A recombinant PTH fusion protein and its construction method and application

Abstract

The present invention relates to the field of protein engineering technology, and discloses a recombinant PTH fusion protein and a construction method and application thereof. The recombinant PTH fusion protein comprises: A1) a signaling domain molecule having at least one domain that can activate the intracellular pathway of PTHR1; A2) a molecule having one or more ECDs that can bind to PTHR1; A3) a human serum albumin nanobody; wherein the amino acid sequence of the human serum albumin nanobody is shown in SEQ ID NO.7. The recombinant PTH fusion protein of the present invention can effectively improve the duration and intensity of drug efficacy, can increase blood calcium within 24 to 48 hours, and return to normal within 48 to 72 hours, and has excellent temperature stability and freeze-thaw stability, and can be used to treat and / or prevent hypoparathyroidism. In addition, the preparation method of the recombinant PTH fusion protein of the present invention is simple and can be used for industrial production.

Description

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 low calcium levels and elevated phosphate levels in the blood due to insufficient circulating concentrations of parathyroid hormone (PTH). The symptoms of this disease are caused by increased neuromuscular hypersensitivity due to hypocalcemia, including tingling, muscle spasms, and epileptic seizures. The most common cause of HP disease 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 Market Forecasts in 2032" report released by DelveInsight on November 7, 2022, as of 2021, there are more than 280,200 patients with hypoparathyroidism in seven European and American countries (80,000 new patients), including more than 81,300 in the United States, more than 34,500 in five European countries, and more than 22,300 in Japan. It is predicted that the compound annual growth rate will be as high as 17.7% in the next ten years 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 involve almost any organ system, including epilepsy, Parkinson's disease or myotonia in the central nervous system, arrhythmia and hypocalcemia-related dilated cardiomyopathy in the cardiovascular system, anxiety and depression in the neuropsychiatric system, cataracts and papilledema in the ophthalmic system, etc. In addition, it also includes nephrocalcinosis, kidney stones and chronic kidney disease in the urinary system and calcification of the central nervous system caused by conventional treatment with large amounts of active vitamin D and / or calcium supplements. In related studies, conventional treatment for HP includes active vitamin D and calcium supplements, and patients take large amounts of calcium to increase intestinal calcium absorption to increase serum calcium concentration. Although conventional treatment with active vitamin D and calcium supplements can restore serum calcium levels, it cannot restore other effects of PTH, such as bone turnover or renal calcium reabsorption. In addition, conventional treatment has a certain risk of high urinary calcium, 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 therapy to achieve better therapeutic effects.

[0004] In order to establish a more physiologically meaningful alternative treatment than traditional therapies, research aimed at replacing parathyroid hormone began with the synthesis of human parathyroid hormone PTH(1–34), which is the biologically active amino-terminal fragment of the full-length parathyroid hormone peptide. Both the active nitrogen-terminal fragment and the full-length 84-amino acid peptide bind to and activate PTH1R. PTH(1–34) is the final active molecular form of parathyroid hormone in the human body, and PTH(1–84) is its precursor form. PTH(1–84) exerts its biological function after being enzymatically processed to form PTH(1–34) in the body. Currently, the drug teriparatide (Eli Lilly) in the form of PTH(1–34) and the drug NATPARA (Takeda) in the form of PTH(1–84) are approved for marketing, with half-lives of 30 minutes and 3 hours in the human body, respectively. Due to its short half-life and the time it takes to increase blood calcium in the body for 2 hours, clinical studies are conducted in the form of pump delivery studies or twice-daily injections. Among them, PTH (1-84) has a half-life of 3 hours. After administration, it can maintain the patient's blood calcium at a normal physiological level within 12 hours. However, since it cannot maintain normal blood calcium levels throughout the day, it is also necessary to take active vitamin D and calcium supplements every day. In addition, there will also be short-term and long-term side effects caused by calcium supplementation therapy. It is only approved for patients who cannot be controlled by conventional calcium supplementation. And because of the poor stability of the PTH molecule itself, the PTH (1-84) drug "NATPARA" launched by Takeda, the PTH (1-34) drug "Xinfutai" launched by Xinlitai in China, and the PTH (1-34) drug "Zhen Gu" launched by United Cell in China are all in the form of lyophilized powder. Patients need to dissolve it with injection water before daily use before injection, which is extremely inconvenient.

[0005] In terms of technology, the above two treatment methods cannot meet the requirements of patients well, especially the short-term and long-term toxic side effects will cause certain harm to the patient's body and affect the patient's quality of life. In addition, patients with hypoparathyroidism who undergo drug intervention generally have permanent hypoparathyroidism and need lifelong drug treatment. Therefore, scientists and pharmaceutical companies in various countries have conducted various studies on the new generation of treatment methods. Among them, the fastest research is Transcon PTH (1-34) of Ascendis Company in Denmark and LA-PTH (1-34) of Amolyt Company in France. Transcon PTH (1-34) is a PEGylated prodrug. Two PEG molecules are connected to PTH (1-34) through lnkers to form an inactive prodrug. After administration, PEG gradually falls off in the body to release PTH molecules, thereby extending the drug half-life of PTH and achieving the purpose of maintaining normal physiological levels of blood calcium within 24 hours. Transcon PTH (1-34) has completed Phase 3 clinical trials abroad in 2022 and submitted an NDA application to the FDA. In China, Ascendis Holdings Shanghai Weisheng Pharmaceutical completed Phase 3 clinical enrollment in June 2022. LA-PTH (1-34) is a parathyroid hormone (PTH) polypeptide analog, which is obtained by fusing the mutated PTH (1-14) with PTHrP (15-36). It can target PTH1R1 with a specific configuration. Compared with PTH (1-34), LA-PTH can increase its binding affinity with PTHR1. Therefore, although it does not change the half-life of the drug, it can increase blood calcium for a long time to safely and effectively maintain the calcium level in the blood for 24 hours, thereby 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 related to hypoparathyroidism has been improved, the preparation of PTH is extremely difficult due to its own characteristics. 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 synthesis of peptides and then PEGylation modification, and the cost of peptide synthesis and coupling are extremely high. LA-PTH is also produced by chemical solid-state synthesis, and the production cost is extremely high. In addition, due to the extremely low solubility of PTHrP, the affected area is mainly in the PTHrP (15-36) segment, that is, the binding area of ​​the ECD of the receptor PTHR1. Therefore, Amolyt has 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 action time, reduce the drug dosage, and has excellent stability and solubility. Summary of the invention

