Long-acting erythropoiesis-stimulating agent

Abstract

The present invention relates to long-acting erythropoietin (EPO), an EPO mimetic peptide, and a use of the EPO.

Description

A long-acting erythropoiesis stimulant Technical Field

[0001] This invention relates to the field of biotechnology. Specifically, this invention relates to Fc and other long-acting modified fusion proteins of human erythropoietin with extended half-life and its mimic peptides, and their uses. Background Technology

[0002] Erythropoietin (EPO) is a hematopoietic growth factor and an essential glycoprotein in cell production. EPO consists of 165 amino acids with a molecular weight of approximately 34 kDa, of which about 40% is glycosylated, located at Asn24, Asn28, Asn83, and Ser126. Naturally occurring EPO exists in two forms: α-type and β-type. While their glycosides differ slightly, they possess the same potency, biological activity, and molecular weight. In the human body, EPO acts on bone marrow hematopoietic cells, stimulating mitosis and differentiation of erythrocyte precursors, promoting the proliferation and differentiation of erythroid stem cells, and ultimately maturing into an endocrine hormone.

[0003] Because EPO promotes erythrocyte proliferation, increases total hemoglobin levels, and improves the body's oxygen-carrying capacity, it is widely used in the diagnosis and treatment of blood diseases caused by insufficient or defective erythrocyte production. However, rhEPO has a relatively short half-life, requiring administration 1-3 times per week, which reduces patient compliance; furthermore, the different dosages for intravenous and subcutaneous administration increase the workload of nursing staff. Therefore, researchers are focusing on designing long-acting EPO drugs. Currently, the main research directions for long-acting EPO include increasing the glycosylation sites of rhEPO, modifying rhEPO with polyethylene glycol, and constructing Fc fusion proteins. For example, Amgen's long-acting erythropoietin product (Aransp) uses genetic engineering to increase the number of glycosylation sites and improve the degree of glycosylation, thereby improving the half-life of erythropoietin in vivo, allowing for a dosing frequency of once every two weeks. However, this product still cannot avoid the enzymatic effects of proteases in vivo, thus limiting its ability to extend the half-life in vivo, and its production cost is relatively high. Roche's polyethylene glycol-modified EPO drug MIRCERA has further reduced its dosing frequency to once a month.

[0004] Furthermore, erythropoietin mimetic peptide (EMP) has shown activity against the EPO receptor (EPOR), making long-acting modification of EMP a research direction. Hansoh Pharmaceutical's long-acting EMP formulation, pemoxatide, has been approved for marketing. This product utilizes polyethylene glycol-modified EMP, allowing for monthly subcutaneous injections. In a five-week in vivo study in rodents and monkeys, no anti-EPO antibodies were observed, indicating lower immunogenicity compared to recombinant EPO protein products.

[0005] Pemoxatide is chemically synthesized through EMP followed by PEG modification, which presents relatively difficulties in process development and quality control.

[0006] Therefore, there is still a need in the field to develop more ideal EMPs, as well as long-acting molecules obtained through further modifications of them. Summary of the Invention

[0007] This invention links human immunoglobulin Fc to EPO or its mimic peptide via a linker, yielding Fc-modified fusion proteins. Some of these proteins are further modified with XTEN (extended recombinant polypeptide), a biocompatible and biodegradable physiologically inert polypeptide, or with CTP (carboxy-terminal peptide), which increases the sialic acid content of the recombinant fusion protein and protects it from protease degradation. This invention provides a more bioactive erythropoiesis stimulant with a longer in vivo half-life, namely, an EPO or EPO mimic peptide fusion protein containing the above modifications.

[0008] In one aspect, this application provides an EPO mimic peptide (EMP) having a sequence selected from SEQ ID NO:5-10.

[0009] In one embodiment, the EPO mimic peptide (EMP) has the sequence shown in SEQ ID NO:9.

[0010] In one embodiment, the EPO mimic peptide (EMP) is used to construct a long-acting erythropoiesis stimulant.

