CN114573715A - Recombinant long-acting human hyaluronidase as well as production method and application thereof - Google Patents
Recombinant long-acting human hyaluronidase as well as production method and application thereof Download PDFInfo
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- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2474—Hyaluronoglucosaminidase (3.2.1.35), i.e. hyaluronidase
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- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
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- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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- C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y302/01035—Hyaluronoglucosaminidase (3.2.1.35), i.e. hyaluronidase
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
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- C12N2800/00—Nucleic acids vectors
- C12N2800/10—Plasmid DNA
- C12N2800/106—Plasmid DNA for vertebrates
- C12N2800/107—Plasmid DNA for vertebrates for mammalian
Abstract
The invention discloses a recombinant long-acting human hyaluronidase and a production method and application thereof, and relates to the technical field of hyaluronidase preparation. The recombinant long-acting human hyaluronidase is a fusion protein of a human immunoglobulin IgG1Fc fragment and the extracellular part of human hyaluronidase PH20, and the amino acid sequence of the recombinant long-acting human hyaluronidase is shown as SEQ ID No. 1. The recombinant long-acting human hyaluronidase provided by the invention has the advantages of high safety, long half-life period and high in-vivo activity, and the fused polypeptide belongs to human protein, has lower immunogenicity and is safer to use in vivo. The invention constructs a eukaryotic expression vector for expressing the PH20-IgFc fusion protein and obtains a genetic engineering CHO cell line for stably expressing the PH20-IgFc fusion protein.
Description
Technical Field
The invention relates to the technical field of hyaluronidase preparation, and in particular relates to a recombinant long-acting human hyaluronidase and a production method and application thereof.
Background
Hyaluronic Acid (HA), also known as hyaluronic acid, is a glycosaminoglycan polymerized from N-acetylglucosamine and D-glucuronic acid as disaccharide units, wherein the N-acetylglucosamine and D-glucuronic acid in the disaccharide units are connected by beta-1, 3 glycosidic bonds, and the disaccharide units are polymerized by beta-1, 4 glycosidic bonds. The molecular weight distribution of hyaluronic acid is relatively wide due to the difference in polymerization degree of disaccharide units, and natural HA is mainly high molecular weight hyaluronic acid (HMW HA, Mr2 × 10)6Da) mode exists. Molecular weight is an important parameter determining the biological function of HA. The main component of extracellular matrix is HMW HA, whose hyperviscosity is the source of extracellular matrix resistance to external compressive forces, and HMW HA is also used in ophthalmic surgery depending on hyperviscosity. Low molecular weight hyaluronic acid (LMW HA, 1X 10)5DaMr2×106Da) has good moisture retention and lubricity and is widely used in cosmetics; it was found that ultra-low molecular weight hyaluronic acid (VLMW HA, Mr 1X 10)5Da) has the functions of promoting angiogenesis, promoting wound healing, regulating immunity and resisting tumor, and has important potential application value in the fields of body healing, immunity enhancement, tumor treatment and the like.
There are three main human hyaluronidases, HAase 1, HAase 2 and pH20, among which pH20 is the main enzyme degrading HA in the human body. The hyaluronidase can degrade artificially produced recombinant high molecular weight hyaluronic acid to produce low molecular weight hyaluronic acid. Currently, most of the commercially available hyaluronidases are monopolized by foreign enterprises and are of a small variety.
In view of the above, the present invention is particularly proposed.
Disclosure of Invention
The invention aims to provide a recombinant long-acting human hyaluronidase, a production method thereof and application thereof to solve the technical problems.
The invention is realized in the following way:
the invention provides a recombinant long-acting human hyaluronidase which is a fusion protein of a human immunoglobulin IgG1Fc fragment and the extracellular part of human hyaluronidase PH20, and the amino acid sequence of the recombinant long-acting human hyaluronidase is shown as SEQ ID No. 1.