[0008] The present invention aims to solve at least one of the technical problems existing in the prior art. To this end, the present invention provides a recombinant PTH fusion protein, which can effectively improve the duration and intensity of drug efficacy, has 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 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 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) molecules with at least one domain that can activate 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 drug effect. The recombinant PTH fusion protein of the present invention is safer and has a longer duration of drug effect 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 to 48 hours and return to normal within 48 to 72 hours, greatly prolonging the half-life of the PTH drug, helping to safely and effectively maintain the calcium level in the blood, thereby controlling hypoparathyroidism.

[0021] (2) Good temperature stability, and can be developed in liquid dosage form. The recombinant PTH fusion protein of the present invention can maintain high stability at 4-37°C. When the fusion protein contains a nano antibody of PTHR1 (such as VHH22A3) or contains three ECD binding domain molecules, its temperature stability is better, and it can maintain good stability at 4-60°C. It can overcome the current inconvenience of using the lyophilized powder of PTH (1-34) and PTH (1-84) drug molecules on the market, 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) to PTH (1-34) include: 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 ECD-binding molecule capable of binding to 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 nanoantibody VHH22A3 of PTHR1 (derived from patent: EP2557090A2).

[0030] In some embodiments of the 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). ), 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 at least one of the nanobodies of 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 present invention, the molecule capable of binding to the ECD of PTHR1 has a binding site different from that of 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 NO.8 to SEQ ID NO.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 separation and purification.

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

[0040] In some embodiments of the 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 described in 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] Other features and advantages of the present invention will be set forth in the description which follows, and in part will be apparent from the description, or may be learned by practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein:

[0052] Figure 1 It is a schematic diagram of the human serum albumin nanobody and PTH protein fusion protein of the present invention.

[0053] Figure 2 It is a schematic diagram of the binding mode between the PTH protein of the present invention and the receptor PTHR1.

[0054] Figure 3 Schematic diagram of the binding of functional molecules of the monovalent and multivalent receptor ECD binding domain modes of the present invention to human serum albumin nanoantibodies.

[0055] Figure 4 The figure is a schematic diagram of the binding and dissociation between the fusion protein of the present invention and the PTHR1 receptor.

[0056] Figure 5 The results of the drug efficacy test of the blank control group of the present invention are shown in Figure 1, where A is 0-6h and B is 0-96h.

[0057] Figure 6 The results of animal efficacy test of the fusion protein T002G-A of the present invention, wherein A is 0-6h and B is 0-96h.

[0058] Figure 7These are the animal efficacy test results of the fusion proteins T002G-B to T002G-I of the present invention, wherein A is the fusion proteins T002G-B and T002G-C, B is the fusion proteins T002G-D and T002G-E, C is the fusion proteins T002G-F and T002G-G, and D is the fusion proteins T002G-H and T002G-I.

[0059] Figure 8 These are the results of animal efficacy assays of the fusion proteins T002G-J ~ T002G-K of the present invention.

[0060] Fig. 9 These are the temperature stability test results of the fusion proteins T002G-A to T002G-K of the present invention, wherein A is the fusion proteins T002G-A to T002G-I, and B is the fusion proteins T002G-J, T002G-K and NbHSA.

[0061] Fig.10 These are the freeze-thaw stability test results of the fusion proteins T002G-A to T002G-K of the present invention, wherein A is the fusion proteins T002G-A to T002G-I, and B is the fusion proteins T002G-J, T002G-K and NbHSA. DETAILED DESCRIPTION

[0062] The following will be combined with the embodiments to clearly and completely describe the concept of the present invention and the technical effects produced, so as to fully understand the purpose, characteristics and effects of the present invention. Obviously, the described embodiments 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.

[0063] When a numerical range is disclosed herein, the above range is considered to be 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 features or characteristics, the ranges can be combined. In other words, unless otherwise indicated, all ranges disclosed herein should be understood to include any and all subranges included therein.

[0064] In the description of the present invention, the description with reference to the terms "one embodiment", "some embodiments", "illustrative embodiments", "examples", "specific examples", or "some examples" means that the specific features, structures, materials, or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example.

[0065] The term "nucleotide" generally refers to a compound formed by a nucleoside linked to an acidic molecule or group through an ester bond. For example, a phosphate ester of a nucleoside usually has one, two, or three phosphate groups covalently linked to the 5-position of the sugar group of the nucleoside. In some cases, the definition of nucleotide also includes homologs or analogs of some typical nucleotides.

[0066] The term "amino acid" refers to the basic unit that constitutes protein, which gives protein a specific molecular structure and makes its molecule have biochemical activity. For example, the "amino acid" used in the present invention includes 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).

[0067] Unless otherwise specified, "room temperature" in the present invention means 25°C±5°C.