[0011] Secondly, this application provides a long-acting erythropoiesis stimulant, wherein the erythropoiesis stimulant includes modified EPO or EMP.

[0012] In one embodiment, the modification is selected from one or more of the Fc modification, CTP modification, and XTEN modification.

[0013] In one implementation, the modification is an Fc modification and a CTP modification.

[0014] In one implementation, the modification is an Fc modification and an XTEN modification.

[0015] In one implementation, the modification is an Fc modification, a CTP modification, and an XTEN modification.

[0016] In one embodiment, the modification is connected to EPO or EMP via a connector.

[0017] In one embodiment, the linker is (GmS)nAp, where n and m are integers from 1 to 10, p is an integer from 0 to 10, G represents glycine (Gly), S represents serine (Ser), and A represents alanine (Ala).

[0018] In one embodiment, the connector is selected from connectors having sequences as shown in SEQ ID NO:12-SEQ ID NO:16 and SEQ ID NO:39.

[0019] In one embodiment, the Fc modification is a human IgG1 or IgG4 wild-type Fc sequence.

[0020] In one embodiment, the Fc modification is a human IgG1 or IgG4 Fc mutant sequence.

[0021] In one embodiment, the Fc modification is a human IgG1 or IgG4 Fc mutant sequence, the mutation giving the protein a terminal half-life extension, a half-antibody formation inhibition effect, and / or a function to reduce effectors.

[0022] In one embodiment, the mutation that prolongs the terminal half-life of the protein is M252Y / S254T / T256E (YTE); the mutation that inhibits half-antibody formation is S228P (for IgG4); the mutation that reduces effector function is selected from one or more combinations of E233P, F234A, L235A, D265A or R409K (for IgG4), or one or more combinations of L234A, L235A, P331S (for IgG1), according to EU index numbers.

[0023] In one embodiment, the Fc has a sequence selected from SEQ ID NO:17-SEQ ID NO:21.

[0024] In one implementation, the CTP sequence has a sequence known to those skilled in the art or a sequence disclosed in the prior art.

[0025] In one embodiment, the CTP sequence is as shown in SEQ ID NO:3.

[0026] In one embodiment, the XTEN sequence has a sequence known to those skilled in the art or a sequence disclosed in the prior art.

[0027] In one embodiment, the XTEN sequence is as shown in SEQ ID NO:4.

[0028] In one embodiment, the Fc sequence is linked to the carboxyl terminus (C-terminus) of EPO or EMP.

[0029] In one embodiment, the Fc sequence is connected to EPO or EMP via a linker.

[0030] In one embodiment, the linker is (GmS)nAp, where n and m are integers from 1 to 10, p is an integer from 0 to 10, G represents glycine (Gly), S represents serine (Ser), and A represents alanine (Ala).

[0031] In one embodiment, the connector is selected from connectors having sequences as shown in SEQ ID NO:12-SEQ ID NO:16 and SEQ ID NO:39.

[0032] In one embodiment, the CTP sequence is linked to the amino terminus (N-terminus) of EPO or EMP.

[0033] In one embodiment, the XTEN sequence is linked to the amino (N-terminus) or carboxyl (C-terminus) end of EPO or EMP.

[0034] In one embodiment, the EPO is a human wild-type EPO sequence, the amino acid sequence of which is shown in SEQ ID NO:1.

[0035] In one embodiment, the EPO is a mutant of the human EPO sequence, the amino acid sequence of which is shown in SEQ ID NO:2.

[0036] In one embodiment, the EMP has an amino acid sequence selected from SEQ ID NO:5-SEQ ID NO:11.

[0037] In one embodiment, the long-acting erythropoiesis stimulant comprises one or two EMP sequences.

[0038] In one embodiment, the long-acting erythropoiesis stimulant includes an EMP sequence, with an XTEN or CTP sequence linked to its N-terminus and an Fc sequence linked to its C-terminus.

