chromatography, was added to enterokinase (EK) at a mass ratio of 1:1000 and digested overnight (18 hours) at 25°C. The digested sample was diluted 10-fold with 20 mmol / L Tris-HCl (pH 8.0). The diluted sample was purified by flow-through using Ni 4FF packing. The digested T002G-A target molecule flowed through, while the undigested T002G-A fusion protein and EK were affinity-captured by the Ni column. The flow-through sample was concentrated using an ultrafiltration concentrator and the buffer exchanged to obtain the single ECD-binding molecule fusion protein, T002G-A.

[0100] The single ECD binding molecule fusion protein T002G-A was sequenced and analyzed, and its amino acid sequence information is as follows:

[0101] The black bold and underlined ones are the connectors.

[0102] The results were consistent with the expected results, indicating that the single ECD binding molecule fusion protein T002G-A was successfully prepared in this example.

[0103] Example 2: PTH(1-34)-PTH(15-34)-NbHSA

[0104] This example provides a dual ECD binding molecule fusion protein T002G-B, which is obtained by sequentially linking PTH (1-34), PTH (15-34) and anti-HSA nanobody, and its amino acid sequence information is as follows:

[0105] The preparation process of the fusion protein T002G-B in this example refers to the preparation of the single ECD binding molecule fusion protein T002G-A in Example 1, wherein the encoding nucleotide sequence information of the fusion protein T002G-B is as follows:

[0106] Example 3: PTH(1-34)-NbHSA-PTH(15-34)

[0107] This example provides a dual ECD binding molecule fusion protein T002G-C, which is obtained by sequentially linking PTH (1-34), anti-HSA nanobody, and PTH (15-34). Its amino acid sequence information is as follows:

[0108] The preparation process of the fusion protein T002G-C in this example refers to the preparation of the single ECD binding molecule fusion protein T002G-A in Example 1, wherein the encoding nucleotide sequence information of the fusion protein T002G-C is as follows:

[0109] Example 4: PTH(1-34)-PTrP(15-36)-NbHSA

[0110] This example provides a dual ECD binding molecule fusion protein T002G-D, which is obtained by sequentially linking PTH (1-34), PTrP (15-36) and anti-HSA nanobody, wherein the linker is SEQ ID NO: 33, and its amino acid sequence information is as follows:

[0111] The preparation process of the fusion protein T002G-D in this example refers to the preparation of the single ECD binding molecule fusion protein T002G-A in Example 1, wherein the encoding nucleotide sequence information of the fusion protein T002G-D is as follows:

[0112] Example 5: PTH(1-34)-NbHSA-PTHrP(15-36)

[0113] This example provides a dual ECD binding molecule fusion protein T002G-E, which is obtained by sequentially linking PTH (1-34), anti-HSA nanobody, and PTrP (15-36). Its amino acid sequence information is as follows:

[0114] The preparation process of the fusion protein T002G-E in this example refers to the preparation of the single ECD binding molecule fusion protein T002G-A in Example 1, wherein the encoding nucleotide sequence information of the fusion protein T002G-E is as follows:

[0115] Example 6: PTH(1-34)-VHH22A3-NbHSA

[0116] This example provides a dual ECD binding molecule fusion protein T002G-F, which is obtained by sequentially linking PTH (1-34), VHH22A3, and an anti-HSA nanobody, wherein VHH22A3 is an anti-PTHR1 nanobody. The amino acid sequence information of the fusion protein T002G-F is as follows:

[0117] The preparation process of the fusion protein T002G-F in this example refers to the preparation of the single ECD binding molecule fusion protein T002G-A in Example 1, wherein the encoding nucleotide sequence information of the fusion protein T002G-F is as follows:

[0118] Example 7: PTH(1-34)-NbHSA-VHH22A3

[0119] This example provides a dual ECD binding molecule fusion protein T002G-G, which is obtained by sequentially linking PTH (1-34), an anti-HSA nanobody, and VHH22A3, wherein VHH22A3 is an anti-PTHR1 nanobody. The amino acid sequence information of the fusion protein T002G-G is as follows:

[0120] The preparation process of the fusion protein T002G-G in this example refers to the preparation of the single ECD binding molecule fusion protein T002G-A in Example 1, wherein the encoding nucleotide sequence information of the fusion protein T002G-G is as follows:

[0121] Example 8: PTH(1-34)-PTrP(15-36)-NbHSA

[0122] This example provides a dual ECD binding molecule fusion protein T002G-H, which is obtained by sequentially linking PTH (1-34), PTrP (15-36) and anti-HSA nanobody, wherein the linker is 5'-GGGGS-3', and its amino acid sequence information is as follows:

[0123] The preparation process of the fusion protein T002G-H in this example refers to the preparation of the single ECD binding molecule fusion protein T002G-A in Example 1, wherein the encoding nucleotide sequence information of the fusion protein T002G-H is as follows:

[0124] Example 9: PTH(1-34)-PTrP(15-36)-NbHSA

[0125] This example provides a dual ECD binding molecule fusion protein T002G-I, which is obtained by sequentially linking PTH (1-34), PTrP (15-36) and anti-HSA nanobody, wherein the linker is SEQ ID NO: 33, and its amino acid sequence information is as follows:

[0126] The preparation process of the fusion protein T002G-I in this example refers to the preparation of the single ECD binding molecule fusion protein T002G-A in Example 1, wherein the encoding nucleotide sequence information of the fusion protein T002G-I is as follows:

[0127] Example 10: PTH(1-34)-NbHSA-PTHrP(15-36)-VHH22A3

[0128] This example provides a three-ECD binding molecule fusion protein T002G-J, which is obtained by sequentially connecting PTH (1-34), anti-HSA nanobody, PTrP (15-36) and VHH22A3 molecules. Its amino acid sequence information is as follows:

[0129] The preparation process of the fusion protein T002G-J in this example refers to the preparation of the single ECD binding molecule fusion protein T002G-A in Example 1, wherein the encoding nucleotide sequence information of the fusion protein T002G-J is as follows:

[0130] Example 11: PTH(1-34)-NbHSA-VHH22A3-PTHrP(15-36)

[0131] This example provides a three-ECD binding molecule fusion protein T002G-K, which is obtained by sequentially connecting PTH (1-34), anti-HSA nanobody, VHH22A3 and PTrP (15-36) molecules. Its amino acid sequence information is as follows:

[0132] The preparation process of the fusion protein T002G-K in this example refers to the preparation of the single ECD binding molecule fusion protein T002G-A in Example 1, wherein the encoding nucleotide sequence information of the fusion protein T002G-K is as follows:

[0133] Test Example 1: Biological Activity Verification

[0134] The biological activities of the fusion proteins and NbHSA prepared in Examples 1-11 were detected according to the teriparatide biological activity detection method, which specifically includes the following steps:

[0135] pGL4.29 was transfected and pressure screened on PTHR1-expressing UMR-106 cells, and the selected stable transfected cell lines were tested for biological activity. The stable transfected cells were cultured at a rate of 2×10 5 The cells were added to a 96-well white transparent bottom plate at a density of 100 μL and cultured at 37°C for 24 hours. The cells were stimulated with different concentrations of fusion protein T002A and teriparatide control (recombinant human parathyroid hormone rhPTH (1-34), hereinafter referred to as TP). Three replicate wells were set for each sample. After 6 hours of stimulation, 50 μL of fluorescent substrate was added. The cells were then placed on a microplate reader for reading and the average value was taken.

[0136] The test results are shown in Table 1.

[0137] Table 1: Biological activity test results

[0138] The results showed that the biological activity of the fusion protein decreased slightly with the increase in the number of ECD binding domains. This may be due to the slight steric hindrance in the spatial structure after the fusion of multiple ECDs. The EC50 of the single-ECD binding domain molecule TP was 24.285pm, while the EC50 of the multi-ECD binding fusion protein ranged from 47.361 to 74.005pm. However, the EC50 of the two-ECD binding fusion protein and the three-ECD binding fusion protein were similar. In addition, the length of the linker had little effect on the activity of the sample.

