CN113563485A - Human long-acting erythropoietin for treating anemia - Google Patents

Human long-acting erythropoietin for treating anemia Download PDF

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Publication number
CN113563485A
CN113563485A CN202110985815.XA CN202110985815A CN113563485A CN 113563485 A CN113563485 A CN 113563485A CN 202110985815 A CN202110985815 A CN 202110985815A CN 113563485 A CN113563485 A CN 113563485A
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epo
terminus
erythropoietin
leu
sequences
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巴萨姆法瑞斯
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Shanghai Yanli Pharmaceutical Co ltd
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Shanghai Yanli Pharmaceutical Co ltd
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • C07K14/505—Erythropoietin [EPO]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—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
    • 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
    • A61K47/62—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 the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/06—Antianaemics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/31—Fusion polypeptide fusions, other than Fc, for prolonged plasma life, e.g. albumin

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Abstract

The invention provides a human erythropoietin with long-acting effect for treating anemia, which is characterized in that a chimeric gene of Erythropoietin (EPO) constructed by using a gene synthesis technology contains one or more CL sequences, wherein the CL sequences can be i) two at C end, ii) one at C end, the other end at N end, iii) one at N end, the other two at C end, iv) two at C end and two at N end, the chimeric genes are sequenced and cloned into eukaryotic expression vectors, the constructed vectors containing the EPO and the CL sequences are transfected into CHO or HEK293 cells, stable clones are selected, culture media of the stable clones are collected, and variants are purified. The long-acting human erythropoietin is developed by linking a carbohydrate linker to the coding sequence of erythropoietin, the long-acting EPO analog is linked to the cloning site of a eukaryotic expression vector, cells are transfected, and clones stably secreting the EPO analog are selected.