[0068] Unless otherwise defined, all scientific or technical terms in this patent are consistent with the common understanding of most general persons in the field.

[0069] If the specific conditions are not specified in the examples, the experiments were carried out under conventional conditions or conditions recommended by the manufacturer. If the manufacturers of the reagents or instruments are not specified, they are all conventional products that can be purchased commercially.

[0070] Invention Concept

[0071] The present invention found that the human serum albumin nanobody NbHSA was fused with the PTH (1-34) molecule (the specific schematic diagram is as follows Figure 1 As shown), it helps to improve the drugability of PTH (1-34) and prolong its therapeutic effect. Secondly, the high stability of nano antibodies 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 analyzes the binding mode of PTH (1-34) molecules and receptor PTHR1 (as shown in Figure 2As shown in Figure 2, it was 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 to be fused with human serum albumin nanoantibodies (such as Figure 3 As shown in the figure, based on the half-life of the drug, the drug action time is increased by enhancing the affinity of the drug molecule to the receptor (such as Figure 4 As shown), further reducing the dosage of the drug and prolonging the time when the drug molecules are elevated.

[0072] The recombinant PTH fusion protein molecules of the present invention are designed to be AC, ABC, ACB, ABCB and other similar structures, wherein:

[0073] A is a signaling domain molecule that can activate PTHR1, including sequence molecules ranging from PTH (1-14) to PTH (1-34);

[0074] 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 nanobodies of PTHR1 (such as VHH22A3), etc., and can be one or more identical or different binding molecules;

[0075] C is a nanobody molecule that binds to human serum protein, such as human serum albumin nanobody NbHSA.

[0076] The amino acid sequence of PTH (1-14) is:

[0077] SVSEIQLMHNLGKH(SEQ ID NO.1);

[0078] The amino acid sequence of PTH (1-34) is:

[0079] SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNF (SEQ ID NO.2);

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

[0081] LNSMERVEWLRKKLQDVHNF(SEQ ID NO.3);

[0082] The amino acid sequence of PTHrP (1-36) is:

[0083] AVSEHQLLHDKGKSIQDLRRRFFLHHLIAEIHTAEI (SEQ ID NO.4);

[0084] The amino acid sequence of PTHrP(15-36) is:

[0085] IQDLRRRFFLHHLIAEIHTAEI (SEQ ID NO.5);

[0086] The amino acid sequence of the PTHR1 nanobody VHH22A3 is:

[0087] EVQLVESGGGLVQAGGSLRLSCAASGNIFANNIMGWYRQPPGKEREFVAHVSHDGDSMYAVSVKGRFAISRKDATNLYLQMNSLKPEDTAIYFCRLLNIPTQGRMEGFWGQGTQVTVSS (SEQ ID NO. 6);

[0088] The amino acid sequence of human serum albumin nanobody NbHSA is:

[0089] QVQLVESGGGLVQTGGSLRLSCAASGSTLVGHSVGFFRQAPGKEREPVVCISSTETVMYYTNSVKGRFTISRDNAKNTVWLQMDRLAPEDTAVYYCAARPQLCSALDYRYNSWGPGTQVTVSS (SEQ ID NO. 7).

[0090] The above A, B and C are connected by a linker, and the linker includes but is not limited to: GGGGSVDDDDK (SEQ ID NO.8), GGGGSGGGS (SEQ ID NO.9) or GGGGSGGGGS (SEQ ID NO.10).

[0091] The above-mentioned inventive concept is described in detail below in conjunction with specific embodiments.

[0092] Example 1: NbHSA and PTH fusion protein

[0093] This embodiment 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 embodiment includes the following process.

[0094] 1. Construction of recombinant strains

[0095] General Biotechnology (Anhui) Co., Ltd. was commissioned to obtain the coding nucleotide sequence of the single ECD binding molecule fusion protein T002G-A (with a his tag) by chemical synthesis. The sequence information is as follows:

[0096] (SEQ ID NO.11).

[0097] pass N and XOt The above fragment was inserted into the prokaryotic expression plasmid pET 30a(+) (Novagen) and sequenced to obtain the recombinant expression plasmid containing the target gene. Then the recombinant expression plasmid containing the target gene was transformed into Escherichia coli BL21(DE3) competent cells. The specific method is as follows:

[0098] Take 100 μL of BL21 competent cells and place them on an ice bath to melt, add the above-mentioned recombinant expression plasmid containing the target gene, shake gently, and place them in an ice bath for 30 minutes. Then heat shock in a 42°C water bath for 30 seconds, then quickly transfer the centrifuge tube to an ice bath and place it for 2 minutes. Do not shake the centrifuge tube during this process. Add 400 μL of sterile 2YT culture medium (containing 16 g / L pancreatic casein, 10 g / L yeast extract, 5 g / L sodium chloride and water, without antibiotics) to the centrifuge tube, mix well, and place it in a shaking incubator at 37°C and 220 rpm for 1 hour to allow the bacteria to recover. Pipette 200 μL of transformed competent cells onto a 2YT agar medium plate containing kanamycin resistance, spread the cells evenly, and culture the plate at 37°C overnight. The next day, a single clone 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.

[0099] 2. Fusion protein expression

[0100] The above recombinant strains were inoculated on 2YT plates containing kanamycin by streaking method and placed in a 37°C constant temperature incubator for overnight culture. On the second day, a monoclonal strain was selected and placed in 20 mL of 2YT liquid culture medium containing kanamycin, and cultured at 37°C with shaking until the OD600 of the bacterial solution was 0.4-0.6, and then the bacterial solution was inoculated into 500 mL of 2YT liquid culture medium containing kanamycin at a ratio of 1:100, and cultured at 37°C with shaking until the OD600 of the bacterial solution was 4-6, and IPTG with a final concentration of 0.5 mM was added for induction, and the bacterial cells were collected after continuing to culture at 18°C ​​overnight to obtain a recombinant strain expressing the fusion protein.