[0039] In one embodiment, the EMP has an amino acid sequence selected from SEQ ID NO:5-SEQ ID NO:11, and preferably, the EMP has an amino acid sequence as shown in SEQ ID NO:9.

[0040] In one embodiment, the XTEN sequence has a sequence known to those skilled in the art or a sequence disclosed in the prior art. Preferably, the XTEN sequence is as shown in SEQ ID NO:4 or has 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:4. The sequence having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:4 has substantially the same function as SEQ ID NO:4.

[0041] In one embodiment, the XTEN sequence is a truncated version of the sequence shown in SEQ ID NO:4, which retains the function of XTEN, which can be detected by detection methods known to those skilled in the art.

[0042] In one embodiment, the CTP sequence has a sequence known to those skilled in the art or a sequence disclosed in the prior art. Preferably, the CTP sequence is as shown in SEQ ID NO:3 or has 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:3. The sequence having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:3 has substantially the same function as SEQ ID NO:3.

[0043] In one embodiment, the CTP sequence is a truncated version of the sequence shown in SEQ ID NO:3, or a polypeptide containing the sequence shown in SEQ ID NO:3, wherein the truncated version or polypeptide retains the function of the CTP, and the function of the CTP can be detected by detection methods known to those skilled in the art.

[0044] In one embodiment, the Fc is a human IgG1 or IgG4 wild-type Fc sequence or a human IgG1 or IgG4 Fc mutant sequence.

[0045] In one embodiment, the Fc is a human IgG1 or IgG4 Fc mutant sequence, the mutation causing the protein to have a terminal half-life extension, a half-antibody formation inhibition effect, and / or a reduced effector function.

[0046] In one embodiment, the mutation that prolongs the terminal half-life of the protein is one or more combinations of mutations of M252Y, S254T, T256E, M428L, and N434S; the mutation that inhibits half-antibody formation is S228P (for IgG4); the mutation that reduces effector function is selected from one or more combinations of mutations of E233P, F234A, L235A, D265A, or R409K (for IgG4), or one or more combinations of mutations of L234A, L235A, and P331S (for IgG1), according to EU index numbers.

[0047] In one embodiment, the Fc has a sequence selected from SEQ ID NO:17-SEQ ID NO:21.

[0048] In one embodiment, the long-acting erythropoiesis stimulant includes an EMP sequence, an XTEN sequence linked to its N-terminus, and an Fc sequence linked to its C-terminus via a linker.

[0049] Preferably, the linker is (GmS)nAp, where n and m are integers from 1 to 10, p is an integer from 0 to 10, G represents glycine (Gly), S represents serine (Ser), and A represents alanine (Ala).

[0050] More preferably, the connector is selected from connectors having the sequences shown in SEQ ID NO:12-SEQ ID NO:16 and SEQ ID NO:39.

[0051] More preferably, the connector has a sequence as shown in SEQ ID NO:13.

[0052] In one embodiment, the long-acting erythropoiesis stimulant has a sequence as shown in SEQ ID NO:27 or SEQ ID NO:30, or has 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence shown in SEQ ID NO:27 or SEQ ID NO:30. The sequence having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence shown in SEQ ID NO:27 or SEQ ID NO:30 has substantially the same function as SEQ ID NO:27 or SEQ ID NO:30.

[0053] Thirdly, this application provides a pharmaceutical composition comprising the aforementioned long-acting erythropoiesis stimulant.

[0054] In one embodiment, the pharmaceutical composition further comprises a pharmaceutically acceptable drug carrier.

[0055] Fourthly, this application provides the use of the aforementioned long-acting erythropoiesis stimulant or a pharmaceutical composition comprising the aforementioned long-acting erythropoiesis stimulant in the preparation of a medicament for treating a disease characterized by a lack of erythropoietin or a deficiency or defect in the red blood cell population.

