CN113717291A

CN113717291A - Ultra-long-acting stable FSH, expression recombination method thereof and assisted reproduction technology

Info

Publication number: CN113717291A
Application number: CN202111093598.XA
Authority: CN
Inventors: 刘建喜
Original assignee: Nantong University
Current assignee: Nantong University
Priority date: 2021-09-17
Filing date: 2021-09-17
Publication date: 2021-11-30
Anticipated expiration: 2041-09-17
Also published as: CN113717291B

Abstract

The invention provides ultra-long-acting stable FSH, an expression recombination method thereof and an assisted reproduction technology, and relates to the technical field of biological pharmacy, wherein the amino terminal of an alpha chain and the carboxyl terminal of a beta chain of the FSH are respectively fused with CTP, Fc and luciferase size subunits. The CTP is the CTP of horse or zebra, the Fc is the heavy chain Fc segment of human immunoglobulin IgG1, and the luciferase size subunit is the large (LgBiT) small subunit (HIBIT) with extremely high affinity for Nano luciferase. The half-life period of the FSH with ultra-long effect and stability in the in-vivo experiment process of a mouse is more than 96 hours, the FSH can meet the requirement that the FSH is only injected for 1 to 2 times in the in-vitro reproductive treatment process within 8 to 12 days, the administration complexity is reduced, the side effect caused by repeated injection of the medicament is avoided, and the real long effect is realized.

Description

Ultra-long-acting stable FSH, expression recombination method thereof and assisted reproduction technology

Technical Field

The invention relates to the technical field of biological pharmacy, in particular to ultra-long-acting stable FSH, an expression recombination method thereof and an assisted reproduction technology.

Background

Recombinant human follicle stimulating hormone is an indispensable tool for assisted reproductive technology. FSH is a highly glycosylated protein synthesized and secreted by the megaphone cells of the anterior pituitary and carries out its biological functions by binding to the receptor (FSHR) and transmitting downstream signals.

In the international market, FSH-related biologics are dominated by recombinant products. At present, Gonal-F of Merck Serono (Merck Serono) in Germany and Puregon of Merck (Merck & Co.) in the United states dominate. Conventional FSH products have a short half-life in humans (about 2 hours), and patients undergoing assisted reproductive therapy require 1 subcutaneous injection of FSH per day over a course of 8-12 days. This is a relatively frequent dosing regimen. The FSH-CTP long-acting FSH produced by America Merck company has a half-life of about 69 hours in vivo, but still cannot meet the requirement of one treatment course (8-12 days) of assisted reproductive treatment such as IVF. The domestic FSH mainly occupies the market by the urine source products of the Lizhu group, the price of the product is lower, the clinical curative effect is equivalent to that of a recombinant product, but the problems of unstable raw material source, safety risk brought by urine donors and the like exist.

Disclosure of Invention

The invention aims to solve the technical problem that the half-life period of the traditional FSH product in a human body is short in the prior art.

In order to achieve the purpose, the invention adopts the following technical scheme:

an ultra-long-acting stable FSH, wherein CTP is fused to the amino terminal of the alpha chain and the carboxyl terminal of the beta chain of the FSH respectively, and the CTP is the CTP of a horse or a zebra.

Preferably, the amino terminus of the alpha chain and the carboxy terminus of the beta chain of said FSH are further fused to Fc.

Preferably, the Fc is incorporated into the α chain on FSH and the β chain on FSH by a combination of HIBIT and LgBIT.

The application also provides an expression recombination method of the FSH with ultra-long-term stability, which is used for the expression recombination method of the FSH with ultra-long-term stability, and the eCGCTP glycosylation is utilized to protect the drug protein from being rapidly degraded by protease; fc is used to increase molecular weight to avoid in vivo rapid filtration, the extremely high affinity of the luciferase subunit is used to increase the yield of the heterobipolar body, and the extremely high affinity of the luciferase subunit is used to increase the stability of the heterobipolar body.

The half-life period of the FSH with ultra-long effect and stability in the in-vivo experiment process of a mouse is more than 96 hours, the FSH can meet the requirement that the FSH is only injected for 1 to 2 times in the in-vitro reproductive treatment process within 8 to 12 days, the administration complexity is reduced, the side effect caused by repeated injection of the medicament is avoided, and the real long effect is realized.

Drawings

FIG. 1 shows the crystal structures of FSH and FSHR; the relative spatial positions of FSH α, FSH β and FSHR are shown.