[0101] 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.

[0102] 3. Purification and enzyme digestion of fusion protein

[0103] The recombinant strain expressing the fusion protein was resuspended in 50 mmol / L Tris-HCl (pH 8.0) at a ratio of 1:30, and then the cells were broken three times with a high-pressure homogenizer at 1000 bar. After the cells were broken, the sample was centrifuged at 8000 rpm for 30 min, the insoluble precipitate was discarded, and the supernatant was retained for the next purification step.

[0104] Ni 4FF filler was used to purify the target protein. The loading buffer was 20mmol / L Tris-HCl (pH 8.0) and balanced with 0.3M NaCl. After loading, the impurities and non-specific binding proteins were washed with 20mmol / L Tris-HCl (pH 8.0), 0.3M NaCl, and 30mM imidazole. Finally, the T002G-A target protein was eluted with 20mmol / L Tris-HCl (pH 8.0), 0.3M NaCl, and 250mM imidazole.

[0105] The T002 GA fusion protein purified by Ni column affinity chromatography was added with enterokinase (EK enzyme) at a mass ratio of 1:1000, and digested overnight (18h) at 25°C. The digested sample was diluted 10 times with 20mmol / L Tris-HCl (pH 8.0). The diluted sample was purified by flow-through of Ni 4FF filler, and the T002G-A target molecule obtained by digestion was flow-through, and the undigested T002G-A fusion protein and EK enzyme were captured by Ni column affinity. The flow-through sample was concentrated and replaced with an ultrafiltration concentration tube to obtain the single ECD binding molecule fusion protein T002G-A.

[0106] The above-mentioned single ECD binding molecule fusion protein T002G-A was sequenced and analyzed, and its amino acid sequence information is as follows:

[0107] MEFKRKRHHHHHH GGGGSVDDDDK SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNF GGGGSGGGS QVQLVESGGGLVQTGGSLRLSCAASGSTLVGHSVGFFRQAPGKEREPVVCISSTETVMYYTNSVKGRFTISRDNAKNTVWLQMDRLAPEDTAVYYCAARPQLCSALDYRYNSWGPGTQVTVSS (SEQ ID NO. 12).

[0108] The ones marked in bold and underlined black are the connectors.

[0109] 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.

[0110] Example 2: PTH(1-34)-PTH(15-34)-NbHSA

[0111] This example provides a dual ECD binding molecule fusion protein T002G-B, which is obtained by sequentially connecting PTH (1-34), PTH (15-34) and NbHSA nanobody, and its amino acid sequence information is as follows:

[0112] MEFKRKRHHHHHHGGGGSVDDDDKSVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFGGGGSGGGSLNSMERVEWLRKKLQDVHNFGGGGSGGGSQVQLVESGGGLVQTG GSLRLSCAASGSTLVGHSVGFFRQAPGKEREPVVCISSTETVMYYTNSVKGRFTISRDNAKNTVWLQMDRLAPEDTAVYYCAARPQLCSALDYRYNSWGPGTQVTVSS (SEQ ID NO.13).

[0113] 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:

[0114] (SEQ ID NO.14).

[0115] Example 3: PTH(1-34)-NbHSA-PTH(15-34)

[0116] This example provides a dual ECD binding molecule fusion protein T002G-C, which is obtained by sequentially connecting PTH (1-34), NbHSA nanobody and PTH (15-34), and its amino acid sequence information is as follows:

[0117] MEFKRKRHHHHHHGGGGSVDDDDKSVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFGGGGSGGGGSQVQLVESGGGLVQTGGSLRLSCAASGSTLVGHSVGFFRQAPGKE REPVVCISSTETVMYYTNSVKGRFTISRDNAKNTVWLQMDRLAPEDTAVYYCAARPQLCSALDYRYNSWGPGTQVTVSSGGGGSGGGSLNSMERVEWLRKKLQDVHNF (SEQ ID NO.15).

[0118] 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:

[0119] (SEQ ID NO.16).

[0120] Example 4: PTH(1-34)-PTHrP(15-36)-NbHSA

[0121] This example provides a dual ECD binding molecule fusion protein T002G-D, which is obtained by sequentially connecting PTH (1-34), PTHrP (15-36) and NbHSA nanobody, wherein the linker is GGGGSGGGS, and its amino acid sequence information is as follows:

[0122] MEFKRKRHHHHHHGGGGSVDDDDKSVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFGGGGSGGGSIQDLRRRFFLHHLIAEIHTAEIGGGGSGGGSQVQLVESGGGLVQT GGSLRLSCAASGSTLVGHSVGFFRQAPGKEREPVVCISSTETVMYYTNSVKGRFTISRDNAKNTVWLQMDRLAPEDTAVYYCAARPQLCSALDYRYNSWGPGTQVTVSS (SEQ ID NO.17).

[0123] 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:

[0124] (SEQ ID NO.18).

[0125] Example 5: PTH(1-34)-NbHSA-PTHrP(15-36)

[0126] This example provides a dual ECD binding molecule fusion protein T002G-E, which is obtained by sequentially connecting PTH (1-34), NbHSA nanobody and PTHrP (15-36), and its amino acid sequence information is as follows:

[0127] MEFKRKRHHHHHHGGGGSVDDDDKSVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFGGGGSGGGGSQVQLVESGGGLVQTGGSLRLSCAASGSTLVGHSVGFFRQAPGKER EPVVCISSTETVMYYTNSVKGRFTISRDNAKNTVWLQMDRLAPEDTAVYYCAARPQLCSALDYRYNSWGPGTQVTVSSGGGGSGGGSIQDLRRRFFLHHLIAEIHTAEI (SEQ ID NO.19).