[0139] Test Example 2: Animal Efficacy Determination

[0140] 8-week-old C57 male mice were selected, and the fusion protein prepared in Example 1-11 and the reference substance TP were diluted with PBS to the required concentrations for the experiment. The fusion protein and the reference substance TP were injected intraperitoneally at two drug doses of 20nmoL / kg and / or 10nmol / kg, with 3 mice in each group. The blank group (KB) was injected with an equal volume of normal saline. Blood was collected at 0h before injection and at 1, 3, 6, 24, 48, and 72 after injection. The blood sample was placed at 4°C for 1 hour, centrifuged at 4000rpm for 5 minutes, and serum was collected after centrifugation. The calcium ion content in mouse serum was quantitatively detected using a calcium azo Ⅲ assay kit (Zhongshan Biaojia Biological) according to the manufacturer's instructions.

[0141] The results of TP efficacy assays are shown in Figures 5A-5B, and the results of single-ECD binding fusion protein T002G-A efficacy assays are shown in Figures 6A-6B. These results show that different doses of TP elevated serum calcium within 2 hours, with return to normal within 6 hours; different doses of T002G-A elevated serum calcium within 24 hours, with return to normal within 48 hours. In the saline-treated blank control group, mice experienced a slight decrease in serum calcium during recovery due to the short interval between blood draws.

[0142] The results of the drug efficacy assay of the double ECD binding molecule fusion protein T002G-B-T002G-I are shown in Figures 7A-7D, 7A is the fusion protein T002G-B and T002G-C, 7B is the fusion protein T002G-D and T002G-E, 7C is the fusion protein T002G-F and T002G-G, and 7D is the fusion protein T002G-H and T002G-I, showing that compared with the fusion protein T002G-A, further fusion with the same ECD binding molecule T002G-B-T002G-I The combined PTH (15-34) could not further increase the duration and intensity of its pharmacodynamic effect (such as T002G-B and T002G-C). Its calcium-elevating time remained at 24 hours, and the maximum blood calcium concentration remained at around 2.5 mmol. This may be because the same ECD binding domain molecules have the same affinity with the receptors on the cell surface. The two ECD binding molecules bind to the receptors at the same time and dissociate from the receptors at the same time, so the action time of the signaling domain activating the receptor is not prolonged.

[0143] Furthermore, different ECD-binding molecules, including PTHrP (15-36) and VHH22A3, can prolong the duration of drug effect to a certain extent. In particular, PTHrP (15-36), whether attached to the front or back of NbHSA (such as T002G-D and T002G-E), and regardless of the length of the linker, can prolong the duration of calcium elevation to 48 hours, and the entire hypercalcemia period is maintained for approximately 72 hours. This may be due to the different affinities of different ECD-binding molecules for receptors on the cell surface. The molecule that first binds to the receptor ECD facilitates the binding of the other ECD-binding molecule. After the ECD-binding molecule dissociates first, the signaling region activates the receptor and continues to activate the receptor. Only after both differentiated ECD-binding molecules dissociate does the signaling region activate the receptor and the activation of the receptor cease. Therefore, overall, the duration of the signaling region activates the receptor is prolonged. However, due to the relationship between the intensity of calcium elevation and the balance of blood calcium regulation in mice, the maximum blood calcium concentration is still maintained at around 2.5 mmol.

[0144] The results of the pharmacodynamic assays of the three ECD-binding molecule fusion proteins T002G-J and T002G-K are shown in FIG8 , indicating that the fusion proteins fused with three different ECDs have stronger pharmacological effects, especially T002G-K, which increased calcium levels in mice within 48 hours and maintained hypercalcemia within 96 hours, with the maximum blood calcium reaching approximately 4.5 mmol.