[0056] Fifthly, this application provides a method for treating a disease characterized by a lack of erythropoietin or a deficiency or defect in erythrocyte populations, comprising administering to a subject in need an effective amount of the aforementioned long-acting erythropoietin stimulant or a pharmaceutical composition comprising said long-acting erythropoietin stimulant.

[0057] Sixthly, this application provides the aforementioned long-acting erythropoiesis stimulant or a pharmaceutical composition containing the aforementioned long-acting erythropoiesis stimulant for treating diseases characterized by a lack of erythropoietin or a deficiency or defect in erythrocyte populations.

[0058] In the foregoing, preferably, the disease is selected from end-stage renal failure or diseases caused by dialysis, AIDS-related anemia, autoimmune diseases, malignant tumors, cystic fibrosis, early anemia of prematurity, anemia associated with chronic inflammatory diseases, spinal cord injury, and acute blood loss.

[0059] More preferably, the disease is kidney-related anemia.

[0060] The present invention also relates to the use of the aforementioned long-acting erythropoiesis stimulant or a pharmaceutical composition comprising the aforementioned long-acting erythropoiesis stimulant in the preparation of a medicament for promoting erythropoiesis.

[0061] definition

[0062] "Effective subfunction"

[0063] These refer to the biological activities attributable to the Fc region of an antibody, which vary across different antibody isotypes. Examples of antibody effector functions include: C1q binding and complement-dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis (ADCP); downregulation of cell surface receptors (e.g., B cell receptors); and B cell activation.

[0064] "Effective therapeutic dose"

[0065] This indicates that the product, when administered in single or multiple doses to a patient (e.g., a person), is effective in treating, preventing, preventing onset, curing, delaying, reducing the severity of, improving, or prolonging the patient’s life beyond what would be expected without such treatment.

[0066] "Pharmaceutical acceptable"

[0067] This refers to non-toxic materials that do not interfere with the bioactivity and effectiveness of the active ingredient.

[0068] "EU Index"

[0069] Unless otherwise stated herein, amino acid residues in the Fc region or constant region are numbered according to the EU numbering system, also known as the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD (1991).

[0070] "XaaaY" (e.g., L234A, etc.)

[0071] "aaa" represents the sequence position of an amino acid or base (when a specific reference sequence exists, "aaa" represents the residue order in that reference sequence; or it can be numbered according to the commonly used position numbering system in the field, such as the EU numbering system, etc.), "X" represents the original amino acid or base at "aaa", and "Y" represents the changed amino acid or base at "aaa". As an example, when describing the L234A mutation in the human heavy chain constant region, it means that, according to the EU numbering system for the human heavy chain constant region, leucine (L) at position 234 is mutated to alanine (A).

[0072] "Identity"

[0073] The percentage of amino acid residues in the candidate sequence that are identical to those in the specific sequence is determined after aligning a candidate sequence with the specific sequence, introducing vacancies where necessary to achieve the maximum percentage of sequence identity, and without treating any conserved substitutions as part of the sequence identity. Alignment for the purpose of determining the percentage of amino acid sequence identity can be performed in various ways within the skill of the art, such as using publicly available computer software like EMBOSS MATCHER, EMBOSS WATER, EMBOSS STRETCHER, EMBOSS NEEDLE, EMBOSS LALIGN, BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine the parameters suitable for measuring alignment, including any algorithm required to achieve maximum alignment across the full length of the compared sequences. Alignment for the purpose of determining the percentage of amino acid sequence identity can be performed, for example, using the publicly available sequence comparison computer program ALIGN-2. The source code for the ALIGN-2 sequence comparison computer program is available with user documentation at the US Copyright Office, Washington DC, 20559, registered under U.S. Copyright Registration No. TXU510087. The ALIGN-2 program can be compiled for use with UNIX operating systems, such as digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not change. Attached Figure Description

[0074] Figure 1. Results of detecting the proliferation activity of erythropoietin mimic peptide (EMP) on UT-7 cells.