[0128] 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:

[0129] (SEQ ID NO. 20).

[0130] Example 6: PTH(1-34)-VHH22A3-NbHSA

[0131] This example provides a dual ECD binding molecule fusion protein T002G-F, which is obtained by sequentially connecting PTH (1-34), VHH22A3 and NbHSA nanobody, wherein VHH22A3 is an anti-PTHR1 nanobody, and the amino acid sequence information of the fusion protein T002G-F is as follows:

[0132] MEFKRKRHHHHHHGGGGSVDDDDKSVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFGGGGSGGGSEVQLVESGGGLVQAGGSLRLSCAASGNIFANNIMGWYRQPPGKEREFVAHVSHDGDSMYAVSVKGRFAISRKDATNLYLQMNSLKPEDTAIYFC RLLNIPTQGRMEGFWGQGTQVTVSSGGGGSGGGSQVQLVESGGGLVQTGGSLRLSCAASGSTLVGHSVGFFRQAPGKEREPVVCISSTETVMYYTNSVKGRFTISRDNAKNTVWLQMDRLAPEDTAVYYCAARPQLCSALDYRYNSWGPGTQVTVSS (SEQ ID NO.21).

[0133] 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:

[0134] ATGGAATTTAAACGTAAACGGCATCACCATCACCATCACGGTGGAGGCGGGAGTGTAGACGATGACGATAAGTCCGTCTCCGAGATTCAGCTGATGCACAACCTGGGCAAACACCTGAACTCCATGGAACGCGTGGAATGGCTGCGTAAAAAGCTGCAGGATGTTCATAACTTCGGTGGTGGCGGTAGCGGCGGCGGTAGTGAAGTTCAGCTGGTTGAAAGCGGTGGTGGCCTGGTTCAGGCAGGTGGCAGTCTGCGCCTGAGTTGTGCCGCAAGCGGTAATATTTTTGCAAATAATATCATGGGCTGGTATCGCCAGCCGCCGGGTAAAGAACGCGAATTTGTGGCCCATGTTAGCCATGATGGTGACAGCATGTATGCAGTTAGTGTTAAAGGTCGCTTTGCAATTAGCCGTAAAGATGCCACCAATCTGTATCTGCAGATGAATAGCCTGAAACCGGAAGATACCGCAATCTATTTTTGTCGTCTGCTGAATATTCCGACCCAGGGCCGTATGGAAGGTTTTTGGGGTCAGGGTACCCAGGTGACCGTTAGTAGTGGTGGTGGCGGTTCCGGTGGCGGTTCTCAGGTGCAGCTGGTGGAAAGCGGCGGTGGCCTGGTTCAGACCGGCGGTAGTCTGCGCCTGAGCTGCGCAGCAAGTGGCAGTACCCTGGTGGGCCATAGTGTGGGCTTTTTCCGTCAGGCACCGGGTAAAGAACGTGAACCGGTTGTTTGCATTAGCAGTACCGAAACCGTTATGTATTATACCAATAGTGTGAAAGGCCGCTTTACCATTAGTCGCGATAATGCAAAAAATACCGTTTGGCTGCAGATGGATCGCCTGGCCCCGGAAGATACCGCAGTGTATTATTGTGCCGCACGCCCGCAGCTGTGTAGCGCCTTAGATTATCGTTATAATAGTTGGGGTCCGGGTACCCAGGTGACCGTTAGTAGT(SEQ ID NO.22)。

[0135] Example 7: PTH(1-34)-NbHSA-VHH22A3

[0136] This example provides a dual ECD binding molecule fusion protein T002G-G, which is obtained by sequentially connecting PTH (1-34), NbHSA nanobody and VHH22A3, wherein VHH22A3 is an anti-PTHR1 nanobody, and the amino acid sequence information of the fusion protein T002G-G is as follows:

[0137] MEFKRKRHHHHHHGGGGSVDDDDKSVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFGGGGSGGGGSQVQLVESGGGLVQTGGSLRLSCAASGSTLVGHSVGFFRQAPGKEREPVVCISSTETVMYYTNSVKGRFTISRDNAKNTVWLQMDRLAPEDTAVY YCAARPQLCSALDYRYNSWGPGTQVTVSSGGGGSGGGSEVQLVESGGGLVQAGGSLRLSCAASGNIFANNIMGWYRQPPGKEREFVAHVSHDGDSMYAVSVKGRFAISRKDATNLYLQMNSLKPEDTAIYFCRLLNIPTQGRMEGFWGQGTQVTVSS (SEQ ID NO.23).