Description

Human long-acting erythropoietin for treating anemia
Technical Field
The invention relates to a human long-acting erythropoietin for treating anemia, belonging to genes
The technical field of construction.
Background
Erythropoietin (EPO) is a 34-kDa glycoprotein hormone produced primarily by the capillary endothelial cells of each duct of the kidney, which regulates erythropoiesis by stimulating erythropoiesis. After reduced oxygenation of kidney tissue, EPO synthesis increases, which binds to specific receptors on erythrocyte precursors in the bone marrow, leading to increased proliferation, differentiation and hematocrit. The biological responses associated with EPO include activation of intracellular signaling molecules (e.g., transcription factors such as signal transducers) and activation of activator of transcription (STAT), resulting in growth and differentiation of cells. EPO receptors belong to the family of homodimeric receptors, which require receptor dimerization to trigger the biological response associated with EPO. Anemia in chronic kidney disease patients is caused by a variety of factors, the most common of which is abnormally low levels of erythropoietin. Anemia of erythropoietin deficiency is considered end-stage renal failure, not early renal disease. The absence of erythropoietin causes anemia in humans and animals. EPO is heavily glycosylated with one o-chain and three n-chain oligosaccharide chains. As a result, it was found that the o-linked oligosaccharide chains had no influence on secretion, receptor binding affinity, and in vivo and in vitro biological activities. On the other hand, n-chain oligosaccharides are not active in vitro, but are crucial for in vivo biological activity. The gene encoding human erythropoietin was cloned in 1985, resulting in recombinant human erythropoietin (rHuEPO). rHuEPO was successfully used to treat anemia associated with chronic kidney disease. It is also approved for the treatment of anemia associated with cancer, HIV infection, and for reduction of blood transfusions in surgical settings. When intravenous, one major problem is that the circulation takes about 5 hours. Thus, the use of stimulants available in clinical treatment regimens to treat patients requires frequent EPO injections. Subcutaneous or intravenous injections are recommended 2-3 times per week. Thus, it is expected that increasing the in vivo half-life of EPO will reduce the number of injections per week. Previous studies have shown that sialic acid carbohydrate content of the molecule is directly related to its serum half-life and in vivo biological activity. Studies have shown that binding of the Carboxy Terminal Peptide (CTP) of the subunits to the four o-chain oligosaccharide chain sites of FSH, TSH, GH and EPO cdnas does not affect secretion, receptor binding affinity and in vitro bioactivity of FSH, TSH, GH and EPO cdnas. On the other hand, in vivo, the addition of o-linked oligosaccharides to the protein backbone can significantly extend half-life and longevity. The present applicants hypothesized that the addition of 4 or more N-chain oligosaccharide chains to the backbone of EPO will significantly extend the life of EPO. Thus, in this study, the applicants have linked a Carbohydrate Linker (CL) comprising 4N-chain oligosaccharide recognition sites to the cDNA of EPO. Such a linkage of one or more analogs will significantly increase the potency of erythropoietin in vivo and the half-life in circulation.
Disclosure of Invention
In order to solve the above technical problems, the present invention aims to develop long-acting human erythropoietin (EPO-LA) by linking Carbohydrate Linker (CL) to the coding sequence of Erythropoietin (EPO). One or more CL will be attached to the N-terminus and/or C-terminus of the EPO coding sequence. And connecting the EPO-LA analogue to a cloning site of a eukaryotic expression vector, transfecting CHO or HEK293 cells, and screening out a clone stably secreting the EPO analogue.
The present invention provides a human erythropoietin having long acting properties for treating anemia by designing analogs by attaching one or more Carbohydrate Linker (CL) sequences to the C-terminus or/and N-terminus of EPO using a means of ligating CL to the coding sequence of erythropoietin.
The invention provides a human long-acting erythropoietin for treating anemia, which comprises one or more CL sequences which can be connected to the N terminal and/or the C terminal of EPO.
The wild type amino acid sequence of the human erythropoietin cDNA is shown as SEQ ID NO. 1;
the amino acid sequence of the carbohydrate linker is shown as SEQ ID NO. 2;
the long-acting human erythropoietin chimeric gene (EPO-CL) containing a CL connector has an amino acid sequence shown in SEQ ID NO. 3;
the invention relates to a method for treating anemia by promoting long-acting erythropoietin, which comprises the following steps: the chimeric genes for construction of Erythropoietin (EPO) using gene synthesis techniques contain one or more CL sequences, where the CL sequences can be i) two at the C-terminus, ii) one at the C-terminus, another N-terminus, iii) one at the N-terminus, another two at the C-terminus, iv) two at the C-terminus, and another two at the N-terminus, will be sequenced and cloned into eukaryotic expression vectors, the constructed vectors containing EPO and CL sequences will be transfected into CHO or HEK293 cells, stable clones selected, culture media for collection of stable clones, and EPO variants purified.
The invention further protects the application of the human long-acting erythropoietin for treating anemia in preparing a medicine for treating anemia.
The invention has the following beneficial effects: long acting human erythropoietin (EPO-LA) was developed by linking a Carbohydrate Linker (CL) to the coding sequence of Erythropoietin (EPO), one or more CLs would be linked to the N-and/or C-terminus of the EPO coding sequence. And connecting the EPO-LA analogue to a cloning site of a eukaryotic expression vector, transfecting CHO or HEK293 cells, and screening out a clone stably secreting the EPO analogue. The biological activity of the selected analogs will be tested in vivo and in vitro. The chimeric gene comprising EPO and CL sequences is stable in circulation and has a long half-life. In the treatment of anemic patients, EPO-LA will only need to be injected once a week.
Drawings
FIG. 1 is a sequence structural diagram of EPO- (CL)2 in example 1 of the present invention;
FIG. 2 is a sequence structural diagram of CL-EPO-CL in example 2 of the present invention;
FIG. 3 is a sequence structural diagram of CL-EPO- (CL)2 in example 3 of the present invention;
FIG. 4 is a sequence structural diagram of (CL)2-EPO- (CL)2 in example 4 of the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention, and it is obvious that the embodiments described are only some representative embodiments of the present invention, rather than all embodiments, and all other embodiments obtained by a person skilled in the art without creative efforts belong to the protection scope of the present invention.
The human erythropoietin for treating anemia of this embodiment comprises one or more CL sequences that can be linked to the N-terminus or/and C-terminus of EPO.
Example 1: referring to FIG. 1, the EPO- (CL)2 is synthesized using two CL sequences and a cassette gene using the EPO sequence as a template, wherein 2 CL sequences are included at the C-terminal of the EPO sequence.
Example 2: referring to FIG. 2, the CL-EPO-CL is a template synthesis in which two CL sequences and a cassette gene using EPO sequence as a template are used, wherein 1 CL and 1 CL sequence are contained at the N-terminal and C-terminal of EPO sequence, respectively.
Example 3: referring to FIG. 3, the CL-EPO- (CL)2 is a cassette gene with three CL sequences and EPO sequence as templates, which contains 1 CL and 2 CL sequences at the N-terminal and C-terminal of EPO sequence, respectively, and is used for template synthesis.
Example 4: referring to FIG. four, the (CL)2-EPO- (CL)2 is a cassette gene with four CL sequences and EPO sequence as templates, which contains 2 CL sequences at the N-and C-terminals of EPO sequence, respectively, and is used for template synthesis.
This example for in vivo studies, male ICR mice will be housed in an air-conditioned cubicle with a 12 hour light/dark schedule, with standard food and water provided ad libitum. Animals will be treated with EPO variants: wherein the EPO-wild type is treated 1-3 times per week and the EPO variant is treated 1 time per week. Animals were weighed and equal amounts of EPO variant per subcutaneous SC (0.2 mL/animal).
The frequency of treatment was three times per week (days 1, 3, 5) or once per week. Hematocrit levels were measured three times a week and the experiment was stopped after three weeks.
Blood pressure determination blood samples obtained from the filling of two heparinized microperfusion tubes from the inferior vena cava under anesthesia were used. In addition, since these cells are present in the blood for about 48 hours before they develop into mature red blood cells, reticulocyte counts will be used.
Reticulocytes were evaluated as appropriate for acute experimental systems. Blood was collected from each puppy by cardiac puncture and placed in EDTA-coated tubes. The brilliant cresyl blue was then mixed and incubated at 37C for 20 minutes. Blood and stain were then smeared on the slides and reticulocyte counts were assessed using a 100-fold oil objective. The metabolic clearance of EPO-WT and EPO-CLs was determined by intravenous injection of 20IU (per animal) into male ICR mice. At selected time intervals following injection, blood samples were collected and EPO immunoreactivity was measured by RIA. EPO-CL analogs stable EPO analog clones were selected from CHO or HEK293 cells transfected with EPO-CL analogs. The biological activity of the selected analogs was tested in vivo and in vitro. The chimeric gene containing the sequences of EPO and CL is capable of producing long-lasting EPO and can be injected into a patient only once a week.
Various modifications may be made to the above without departing from the spirit and scope of the invention as defined by the appended claims. The scope of the invention is therefore intended to be limited not by the above description, but rather by the scope of the appended claims.
Sequence listing
<110> Shanghai Yanli pharmaceutical Co., Ltd
<120> a long-acting erythropoietin for treating anemia
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Claims (3)