[0075] Figure 2A shows the results of detecting the proliferation activity of EPO fusion protein in UT-7 cells.

[0076] Figure 2B shows the results of detecting the proliferation activity of EMP fusion protein in UT-7 cells.

[0077] Figure 3A shows the effects of the #6 and #9 fusion proteins and pemoxacin on mouse erythropoiesis.

[0078] Figure 3B shows the effects of the #6 and #9 fusion proteins and pemoxacin on hemoglobin production in mice.

[0079] Figure 3C shows the effects of the #6 and #9 fusion proteins and pemoxacin on mouse hematocrit.

[0080] Figure 4A shows the effects of #9 fusion protein and pemoxacin on mouse erythropoiesis.

[0081] Figure 4B shows the effects of #9 fusion protein and pemoxacin on hemoglobin production in mice.

[0082] Figure 4 shows the effects of C #9 fusion protein and pemoxacin on mouse hematocrit.

[0083] Figure 5A shows the results of the high-temperature stability test of the #6-1 fusion protein at 25℃.

[0084] Figure 5B shows the results of the high-temperature stability test of the #6-1 fusion protein at 40℃. Detailed Implementation

[0085] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions or as recommended by the manufacturer. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Furthermore, any methods and materials similar to or equivalent to those described herein may be applied to the methods of the present invention. The preferred embodiments and materials shown herein are for illustrative purposes only.

[0086] The starting materials used in the embodiments of the present invention are known and commercially available, or can be synthesized using or in accordance with methods known in the art.

[0087] Example 1: Design and Synthesis of Erythropoietin Mimic Peptide (EMP)

[0088] Based on molecular design requirements and feasibility analysis, design molecules named JS-EMP-1~JS-EMP-6 and JS-EMP-PM (pemoxanide monovalent) as shown in Table 1 below, and chemically synthesize the corresponding amino acid sequences:

[0089] Table 1 Molecular Design Sequence List

[0090] Example 2: Fortebio detects the affinity of EMP for human EPOR protein.

[0091] In this experiment, the HIS1K biosensor was used to bind ligands, followed by analytes. Human EPOR protein (purchased from Nearshore Proteins, catalog number CW05) was immobilized on the biosensor to a binding response threshold of 0.3 nm. After a 120-s baseline step, the sensor was immersed in the test sample diluted with 0.02% PBST buffer (initial sample concentration 100 μM, 3-fold serial dilution, 7 gradients). The binding and dissociation times of the immobilized protein and analyte were 120 s and 300 s, respectively. Affinity data for the protein receptor were calculated using regression analysis software based on the binding curves. The experimental results are shown in Table 2. JS-EMP-3 and JS-EMP-5 exhibited affinity comparable to JS-EMP-PM.

[0092] Table 2. Affinity of EMP to human EPOR protein as determined by Fortebio.

[0093] Note: ND indicates not detected.

[0094] Example 3: Evaluation of EPO receptor-mediated UT-7 cell proliferation activity

[0095] The bioactivity of EMP was assessed using a cell proliferation assay in the erythropoietin-dependent cell line UT-7. UT-7 cells were cultured in complete medium (CBP60809M, EPO-added) until 1*10⁶ cells / year. 6 Cells were then counted at 4*10^6 cells / ml, washed twice with EPO-free medium (RPMI 1640, 10% FBS), resuspended, and subjected to 24-hour starvation. Cells were counted the following day, and the cell density was adjusted to 4*10^6 cells / ml. 5 Cells / ml, 50 μL of cell suspension was added to each well of a 96-well plate. Serially diluted samples (starting concentration 20 μM, four-fold serial dilutions) with RPMI 1640 (10% FBS) were added to each well, 50 μL / well, and the plates were incubated for 48 h. 100 μL of CTG was added to each well. The Luminescent Cell Viability Assay (purchased from Promega, catalog number G7572) was incubated at room temperature for 3-5 minutes before being read using a microplate reader. Data analysis was performed using GraphPad. The experimental results are shown in Figure 1 and Table 3. The results showed that the proliferation activities of JS-EMP-3 and JS-EMP-5 were weaker than those of JS-EMP-PM, while JS-EMP-4 and JS-EMP-6 showed no proliferation activity.