[0138] 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:

[0139] ATGGAATTTAAACGTAAACGGCATCACCATCACCATCACGGTGGAGGCGGGAGTGTAGACGATGACGATAAGTCCGTCTCCGAGATTCAGCTGATGCACAACCTGGGCAAACACCTGAACTCCATGGAACGCGTGGAATGGCTGCGTAAAAAGCTGCAGGATGTTCATAACTTCGGTGGTGGCGGTTCCGGTGGCGGTTCTCAGGTGCAGCTGGTGGAAAGCGGCGGTGGCCTGGTTCAGACCGGCGGTAGTCTGCGCCTGAGCTGCGCAGCAAGTGGCAGTACCCTGGTGGGCCATAGTGTGGGCTTTTTCCGTCAGGCACCGGGTAAAGAACGTGAACCGGTTGTTTGCATTAGCAGTACCGAAACCGTTATGTATTATACCAATAGTGTGAAAGGCCGCTTTACCATTAGTCGCGATAATGCAAAAAATACCGTTTGGCTGCAGATGGATCGCCTGGCCCCGGAAGATACCGCAGTGTATTATTGTGCCGCACGCCCGCAGCTGTGTAGCGCCTTAGATTATCGTTATAATAGTTGGGGTCCGGGTACCCAGGTGACCGTTAGTAGTGGTGGTGGCGGTAGCGGCGGCGGTAGTGAAGTTCAGCTGGTTGAAAGCGGTGGTGGCCTGGTTCAGGCAGGTGGCAGTCTGCGCCTGAGTTGTGCCGCAAGCGGTAATATTTTTGCAAATAATATCATGGGCTGGTATCGCCAGCCGCCGGGTAAAGAACGCGAATTTGTGGCCCATGTTAGCCATGATGGTGACAGCATGTATGCAGTTAGTGTTAAAGGTCGCTTTGCAATTAGCCGTAAAGATGCCACCAATCTGTATCTGCAGATGAATAGCCTGAAACCGGAAGATACCGCAATCTATTTTTGTCGTCTGCTGAATATTCCGACCCAGGGCCGTATGGAAGGTTTTTGGGGTCAGGGTACCCAGGTGACCGTTAGTAGT(SEQ ID NO.24)。

[0140] Example 8: PTH(1-34)-PTHrP(15-36)-NbHSA

[0141] This example provides a dual ECD binding molecule fusion protein T002G-H, which is obtained by sequentially connecting PTH (1-34), PTHrP (15-36) and NbHSA nanobody, wherein the linker is GGGGS, and its amino acid sequence information is as follows:

[0142] MEFKRKRHHHHHHGGGGSVDDDDKSVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNF GGGGS IQDLRRRFFLHHLIAEIHTAEIGGGGSGGGSQVQLVESGGGLVQTGGSLRLSCAASGSTLVGHSVGFFRQAPGKEREPVVCISSTETVMYYTNSVKGRFTISRDNAKNTVWLQMDRLAPEDTAVYYCAARPQLCSALDYRYNSWGPGTQVTVSS (SEQ ID NO. 25).

[0143] 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:

[0144] (SEQ ID NO.26).

[0145] Example 9: PTH(1-34)-PTHrP(15-36)-NbHSA

[0146] This example provides a dual ECD binding molecule fusion protein T002G-I, which is obtained by sequentially connecting PTH (1-34), PTHrP (15-36) and NbHSA nanobody, wherein the linker is GGGGSGGGGSGGGGS, and its amino acid sequence information is as follows:

[0147] MEFKRKRHHHHHHGGGGSVDDDDKSVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNF GGGGSGGGGS GGGGSIQDLRRRFFLHHLIAEIHTAEIGGGGSGGGSQVQLVESGGGLVQTGGSLRLSCAASGSTLVGHSVGFFRQAPGKEREPVVCISSTETVMYYTNSVKGRFTISRDNAKNTVWLQMDRLAPEDTAVYYCAARPQLCSALDYRYNSWGPGTQVTVSS (SEQ ID NO.27).

[0148] 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. The coding nucleotide sequence information of the fusion protein T002G-I is as follows:

[0149] ATGGAATTTAAACGTAAACGGCATCACCATCACCATCACGGTGGAGGCGGGAGTGTAGACGATGACGATAAGTCCGTCTCCGAGATTCAGCTGATGCACAACCTGGGCAAACACCTGAACTCCATGGAACGCGTGGAATGGCTGCGTAAAAAGCTGCAGGATGTTCATAACTTCGGCGGTGGTGGCAGTGGTGGCGGTGGCAGTGGCGGTGGTGGTAGTATTCAGGATCTGCGCCGTCGCTTTTTCCTGCATCATCTGATTGCAGAAATTCATACCGCCGAAATTGGTGGTGGCGGTTCCGGTGGCGGTTCTCAGGTGCAGCTGGTGGAAAGCGGCGGTGGCCTGGTTCAGACCGGCGGTAGTCTGCGCCTGAGCTGCGCAGCAAGTGGCAGTACCCTGGTGGGCCATAGTGTGGGCTTTTTCCGTCAGGCACCGGGTAAAGAACGTGAACCGGTTGTTTGCATTAGCAGTACCGAAACCGTTATGTATTATACCAATAGTGTGAAAGGCCGCTTTACCATTAGTCGCGATAATGCAAAAAATACCGTTTGGCTGCAGATGGATCGCCTGGCCCCGGAAGATACCGCAGTGTATTATTGTGCCGCACGCCCGCAGCTGTGTAGCGCCTTAGATTATCGTTATAATAGTTGGGGTCCGGGTACCCAGGTGACCGTTAGTAGT (SEQ ID NO.28).

[0150] Example 10: PTH(1-34)-VHH22A3-NbHSA-PTHrP(15-36)

[0151] This example provides a three-ECD binding molecule fusion protein T002G-J, which is obtained by sequentially connecting PTH (1-34), VHH22A3, NbHSA nanobody and PTHrP (15-36) molecules, and its amino acid sequence information is as follows:

[0152] MEFKRKRHHHHHHGGGGSVDDDDKSVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFGGGGSGGGSEVQLVESGGGLVQAGGSLRLSCAASGNIFANNIMGWYRQPPGKEREFVAHVSHDGDSMYAVSVKGRFAISRKDATNLYLQMNSLKPEDTAIYFCRLLNIPTQGRMEGFW GQGTQVTVSSGGGGSGGGSQVQLVESGGGLVQTGGSLRLSCAASGSTLVGHSVGFFRQAPGKEREPVVCISSTETVMYYTNSVKGRFTISRDNAKNTVWLQMDRLAPEDTAVYYCAARPQLCSALDYRYNSWGPGTQVTVSSGGGGSGGGSIQDLRRRFFLHHLIAEIHTAEI (SEQ ID NO.29).