The recombinant long-acting human hyaluronidase provided by the invention has the advantages of high safety, long half-life period and high in-vivo activity, belongs to human protein, and is safer to use in vivo.
The invention also provides a coding gene of the recombinant long-acting human hyaluronidase, which has a sequence shown as SEQ ID No. 2.
The invention also provides an expression vector which comprises the coding gene of the recombinant long-acting human hyaluronidase.
The invention also provides a host cell which comprises the coding gene of the recombinant long-acting human hyaluronidase. In an alternative embodiment, the host cell is selected from the group consisting of CHO cells.
The invention also provides a method for producing recombinant long-acting human hyaluronidase, which comprises the following steps: expressing a gene sequence SEQ ID No.2 in Chinese hamster CHO by using an animal cell expression vector pMH2 or pMH7 to obtain PH20-IgFc fusion protein, and then separating and purifying to obtain the recombinant human serum albumin; the gene sequences of the expression vectors pMH2 and pMH7 are referenced to the vector sequence provided by http:// www.biovector.net/and are commercially available or synthetically available.
In a preferred embodiment of the present invention, the PH20-IgFc is isolated and purified by Protein G affinity purification.
The invention also provides a recombinant long-acting human hyaluronidase preparationThe recombinant long-acting human hyaluronidase or the recombinant long-acting human hyaluronidase prepared by the method comprises 200-1200IU of recombinant long-acting human hyaluronidase and 10-15mM Na2HPO4， 0.01-0.03％CaCl2，0.1-0.2％EDTA-Na2145-150mM NaCl, 0.1-0.2% HSA, 0.1-0.15% mannitol, and a pH of 5-6.
For example: 200. 500, 700, 800, 900, 1000, 1200IU of recombinant long-acting human hyaluronidase.
The invention also provides a compound preparation, which comprises a recombinant long-acting human hyaluronidase preparation and a medicament.
The compound preparation is in the dosage form of tablet, pill, powder, suspension, gel, emulsion, cream, granule, nanoparticle, capsule, suppository, injection, spray and injection.
In an optional embodiment, the above compound preparation further comprises a pharmaceutically acceptable salt or excipient.
In an alternative embodiment, the drug is a solid tumor treatment drug. Solid tumors include, but are not limited to, head and neck tumors (e.g., laryngeal carcinoma), breast tumors (e.g., breast cancer, lung cancer), digestive system tumors (e.g., gastric cancer, colon cancer, rectal cancer, liver cancer, pancreatic cancer, biliary tract cancer), genitourinary system tumors (e.g., ovarian cancer, cervical cancer, prostate cancer), bone tumors, central nervous system tumors, soft tissue tumors, skin and adnexal tumors, and the like.
The invention also provides the application of the recombinant long-acting human hyaluronidase or the recombinant long-acting human hyaluronidase prepared by the method in preparing a co-used medicament, wherein the co-used medicament prepared from the human hyaluronidase is used for transferring to tissues or organs combined with human immunoglobulin under the condition that the activity of the co-used medicament is not degraded; or for delivering specific therapeutic agents to the tissue or organ in which the disease is located.
Uses of the above co-administered drugs include, but are not limited to: chemotherapy, radiotherapy, photosensitizers, photothermal agents or immunotherapy.
The invention also provides the application of the recombinant long-acting human hyaluronidase or the recombinant long-acting human hyaluronidase prepared by the method in preparing a chemical coupling agent, and the chemical coupling agent prepared by the human hyaluronidase is used for providing non-functional sites for chemically coupling diagnostic agents, developing agents, medicines, toxins for treatment and genes into tissues and organs of diseases through the IgFc of the recombinant long-acting hyaluronidase.
In an alternative embodiment, the recombinant long-acting human hyaluronidase prepared by the above method is used for subcutaneous infusion, periocular surgical anesthesia, chemo-and bio-drug permeation promotion, and contrast agent absorption in urography.