[0145] Test Example 3: Temperature Stability Study

[0146] This test example tests the temperature stability of the fusion proteins and NbHSA prepared in Examples 1-11 above. The specific method is as follows:

[0147] First, the fusion protein stock solution prepared in Example 1 was diluted to 1 mg / mL (OD concentration) with the corresponding buffer and filtered using a 0.22 μm filter membrane. Then it was divided into 8 equal parts, corresponding to: 4°C, 25°C, 37°C, 50°C, 60°C, 70°C, 80°C and 90°C, and incubated in a water bath for 4 hours, then placed at room temperature for 2 hours, and OD was measured. 280 The concentration was measured by nm. Finally, the concentration of the protein stock solution in the solution under each temperature condition was measured, that is, its temperature stability was tested. Three replicates were set for each group, and the average value was taken.

[0148] The test results are shown in Table 2 and Figures 9A-9B.

[0149] Table 2: Temperature stability test results

[0150] Results showed that the temperature stability of the single ECD-binding molecule T002G-A was average, stable below 37°C and exhibiting significant protein loss at 50°C. Among the dual-ECD-binding molecule fusion proteins, PTH(15-34) and PTHrP(15-36) and different linkers did not significantly improve the temperature stability of the fusion proteins. However, the addition of the PTHrP-binding nanobody VHH22A3 significantly improved the temperature stability of the fusion protein, regardless of whether it was fused before (T002G-F) or after the human serum albumin nanobody NbHSA. This may be related to the strong stability of the nanobody itself, resulting in a significant improvement in the stability of the smaller PTH(1-34) after fusion expression.

[0151] Furthermore, the test results of the three ECD binding domain fusion proteins T002G-J and T002G-K showed that the addition of PTHrP nanoantibody VHH22A3 can improve the temperature stability of the fusion protein molecule. Regardless of whether it is fused before (T002G-J) or after (T002G-K) the human serum protein nanoantibody NbHSA, it maintains a strong temperature stability, that is, after being treated at 60°C for 4 hours, there is almost no loss of the protein stock solution.

[0152] Test Example 4: Freeze-thaw stability study

[0153] This test example tests the freeze-thaw stability of the fusion protein prepared in Examples 1-11 above. The specific method is as follows:

[0154] The protein stock solutions prepared in Examples 1-11 were diluted to 1 mg / mL with the corresponding buffer and filtered through a 0.22 μm filter. The solutions were then divided into three portions and frozen and thawed at -80°C for 30 minutes each time. The portions were then thawed naturally at room temperature. Samples were collected at the 0th, 1st, 3rd, and 5th times, and their properties were observed. OD values ​​were measured at a wavelength of 280 nm.

[0155] The test results are shown in Table 3 and Figures 10A-10B.

[0156] Table 3: Freeze-thaw stability test results

[0157] The results showed that the fusion proteins T002G-A-T002G-K maintained the stability of the protein stock solution during repeated freezing and thawing from -80°C to room temperature, without protein loss or precipitation, indicating that they have excellent freeze-thaw stability.

[0158] Test Example 5: Protein Concentration Study

[0159] In this test, two recombinant proteins (T002G-J and T002G-K) were selected and their solubility was tested. The specific method is as follows:

[0160] The protein stock solutions obtained in the above-mentioned embodiments 10-11 were diluted to 1 mg / mL with the corresponding buffer, and 150 mL of the solution was centrifuged at 3000 g using a Sartorius 3000D pore size ultrafiltration concentration tube. Concentration tests and appearance inspections (to check whether a solid precipitate was produced) were performed at volumes of 30 mL, 15 mL, 10 mL, 5 mL, 3 mL, and 1.5 mL, respectively.

[0161] The test results are shown in Table 4 and Figure 10B.

[0162] Table 4 Fusion protein solubility test data

[0163] The results showed that both T002G-J and T002G-K had good solubility and remained clear without precipitation even at a concentration of 100 mg / mL.

[0164] While the embodiments of the present invention have been described in detail above, the present invention is not limited to the embodiments described above. Various modifications may be made within the scope of knowledge possessed by a person skilled in the art without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof may be combined with one another unless there is a conflict.

Claims

1. A recombinant PTH fusion protein, characterized in that: Include: A1) molecules with at least one signaling domain that can activate the PTHR1 intracellular pathway; A2) molecules having one or more ECDs capable of binding to PTHR1; A3) human serum albumin nanobody; The amino acid sequence of the human serum albumin nanobody is shown in SEQ ID NO:

7.