[0153] 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:

[0154]

[0155] Example 11: PTH(1-34)-PTHrP(15-36)-NbHSA-VHH22A3

[0156] This example provides a three-ECD binding molecule fusion protein T002G-K, which is obtained by sequentially connecting PTH (1-34), PTHrP (15-36), NbHSA nanobody and VHH22A3 molecules, and its amino acid sequence information is as follows:

[0157] MEFKRKRHHHHHHGGGGSVDDDDKSVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFGGGGSGGGSIQDLRRRFFLHHLIAEIHTAEIGGGGSGGGSQVQLVESGGGLVQTGGSLRLSCAASGSTLVGHSVGFFRQAPGKEREPVVCISSTETVMYYTNSVKGRFTISRDNAKNT VWLQMDRLAPEDTAVYYCAARPQLCSALDYRYNSWGPGTQVTVSSGGGGSGGGSEVQLVESGGGLVQAGGSLRLSCAASGNIFANNIMGWYRQPPGKEREFVAHVSHDGDSMYAVSVKGRFAISRKDATNLYLQMNSLKPEDTAIYFCRLLNIPTQGRMEGFWGQGTQVTVSS (SEQ ID NO.31).

[0158] 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:

[0159]

[0160] Test example 1: Biological activity verification

[0161] The biological activities of the fusion proteins and NbHSA prepared in Examples 1 to 11 were detected according to the teriparatide biological activity detection method, which specifically includes the following steps:

[0162] pGL4.29 was transfected and screened on UMR-106 cells expressing PTHR1, and the selected stable transfected cell lines were tested for biological activity. Stable transfected cells were cultured at 2×10 5 The cells were cultured at a density of 1.5 × 10 μL in 96-well white transparent bottom plates at 37°C for 24 hours, and then 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, and 50uL of fluorescent substrate was added after 6 hours of stimulation. The cells were then placed on a microplate reader for reading and the average value was taken.

[0163] The test results are shown in Table 1.

[0164] Table 1: Biological activity test results

[0165]

[0166] The results showed that the biological activity of the fusion protein decreased slightly with the increase of ECD binding domains, which may be related 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 24.285 pm, and the EC50 of the fusion protein of multiple ECD binding molecules was between 47.361-74.005 pm, but the EC50 of the two ECD binding fusion protein and the three ECD binding fusion protein was not much different, and the length of the Linker had little effect on the activity of the sample.

[0167] Test Example 2: Animal Efficacy Determination

[0168] 8-week-old C57 male mice were selected, and the fusion proteins and reference substance TP prepared in Examples 1 to 11 were diluted with PBS to the required concentrations of the experiment. The fusion protein and reference substance TP were injected intraperitoneally at two drug doses of 20 nmoL / kg and / or 10 nmol / kg, with 3 mice in each group, and 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 the calcium azo Ⅲ method detection kit (Zhongshan Biaojia Biology) according to the manufacturer's instructions.

[0169] TP efficacy test results are as follows Figure 5 As shown, the results of the efficacy test of the single ECD binding molecule fusion protein T002G-A Figure 6 As shown. It shows that different doses of TP can increase blood calcium within 2 hours and return to normal within 6 hours; different doses of T002G-A can increase blood calcium within 24 hours and return to normal within 48 hours. In the blank control group treated with normal saline, the blood calcium of mice slightly decreased during the recovery process because the blood collection interval was too short.

[0170] The results of the drug efficacy assay of the dual ECD binding molecule fusion protein T002G-B~T002G-I are as follows Figure 7 As shown, A is the fusion protein T002G-B and T002G-C, B is the fusion protein T002G-D and T002G-E, C is the fusion protein T002G-F and T002G-G, and D is the fusion protein T002G-H and T002G-I. It shows that compared with the fusion protein T002G-A, further fusion of PTH (15-34) with the same ECD binding cannot further increase its pharmacodynamic action time and intensity (such as T002G-B and T002G-C). Its calcium rise time remains at 24 hours, and the maximum blood calcium concentration remains at around 2.5mmol. This may be due to the same ECD binding region molecule having the same affinity with the cell surface receptor, and the two ECD binding molecules bind to the receptor at the same time and dissociate from the receptor at the same time, so the action time of the signaling region activating the receptor is not prolonged.

[0171] Furthermore, different ECD binding molecules, including PTHrP (15-36) and VHH22A3, can prolong the duration of drug efficacy to a certain extent, especially PTHrP (15-36), whether it is linked to the front or back of NbHSA (such as T002G-D and T002G-E), regardless of the length of its linker, it can prolong the calcium rise time to 48 hours, and the entire hypercalcemia time is maintained for about 72 hours. This may be due to the difference in the affinity between different ECD binding molecules and the receptors on the cell surface. The molecule that first binds to the receptor ECD helps the binding of another ECD binding molecule. After the dissociation of the first ECD binding molecule, the signaling region still activates the receptor. Only when the two differentiated ECD binding molecules are dissociated, the signaling region activates the receptor. The action of receptor activation is terminated. Therefore, overall, the duration of receptor activation in the signaling region is prolonged, but due to the relationship between its calcium rise intensity and the balance of blood calcium regulation in mice, the maximum blood calcium concentration is still maintained at about 2.5mmol.