[0096] Table 3. EC2 cells used for EMP detection of cell proliferation activity 50 Value (nM)

[0097] Note: ND indicates not detected.

[0098] Example 4: Design and Obtaining of Long-Lasting Modified EPO and EMP Fusion Proteins

[0099] Based on the amino acid sequences of fusion proteins #1-#15 (where #1-#5 are EPO fusion proteins and #6-#15 are EMP fusion proteins (all fusion proteins are dimers, consisting of two identical single strands, the sequence of each single strand is shown in Table 4) and the control Efepoetin alfa (CN 114159552A), the corresponding genes were chemically synthesized and cloned into the pcDNA3.1 vector after being coupled with Fc, XTEN, and CTP via a linker (Table 4). Protein expression was performed using a mammalian cell CHO expression system. After 7 days of culture, the cell lysate was centrifuged, and the supernatant was centrifuged at high speed to remove the precipitate and collected. The Protein A / G column was pre-equilibrated with PBS buffer, washed with 2-5 column volumes, and the supernatant sample was loaded onto the column. The column was washed with PBS buffer until the A280 reading dropped to baseline. The target protein was eluted with 0.1M glycine solution at pH 2.7 to 1M solution pre-added to pH 9.0. The fusion protein was neutralized in Tris-HCl buffer. The collected eluent was then exchanged for PBS buffer. The purity of the fusion protein was determined by HPLC-SEC, and the content of the fusion protein was determined by Nanodrop.

[0100] Table 4 Molecular Design Sequence List

[0101] Human EPO wild-type sequence:

[0102] Human EPO mutant sequence:

[0103] Preferred CTP sequences:

[0104] Preferred XTEN sequences:

[0105] Preferred linker sequences:

[0106] Preferred Fc mutant sequence:

[0107] Example 5: Evaluation of the fusion protein's EPO receptor-mediated proliferation activity in UT-7 cells.

[0108] UT-7 was cultured to 1*10⁻⁷ in complete medium (with EPO added). 6Cells were then counted at 4*10^6 cells / ml, washed twice with EPO-free medium (RPMI 1640, 10% FBS), resuspended, and subjected to 24-hour starvation. Cells were counted the following day, and the cell density was adjusted to 4*10^6 cells / ml. 5 Cells / ml, 50 μl of cell suspension was added to each well of a 96-well plate. Serially diluted samples (RPMI 1640, 10% FBS) and the positive control pemoxacin (purchased from Zihaosen Pharmaceutical Co., Ltd., National Drug Approval Number H20230020) were added to each well, mixed with cells at 50 μl / well, and incubated for 48 h. 100 μl of CTG was added to each well. The Luminescent Cell Viability Assay (purchased from Promega, catalog number G7572) was incubated at room temperature for 3-5 minutes before being read using a microplate reader. Data analysis was performed using GraphPad. The experimental results are shown in Figures 2A and 2B and Table 5. EMP fusion proteins #6 and #6-1 showed comparable proliferation activity, but were weaker than the positive control pemoxacin. Fusion proteins #9 and #10 showed even weaker activity.

[0109] Table 5. EC5 assay for fusion protein-mediated cell proliferation activity. 50 Value (nM)

[0110] Note: ND indicates not detected.