2. The recombinant PTH fusion protein according to claim 1, characterized in that The amino acid sequence of the signaling activation domain molecule capable of activating the PTHR1 intracellular pathway comprises the sequence shown in SEQ ID NO: 1; Preferably, the signaling activation domain molecule capable of activating the PTHR1 intracellular pathway is selected from at least one of PTH (1-14) to PTH (1-34).

3. The recombinant PTH fusion protein according to claim 1 or 2, characterized in that The amino acid sequence of the molecule capable of binding to the ECD of PTHR1 comprises at least one of the sequences shown in SEQ ID NO: 3, SEQ ID NO: 5, and SEQ ID NO:

6.

4. The recombinant PTH fusion protein according to claim 1 or 2, characterized in that The molecule capable of binding to the ECD of PTHR1 is selected from the group consisting of PTH (15-34), PTH (14-34), PTH (13-34), PTH (12-34), PTH (11-34), PTH (10-34), PTH (9-34), PTH (8-34), PTH (7-34), PTH (6-34), PTH (5-34), PTH (4-34), PTH (2-34), PTH (15-84), PTH (14-84), PTH (13-84), PTH (12-84), PTH (11-84), PTH (10-84), PTH (9-34), PTH (8-34), PTH (7-34), PTH (6-34), PTH (5-34), PTH (4-34), PTH (2-34), PTH (15-84), PTH (14-84), PTH (13-84), PTH (12-84), PTH (11-84), PTH (10-84), PTH (9-8 ...8-34), PTH (7-34), PTH (8-34), PTH (8-34), PTH (7-34), PTH (8-34), PTH (7-34), PTH (5-34), PTH (4-34), PTH (2-34), P -84), PTH(6-84), PTH(5-84), PTH(4-84), PTH(3-84), PTH(2-84), PTHrP(15-36), PTHrP(14-36), PTHrP(13-36), PTHrP(12-36), PTHrP(11-36), PTHrP(10-36), PTHrP(9-36), PTHrP(8-36), PTHrP(7-36), PTHrP(6-36), PTHrP(5-36), PTHrP(4-36), PTHrP(3-36), PTHrP(2-36) and at least one of the nanobodies of PTHr1.

5. The recombinant PTH fusion protein according to claim 1, characterized in that The signaling domain molecule capable of activating the PTHR1 intracellular pathway in the recombinant PTH fusion protein, the molecule capable of binding to the ECD of PTHR1 or the human serum albumin nanobody are connected via a linker; Preferably, the linker comprises an amino acid sequence as shown in any one of SEQ ID NO: 8 to SEQ ID NO:

10.

6. The method for constructing the recombinant PTH fusion protein according to any one of claims 1 to 5, characterized in that: The method comprises introducing an expression vector containing a gene encoding the recombinant PTH fusion protein according to any one of claims 1 to 5 into a host cell, allowing the encoding gene to be expressed, and isolating and purifying the recombinant PTH fusion protein to obtain the fusion protein.

7. Biomaterial, characterized in that The biological material is any one of B1) to B4): B1), a nucleic acid molecule encoding the recombinant PTH fusion protein according to any one of claims 1 to 5; B2), an expression cassette containing the nucleic acid molecule described in B1); B3) a recombinant vector containing the nucleic acid molecule described in B1) or the expression cassette described in B2); B4) A host cell containing the nucleic acid molecule described in B1), the expression cassette described in B2) or the recombinant vector described in B3).

8. Use of the recombinant PTH fusion protein according to any one of claims 1 to 5 in the preparation of a product for treating and / or preventing hypoparathyroidism.

9. A medicament comprising the recombinant PTH fusion protein according to any one of claims 1 to 5 and pharmaceutically acceptable excipients.

10. A combined drug comprising the recombinant PTH fusion protein according to any one of claims 1 to 5 and at least one of the drugs according to claim 9, and a tumor-targeting drug.

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