[0172] The results of the drug efficacy assay of the three ECD binding molecule fusion proteins T002G-J and T002G-K are as follows Figure 8 As shown, the fusion protein with three different ECDs has a stronger efficacy, especially T002G-K, which increases calcium in mice within 48 hours and maintains hypercalcemia within 96 hours, and the maximum blood calcium can reach about 4.5mmol.

[0173] Test Example 3: Temperature Stability Study

[0174] This test example tests the temperature stability of the fusion proteins and NbHSA prepared in Examples 1 to 11 above, and the specific method is as follows:

[0175] First, the fusion protein stock solution prepared in the example 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, and then placed at room temperature for 2 hours to measure OD 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 measured. Three replicates were set for each group, and the average was taken.

[0176] The test results are shown in Table 2 and Fig. 9 described.

[0177] Table 2: Temperature stability test results

[0178]

[0179] The results showed that the temperature stability of the single ECD binding molecule T002G-A was average, stable below 37°C, and a large amount of protein loss occurred 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 protein. However, after the addition of the PTHrP nanobody VHH22A3, the temperature stability of the fusion protein was significantly improved regardless of whether it was fused before (T002G-F) or after the human serum protein protein nanobody NbHSA. This may be related to the strong stability of the nanobody itself. Therefore, after fusion expression, the stability of PTH (1-34), which is smaller than it, was significantly improved.

[0180] Furthermore, the test results of the three ECD binding domain fusion proteins T002G-J and T002G-K showed that the addition of PTHrP to the nanoantibody VHH22A3 could improve the temperature stability of the fusion protein molecule, and maintained a strong temperature stability regardless of whether it was fused with the human serum protein nanoantibody NbHSA before (T002G-J) or after (T002G-K), that is, after being treated at 60°C for 4 hours, there was almost no loss of the protein stock solution.

[0181] Test Example 4: Freeze-thaw stability study

[0182] This test example tests the freeze-thaw stability of the fusion proteins prepared in Examples 1 to 11 above, and the specific method is as follows:

[0183] The protein stock solutions prepared in Examples 1 to 11 were diluted to 1 mg / mL with the corresponding buffer and filtered using a 0.22 μm filter membrane. They were then divided into three portions and placed in a -80°C refrigerator for freeze-thaw, with each freeze lasting 30 minutes and thawed naturally at room temperature. Samples were taken at the 0th, 1st, 3rd and 5th times to observe the properties and measure the OD value at a wavelength of 280 nm.

[0184] The test results are shown in Table 3 and Fig.10 shown.

[0185] Table 3: Freeze-thaw stability test results

[0186]

[0187] 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℃ to room temperature, without protein loss or precipitation, indicating that they have excellent freeze-thaw stability.

[0188] Test Example 5: Protein Concentration Study

[0189] In this test example, two recombinants (T002G-J and T002G-K proteins) were selected and their solubility was tested. The specific method is as follows:

[0190] The protein stock solutions obtained in the above-mentioned embodiments 10-11 were diluted to 1 mg / mL with corresponding buffers, 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 solid precipitates were produced) were performed at volumes of 30 mL, 15 mL, 10 mL, 5 mL, 3 mL and 1.5 mL, respectively.

[0191] The test results are shown in Table 4 and Fig.10 shown.

[0192] Table 4 Fusion protein solubility test data

[0193]

[0194] 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.

[0195] The above is a detailed description of the embodiments of the present invention, but the present invention is not limited to the above embodiments. Various changes can be made within the knowledge of ordinary technicians in the relevant technical field without departing from the purpose of the present invention. In addition, the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

Claims

1. A recombinant PTH fusion protein, characterized in that: Include: A1) A signaling domain molecule that activates the PTHR1 intracellular pathway; A2) one or two molecules that can bind to the ECD of PTHR1; A3) Human serum albumin nanobody; Wherein, the amino acid sequence of the signaling domain molecule capable of activating the PTHR1 intracellular pathway is shown in SEQ ID NO.2; When there is one molecule capable of binding to the ECD of PTHR1, the amino acid sequence of the molecule capable of binding to the ECD of PTHR1 is a sequence shown in SEQ ID NO.3, SEQ ID NO.5 or SEQ ID NO.6; When there are two molecules capable of binding to the ECD of PTHR1, the molecules capable of binding to the ECD of PTHR1 are different, and the amino acid sequences of the molecules capable of binding to the ECD of PTHR1 are independently the sequences shown in SEQ ID NO.5 or SEQ ID NO.6; 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 signaling domain molecule capable of activating the intracellular pathway of PTHR1 in the recombinant PTH fusion protein, the molecule capable of binding to the ECD of PTHR1 or the human serum albumin nanobody are connected through a linker.

3. The recombinant PTH fusion protein according to claim 2, characterized in that: The linker comprises an amino acid sequence as shown in any one of SEQ ID NO.8 to SEQ ID NO.

10.

4. The method for constructing a recombinant PTH fusion protein according to any one of claims 1 to 3, 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 3 into a host cell, allowing the encoding gene to be expressed, and obtaining the fusion protein after separation and purification.

5. 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 3; 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).

6. Use of the recombinant PTH fusion protein according to any one of claims 1 to 3 in the preparation of a product for treating and / or preventing hypoparathyroidism.

7. A medicine comprising the recombinant PTH fusion protein according to any one of claims 1 to 3 and a pharmaceutically acceptable excipient.

8. A combined drug comprising the recombinant PTH fusion protein according to any one of claims 1 to 3 and at least one of the drugs according to claim 7, and a tumor targeting drug.

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