[0111] Example 6: Activity assay of the fusion protein in mice

[0112] The effects of #6 and #9 fusion proteins and pemoxacin on erythropoiesis in mice were evaluated by measuring erythrocyte, hemoglobin, and hematocrit levels. Six-week-old male ICR mice were randomly divided into five groups based on baseline erythrocyte, hemoglobin, and hematocrit levels (see Table 6). During the experimental period, whole blood samples were collected and analyzed using an automated blood cell counter to measure cell count, hemoglobin, and hematocrit. The results are shown in Figures 3A-3C and Tables 7-9. A single subcutaneous injection of #6, #9 fusion proteins, and pemoxacin all led to increased peripheral blood erythrocyte count (RBC), hemoglobin (HGB), and hematocrit (HCT) in ICR mice. The stimulatory effect of #6 fusion protein at the 55 nmol / kg dose group on erythrocyte, hemoglobin, and hematocrit in mice was comparable to that of pemoxacin at the 110 nmol / kg dose group; the #9 fusion protein at both dose groups was superior to pemoxacin.

[0113] Table 6 Experimental Groups

[0114] Table 7 Effects of fusion protein and pemoxanide on erythropoiesis in mice

[0115] Table 8. Effects of fusion protein and pemoxacin on hemoglobin production in mice.

[0116] Table 9. Effects of fusion protein and pemoxacin on mouse hematocrit.

[0117] Example 7: Activity assay of #9 fusion protein in mice

[0118] The effects of multiple doses of #9 fusion protein and pemoxacin on erythropoiesis in mice were evaluated. Six-week-old male ICR mice were randomly divided into five groups based on baseline erythrocyte, hemoglobin, and hematocrit levels (see Table 10). During the experimental period, whole blood samples were collected and analyzed for erythrocyte count, hemoglobin, and hematocrit using an automated hematology analyzer. The results are shown in Figures 4A-C and Tables 11-13. A single subcutaneous injection of either #9 fusion protein or pemoxacin resulted in increased peripheral blood erythrocyte count (RBC), hemoglobin (HGB), and hematocrit (HCT) in ICR mice. A single subcutaneous injection of #9 led to a dose-dependent increase in erythrocyte, hemoglobin, and hematocrit levels. The 13.75 nmol / kg dose group of #9 fusion protein showed significantly better stimulatory effects on erythrocytes, hemoglobin, and hematocrit in mice than the 110 nmol / kg dose group of pemoxacin. The 6.875 nmol / kg dose group of #9 fusion protein showed comparable stimulatory effects on erythrocytes, hemoglobin, and hematocrit in mice to the 110 nmol / kg dose group of pemoxacin, but its duration of stimulation was better than that of pemoxacin.

[0119] Table 10 Experimental Groups

[0120] Table 11 Effects of fusion protein and pemoxetine on erythropoiesis in mice

[0121] Table 12 Effects of fusion protein and pemoxacin on hemoglobin production in mice

[0122] Table 13 Effects of fusion protein and pemoxacin on mouse hematocrit

[0123] Example 8: High-Temperature Stability Detection of Fusion Protein

[0124] To assess the stability of the fusion protein under different buffer systems and temperatures, molecule #6-1 was selected for high-temperature stability testing. A certain amount of #6-1 sample was taken and ultrafiltered and concentrated using PBS buffer and His / His-HCl buffer, with a displacement factor exceeding 1000-fold. After displacement, the sample concentration was diluted to 5 g / L. The sample was filtered and aliquoted into vials (200 μL / vial) and placed at 25°C for stability testing. Samples were taken at weeks 0, 1, 2, and 4 for SEC purity determination. The experimental results, shown in Figures 5A and 5B, indicate that #6-1 exhibits good stability under different buffer conditions.

Claims

1. An erythropoietin mimic peptide (EMP) comprising a sequence selected from SEQ ID NO:5-10.

2. The EMP of claim 1, comprising the sequence shown in SEQ ID NO:

9.

3. The EMP as described in any one of claims 1-2, used to construct a long-acting erythropoiesis stimulant.

4. A long-acting erythropoiesis stimulant comprising the EMP of any one of claims 1-2.

5. The long-acting erythropoiesis stimulant of claim 4, further comprising one or more modifications selected from Fc, CTP, and XTEN; preferably, the long-acting erythropoiesis stimulant comprises Fc modification and CTP modification or Fc modification and XTEN modification; more preferably, the Fc modification is linked to the carboxyl terminus of EMP, and the CTP or XTEN sequence is linked to the amino terminus of EMP. Optionally, it is a dimer formed by two identical single chains through Fc.