The invention has the following beneficial effects:
the recombinant long-acting human hyaluronidase provided by the invention has the advantages of high safety, long half-life period and high in-vivo activity, and the two sections of polypeptides belong to human proteins, so that the recombinant long-acting human hyaluronidase has lower immunogenicity and is safer to use in vivo. The invention constructs a eukaryotic expression vector for expressing the PH20-IgFc fusion protein and obtains a genetic engineering CHO cell line for stably expressing the PH20-IgFc fusion protein.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and those skilled in the art can also obtain other related drawings based on the drawings without inventive efforts.
FIG. 1 is a schematic diagram of the test result of the stability of the fusion protein PH20-IgFc at 37 ℃.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are conventional products which are not indicated by manufacturers and are commercially available.
The features and properties of the present invention are described in further detail below with reference to examples.
Example 1
The embodiment provides a construction method of a gene expression vector of a fusion protein PH20-IgFc, expression of the fusion protein, immunological detection, detection of the activity of human hyaluronidase and reactor production.
The construction method of the gene expression vector comprises the following steps:
the structural gene PH20-IgFc (SEQ ID No.2) is respectively constructed into expression vectors pMH2 and pMH7 by a PCR method, both are ultrahigh-capacity mammalian cell expression vector plasmids based on a GC-Rich mechanism, and are synthesized by a reagent company. And miniprep of both expression plasmids was performed.
The two expression plasmids prepared above were stably transfected into serum-free suspension cultured CHO cells. Stable clones were manually picked by tip into 96-well plates by G418 selection. When the cell confluency is more than 55%, replacing the serum-free fresh culture medium; collecting culture medium samples after 3-6 hours, detecting expression quantity by anti-IgG 1Fc antibody dot hybridization or ELISA method, selecting multiple monoclonals with highest expression quantity, and continuing to perform serum-free culture and subculture stability study in a small shake flask with a sharp bottom; clones with stable generation were selected for amplification in a torrent reactor.
The results show that: (1) the fusion protein PH20-IgFc is successfully expressed in CHO cells, and dot hybridization and western blot results show that the molecular weight of a band generated by the screened fusion protein PH20-IgFc stable high-expression clone is correct; (2) high expression cloning is carried out serum-free culture and stable passage in a small shake flask with a sharp bottom; (3) the stable high expression clone is subjected to fed-batch culture amplification in a torrent reactor, the activity of the hyaluronidase in each batch of harvest liquid reaches 200-1200 activity units/ml, and the industrial production level is reached.
Example 2
This example provides a method for the isolation and purification of the fusion protein PH 20-IgFc. Specifically, Protein G affinity purification is adopted.
The clone with high expression of PH20-IgFc is harvested, and is centrifuged at 6000rpm for 8min, and the centrifuged supernatant is filtered. The sample was applied to Native Protein G (Alkaline phospholipid) (ab7461) equilibrated with equilibration solution A (20mM Tris +100mM NaCl, pH 7.4), and eluted with eluent B (100mM Gly +50mM Arg + 0.01% Tween 80, pH 3.5) after washing until the baseline was unchanged. The separation and purification of the product was checked by SDS-PAGE.
The purity of the fusion protein PH20-IgFc is more than 95%, and the yield is more than 30%.
Example 3
The embodiment provides a recombinant long-acting human hyaluronidase preparation of a fusion protein PH20-IgFc and in vitro and in vivo activity research.
The recombinant long-acting human hyaluronidase preparation is an injection, and the formula is as follows:
PH20-IgFc+12mM Na2HPO4+0.01％CaCl2+0.1％EDTA－ Na2+150mM NaCl + 0.2% HAS + 0.1% mannitol, pH 5-6.