Fig. 2 is a comparison of the biological activities of the amino terminus of the incorporation of eCGCTP into the FSH α chain and the carboxy terminus of the α chain in the present application, wherein a represents the amino terminus of the incorporation of eCGCTP into the FSH α chain, and ● represents the carboxy terminus of the incorporation of eCGCTP into the FSH α chain;

fig. 3 is a schematic structural diagram of an ultra-long effective stable FSH according to an embodiment of the present invention; the structure diagram specifically shows the specific position of the eCGCTP fused into the FSH alpha chain

Fig. 4 is a schematic structural diagram of an ultra-long effective stable FSH according to another embodiment of the present invention; the structure contains Fc and eCGCTP.

Fig. 5 is a schematic structural diagram of ultra-long effective stable FSH according to still another embodiment of the present invention; in the structure, high-affinity luciferase large-size subunits HIBIT and LgBIT are added on the basis of a structure III.

FIG. 6 is a graph comparing the efficiency and potency of an ultra-long acting stable FSH according to one embodiment of the present invention; ● is standard substance, and tangle-solidup is FSH containing Fc and eCGCTP, and the specific structure is structure three in FIG. 4.

FIG. 7 is a bar graph of the recovery efficiency of FSH of structure three and structure four in accordance with the present invention;

fig. 8 is a line graph showing half-life of ultra-long-acting stable FSH according to an embodiment of the present invention. Specifically, expression of purified structure four, half-life in vivo assay.

Part of English explanation:

FSH: follicle stimulating hormone, a hormone secreted by anterior pituitary basophilic cells, and the component of follicle stimulating hormone is glycoprotein;

FSHR: FSH receptor

eCG: pregnant mare serum gonadotropin

CTP: human chorionic gonadotropin beta subunit carboxy terminal peptide

Fc human immunoglobulin IGG heavy chain Fc segment

HIBIT: luciferase high affinity small subunit;

LgBIT: luciferase large subunit.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments.

In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention, however, the present invention may be practiced in other ways than those specifically described herein, and thus the present invention is not limited to the specific embodiments of the present disclosure.

In view of the fact that the crystal structures of FSH and FSHR have been resolved, it can be seen from fig. 1 that the carboxyl terminal of FSH α is deep into the FSHR structure, and that the addition of a fusion protein at the carboxyl terminal apparently pushes away FSHR and reduces the affinity of FSH to FSHR. While the amino terminus of FSH α is free to extend beyond the entire structure. The addition of groups in this direction has been shown to have less effect on the trimer structure. According to molecular design, the free space at the amino terminal of the FSH alpha chain is larger than that at the carboxyl terminal, and the fusion tag has smaller influence on the combination of FSH and a receptor FSHR thereof. The free spaces of the amino terminal and the carboxyl terminal of the FSH beta chain are both larger, and the influence of other tags fused into any direction on FSHR combination of FSH is not large. Referring to fig. 2, it can be seen that the biological activity of the fusion protein at the amino terminus of the FSH α chain is better than that of the fusion protein at the carboxy terminus of the FSH α chain.

Therefore, the present application provides an ultra-long-acting stable FSH, please refer to fig. 3, wherein the amino terminal of the α chain and the carboxy terminal of the β chain of the FSH are fused with CTPs, respectively, and the CTPs are CTPs of horse or zebra, to form FSH of structure one and structure two. According to literature, FSH has a half-life of about 2 hours, human chorionic gonadotropin (hCG) has a half-life of about 35 hours, but pregnant mare serum gonadotropin (eCG) has a half-life of up to 4 days. Alignment found that the beta subunit of human, equine and zebra chorionic gonadotropin is more abundant in amino acids that can be glycosylated at the carboxy terminus, that is twice as much amino acids that can be glycosylated on the beta subunit of human chorionic gonadotropin as compared to human chorionic gonadotropin, and that the use of the carboxy terminus of the subunits of equine or zebra chorionic gonadotropin greatly increases the half-life in FSH.

In one embodiment, referring to fig. 4, the amino terminal of the α chain and the carboxy terminal of the β chain of the FSH are fused with Fc, respectively, to form FSH as shown in structure three, and Fc is grafted into the FSH α chain and the FSH β chain to prolong the half-life of the FSH.