1. A human long acting erythropoietin for treating anemia, comprising: one or more CL sequences may be attached to the N-terminus or/and C-terminus of EPO;
the wild type amino acid sequence of the human erythropoietin cDNA is shown as SEQ ID NO. 1;
the amino acid sequence of the carbohydrate linker is shown as SEQ ID NO. 2;
the long-acting human erythropoietin chimeric gene (EPO-CL) amino acid sequence containing a CL connector is shown in SEQ ID NO. 3.
2. A method of preparing human erythropoietin having long-acting activity for treating anemia according to claim 1, wherein the method comprises the steps of: the specific method comprises the following steps: the chimeric genes for construction of Erythropoietin (EPO) using gene synthesis techniques contain one or more CL sequences, where the CL sequences can be i) two at the C-terminus, ii) one at the C-terminus, another N-terminus, iii) one at the N-terminus, another two at the C-terminus, iv) two at the C-terminus, and another two at the N-terminus, will be sequenced and cloned into eukaryotic expression vectors, the constructed vectors containing EPO and CL sequences will be transfected into CHO or HEK293 cells, stable clones selected, culture media for collection of stable clones, and EPO variants purified.
3. Use of the human long-acting erythropoietin for treating anemia according to claim 1 in the preparation of a medicament for treating anemia.
CN202110985815.XA 2021-08-26 2021-08-26 Human long-acting erythropoietin for treating anemia Pending CN113563485A (en)

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Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
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Publications (1)

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CN113563485A true CN113563485A (en) 2021-10-29

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