6. The long-acting erythropoiesis stimulant of claim 5, wherein the Fc modification comprises a human IgG1 or IgG4 Fc mutant sequence, the mutation giving the protein a terminal half-life extension, a half-antibody formation inhibition effect, and / or an effector reduction function; preferably, the Fc comprises a sequence selected from SEQ ID NO:17-SEQ ID NO:

21.

7. The long-acting erythropoiesis stimulant of claim 5 or 6, wherein the CTP comprises the sequence shown in SEQ ID NO:3, or comprises a sequence having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence shown in SEQ ID NO:

3.

8. The long-acting erythropoiesis stimulant according to any one of claims 5-7, wherein the XTEN comprises the sequence shown in SEQ ID NO:4, or comprises a sequence having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence shown in SEQ ID NO:

3.

9. The long-acting erythropoiesis stimulant according to any one of claims 5-8, wherein the Fc sequence is linked to EMP via a linker, the linker being (GmS)nAp, where n and m are integers from 1 to 10, and p is an integer from 0 to 10; preferably, the linker comprises a sequence selected from SEQ ID NO:12-SEQ ID NO:16 and SEQ ID NO:39; more preferably, the linker comprises the sequence shown in SEQ ID NO:

13.

10. A long-acting erythropoiesis stimulant comprising a sequence as shown in SEQ ID NO:27, SEQ ID NO:30 or SEQ ID NO:37, or comprising a sequence having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the sequence shown in SEQ ID NO:27, SEQ ID NO:30 or SEQ ID NO:37; Optionally, it is a dimer formed by two identical single strands through Fc, and each single strand contains any of the above sequences.

11. A pharmaceutical composition comprising the long-acting erythropoiesis stimulant according to any one of claims 3-10.

12. The pharmaceutical composition of claim 11, further comprising a pharmaceutically acceptable pharmaceutical carrier.

13. Use of the EMP of any one of claims 1-3, the long-acting erythropoiesis stimulant of any one of claims 4-10, or the pharmaceutical composition of claim 11 or 12 in the preparation of a medicament for treating a disease characterized by a lack of erythropoietin or a deficiency or defect in the red blood cell population; preferably, the disease is selected from end-stage renal failure or a disease caused by dialysis, AIDS-related anemia, autoimmune diseases, malignant tumors, cystic fibrosis, anemia of early preterm infants, anemia associated with chronic inflammatory diseases, spinal cord injury, and acute blood loss; more preferably, the disease is kidney-related anemia.

14. A method of treating a disease characterized by a lack of erythropoietin or a deficiency or defect in the red blood cell population, comprising administering to a subject in need a therapeutically effective amount of the EMP of any one of claims 1-3, the long-acting erythropoietin stimulant of any one of claims 4-10, or the pharmaceutical composition of claim 11 or 12; preferably, the disease is selected from end-stage renal failure or a disease caused by dialysis, AIDS-related anemia, autoimmune diseases, malignancies, cystic fibrosis, anemia of early preterm infants, anemia associated with chronic inflammatory diseases, spinal cord injury, and acute blood loss; more preferably, the disease is kidney-related anemia.

15. The EMP of any one of claims 1-3, the long-acting erythropoiesis stimulant of any one of claims 4-10, or the pharmaceutical composition of claim 11 or 12, for treating a disease characterized by a lack of erythropoietin or a deficiency or defect in the red blood cell population; preferably, the disease is selected from end-stage renal failure or diseases caused by dialysis, AIDS-related anemia, autoimmune diseases, malignant tumors, cystic fibrosis, anemia in early preterm infants, anemia associated with chronic inflammatory diseases, spinal cord injury, and acute blood loss; more preferably, the disease is kidney-related anemia.

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