Because of the strong affinity between Fc and Fc, theoretically, three combinations of FSH α Fc-FSH α Fc, FSH β Fc-FSH β Fc and FSH α Fc-FSH β Fc can be used in the actual production process. About half of the dimers are homodimers, which are nonfunctional. In the actual production process, the production efficiency is reduced; in one embodiment, referring to fig. 5, the Fc is integrated into the α chain of FSH and the β chain of FSH by a combination of hilbit and LgBIT to form FSH as structure four, and by adding a combination of high affinity luciferase hilbit and LgBIT size subunits to the dimer structure, the efficiency of heterodimer polymerization is greatly improved, and the inefficient production of homodimers is reduced (fig. 7); specifically, referring to fig. 7, the yield of functional heterodimers in structure three was about 60% when both Fc was incorporated into FSH α and FSH β chains, and the other 40% were α - α, β - β nonfunctional homodimers. However, when high affinity luciferase large and small subunits HIBIT and LgBIT were further introduced based on structure three, structure four was obtained, where the yield of functional heterodimers expressed by the cells was higher than 90% and the amount of non-functional homodimers of α - α, β - β would be less than 10%. Meanwhile, the binding between FSH α and FSH β chains does not involve disulfide bonds, and relies mainly on weak bond binding, relatively loose. By respectively fusing the luciferase HIBIT and the LgBiT large-small subunit into an Fc alpha chain and an Fc beta chain, the affinity of the luciferase HIBIT and the LgBiT large-small subunit is extremely strong. Thus the stability of the heterodimer can be increased.

And (3) verification experiment:

1 protein expression purification

HEK 293 cells were co-transfected with FSH alpha and FSH beta chain expression plasmids, cultured for 48 hours, and the culture supernatant was taken and added with 2M imidazole solution to a final concentration of 20 mM. The impurities were removed by simple filtration through a 0.45 μm filter. Loading 1ml nickel agarose gel nickel NTA agarose gel into column, balancing, loading, eluting, and collecting fractions. And determining a target protein collecting solution by using an ELISA His tag antibody, and carrying out ultrafiltration by using normal saline to remove imidazole. The concentration of the target protein is further determined by ELISA His-tag antibody for use. The structures of partially expressed FSH proteins are shown in fig. 3, 4 and 5. The effects of FSH protein are shown in figures 2, 6, 7 and 8.

2 in vitro FSH Activity assay

Firstly, constructing a stable FSH receptor internal expression cell strain, inducing the internal internalization of the FSH receptor by using FSH, further dyeing the FSH receptor, and calculating the number of FSH receptor particles in cells by using high content image analysis to determine the activity of FSH. The results of the comparative activity experiments for structure three and the standard are shown in fig. 6.

3 measurement of half-life by in vivo administration in mice

Approximately 30 grams of diluted FSH per mouse, 6 per group, was injected subcutaneously at approximately 200 μ l to a final concentration of approximately 33 μ g/kg.

4 blood collection and determination of half-life of FSH concentration in blood in vivo

The injection was set at 0 hours before injection and 40 μ l of tail vein blood was collected at 2, 4, 6, 24, 48, 72, 96, and 120 h post injection intervals.

The HIBIT fluorescein detection kit is used according to the instruction. The higher the chemiluminescent fluorescence value, the greater the amount of FSH in the corresponding sample.

It can be seen from figure 8 that the half-life of FSH described in structure four was greater than 96 hours during the in vivo experiments in mice. Is the most excellent in the long-acting FSH known so far. Can meet the requirement that only 1-2 times of injection is carried out in the process of in vitro reproductive therapy (such as IVF) within 8-12 days, reduces the administration complexity, avoids the side effect caused by repeated injection of the medicine and realizes real long-acting effect.

The above description is only an example of the present invention, and is not intended to limit the present invention, and it is obvious to those skilled in the art that various modifications and variations can be made in the present invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the scope of the claims of the present invention.

Claims

1. An ultra-long acting stable FSH, characterized by: and the amino terminal of the alpha chain and the carboxyl terminal of the beta chain of the FSH are respectively fused with CTP, and the CTP is the CTP of horse or zebra.

2. The ultralong-acting stable FSH of claim 1, wherein: the amino-terminus of the alpha chain and the carboxy-terminus of the beta chain of the FSH are also fused to Fc.

3. The ultralong-acting stable FSH of claim 1, wherein: the Fc is incorporated into the alpha chain on the FSH and the beta chain on FSH by a combination of HIBIT and LgBIT.

4. An expression recombination method of ultra-long-term stable FSH is characterized in that: the recombinant method for expression of ultra-long-acting stable FSH according to any of claims 1 to 3, wherein the drug proteins are protected against rapid degradation by proteases by eCGCTP glycosylation; fc is used to increase molecular weight to avoid in vivo rapid filtration, the extremely high affinity of the luciferase subunit is used to increase the yield of the heterobipolar body, and the extremely high affinity of the luciferase subunit is used to increase the stability of the heterobipolar body.