600U/ml of the solution after the PH20-IgFc filtration was dispensed into 2ml ampoules, and 25 ampoules were placed in an accelerated test incubator at 37 ℃ and a humidity of 75. + -. 5%. Samples were taken on days 0, 7, 14, 21, 28, 35, and 42, respectively, and the stability test results (table 1, fig. 1) showed that a significant decrease (about 12%) in hyaluronidase activity occurred from day 42.
TABLE 1 results of stability test at 37 ℃ for PH20-IgFc
| Time (sky) | Repetition of 1 | Repetition 2 | Repetition 3 | Mean value (U/mL) | Deviation of | |
| 0 | 582 | 609 | 573 | 588.00 | 18.73 | 0.03 |
| 7 | 610 | 576 | 615 | 600.33 | 21.22 | 0.04 |
| 14 | 593 | 559 | 585 | 579.00 | 17.78 | 0.03 |
| 21 | 576 | 542 | 622 | 580.00 | 40.15 | 0.07 |
| 28 | 554 | 548 | 579 | 560.33 | 16.44 | 0.03 |
| 35 | 548 | 541 | 570 | 553.00 | 15.13 | 0.03 |
| 42 | 543 | 521 | 536 | 533.33 | 11.24 | 0.02 |
The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
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ggagtgggag agcaatgggc agccggagaa caactacaag accacgcctc ccgtgctgga 1920
ctccgacggc tccttcttcc tctacagcaa gctcaccgtg gacaagagca ggtggcagca 1980
ggggaacgtc ttctcatgct ccgtgatgca tggggtctgc acaaccacta cacgcagaag 2040
agcctctccc tgtctccggg taaa 2064
Claims (10)
1. The recombinant long-acting human hyaluronidase is a fusion protein of a human immunoglobulin IgG1Fc fragment and the extracellular part of human hyaluronidase PH20, and the amino acid sequence of the recombinant long-acting human hyaluronidase is shown as SEQ ID No. 1.
2. A coding gene of recombinant long-acting human hyaluronidase is characterized by having a sequence shown as SEQ ID No. 2.
3. An expression vector comprising a gene encoding the recombinant long-acting human hyaluronidase of claim 2.
4. A host cell comprising a gene encoding the recombinant long-acting human hyaluronidase of claim 2.
5. A method of producing a recombinant long-acting human hyaluronidase, comprising: expressing a gene sequence SEQ ID No.2 in Chinese hamster CHO cells by using an animal cell expression vector pMH2 or pMH7 to obtain PH20-IgFc fusion protein, and then separating and purifying to obtain the recombinant human immunodeficiency virus (IgFc).
6. The method of claim 5, wherein the PH20-IgFc is isolated and purified using Protein G affinity purification.
7. A recombinant long-acting human hyaluronidase preparation comprising the recombinant long-acting human hyaluronidase of claim 1 or the recombinant long-acting human hyaluronidase prepared by the method of any one of claims 5-6, wherein the preparation comprises 200-1200IU of recombinant long-acting human hyaluronidase, 10-15mM Na2HPO4，0.01-0.03％CaCl2，0.1-0.2％EDTA-Na2145-150mM NaCl, 0.1-0.2% HSA, 0.1-0.15% mannitol, and a pH of 5-6.
8. A combination formulation comprising the recombinant long-acting human hyaluronidase formulation of claim 7 and a drug;
preferably, the drug is a solid tumor treatment drug.
9. Use of the recombinant long-acting human hyaluronidase of claim 1 or prepared by the method of any one of claims 5-6 in the preparation of a co-administered medicament for delivery to a tissue or organ to which a human immunoglobulin binds without degradation of its activity; or for delivering specific therapeutic agents to the tissue or organ in which the disease is located.
10. Use of the recombinant long-acting human hyaluronidase of claim 1 or prepared by the method of any one of claims 5-6 for the preparation of a chemical coupling agent for the chemical coupling of diagnostic agents, imaging agents, drugs, therapeutic toxins, genes into diseased tissues and organs by providing a non-functional site through the IgFc of the recombinant long-acting hyaluronidase.
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